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Henry Schmitt -

All Publications since 2000


2023 Scientific Article in Neural regeneration research Neural Regen. Res. 18, 652-656 (2023)

Mayer, J.M.# ; Krug, C.# ; Saller, M.M. ; Feuchtinger, A. ; Giunta, R.E. ; Volkmer, E. ; Holzbach, T.

Hypoxic pre-conditioned adipose-derived stem/progenitor cells embedded in fibrin conduits promote peripheral nerve regeneration in a sciatic nerve graft model.

Recent results emphasize the supportive effects of adipose-derived multipotent stem/progenitor cells (ADSPCs) in peripheral nerve recovery. Cultivation under hypoxia is considered to enhance the release of the regenerative potential of ADSPCs. This study aimed to examine whether peripheral nerve regeneration in a rat model of autologous sciatic nerve graft benefits from an additional custom-made fibrin conduit seeded with hypoxic pre-conditioned (2% oxygen for 72 hours) autologous ADSPCs (n = 9). This treatment mode was compared with three others: fibrin conduit seeded with ADSPCs cultivated under normoxic conditions (n = 9); non-cell-carrying conduit (n = 9); and nerve autograft only (n = 9). A 16-week follow-up included functional testing (sciatic functional index and static sciatic index) as well as postmortem muscle mass analyses and morphometric nerve evaluations (histology, g-ratio, axon density, and diameter). At 8 weeks, the hypoxic pre-conditioned group achieved significantly higher sciatic functional index/static sciatic index scores than the other three groups, indicating faster functional regeneration. Furthermore, histologic evaluation showed significantly increased axon outgrowth/branching, axon density, remyelination, and a reduced relative connective tissue area. Hypoxic pre-conditioned ADSPCs seeded in fibrin conduits are a promising adjunct to current nerve autografts. Further studies are needed to understand the underlying cellular mechanism and to investigate a potential application in clinical practice.

2022 Scientific Article in Clinical Chemistry Clin. Chem., DOI: 10.1093/clinchem/hvac191 (2022)

Murakami, M. ; Sun, N. ; Li, F. ; Feuchtinger, A. ; Gomez-Sanchez, C. ; Fassnacht, M. ; Reincke, M. ; Bancos, I. ; Walch, A.K. ; Kroiss, M. ; Beuschlein, F.

In situ metabolomics of cortisol-producing adenomas.

BACKGROUND: Recent advances in omics techniques have allowed detailed genetic characterization of cortisol-producing adrenal adenoma (CPA). In contrast, the pathophysiology of CPAs has not been elucidated in detail on the level of tumor metabolic alterations. METHODS: The current study conducted a comprehensive mass spectrometry imaging (MSI) map of CPAs in relation to clinical phenotypes and immunohistochemical profiles of steroidogenic enzymes. The study cohort comprised 46 patients with adrenal tumors including CPAs (n = 35) and nonfunctional adenomas (n = 11). RESULTS: Severity of cortisol hypersecretion was significantly correlated with 29 metabolites (adjusted P < 0.05). Adrenal androgens derived from the classic androgen pathway were inversely correlated with both cortisol secretion (rs = -0.41, adjusted P = 0.035) and CYP11B1 expression (rs = -0.77, adjusted P = 2.00E-08). The extent of cortisol excess and tumor CYP11B1 expression further correlated with serotonin (rs = 0.48 and 0.62, adjusted P = 0.008 and 2.41E-05). Tumor size was found to be correlated with abundance of 13 fatty acids (adjusted P < 0.05) and negatively associated with 9 polyunsaturated fatty acids including phosphatidic acid 38:8 (rs = -0.56, adjusted P = 0.009). CONCLUSIONS: MSI reveals novel metabolic links between endocrine function and tumorigenesis, which will further support the understanding of CPA pathophysiology.

2022 Scientific Article in Oncology Oncology, DOI: 10.1159/000526436 (2022)

Erlmeier, F.# ; Sun, N.# ; Shen, J. ; Feuchtinger, A. ; Buck, A. ; Prade, V.M. ; Kunzke, T. ; Schraml, P. ; Moch, H. ; Autenrieth, M. ; Weichert, W. ; Hartmann, A. ; Walch, A.K.

MALDI mass spectrometry imaging-Diagnostic pathways and metabolites for renal tumor entities.

Background: Correct tumor subtyping of primary renal tumors is essential for treatment decision in daily routine. Most of the tumors can be classified based on morphology alone. Nevertheless, some diagnoses are difficult, and further investigations are needed for correct tumor subtyping. Besides histochemical investigations, high-mass-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can detect new diagnostic biomarkers and hence improve the diagnostic. Patients and Methods: Formalin-fixed paraffin embedded tissue specimens from clear cell renal cell carcinoma (ccRCC, n = 552), papillary renal cell carcinoma (pRCC, n = 122), chromophobe renal cell carcinoma (chRCC, n = 108), and renal oncocytoma (rO, n = 71) were analyzed by high-mass-resolution MALDI fourier-transform ion cyclotron resonance (FT-ICR) MSI. The SPACiAL pipeline was executed for automated co-registration of histological and molecular features. Pathway enrichment and pathway topology analysis were performed to determine significant differences between RCC subtypes. Results: We discriminated the four histological subtypes (ccRCC, pRCC, chRCC, and rO) and established the subtype-specific pathways and metabolic profiles. rO showed an enrichment of pentose phosphate, taurine and hypotaurine, glycerophospholipid, amino sugar and nucleotide sugar, fructose and mannose, glycine, serine, and threonine pathways. ChRCC is defined by enriched pathways including the amino sugar and nucleotide sugar, fructose and mannose, glycerophospholipid, taurine and hypotaurine, glycine, serine, and threonine pathways. Pyrimidine, amino sugar and nucleotide sugar, glycerophospholipids, and glutathione pathways are enriched in ccRCC. Furthermore, we detected enriched phosphatidylinositol and glycerophospholipid pathways in pRCC. Conclusion: In summary, we performed a classification system with a mean accuracy in tumor discrimination of 85.13%. Furthermore, we detected tumor-specific biomarkers for the four most common primary renal tumors by MALDI-MSI. This method is a useful tool in differential diagnosis and biomarker detection.

2022 Scientific Article in JCI insight JCI insight 7:e162138 (2022)

Buck, A.# ; Prade, V.M.# ; Kunzke, T. ; Erben, R.G.&deg ; Walch, A.K.&deg

Spatial metabolomics reveals upregulation of several pyrophosphate-producing pathways in cortical bone of Hyp mice.

Patients with the renal phosphate-wasting disease X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of XLH, are characterized by loss-of-function mutations in phosphate-regulating endopeptidase homolog X-linked (PHEX), leading to excessive secretion of the bone-derived phosphotropic hormone FGF23. The mineralization defect in patients with XLH and Hyp mice is caused by a combination of hypophosphatemia and local accumulation of mineralization-inhibiting molecules in bone. However, the mechanism by which PHEX deficiency regulates bone cell metabolism remains elusive. Here, we used spatial metabolomics by employing matrix-assisted laser desorption/ionization (MALDI) Fourier-transform ion cyclotron resonance mass spectrometry imaging (MSI) of undecalcified bone cryosections to characterize in situ metabolic changes in bones of Hyp mice in a holistic, unbiased manner. We found complex changes in Hyp bone metabolism, including perturbations in pentose phosphate, purine, pyrimidine, and phospholipid metabolism. Importantly, our study identified an upregulation of several biochemical pathways involved in intra- and extracellular production of the mineralization inhibitor pyrophosphate in the bone matrix of Hyp mice. Our data emphasize the utility of MSI-based spatial metabolomics in bone research and provide holistic in situ insights as to how Phex deficiency-induced changes in biochemical pathways in bone cells are linked to impaired bone mineralization.

2022 Scientific Article in Scientific Reports Sci. Rep. 12:19793 (2022)

Klein-Rodewald, T. ; Micklich, K. ; Sanz-Moreno, A. ; Tost, M. ; Calzada-Wack, J. ; Adler, T. ; Klaften, M. ; Sabrautzki, S. ; Aigner, B. ; Kraiger, M.J. ; Gailus-Durner, V. ; Fuchs, H. ; German Mouse Clinic Consortium (Aguilar-Pimentel, J.A. ; Becker, L. ; Garrett, L. ; Hölter, S.M. ; Prehn, C. ; Rácz, I. ; Rozman, J. ; Puk, O. ; Schrewe, A. ; Adamski, J. ; Esposito, I. ; Wurst, W. ; Stöger, C.) ; Gründer, A. ; Pahl, H. ; Wolf, E. ; Hrabě de Angelis, M. ; Rathkolb, B.

New C3H KitN824K/WT cancer mouse model develops late-onset malignant mammary tumors with high penetrance.

Gastro-intestinal stromal tumors and acute myeloid leukemia induced by activating stem cell factor receptor tyrosine kinase (KIT) mutations are highly malignant. Less clear is the role of KIT mutations in the context of breast cancer. Treatment success of KIT-induced cancers is still unsatisfactory because of primary or secondary resistance to therapy. Mouse models offer essential platforms for studies on molecular disease mechanisms in basic cancer research. In the course of the Munich N-ethyl-N-nitrosourea (ENU) mutagenesis program a mouse line with inherited polycythemia was established. It carries a base-pair exchange in the Kit gene leading to an amino acid exchange at position 824 in the activation loop of KIT. This KIT variant corresponds to the N822K mutation found in human cancers, which is associated with imatinib-resistance. C3H KitN824K/WT mice develop hyperplasia of interstitial cells of Cajal and retention of ingesta in the cecum. In contrast to previous Kit-mutant models, we observe a benign course of gastrointestinal pathology associated with prolonged survival. Female mutants develop mammary carcinomas at late onset and subsequent lung metastasis. The disease model complements existing oncology research platforms. It allows for addressing the role of KIT mutations in breast cancer and identifying genetic and environmental modifiers of disease progression.

2022 Scientific Article in Nature Communications Nat. Commun. 13:4689 (2022)

Koch, J. ; Schober, S.J. ; Hindupur, S.V. ; Klein, F.G. ; Mantwill, K. ; Ehrenfeld, M. ; Schillinger, U. ; Hohnecker, T. ; Qi, P. ; Steiger, K. ; Aichler, M. ; Gschwend, J.E. ; Nawroth, R.&deg ; Holm, P.S.&deg

Targeting the Retinoblastoma/E2F repressive complex by CDK4/6 inhibitors amplifies oncolytic potency of an oncolytic adenovirus.

CDK4/6 inhibitors (CDK4/6i) and oncolytic viruses are promising therapeutic agents for the treatment of various cancers. As single agents, CDK4/6 inhibitors that are approved for the treatment of breast cancer in combination with endocrine therapy cause G1 cell cycle arrest, whereas adenoviruses induce progression into S-phase in infected cells as an integral part of the their life cycle. Both CDK4/6 inhibitors and adenovirus replication target the Retinoblastoma protein albeit for different purposes. Here we show that in combination CDK4/6 inhibitors potentiate the anti-tumor effect of the oncolytic adenovirus XVir-N-31 in bladder cancer and murine Ewing sarcoma xenograft models. This increase in oncolytic potency correlates with an increase in virus-producing cancer cells, enhanced viral genome replication, particle formation and consequently cancer cell killing. The molecular mechanism that regulates this response is fundamentally based on the reduction of Retinoblastoma protein expression levels by CDK4/6 inhibitors.

2022 Scientific Article in Frontiers in Immunology Front. Immunol. 13:913275 (2022)

Kuhn, L.# ; Valentin, S.# ; Stojanovic, K.# ; Strobl, D.C. ; Babushku, T. ; Wang, Y. ; Rambold, U. ; Scheffler, L. ; Grath, S. ; John-Robbert, D. ; Blum, H. ; Feuchtinger, A. ; Blutke, A. ; Weih, F. ; Kitamura, D. ; Rad, R. ; Strobl, L.J. ; Zimber-Strobl, U.

RelB contributes to the survival, migration and lymphomagenesis of B cells with constitutively active CD40 signaling.

Activation of CD40-signaling contributes to the initiation, progression and drug resistance of B cell lymphomas. We contributed to this knowledge by showing that constitutive CD40-signaling in B cells induces B cell hyperplasia and finally B cell lymphoma development in transgenic mice. CD40 activates, among others, the non-canonical NF-ĸB signaling, which is constitutively activated in several human B cell lymphomas and is therefore presumed to contribute to lymphopathogenesis. This prompted us to study the regulatory role of the non-canonical NF-ĸB transcription factor RelB in lymphomagenesis. To this end, we crossed mice expressing a constitutively active CD40 receptor in B cells with conditional RelB-KO mice. Ablation of RelB attenuated pre-malignant B cell expansion, and resulted in an impaired survival and activation of long-term CD40-stimulated B cells. Furthermore, we found that hyperactivation of non-canonical NF-кB signaling enhances the retention of B cells in the follicles of secondary lymphoid organs. RNA-Seq-analysis revealed that several genes involved in B-cell migration, survival, proliferation and cytokine signaling govern the transcriptional differences modulated by the ablation of RelB in long-term CD40-stimulated B cells. Inactivation of RelB did not abrogate lymphoma development. However, lymphomas occurred with a lower incidence and had a longer latency period. In summary, our data suggest that RelB, although it is not strictly required for malignant transformation, accelerates the lymphomagenesis of long-term CD40-stimulated B cells by regulating genes involved in migration, survival and cytokine signaling.

2022 Scientific Article in Molecular Metabolism Mol. Metab. 66:101616 (2022)

Maity-Kumar, G.# ; Ständer, L.# ; de Angelis, M. ; Lee, S. ; Molenaar, A. ; Becker, L. ; Garrett, L. ; Amarie, O.V. ; Hölter, S.M. ; Wurst, W. ; Fuchs, H. ; Feuchtinger, A. ; Gailus-Durner, V. ; García-Cáceres, C. ; Othman, A.E. ; Brockmann, C. ; Schöffling, V.I. ; Beiser, K. ; Krude, H. ; Mroz, P.A. ; Hofmann, S.M. ; Tuckermann, J. ; DiMarchi, R.D. ; Hrabě de Angelis, M. ; Tschöp, M.H. ; Pfluger, P.T. ; Müller, T.D.

Validation of Mct8/Oatp1c1 dKO mice as a model organism for the Allan-Herndon-Dudley Syndrome.

OBJECTIVE: The Allan-Herndon-Dudley syndrome (AHDS) is a severe disease caused by dysfunctional central thyroid hormone transport due to functional loss of the monocarboxylate transporter 8 (MCT8). In this study, we assessed whether mice with concomitant deletion of the thyroid hormone transporters Mct8 and the organic anion transporting polypeptide (Oatp1c1) represent a valid preclinical model organism for the AHDS. METHODS: We generated and metabolically characterized a new CRISPR/Cas9 generated Mct8/Oatp1c1 double-knockout (dKO) mouse line for the clinical features observed in patients with AHDS. RESULTS: We show that Mct8/Oatp1c1 dKO mice mimic key hallmarks of the AHDS, including decreased life expectancy, central hypothyroidism, peripheral hyperthyroidism, impaired neuronal myelination, impaired motor abilities and enhanced peripheral thyroid hormone action in the liver, adipose tissue, skeletal muscle and bone. CONCLUSIONS: We conclude that Mct8/Oatp1c1 dKO mice are a valuable model organism for the preclinical evaluation of drugs designed to treat the AHDS.

2022 Scientific Article in Nature metabolism Nat. Metab. 4, 1071-1083 (2022)

Quarta, C.# ; Stemmer, K.# ; Novikoff, A.# ; Yang, B. ; Klingelhuber, F. ; Harger, A. ; Bakhti, M. ; Bastidas-Ponce, A. ; Baugé, E. ; Campbell, J.E. ; Capozzi, M.E. ; Clemmensen, C. ; Collden, G. ; Cota, P. ; Douros, J. ; Drucker, D.J. ; Dubois, B. ; Feuchtinger, A. ; García-Cáceres, C. ; Grandl, G. ; Hennuyer, N. ; Herzig, S. ; Hofmann, S.M. ; Knerr, P.J. ; Kulaj, K. ; Lalloyer, F. ; Lickert, H. ; Liskiewicz, A. ; Liskiewicz, D. ; Maity-Kumar, G. ; Perez-Tilve, D. ; Prakash, S. ; Sanchez-Garrido, M.A. ; Zhang, Q. ; Staels, B. ; Krahmer, N. ; DiMarchi, R.D. ; Tschöp, M.H. ; Finan, B.&deg ; Müller, T.D.&deg

GLP-1-mediated delivery of tesaglitazar improves obesity and glucose metabolism in male mice.

Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARɑ/ɣ) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARɑ/ɣ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARɣ-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography-mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance.

2022 Scientific Article in Science Advances Sci. Adv. 8:eabo5555 (2022)

Demir, S. ; Wolff, G. ; Wieder, A. ; Maida, A. ; Bühler, L. ; Brune, M. ; Hautzinger, O. ; Feuchtinger, A. ; Poth, T. ; Szendroedi, J. ; Herzig, S. ; Ekim Üstünel, B.

TSC22D4 interacts with Akt1 to regulate glucose metabolism.

Maladaptive insulin signaling is a key feature in the pathogenesis of severe metabolic disorders, including obesity and diabetes. Enhancing insulin sensitivity represents a major goal in the treatment of patients affected by diabetes. Here, we identify transforming growth factor-β1 stimulated clone 22 D4 (TSC22D4) as a novel interaction partner for protein kinase B/Akt1, a critical mediator of insulin/phosphatidylinositol 3-kinase signaling pathway. While energy deprivation and oxidative stress promote the TSC22D4-Akt1 interaction, refeeding mice or exposing cells to glucose and insulin impairs this interaction, which relies on an intrinsically disordered region (D2 domain) within TSC22D4. Functionally, the interaction with TSC22D4 reduces basal phosphorylation of Akt and its downstream targets during starvation, thereby promoting insulin sensitivity. Genetic, liver-specific reconstitution experiments in mice demonstrate that the interaction between TSC22D4 and Akt1 improves glucose handling and insulin sensitivity. Overall, our findings postulate a model whereby TSC22D4 acts as an environmental sensor and interacts with Akt1 to regulate insulin signaling and glucose metabolism.

2022 Scientific Article in EBioMedicine EBioMedicine 85:104296 (2022)

Ackermann, M. ; Kamp, J.C. ; Werlein, C. ; Walsh, C.L. ; Stark, H. ; Prade, V.M. ; Surabattula, R. ; Wagner, W.L. ; Disney, C. ; Bodey, A.J. ; Illig, T. ; Leeming, D.J. ; Karsdal, M.A. ; Tzankov, A. ; Boor, P. ; Kühnel, M.P. ; Länger, F.P. ; Verleden, S.E. ; Kvasnicka, H.M. ; Kreipe, H.H. ; Haverich, A. ; Black, S.M. ; Walch, A. ; Tafforeau, P. ; Lee, P.D. ; Hoeper, M.M. ; Welte, T. ; Seeliger, B. ; David, S.P. ; Schuppan, D. ; Mentzer, S.J. ; Jonigk, D.D.

The fatal trajectory of pulmonary COVID-19 is driven by lobular ischemia and fibrotic remodelling.

BACKGROUND: COVID-19 is characterized by a heterogeneous clinical presentation, ranging from mild symptoms to severe courses of disease. 9-20% of hospitalized patients with severe lung disease die from COVID-19 and a substantial number of survivors develop long-COVID. Our objective was to provide comprehensive insights into the pathophysiology of severe COVID-19 and to identify liquid biomarkers for disease severity and therapy response. METHODS: We studied a total of 85 lungs (n = 31 COVID autopsy samples; n = 7 influenza A autopsy samples; n = 18 interstitial lung disease explants; n = 24 healthy controls) using the highest resolution Synchrotron radiation-based hierarchical phase-contrast tomography, scanning electron microscopy of microvascular corrosion casts, immunohistochemistry, matrix-assisted laser desorption ionization mass spectrometry imaging, and analysis of mRNA expression and biological pathways. Plasma samples from all disease groups were used for liquid biomarker determination using ELISA. The anatomic/molecular data were analyzed as a function of patients' hospitalization time. FINDINGS: The observed patchy/mosaic appearance of COVID-19 in conventional lung imaging resulted from microvascular occlusion and secondary lobular ischemia. The length of hospitalization was associated with increased intussusceptive angiogenesis. This was associated with enhanced angiogenic, and fibrotic gene expression demonstrated by molecular profiling and metabolomic analysis. Increased plasma fibrosis markers correlated with their pulmonary tissue transcript levels and predicted disease severity. Plasma analysis confirmed distinct fibrosis biomarkers (TSP2, GDF15, IGFBP7, Pro-C3) that predicted the fatal trajectory in COVID-19. INTERPRETATION: Pulmonary severe COVID-19 is a consequence of secondary lobular microischemia and fibrotic remodelling, resulting in a distinctive form of fibrotic interstitial lung disease that contributes to long-COVID. FUNDING: This project was made possible by a number of funders. The full list can be found within the Declaration of interests / Acknowledgements section at the end of the manuscript.

2022 Scientific Article in European Journal of Cancer Eur. J. Cancer 176, 41-49 (2022)

Lombardo, E.# ; Hess-Rieger, J.# ; Kurz, C. ; Riboldi, M. ; Marschner, S. ; Baumeister, P. ; Lauber, K. ; Pflugradt, U. ; Walch, A.K. ; Canis, M. ; Klauschen, F. ; Zitzelsberger, H. ; Belka, C. ; Landry, G. ; Unger, K.

DeepClassPathway: Molecular pathway aware classification using explainable deep learning.

OBJECTIVE: HPV-associated head and neck cancer is correlated with favorable prognosis; however, its underlying biology is not fully understood. We propose an explainable convolutional neural network (CNN) classifier, DeepClassPathway, that predicts HPV-status and allows patient-specific identification of molecular pathways driving classifier decisions. METHODS: The CNN was trained to classify HPV-status on transcriptome data from 264 (13% HPV-positive) and tested on 85 (25% HPV-positive) head and neck squamous carcinoma patients after transformation into 2D-treemaps representing molecular pathways. Grad-CAM saliency was used to quantify pathways contribution to individual CNN decisions. Model stability was assessed by shuffling pathways within 2D-images. RESULTS: The classification performance of the CNN-ensembles achieved ROC-AUC/PR-AUC of 0.96/0.90 for all treemap variants. Quantification of the averaged pathway saliency heatmaps consistently identified KRAS, spermatogenesis, bile acid metabolism, and inflammation signaling pathways as the four most informative for classifying HPV-positive patients and MYC targets, epithelial-mesenchymal transition, and protein secretion pathways for HPV-negative patients. CONCLUSION: We have developed and applied an explainable CNN classification approach to transcriptome data from an oncology cohort with typical sample size that allows classification while accounting for the importance of molecular pathways in individual-level decisions.

2022 Scientific Article in Journal of Pathology, The J. Pathol. 258, 189-198 (2022)

Lansink Rotgerink, L. ; Felchle, H. ; Feuchtinger, A. ; Nefzger, S.M. ; Walther, C.N. ; Gissibl, J. ; Steiger, K. ; Schmid, T.E. ; Heidegger, S. ; Combs, S.E. ; Fischer, J.C.

Experimental investigation of skin toxicity after immune checkpoint inhibition in combination with radiation therapy.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. However, structured knowledge to mitigate a patient's specific risk of developing adverse events are limited. Nevertheless, there is an exponential growth of clinical studies combining conventional therapies such as radiation therapy (RT) with ICIs. Cutaneous reactions are amongst the most common adverse events after monotherapy with either ICIs or RT. So far, little is known about inter-individual differences in the risk of developing severe tissue toxicity after the combination of RT with ICIs, and the underlying biological mechanisms are ill defined. We used experimental models of RT-induced skin injury to analyze skin toxicity after simultaneous application of ICIs. We compared different RT regimens such as fractionated or stereotactic RT with varying dose intensity. Strikingly, we found that simultaneous application of RT and ICIs did not significantly aggravate acute skin injury in two different mouse strains. Detailed examination of long-term tissue damage of the skin revealed similar signs of epidermal hyperplasia, dermal fibrosis, and adnexal atrophy. In summary, we here present the first experimental study demonstrating excellent safety profiles of concurrent treatment with RT and ICIs. These findings will help to interpret the development of adverse events of the skin after radioimmunotherapy and guide the design of new clinical trials and clinical decision making in individual cases. This article is protected by copyright. All rights reserved.

2022 Scientific Article in Cancers Cancers 14:3745 (2022)

Hess-Rieger, J.# ; Unger, K.# ; Maihoefer, C. ; Schuettrumpf, L. ; Weber, P. ; Marschner, S. ; Wintergerst, L. ; Pflugradt, U. ; Baumeister, P. ; Walch, A.K. ; Woischke, C. ; Kirchner, T. ; Werner, M. ; Soerensen, K. ; Baumann, M. ; Tinhofer, I. ; Combs, S.E. ; Debus, J. ; Schaefer, H. ; Krause, M. ; Linge, A. ; von der Gruen, J. ; Stuschke, M. ; Zips, D. ; Canis, M. ; Lauber, K. ; Ganswindt, U. ; Henke, M. ; Zitzelsberger, H. ; Belka, C.

Integration of p16/HPV DNA status with a 24-miRNA-defined molecular phenotype improves clinically relevant stratification of head and neck cancer patients.

Human papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC) generally have a more favourable prognosis. We hypothesized that HPV-associated HNSCC may be identified by an miRNA-signature according to their specific molecular pathogenesis, and be characterized by a unique transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of two p16/HPV DNA characterized HNSCC cohorts of patients treated by adjuvant radio(chemo)therapy (multicentre DKTK-ROG n = 128, single-centre LMU-KKG n = 101). A linear model predicting HPV status built in DKTK-ROG using lasso-regression was tested in LMU-KKG. LMU-KKG tumours (n = 30) were transcriptome profiled for differential gene expression and miRNA-integration. A 24-miRNA signature predicted HPV-status with 94.53% accuracy (AUC: 0.99) in DKTK-ROG, and 86.14% (AUC: 0.86) in LMU-KKG. The prognostic values of 24-miRNA- and p16/HPV DNA status were comparable. Combining p16/HPV DNA and 24-miRNA status allowed patient sub-stratification and identification of an HPV-associated patient subgroup with impaired overall survival. HPV-positive tumours showed downregulated MAPK, Estrogen, EGFR, TGFbeta, WNT signaling activity. miRNA-mRNA integration revealed HPV-specific signaling pathway regulation, including PD-L1 expression/PD-1 checkpoint pathway in cancer in HPV-associated HNSCC. Integration of clinically established p16/HPV DNA with 24-miRNA signature status improved clinically relevant risk stratification, which might be considered for future clinical decision-making with respect to treatment de-escalation in HPV-associated HNSCC.

2022 Scientific Article in Laboratory Investigation Lab. Invest. 102, 1400-1405 (2022)

Kreutzer, L. ; Weber, P. ; Heider, T. ; Heikenwaelder, M. ; Riedl, T. ; Baumeister, P. ; Klauschen, F. ; Belka, C. ; Walch, A.K. ; Zitzelsberger, H. ; Hess-Rieger, J. ; Unger, K.

Simultaneous metabolite MALDI-MSI, whole exome and transcriptome analysis from formalin-fixed paraffin-embedded tissue sections.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) allows spatial analysis of proteins, metabolites, or small molecules from tissue sections. Here, we present the simultaneous generation and analysis of MALDI-MSI, whole-exome sequencing (WES), and RNA-sequencing data from the same formalin-fixed paraffin-embedded (FFPE) tissue sections. Genomic DNA and total RNA were extracted from (i) untreated, (ii) hematoxylin-eosin (HE) stained, and (iii) MALDI-MSI-analyzed FFPE tissue sections from three head and neck squamous cell carcinomas. MALDI-MSI data were generated by a time-of-flight analyzer prior to preprocessing and visualization. WES data were generated using a low-input protocol followed by detection of single-nucleotide variants (SNVs), tumor mutational burden, and mutational signatures. The transcriptome was determined using 3’-RNA sequencing and was examined for similarities and differences between processing stages. All data met the commonly accepted quality criteria. Besides SNVs commonly identified between differently processed tissues, FFPE-typical artifactual variants were detected. Tumor mutational burden was in the same range for tissues from the same patient and mutational signatures were highly overlapping. Transcriptome profiles showed high levels of correlation. Our data demonstrate that simultaneous molecular profiling of MALDI-MSI-processed FFPE tissue sections at the transcriptome and exome levels is feasible and reliable.

2022 Scientific Article in International Journal of Biological Sciences Int. J. Biol. Sci. 18, 5230-5240 (2022)

Hui, B. ; Lu, C. ; Li, H. ; Hao, X. ; Liu, H. ; Zhuo, D. ; Wang, Q. ; Li, Z. ; Liu, L.&deg ; Wang, X.&deg ; Gu, Y.&deg ; Tang, W.&deg

Inhibition of APOE potentiates immune checkpoint therapy for cancer.

Checkpoint immunotherapy is capable of unleashing T cells for controlling tumor, whereas it is destroyed by immunosuppressive myeloid cell. Apoprotein E (APOE) refers to a ligand in terms of the members of low-density lipoprotein (LDL) receptor family for mediating Apoprotein B-involving atherogenic lipoprotein clearance. Besides, tumor-infiltration macrophage can express APOE. The present study reported Apoe-/- mice to exhibit higher resistance toward the development of three types of carcinomas as compared with mice with wild type and to have greater responses to αPD-1 (anti-PD-1) immunotherapy. Moreover, treatment by exploiting APOE inhibitor (COG 133TFA, αAPOE) was capable of curbing tumor development and fostering regression if in combination of αPD-1. According to single-cell RNA sequencing (scRNA-seq), Apoe deletion was correlated with the decline of C1QC+ and CCR2+ macrophage within tumor infiltration, and mass spectrometry results noticeably showed down-regulated the number of M2 macrophages as well. Furthermore, APOE expression in cancer patients resistant to αPD-1 treatment significantly exceeded that in the sensitive group. For this reason, APOE is likely to be targeted for modifying tumor macrophage infiltrate and augmenting checkpoint immunotherapy.

2022 Scientific Article in Cancers Cancers 14:1763 (2022)

Erlmeier, F.#&deg ; Sun, N.#&deg ; Shen, J. ; Feuchtinger, A. ; Buck, A. ; Prade, V.M. ; Kunzke, T. ; Schraml, P. ; Moch, H. ; Autenrieth, M. ; Weichert, W. ; Hartmann, A. ; Walch, A.K.

MALDI mass spectrometry imaging—prognostic pathways and metabolites for renal cell carcinomas.

High mass resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a suitable method for biomarker detection for several tumor entities. Renal cell carcinoma (RCC) is the seventh most common cancer type and accounts for more than 80% of all renal tumors. Prognostic biomarkers for RCC are still missing. Therefore, we analyzed a large, multicenter cohort including the three most common RCC subtypes (clear cell RCC (ccRCC), papillary RCC (pRCC) and chromophobe RCC (chRCC)) by MALDI for prognostic biomarker detection. MALDI-Fourier-transform ion cyclotron resonance (FT-ICR)-MSI analysis was performed for renal carcinoma tissue sections from 782 patients. SPACiAL pipeline was integrated for automated co-registration of histological and molecular features. Kaplan–Meier analyses with overall survival as endpoint were executed to determine the metabolic features associated with clinical outcome. We detected several pathways and metabolites with prognostic power for RCC in general and also for different RCC subtypes.

2022 Scientific Article in Histochemistry and Cell Biology Histochem. Cell Biol. 157, 595–605 (2022)

Wang, Q. ; Sun, N. ; Kunzke, T. ; Buck, A. ; Shen, J. ; Prade, V.M. ; Stöckl, B. ; Wang, J. ; Feuchtinger, A. ; Walch, A.K.

A simple preparation step to remove excess liquid lipids in white adipose tissue enabling improved detection of metabolites via MALDI-FTICR imaging MS.

Matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) imaging mass spectrometry (MS) is a powerful technology used to analyze metabolites in various tissues. However, it faces significant challenges in studying adipose tissues. Poor matrix distribution and crystallization caused by excess liquid lipids on the surface of tissue sections hamper m/z species detection, an adverse effect that particularly presents in lipid-rich white adipose tissue (WAT). In this study, we integrated a simple and low-cost preparation step into the existing MALDI-FTICR imaging MS pipeline. The new method—referred to as filter paper application—is characterized by an easy sample handling and high reproducibility. The aforementioned filter paper is placed onto the tissue prior to matrix application in order to remove the layer of excess liquid lipids. Consequently, MALDI-FTICR imaging MS detection was significantly improved, resulting in a higher number of detected m/z species and higher ion intensities. After analyzing various durations of filter paper application, 30 s was found to be optimal, resulting in the detection of more than 3700 m/z species. Apart from the most common lipids found in WAT, other molecules involved in various metabolic pathways were detected, including nucleotides, carbohydrates, and amino acids. Our study is the first to propose a solution to a specific limitation of MALDI-FTICR imaging MS in investigating lipid-rich WAT. The filter paper approach can be performed quickly and is particularly effective for achieving uniform matrix distribution on fresh frozen WAT while maintaining tissue integrity. It thus helps to gain insight into the metabolism in WAT.

2022 Scientific Article in BMC Cancer BMC Cancer 22:254 (2022)

Ebert, K. ; Haffner, I. ; Zwingenberger, G. ; Keller, S. ; Raimúndez, E. ; Geffers, R. ; Wirtz, R. ; Barbaria, E. ; Hollerieth, V. ; Arnold, R. ; Walch, A.K. ; Hasenauer, J. ; Maier, D. ; Lordick, F. ; Luber, B.

Combining gene expression analysis of gastric cancer cell lines and tumor specimens to identify biomarkers for anti-HER therapies-the role of HAS2, SHB and HBEGF.

BACKGROUND: The standard treatment for patients with advanced HER2-positive gastric cancer is a combination of the antibody trastuzumab and platin-fluoropyrimidine chemotherapy. As some patients do not respond to trastuzumab therapy or develop resistance during treatment, the search for alternative treatment options and biomarkers to predict therapy response is the focus of research. We compared the efficacy of trastuzumab and other HER-targeting drugs such as cetuximab and afatinib. We also hypothesized that treatment-dependent regulation of a gene indicates its importance in response and that it can therefore be used as a biomarker for patient stratification. METHODS: A selection of gastric cancer cell lines (Hs746T, MKN1, MKN7 and NCI-N87) was treated with EGF, cetuximab, trastuzumab or afatinib for a period of 4 or 24 h. The effects of treatment on gene expression were measured by RNA sequencing and the resulting biomarker candidates were tested in an available cohort of gastric cancer patients from the VARIANZ trial or functionally analyzed in vitro. RESULTS: After treatment of the cell lines with afatinib, the highest number of regulated genes was observed, followed by cetuximab and trastuzumab. Although trastuzumab showed only relatively small effects on gene expression, BMF, HAS2 and SHB could be identified as candidate biomarkers for response to trastuzumab. Subsequent studies confirmed HAS2 and SHB as potential predictive markers for response to trastuzumab therapy in clinical samples from the VARIANZ trial. AREG, EREG and HBEGF were identified as candidate biomarkers for treatment with afatinib and cetuximab. Functional analysis confirmed that HBEGF is a resistance factor for cetuximab. CONCLUSION: By confirming HAS2, SHB and HBEGF as biomarkers for anti-HER therapies, we provide evidence that the regulation of gene expression after treatment can be used for biomarker discovery. TRIAL REGISTRATION: Clinical specimens of the VARIANZ study (NCT02305043) were used to test biomarker candidates.

2022 Scientific Article in Cancer Cell Cancer Cell 40, 639-655.e13 (2022)

Ravi, V.M.# ; Will, P.# ; Kueckelhaus, J. ; Sun, N. ; Joseph, K. ; Salié, H.# ; Vollmer, L.# ; Kuliesiute, U.# ; von Ehr, J. ; Benotmane, J.K. ; Neidert, N. ; Follo, M. ; Scherer, F. ; Goeldner, J.M. ; Behringer, S.P. ; Franco, P. ; Khiat, M. ; Zhang, J. ; Hofmann, U.G. ; Fung, C. ; Ricklefs, F.L. ; Lamszus, K. ; Boerries, M. ; Ku, M.C. ; Beck, J. ; Sankowski, R. ; Schwabenland, M. ; Prinz, M. ; Schüller, U. ; Killmer, S. ; Bengsch, B. ; Walch, A.K. ; Delev, D. ; Schnell, O. ; Heiland, D.H.

Spatially resolved multi-omics deciphers bidirectional tumor-host interdependence in glioblastoma.

Glioblastomas are malignant tumors of the central nervous system hallmarked by subclonal diversity and dynamic adaptation amid developmental hierarchies. The source of dynamic reorganization within the spatial context of these tumors remains elusive. Here, we characterized glioblastomas by spatially resolved transcriptomics, metabolomics, and proteomics. By deciphering regionally shared transcriptional programs across patients, we infer that glioblastoma is organized by spatial segregation of lineage states and adapts to inflammatory and/or metabolic stimuli, reminiscent of the reactive transformation in mature astrocytes. Integration of metabolic imaging and imaging mass cytometry uncovered locoregional tumor-host interdependence, resulting in spatially exclusive adaptive transcriptional programs. Inferring copy-number alterations emphasizes a spatially cohesive organization of subclones associated with reactive transcriptional programs, confirming that environmental stress gives rise to selection pressure. A model of glioblastoma stem cells implanted into human and rodent neocortical tissue mimicking various environments confirmed that transcriptional states originate from dynamic adaptation to various environments.

2022 Scientific Article in Annals of Translational Medicine Ann. Transl. Med. 10:625 (2022)

Tang, W. ; Zhou, Y. ; Zhao, H. ; Sun, G. ; Rong, D. ; Li, Z. ; Hu, M. ; Han, L.F. ; He, X. ; Zhao, S. ; Chen, X. ; Yuan, H. ; Chen, S. ; Wang, Q. ; Gu, J.&deg ; Wang, X.&deg ; Song, J.&deg

A 3D multi-modal intelligent intervention system using electromagnetic navigation for real-time positioning and ultrasound images: A prospective randomized controlled trial.

Background: Anesthesia, nerve block, therapeutic injections, and biopsies all require an acupuncture intervention. However, traditional two-dimensional (2D) ultrasound-guided needle puncture is often challenging and therefore requires the use of three-dimensional (3D) ultrasound images to accurately identify and evaluate the patient’s anatomical structure. Methods: In this study, a 3D multi-modal intelligent intervention system using electromagnetic navigation for real-time positioning and ultrasound images was described. A total of 190 cases requiring puncture were randomly divided into control (conventional 2D ultrasound instrument) and experimental (novel 3D ultrasound imedis9000) groups. The advantages and disadvantages of the two puncture methods were prospectively analyzed in the 190 cases, and the feasibility of electromagnetic navigation real-time positioning was compared to ultrasound imaging. Results: This study included 190 cases from two centers that required puncture treatment and were randomly assigned to the control (conventional 2D ultrasound instrument; n=95) or the experimental (novel 3D ultrasound imedis9000; n=95) groups. Percutaneous vascular puncture, percutaneous biopsy, percutaneous bile duct puncture, thoracic paravertebral nerve block, and sciatic nerve block operations were performed separately. The results indicated that the puncture time and number of trials in the experimental group were significantly lower than those in the control group. No significant difference was identified in the basic vital signs between the two groups before and after surgery. The success rate of the novel 3D ultrasound imedis9000 was 100%, and the success rate of the conventional 2D ultrasound instrument was 95.7%. Furthermore, the results also showed that the novel 3D ultrasound imedis9000 and the matching coaxial positioning channel puncture needle had low pain, good toughness and strength, and great convenience. Conclusions: The new 3D multi-modal intelligent intervention system using electromagnetic navigation real-time positioning and ultrasound images has significant advantages compared with conventional 2D ultrasound in terms of puncture time, number of trials, operation difficulty, and convenience, and is worthy of further promotion and use in clinics. Trial Registration: Beijing Municipal Drug Administration, 20190015.

2022 Scientific Article in Cancer communications Cancer Comm. 42, 517-535 (2022)

Shen, J.# ; Sun, N.# ; Zens, P. ; Kunzke, T. ; Buck, A. ; Prade, V.M. ; Wang, J. ; Wang, Q. ; Hu, R. ; Feuchtinger, A. ; Berezowska, S.&deg ; Walch, A.K.&deg

Spatial metabolomics for evaluating response to neoadjuvant therapy in non-small cell lung cancer patients.

BACKGROUND: The response to neoadjuvant chemotherapy (NAC) differs substantially among individual patients with non-small cell lung cancer (NSCLC). Major pathological response (MPR) is a histomorphological read-out used to assess treatment response and prognosis in patients NSCLC after NAC. Although spatial metabolomics is a promising tool for evaluating metabolic phenotypes, it has not yet been utilized to assess therapy responses in patients with NSCLC. We evaluated the potential application of spatial metabolomics in cancer tissues to assess the response to NAC, using a metabolic classifier that utilizes mass spectrometry imaging combined with machine learning. METHODS: Resected NSCLC tissue specimens obtained after NAC (n = 88) were subjected to high-resolution mass spectrometry, and these data were used to develop an approach for assessing the response to NAC in patients with NSCLC. The specificities of the generated tumor cell and stroma classifiers were validated by applying this approach to a cohort of biologically matched chemotherapy-naïve patients with NSCLC (n = 85). RESULTS: The developed tumor cell metabolic classifier stratified patients into different prognostic groups with 81.6% accuracy, whereas the stroma metabolic classifier displayed 78.4% accuracy. By contrast, the accuracies of MPR and TNM staging for stratification were 62.5% and 54.1%, respectively. The combination of metabolic and MPR classifiers showed slightly lower accuracy than either individual metabolic classifier. In multivariate analysis, metabolic classifiers were the only independent prognostic factors identified (tumor: P = 0.001, hazards ratio [HR] = 3.823, 95% confidence interval [CI] = 1.716-8.514; stroma: P = 0.049, HR = 2.180, 95% CI = 1.004-4.737), whereas MPR (P = 0.804; HR = 0.913; 95% CI = 0.445-1.874) and TNM staging (P = 0.078; HR = 1.223; 95% CI = 0.977-1.550) were not independent prognostic factors. Using Kaplan-Meier survival analyses, both tumor and stroma metabolic classifiers were able to further stratify patients as NAC responders (P < 0.001) and non-responders (P < 0.001). CONCLUSIONS: Our findings indicate that the metabolic constitutions of both tumor cells and the stroma are valuable additions to the classical histomorphology-based assessment of tumor response.

2022 Scientific Article in Nature Communications Nat. Commun. 13:1589 (2022)

Paul, T.# ; Ledderose, S.# ; Bartsch, H. ; Sun, N. ; Soliman, S. ; Märkl, B. ; Ruf, V. ; Herms, J. ; Stern, M. ; Keppler, O.T. ; Delbridge, C. ; Müller, S. ; Piontek, G. ; Kimoto, Y.S. ; Schreiber, F. ; Williams, T.A. ; Neumann, J. ; Knösel, T. ; Schulz, H. ; Spallek, R. ; Graw, M. ; Kirchner, T. ; Walch, A.K. ; Rudelius, M.

Adrenal tropism of SARS-CoV-2 and adrenal findings in a post-mortem case series of patients with severe fatal COVID-19.

Progressive respiratory failure and hyperinflammatory response is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. Despite mounting evidence of disruption of the hypothalamus-pituitary-adrenal axis in COVID-19, relatively little is known about the tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to adrenal glands and associated changes. Here we demonstrate adrenal viral tropism and replication in COVID-19 patients. Adrenal glands showed inflammation accompanied by inflammatory cell death. Histopathologic analysis revealed widespread microthrombosis and severe adrenal injury. In addition, activation of the glycerophospholipid metabolism and reduction of cortisone intensities were characteristic for COVID-19 specimens. In conclusion, our autopsy series suggests that SARS-CoV-2 facilitates the induction of adrenalitis. Given the central role of adrenal glands in immunoregulation and taking into account the significant adrenal injury observed, monitoring of developing adrenal insufficiency might be essential in acute SARS-CoV-2 infection and during recovery.

2022 Scientific Article in Cell Metabolism Cell Metab. 34, 473-486.e9 (2022)

Loft, A.# ; Schmidt, S.F.#&deg ; Caratti, G. ; Stifel, U. ; Havelund, J.F. ; Sekar, R. ; Kwon, Y. ; Sulaj, A. ; Chow, K.K. ; Alfaro, A.J. ; Schwarzmayr, T. ; Rittig, N. ; Svart, M. ; Tsokanos, F.-F. ; Maida, A. ; Blutke, A. ; Feuchtinger, A. ; Møller, N. ; Blüher, M. ; Nawroth, P. ; Szendrödi, J. ; Færgeman, N.J. ; Zeigerer, A. ; Tuckermann, J.&deg ; Herzig, S.&deg

A macrophage-hepatocyte glucocorticoid receptor axis coordinates fasting ketogenesis.

Fasting metabolism and immunity are tightly linked; however, it is largely unknown how immune cells contribute to metabolic homeostasis during fasting in healthy subjects. Here, we combined cell-type-resolved genomics and computational approaches to map crosstalk between hepatocytes and liver macrophages during fasting. We identified the glucocorticoid receptor (GR) as a key driver of fasting-induced reprogramming of the macrophage secretome including fasting-suppressed cytokines and showed that lack of macrophage GR impaired induction of ketogenesis during fasting as well as endotoxemia. Mechanistically, macrophage GR suppressed the expression of tumor necrosis factor (TNF) and promoted nuclear translocation of hepatocyte GR to activate a fat oxidation/ketogenesis-related gene program, cooperatively induced by GR and peroxisome proliferator-activated receptor alpha (PPARα) in hepatocytes. Together, our results demonstrate how resident liver macrophages directly influence ketogenesis in hepatocytes, thereby also outlining a strategy by which the immune system can set the metabolic tone during inflammatory disease and infection.

2022 Nature Genetics Nat. Genet. 54, 358-360 (2022)

Eumorphia Consortium (Hrabě de Angelis, M. ; Wurst, W. ; Abe, K. ; Beckers, J. ; Busch, D.H. ; Dalke, C. ; Gailus-Durner, V. ; Fuchs, H. ; Graw, J. ; Hölter, S.M. ; Kallnik, M. ; Lengger, C. ; Pedersen, V. ; Puk, O. ; Vogt Weisenhorn, D.M. ; Wagner, S.) ; Quintanilla-Fend, L.

EMPReSS: Standardized phenotype screens for functional annotation of the mouse genome (vol 37, pg 1155, 2005).

In the version of this article initially published, members of the Eumorphia Consortium appeared in the Supplementary Information but were not included in the main article. The full list of members appears below.

2022 Scientific Article in Clinical Cancer Research Clin. Cancer Res. 28, 2865-2877 (2022)

Wang, J. ; Kunzke, T. ; Prade, V.M. ; Shen, J. ; Buck, A. ; Feuchtinger, A. ; Haffner, I. ; Luber, B. ; Liu, D.H.W. ; Langer, R. ; Lordick, F. ; Sun, N. ; Walch, A.K.

Spatial metabolomics identifies distinct tumor-specific subtypes in gastric cancer patients.

PURPOSE: Current systems of gastric cancer (GC) molecular classification include genomic, molecular, and morphological features. GC classification based on tissue metabolomics remains lacking. This study aimed to define metabolically distinct GC subtypes and identify their clinicopathological and molecular characteristics. EXPERIMENTAL DESIGN: Spatial metabolomics by high mass resolution imaging mass spectrometry was performed in 362 GC patients. K-means clustering was used to define tumor and stroma-related subtypes based on tissue metabolites. The identified subtypes were linked with clinicopathological characteristics, molecular features, and metabolic signatures. Responses to trastuzumab treatment were investigated across the subtypes by introducing an independent patient cohort with HER2-positive GC from a multicenter observational study. RESULTS: Three tumor- and three stroma-specific subtypes with distinct tissue metabolite patterns were identified. Tumor-specific subtype T1(HER2+MIB+CD3+) positively correlated with HER2, MIB1, DEFA-1, CD3, CD8, FOXP3, but negatively correlated with MMR. Tumor-specific subtype T2(HER2-MIB-CD3-) negatively correlated with HER2, MIB1, CD3, FOXP3, but positively correlated with MMR. Tumor-specific subtype T3(pEGFR+) positively correlated with pEGFR. Patients with tumor subtype T1(HER2+MIB+CD3+) had elevated nucleotide levels, enhanced DNA metabolism, and a better prognosis than T2(HER2-MIB-CD3-) and T3(pEGFR+). An independent validation cohort confirmed that the T1 subtype benefited from trastuzumab therapy. Stroma-specific subtypes had no association with clinicopathological characteristics, however linked to distinct metabolic pathways and molecular features. CONCLUSIONS: Patient subtypes derived by tissue-based spatial metabolomics are a valuable addition to existing GC molecular classification systems. Metabolic differences between the subtypes and their associations with molecular features could provide a valuable tool to aid in selecting specific treatment approaches.

2022 Scientific Article in Cell Metabolism Cell Metab. 34, 329-345.e8 (2022)

Sato, S.# ; Dyar, K.A.# ; Treebak, J.T.# ; Jepsen, S.L. ; Ehrlich, A.M. ; Ashcroft, S.P. ; Trost, K. ; Kunzke, T. ; Prade, V.M. ; Small, L. ; Basse, A.L. ; Schönke, M. ; Chen, S. ; Samad, M. ; Baldi, P. ; Barrès, R. ; Walch, A.K. ; Moritz, T. ; Holst, J.J. ; Lutter, D.&deg ; Zierath, J.R.&deg ; Sassone-Corsi, P.

Atlas of exercise metabolism reveals time-dependent signatures of metabolic homeostasis.

Tissue sensitivity and response to exercise vary according to the time of day and alignment of circadian clocks, but the optimal exercise time to elicit a desired metabolic outcome is not fully defined. To understand how tissues independently and collectively respond to timed exercise, we applied a systems biology approach. We mapped and compared global metabolite responses of seven different mouse tissues and serum after an acute exercise bout performed at different times of the day. Comparative analyses of intra- and inter-tissue metabolite dynamics, including temporal profiling and blood sampling across liver and hindlimb muscles, uncovered an unbiased view of local and systemic metabolic responses to exercise unique to time of day. This comprehensive atlas of exercise metabolism provides clarity and physiological context regarding the production and distribution of canonical and novel time-dependent exerkine metabolites, such as 2-hydroxybutyrate (2-HB), and reveals insight into the health-promoting benefits of exercise on metabolism.

2022 Scientific Article in Clinical Cancer Research Clin. Cancer Res. 28, 1038-1052 (2022)

Weber, P.# ; Künstner, A.# ; Hess-Rieger, J.# ; Unger, K.# ; Marschner, S. ; Idel, C. ; Ribbat-Idel, J. ; Walz, C. ; Rietzler, S. ; Valeanu, L. ; Herkommer, T. ; Kreutzer, L. ; Klymenko, O. ; Kirchner, T. ; Ganswindt, U. ; Walch, A.K. ; Sterr, M. ; Lickert, H. ; Canis, M. ; Rades, D. ; Perner, S. ; Berriel Diaz, M. ; Herzig, S. ; Wollenberg, B. ; Busch, H. ; Zitzelsberger, H.

Therapy-related transcriptional subtypes in matched primary and recurrent head and neck cancer.

PURPOSE: The genetic relatedness between primary and recurrent head and neck squamous cell carcinomas (HNSCC) reflects the extent of heterogeneity and therapy-driven selection of tumor subpopulations. Yet, current treatment of recurrent HNSCC ignores the molecular characteristics of therapy-resistant tumor populations. EXPERIMENTAL DESIGN: From 150 tumors, 74 primary HNSCCs were RNA-sequenced and 38 matched primary/recurrent tumor pairs were both, whole-exome and RNA-sequenced. Transcriptome analysis determined the predominant classical (CL), basal (BA) and inflamed-mesenchymal (IMS) transcriptional subtypes according to an established classification. Genomic alterations and clonal compositions of tumors were evaluated from whole-exome data. RESULTS: While CL and IMS subtypes were more common in primary HNSCC with low recurrence rates, the BA subtype was more prevalent and stable in recurrent tumors. The BA subtype was associated with a transcriptional signature of partial epithelial-to-mesenchymal transition (p-emt) and early recurrence. In 44% of matched cases, the dominant subtype changed from primary to recurrent tumors, preferably from IMS to BA or CL. Gene set enrichment analysis identified upregulation of Hypoxia, p-emt and radiation resistance signatures and downregulation of tumor inflammation in recurrences compared to index tumors. A relevant subset of primary/recurrent tumor pairs presented no evidence for a common clonal origin. CONCLUSIONS: Our study showed a high degree of genetic and transcriptional heterogeneity between primary/recurrent tumors, suggesting therapy-related selection of a transcriptional subtype with characteristics unfavorable for therapy. We conclude that therapy decisions should be based on genetic and transcriptional characteristics of recurrences rather than primary tumors to enable optimally tailored treatment strategies.

2022 Scientific Article in Journal of Pathology, The J. Pathol. 256, 202-213 (2022)

Buck, A.# ; Prade, V.M.# ; Kunzke, T. ; Feuchtinger, A. ; Kröll, D. ; Feith, M. ; Dislich, B. ; Balluff, B. ; Langer, R.&deg ; Walch, A.K.&deg

Metabolic tumor constitution is superior to tumor regression grading for evaluating response to neoadjuvant therapy of esophageal adenocarcinoma patients.

The response to neoadjuvant therapy can vary widely between individual patients. Histopathological tumor regression grading (TRG) is a strong factor for treatment response and survival prognosis of esophageal adenocarcinoma (EAC) patients following neoadjuvant treatment and surgery. However, TRG systems are usually based on the estimation of residual tumor but do not consider stromal or metabolic changes after treatment. Spatial metabolomics analysis is a powerful tool for molecular tissue phenotyping but has not been used so far in the context of neoadjuvant treatment of esophageal cancer. We used imaging mass spectrometry to assess the potential of spatial metabolomics on tumor and stroma tissue for evaluating therapy response of neoadjuvant-treated EAC patients. With an accuracy of 89.7%, the binary classifier trained on spatial tumor metabolite data proved to be superior for stratifying patients when compared to histopathological response assessment which had an accuracy of 70.5%. Sensitivities and specificities for the poor and favorable survival patient groups ranged from 84.9 to 93.3% using the metabolic classifier and from 62.2 to 78.1% using TRG. The tumor classifier was the only significant prognostic factor (HR 3.38, 95% CI = 1.40-8.12, P = 0.007) when adjusted for clinicopathological parameters such as TRG (HR 1.01, 95% CI = 0.67-1.53, P = 0.968) or stromal classifier (HR 1.856, 95% CI = 0.81-4.25, P = 0.143). The classifier even allowed to further stratify patients within the TRG1-3 categories. The underlying mechanisms of response to treatment has been figured out through network analysis. In summary, metabolic response evaluation outperformed histopathological response evaluation in our study with regard to prognostic stratification. This finding indicates that the metabolic constitution of tumor may have a greater impact on patient survival than the quantity of residual tumor cells or the stroma. This article is protected by copyright. All rights reserved.

In: (European Conference on Biomedical Optics, 20–24 June 2021, Munich Germany). 2021. DOI: 10.1117/12.2615998 ( ; 11923)

Liu, N. ; O'Connor, P. ; Gujrati, V. ; Gorpas, D. ; Glasl, S. ; Blutke, A. ; Walch, A.K. ; Kleigrewe, K. ; Sattler, M. ; Plettenburg, O. ; Ntziachristos, V.

Facile synthesis of a croconaine-based nanoformulation for optoacoustic imaging and photothermal therapy.

CR760, a croconaine dye with excellent optical properties, was synthesized in a single step and subsequently nano-formulated for optoacoustic imaging and photothermal therapy of cancer.

2021 Review in Frontiers in Oncology Front. Oncol. 11:771335 (2021)

Li, Z.# ; Sun, G.# ; Sun, G.# ; Cheng, Y. ; Wu, L. ; Wang, Q. ; Lv, C.&deg ; Zhou, Y.&deg ; Xia, Y.&deg ; Tang, W.&deg

Various uses of PD1/PD-L1 inhibitor in oncology: Opportunities and challenges.

The occurrence and development of cancer are closely related to the immune escape of tumor cells and immune tolerance. Unlike previous surgical, chemotherapy, radiotherapy and targeted therapy, tumor immunotherapy is a therapeutic strategy that uses various means to stimulate and enhance the immune function of the body, and ultimately achieves the goal of controlling tumor cells.With the in-depth understanding of tumor immune escape mechanism and tumor microenvironment, and the in-depth study of tumor immunotherapy, immune checkpoint inhibitors represented by Programmed Death 1/Programmed cell Death-Ligand 1(PD-1/PD-L1) inhibitors are becoming increasingly significant in cancer medication treatment. employ a variety of ways to avoid detection by the immune system, a single strategy is not more effective in overcoming tumor immune evasion and metastasis. Combining different immune agents or other drugs can effectively address situations where immunotherapy is not efficacious, thereby increasing the chances of success and alternative access to alternative immunotherapy. Immune combination therapies for cancer have become a hot topic in cancer treatment today. In this paper, several combination therapeutic modalities of PD1/PD-L1 inhibitors are systematically reviewed. Finally, an analysis and outlook are provided in the context of the recent advances in combination therapy with PD1/PD-L1 inhibitors and the pressing issues in this field.

2021 Scientific Article in EJNMMI Research EJNMMI Res. 11:120 (2021)

Sun, N.# ; Trajkovic-Arsic, M.# ; Li, F.# ; Wu, Y. ; Münch, C. ; Kunzke, T. ; Feuchtinger, A. ; Steiger, K. ; Schlitter, A.M. ; Weichert, W. ; Esposito, I. ; Siveke, J.T.&deg ; Walch, A.K.&deg

Native glycan fragments detected by MALDI mass spectrometry imaging are independent prognostic factors in pancreatic ductal adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies to date. The impressively developed stroma that surrounds and modulates the behavior of cancer cells is one of the main factors regulating the PDAC growth, metastasis and therapy resistance. Here, we postulate that stromal and cancer cell compartments differentiate in protein/lipid glycosylation patterns and analyze differences in glycan fragments in those compartments with clinicopathologic correlates. Results: We analyzed native glycan fragments in 109 human FFPE PDAC samples using high mass resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometric imaging (MALDI-FT-ICR-MSI). Our method allows detection of native glycan fragments without previous digestion with PNGase or any other biochemical reaction. With this method, 8 and 18 native glycans were identified as uniquely expressed in only stromal or only cancer cell compartment, respectively. Kaplan–Meier survival model identified glycan fragments that are expressed in cancer cell or stromal compartment and significantly associated with patient outcome. Among cancer cell region-specific glycans, 10 predicted better and 6 worse patient survival. In the stroma, 1 glycan predicted good and 4 poor patient survival. Using factor analysis as a dimension reduction method, we were able to group the identified glycans in 2 factors. Multivariate analysis revealed that these factors can be used as independent survival prognostic elements with regard to the established Union for International Cancer Control (UICC) classification both in tumor and stroma regions. Conclusion: Our method allows in situ detection of naturally occurring glycans in FFPE samples of human PDAC tissue and highlights the differences among glycans found in stromal and cancer cell compartment offering a basis for further exploration on the role of specific glycans in cancer–stroma communication.

2021 Scientific Article in Cellular Microbiology Cell. Microbiol. 23:e13399 (2021)

Zhao, L.# ; Chen, F.# ; Quitt, O.# ; Festag, M.# ; Ringelhan, M. ; Wisskirchen, K. ; Festag, J. ; Yakovleva, L. ; Sureau, C. ; Bohne, F. ; Aichler, M. ; Bruss, V. ; Shevtsov, M. ; van de Klundert, M. ; Momburg, F. ; Möhl, B.S. ; Protzer, U.

Hepatitis B virus envelope proteins can serve as therapeutic targets embedded in the host cell plasma membrane.

Hepatitis B virus (HBV) infection is a major health threat causing 880,000 deaths each year. Available therapies control viral replication, but do not cure HBV leaving patients at risk to develop hepatocellular carcinoma. Here we show that HBV envelope proteins (HBs) - besides their integration into endosomal membranes - become embedded in the plasma membrane where they can be targeted by redirected T-cells. HBs was detected on the surface of HBV-infected cells, in livers of mice replicating HBV and in HBV-induced hepatocellular carcinoma. Staining with HBs-specific recombinant antibody MoMab recognizing a conformational epitope indicated that membrane-associated HBs remains correctly folded in HBV-replicating cells in cell culture and in livers of HBV-transgenic mice in vivo. MoMab coated onto superparamagnetic iron oxide nanoparticles allowed to detect membrane-associated HBs after HBV infection by electron microscopy in distinct stretches of the hepatocyte plasma membrane. Last not least we demonstrate that HBs located to the cell surface allows therapeutic targeting of HBV-positive cells by T-cells either engrafted with a chimeric antigen receptor or redirected by bispecific, T-cell engager antibodies. This article is protected by copyright. All rights reserved.

2021 Scientific Article in Cell Discovery Cell Discov. 7:105 (2021)

Yuan, S.# ; Liao, G.# ; Zhang, M.# ; Zhu, Y.# ; Xiao, W.# ; Wang, K. ; Li, C. ; Jia, C. ; Sun, N. ; Walch, A.K. ; Gao, D. ; Xu, P.&deg ; Deng, Q.&deg ; Zhang, J.&deg ; Wang, H.&deg ; Hu, R.&deg

Multiomics interrogation into HBV (Hepatitis B virus)-host interaction reveals novel coding potential in human genome, and identifies canonical and non-canonical proteins as host restriction factors against HBV.

Hepatitis B Virus (HBV) constitutes a major threat to global public health. Current understanding of HBV-host interaction is yet limited. Here, ribosome profiling, quantitative mass spectrometry and RNA-sequencing were conducted on a recently established HBV replication system, through which we identified multiomic differentially expressed genes (DEGs) that HBV orchestrated to remodel host proteostasis networks. Our multiomics interrogation revealed that HBV induced significant changes in both transcription and translation of 35 canonical genes including PPP1R15A, PGAM5 and SIRT6, as well as the expression of at least 15 non-canonical open reading frames (ncORFs) including ncPON2 and ncGRWD1, thus revealing an extra coding potential of human genome. Overexpression of these five genes but not the enzymatically deficient SIRT6 mutants suppressed HBV replication while knockdown of SIRT6 had opposite effect. Furthermore, the expression of SIRT6 was down-regulated in patients, cells or animal models of HBV infection. Mechanistic study further indicated that SIRT6 directly binds to mini-chromosome and deacetylates histone H3 lysine 9 (H3K9ac) and histone H3 lysine 56 (H3K56ac), and chemical activation of endogenous SIRT6 with MDL800 suppressed HBV infection in vitro and in vivo. By generating the first multiomics landscape of host-HBV interaction, our work is thus opening a new avenue to facilitate therapeutic development against HBV infection.

2021 Scientific Article in Physics & Imaging in Radiation Oncology Phys. Imag. Radiat. Oncology 20, 11-16 (2021)

Burkhardt, R.# ; Gora, T.# ; Fingerle, A.A. ; Sauter, A.P. ; Meurer, F. ; Gassert, F.T. ; Dobiasch, S. ; Schilling, D. ; Feuchtinger, A. ; Walch, A.K. ; Multhoff, G. ; Herzen, J. ; Noel, P.B. ; Rummeny, E.J. ; Combs, S.E. ; Schmid, T.E. ; Pfeiffer, F. ; Wilkens, J.J.

In-vivo X-ray dark-field computed tomography for the detection of radiation-induced lung damage in mice.

Background and Purpose: Radiotherapy of thoracic tumours can lead to side effects in the lung, which may benefit from early diagnosis. We investigated the potential of X-ray dark-field computed tomography by a proof-of-principle murine study in a clinically relevant radiotherapeutic setting aiming at the detection of radiation-induced lung damage. Material and Methods: Six mice were irradiated with 20 Gy to the entire right lung. Together with five unirradiated control mice, they were imaged using computed tomography with absorption and dark-field contrast before and 16 weeks post irradiation. Mean pixel values for the right and left lung were calculated for both contrasts, and the right-to-left-ratio R of these means was compared. Radiologists also assessed the tomograms acquired 16 weeks post irradiation. Sensitivity, specificity, inter- and intra-reader accuracy were evaluated. Results: In absorption contrast the group-average of R showed no increase in the control group and increased by 7% (p = 0.005) in the irradiated group. In dark-field contrast, it increased by 2% in the control group and by 14% (p = 0.005) in the irradiated group. Specificity was 100% for both contrasts but sensitivity was almost four times higher using dark-field tomography. Two cases were missed by absorption tomography but were detected by dark-field tomography. Conclusions: The applicability of X-ray dark-field computed tomography for the detection of radiation-induced lung damage was demonstrated in a pre-clinical mouse model. The presented results illustrate the differences between dark-field and absorption contrast and show that dark-field tomography could be advantageous in future clinical settings.

2021 Scientific Article in Cancer Research Cancer Res. 81, 5862-5875 (2021)

Kunzke, T. ; Prade, V.M. ; Buck, A. ; Sun, N. ; Feuchtinger, A. ; Matzka, M. ; Fernandez, I.E. ; Wuyts, W.A. ; Ackermann, M. ; Jonigk, D. ; Aichler, M. ; Schmid, R.A. ; Eickelberg, O. ; Berezowska, S. ; Walch, A.K.

Patterns of carbon-bound exogenous compounds in lung cancer patients and association with disease pathophysiology.

Asymptomatic anthracosis is the accumulation of black carbon particles in adult human lungs. It is a common occurrence, but the pathophysiological significance of anthracosis is debatable. Using in situ high mass resolution matrix-assisted laser desorption/ionization (MALDI) fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry imaging analysis, we discovered noxious carbon-bound exogenous compounds, such as polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines, or aromatic amines, in a series of 330 lung cancer patients in highly variable and unique patterns. The characteristic nature of carbon-bound exogenous compound had a strong association with patient outcome, tumor progression, the tumor immune microenvironment, PD-L1 expression, and DNA damage. Spatial correlation network analyses revealed substantial differences in the metabolome of tumor cells compared to tumor stroma depending on carbon-bound exogenous compounds. Overall, the bioactive pool of exogenous compounds is associated with several changes in lung cancer pathophysiology and correlates with patient outcome. Given the high prevalence of anthracosis in the lungs of adult humans, future work should investigate the role of carbon-bound exogenous compounds in lung carcinogenesis and lung cancer therapy.

2021 Scientific Article in Molecular Metabolism Mol. Metab. 54:101330 (2021)

Oppenländer, L. ; Palit, S. ; Stemmer, K. ; Greisle, T. ; Sterr, M. ; Salinno, C. ; Bastidas-Ponce, A. ; Feuchtinger, A. ; Böttcher, A. ; Ansarullah&deg ; Theis, F.J.&deg ; Lickert, H.&deg

Vertical sleeve gastrectomy triggers fast β-cell recovery upon overt diabetes.

While the effectiveness of bariatric surgery in restoring β-cell function has been described in type-2 diabetes (T2D) patients and animal models for years, the mechanistic underpinnings are largely unknown. The possibility of vertical sleeve gastrectomy (VSG) to rescue a clinically-relevant, late-stage T2D condition and to promote β-cell recovery has not been investigated on a single-cell level. Nevertheless, characterization of the heterogeneity and functional states of β-cells after VSG is a fundamental step to understand mechanisms of glycaemic recovery and to ultimately develop alternative, less-invasive therapies. Here, we report that VSG was superior to calorie restriction in late-stage T2D and rapidly restored normoglycaemia in morbidly obese and overt diabetic db/db mice. Single-cell profiling of islets of Langerhans showed that VSG induced distinct, intrinsic changes in the β-cell transcriptome, but not in that of α-, δ-, and PP-cells. VSG triggered fast β-cell redifferentiation and functional improvement within only two weeks of intervention, which is not seen upon calorie restriction. Furthermore, VSG expanded β-cell area by means of redifferentiation and by creating a proliferation competent β-cell state. Collectively, our study reveals the superiority of VSG in the remission of far-progressed T2D and presents paths of β-cell regeneration and molecular pathways underlying the glycaemic benefits of VSG.

2021 Scientific Article in Nature metabolism Nat. Metab. 3, 1202-1216 (2021)

Aliluev, A.# ; Tritschler, S.# ; Sterr, M. ; Oppenländer, L. ; Hinterdobler, J. ; Greisle, T. ; Irmler, M. ; Beckers, J. ; Sun, N. ; Walch, A.K. ; Stemmer, K. ; Kindt, A. ; Krumsiek, J. ; Tschöp, M.H. ; Luecken, M. ; Theis, F.J.&deg ; Lickert, H.&deg ; Böttcher, A.&deg

Diet-induced alteration of intestinal stem cell function underlies obesity and prediabetes in mice.

Excess nutrient uptake and altered hormone secretion in the gut contribute to a systemic energy imbalance, which causes obesity and an increased risk of type 2 diabetes and colorectal cancer. This functional maladaptation is thought to emerge at the level of the intestinal stem cells (ISCs). However, it is not clear how an obesogenic diet affects ISC identity and fate. Here we show that an obesogenic diet induces ISC and progenitor hyperproliferation, enhances ISC differentiation and cell turnover and changes the regional identities of ISCs and enterocytes in mice. Single-cell resolution of the enteroendocrine lineage reveals an increase in progenitors and peptidergic enteroendocrine cell types and a decrease in serotonergic enteroendocrine cell types. Mechanistically, we link increased fatty acid synthesis, Ppar signaling and the Insr-Igf1r-Akt pathway to mucosal changes. This study describes molecular mechanisms of diet-induced intestinal maladaptation that promote obesity and therefore underlie the pathogenesis of the metabolic syndrome and associated complications.

2021 Scientific Article in EMBO Molecular Medicine EMBO Mol. Med. 13:e13490 (2021)

Huang, S. ; Blutke, A. ; Feuchtinger, A. ; Klemm, U. ; Zachariah Tom, R. ; Hofmann, S.M. ; Stiel, A.C. ; Ntziachristos, V.

Functional multispectral optoacoustic tomography imaging of hepatic steatosis development in mice.

The increasing worldwide prevalence of obesity, fatty liver diseases and the emerging understanding of the important roles lipids play in various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. We investigated the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non-invasively visualize lipids in laboratory mice around the 930nm spectral range. Using an obesity-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label-free mode. Moreover, we demonstrate the efficacy of using the real-time clearance kinetics of indocyanine green (ICG) in the liver, monitored by MSOT, as a biomarker to evaluate the organ’s function and assess the severity of NAFLD. This study establishes MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD.

2021 Scientific Article in Molecular Metabolism Mol. Metab. 54:101334 (2021)

Chhabra, N.F.# ; Amend, A.-L.# ; Bastidas-Ponce, A. ; Sabrautzki, S. ; Tarquis Medina, M. ; Sachs, S. ; Rubey, M. ; Lorenz-Depiereux, B. ; Feuchtinger, A. ; Bakhti, M. ; Lickert, H. ; Przemeck, G.K.H. ; Hrabě de Angelis, M.

A point mutation in the Pdia6 gene results in loss of pancreatic β-cell identity causing overt diabetes.

OBJECTIVE: Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to carry a vital role in β-cell dysfunction and diabetes. However, very little is known about the function of this protein in β-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on β-cell development and function. METHODS: We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the β-cell development at embryonic day (E)18.5 and β-cell identity as well as function at postnatal stages. RESULTS: Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although, no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing β-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP as well as PDI family member PDIA4, however without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of β-cell maturation and function, such as Ins2, Mafa and Slc2a2 along with increased expression of α-cell markers, Mafb and glucagon was observed in adult mice, suggesting loss of β-cell identity. CONCLUSIONS: The data demonstrates that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic β-cell function and identity, suggesting a critical role of PDIA6 specifically for β-cells.

2021 Scientific Article in Cell Death & Disease Cell Death Dis. 12:723 (2021)

Maier, J.P. ; Kueckelhaus, J. ; Behringer, S.P. ; Garrelfs, N. ; Will, P. ; Sun, N. ; von Ehr, J. ; Goeldner, J.M. ; Pfeifer, D. ; Follo, M. ; Hannibal, L. ; Walch, A.K. ; Hofmann, U.G. ; Beck, J. ; Heiland, D.H. ; Schnell, O.

Inhibition of metabotropic glutamate receptor III facilitates sensitization to alkylating chemotherapeutics in glioblastoma.

Glioblastoma (GBM), the most malignant tumor of the central nervous system, is marked by its dynamic response to microenvironmental niches. In particular, this cellular plasticity contributes to the development of an immediate resistance during tumor treatment. Novel insights into the developmental trajectory exhibited by GBM show a strong capability to respond to its microenvironment by clonal selection of specific phenotypes. Using the same mechanisms, malignant GBM do develop intrinsic mechanisms to resist chemotherapeutic treatments. This resistance was reported to be sustained by the paracrine and autocrine glutamate signaling via ionotropic and metabotropic receptors. However, the extent to which glutamatergic signaling modulates the chemoresistance and transcriptional profile of the GBM remains unexplored. In this study we aimed to map the manifold effects of glutamate signaling in GBM as the basis to further discover the regulatory role and interactions of specific receptors, within the GBM microenvironment. Our work provides insights into glutamate release dynamics, representing its importance for GBM growth, viability, and migration. Based on newly published multi-omic datasets, we explored the and characterized the functions of different ionotropic and metabotropic glutamate receptors, of which the metabotropic receptor 3 (GRM3) is highlighted through its modulatory role in maintaining the ability of GBM cells to evade standard alkylating chemotherapeutics. We addressed the clinical relevance of GRM3 receptor expression in GBM and provide a proof of concept where we manipulate intrinsic mechanisms of chemoresistance, driving GBM towards chemo-sensitization through GRM3 receptor inhibition. Finally, we validated our findings in our novel human organotypic section-based tumor model, where GBM growth and proliferation was significantly reduced when GRM3 inhibition was combined with temozolomide application. Our findings present a new picture of how glutamate signaling via mGluR3 interacts with the phenotypical GBM transcriptional programs in light of recently published GBM cell-state discoveries.

2021 Scientific Article in Cell Metabolism Cell Metab. 33, 1685-1700.e9 (2021)

Loft, A.&deg ; Alfaro, A.J. ; Schmidt, S.F. ; Pedersen, F.B. ; Terkelsen, M.K. ; Puglia, M. ; Chow, K.K. ; Feuchtinger, A. ; Troullinaki, M. ; Maida, A. ; Wolff, G. ; Sakurai, M. ; Berutti, R. ; Ekim Üstünel, B. ; Nawroth, P.P. ; Ravnskjaer, K. ; Diaz, M.B. ; Blagoev, B. ; Herzig, S.&deg

Liver-fibrosis-activated transcriptional networks govern hepatocyte reprogramming and intra-hepatic communication.

Liver fibrosis is a strong predictor of long-term mortality in individuals with metabolic-associated fatty liver disease; yet, the mechanisms underlying the progression from the comparatively benign fatty liver state to advanced non-alcoholic steatohepatitis (NASH) and liver fibrosis are incompletely understood. Using cell-type-resolved genomics, we show that comprehensive alterations in hepatocyte genomic and transcriptional settings during NASH progression, led to a loss of hepatocyte identity. The hepatocyte reprogramming was under tight cooperative control of a network of fibrosis-activated transcription factors, as exemplified by the transcription factor Elf-3 (ELF3) and zinc finger protein GLIS2 (GLIS2). Indeed, ELF3- and GLIS2-controlled fibrosis-dependent hepatokine genes targeting disease-associated hepatic stellate cell gene programs. Thus, interconnected transcription factor networks not only promoted hepatocyte dysfunction but also directed the intra-hepatic crosstalk necessary for NASH and fibrosis progression, implying that molecular "hub-centered" targeting strategies are superior to existing mono-target approaches as currently used in NASH therapy.

2021 Scientific Article in European Journal of Endocrinology Eur. J. Endocrinol. 185, 179-191 (2021)

Murakami, M.# ; Sun, N.# ; Greunke, C. ; Feuchtinger, A. ; Kircher, S. ; Deutschbein, T. ; Papathomas, T. ; Bechmann, N. ; Wallace, P.W. ; Peitzsch, M. ; Korpershoek, E. ; Friemel, J. ; Gimenez Roqueplo, A.P. ; Robledo, M. ; Timmers, H.J. ; Canu, L. ; Weber, A. ; de Krijger, R.R. ; Fassnacht, M. ; Knösel, T. ; Kirchner, T. ; Reincke, M. ; Walch, A.K. ; Kroiss, M. ; Beuschlein, F.

Mass spectrometry imaging identifies metabolic patterns associated with malignant potential in pheochromocytoma and paraganglioma.

OBJECTIVE: Within the past decade, important genetic drivers of pheochromocytoma and paraganglioma (PPGLs) development have been identified. The pathophysiological mechanism that translate these alterations into functional autonomy and potentially malignant behavior have not been elucidated in detail. Here we used MALDI-mass spectrometry imaging (MALDI-MSI) of formalin-fixed paraffin-embedded tissue specimens to comprehensively characterize the metabolic profiles of PPGLs. DESIGN AND METHODS: MALDI-MSI was conducted in 344 PPGLs and results correlated with genetic and phenotypic information. We experimentally silenced genetic drivers by siRNA in PC12 cells to confirm their metabolic impact in vitro. RESULTS: Tissue abundance of kynurenine pathway metabolites such as xanthurenic acid was significantly lower (P = 5.06E-11) in the pseudohypoxia pathway cluster 1 compared to PPGLs of the kinase-driven PPGLs cluster 2. Lower abundance of xanthurenic acid was associated with shorter metastasis-free survival (log-rank tests P = 7.96E-06) and identified as a risk factor for metastasis independent of the genetic status (hazard ratio, 32.6, P = 0.002). Knock-down of Sdhb and Vhl in an in vitro model demonstrated that inositol metabolism and sialic acids were similarly modulated as in tumors of the respective cluster. CONCLUSIONS: The present study has identified distinct tissue metabolomic profiles of PPGLs in relation to tumor genotypes. In addition, we revealed significantly altered metabolites in the kynurenine pathway in metastatic PPGLs, which can aid in the prediction of its malignant potential. However, further validation studies will be required to confirm our findings.

2021 Scientific Article in Nature Communications Nat. Commun. 12:2999 (2021)

Georgiadi, A.#&deg ; Lopez Salazar, V.# ; El-Merahbi, R. ; Karikari, R.A. ; Ma, X. ; Mourao, A. ; Klepac, K. ; Bühler, L. ; Alfaro, A.J. ; Kaczmarek, I. ; Linford, A. ; Bosma, M. ; Shilkova, O. ; Ritvos, O. ; Nakamura, N. ; Hirose, S. ; Lassi, M. ; Teperino, R. ; Machado, J. ; Scheideler, M. ; Dietrich, A. ; Geerlof, A. ; Feuchtinger, A. ; Blutke, A. ; Fischer, K. ; Müller, T.D. ; Kessler, K. ; Schöneberg, T. ; Thor, D. ; Hornemann, S. ; Kruse, M. ; Nawroth, P.P. ; Pivovarova-Ramich, O. ; Pfeiffer, A.F.H. ; Sattler, M. ; Blüher, M. ; Herzig, S.&deg

Orphan GPR116 mediates the insulin sensitizing effects of the hepatokine FNDC4 in adipose tissue.

The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes.

2021 Scientific Article in Diabetologia Diabetologia 64, 1850-1865 (2021)

Giroud, M. ; Tsokanos, F.-F. ; Caratti, G. ; Kotschi, S. ; Khani, S. ; Jouffe, C. ; Vogl, E.S. ; Irmler, M. ; Glantschnig, C. ; Gil Lozano, M. ; Haß, D. ; Khan, A.A. ; Rios Garcia, M. ; Mattijssen, F. ; Maida, A. ; Tews, D. ; Fischer-Posovszky, P. ; Feuchtinger, A. ; Virtanen, K.A. ; Beckers, J. ; Wabitsch, M. ; Uhlenhaut, N.H. ; Blüher, M. ; Tuckermann, J. ; Scheideler, M. ; Bartelt, A. ; Herzig, S.

HAND2 is a novel obesity-linked adipogenic transcription factor regulated by glucocorticoid signalling.

Aims/hypothesis: Adipocytes are critical cornerstones of energy metabolism. While obesity-induced adipocyte dysfunction is associated with insulin resistance and systemic metabolic disturbances, adipogenesis, the formation of new adipocytes and healthy adipose tissue expansion are associated with metabolic benefits. Understanding the molecular mechanisms governing adipogenesis is of great clinical potential to efficiently restore metabolic health in obesity. Here we investigate the role of heart and neural crest derivatives-expressed 2 (HAND2) in adipogenesis. Methods: Human white adipose tissue (WAT) was collected from two cross-sectional studies of 318 and 96 individuals. In vitro, for mechanistic experiments we used primary adipocytes from humans and mice as well as human multipotent adipose-derived stem (hMADS) cells. Gene silencing was performed using siRNA or genetic inactivation in primary adipocytes from loxP and or tamoxifen-inducible Cre-ERT2 mouse models with Cre-encoding mRNA or tamoxifen, respectively. Adipogenesis and adipocyte metabolism were measured by Oil Red O staining, quantitative PCR (qPCR), microarray, glucose uptake assay, western blot and lipolysis assay. A combinatorial RNA sequencing (RNAseq) and ChIP qPCR approach was used to identify target genes regulated by HAND2. In vivo, we created a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter (Hand2 ) and performed a large panel of metabolic tests. Results: We found that HAND2 is an obesity-linked white adipocyte transcription factor regulated by glucocorticoids that was necessary but insufficient for adipocyte differentiation in vitro. In a large cohort of humans, WAT HAND2 expression was correlated to BMI. The HAND2 gene was enriched in white adipocytes compared with brown, induced early in differentiation and responded to dexamethasone (DEX), a typical glucocorticoid receptor (GR, encoded by NR3C1) agonist. Silencing of NR3C1 in hMADS cells or deletion of GR in a transgenic conditional mouse model results in diminished HAND2 expression, establishing that adipocyte HAND2 is regulated by glucocorticoids via GR in vitro and in vivo. Furthermore, we identified gene clusters indirectly regulated by the GR–HAND2 pathway. Interestingly, silencing of HAND2 impaired adipocyte differentiation in hMADS and primary mouse adipocytes. However, a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter did not mirror these effects on adipose tissue differentiation, indicating that HAND2 was required at stages prior to Adipoq expression. Conclusions/interpretation: In summary, our study identifies HAND2 as a novel obesity-linked adipocyte transcription factor, highlighting new mechanisms of GR-dependent adipogenesis in humans and mice. Data availability: Array data have been submitted to the GEO database at NCBI (GSE148699). Graphical abstract: [Figure not available: see fulltext.] AdipoqCre

2021 Scientific Article in Photoacoustics Photoacoustics 22:100263 (2021)

Liu, N. ; Gujrati, V. ; Malekzadeh-Najafabadi, J. ; Werner, J.P,F. ; Klemm, U. ; Tang, L. ; Chen, Z. ; Prakash, J. ; Huang, Y. ; Stiel, A.-C. ; Mettenleiter, G. ; Aichler, M. ; Blutke, A. ; Walch, A.K. ; Kleigrewe, K. ; Razansky, D. ; Sattler, M. ; Ntziachristos, V.

Croconaine-based nanoparticles enable efficient optoacoustic imaging of murine brain tumors.

Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors.

2021 Scientific Article in Frontiers in Oncology Front. Oncol. 11:612354 (2021)

Orth, M. ; Albrecht, V. ; Seidl, K. ; Kinzel, L. ; Unger, K. ; Hess-Rieger, J. ; Kreutzer, L. ; Sun, N. ; Stegen, B. ; Nieto, A. ; Maas, J. ; Winssinger, N. ; Friedl, A.A. ; Walch, A.K. ; Belka, C. ; Zitzelsberger, H. ; Niyazi, M. ; Lauber, K.

Inhibition of HSP90 as a strategy to radiosensitize glioblastoma: Targeting the DNA damage response and beyond.

Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damage response factors on the protein level, distinct transcriptomic alterations, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in glioblastoma cells in vitro. In vivo, HSP90 inhibition by NW457 improved the therapeutic outcome of fractionated CBCT-based irradiation in an orthotopic, syngeneic GBM mouse model, both in terms of tumor progression and survival. Nevertheless, in view of the promising in vitro results the in vivo efficacy was not as strong as expected, although apart from the radiosensitizing effects HSP90 inhibition also reduced irradiation-induced GBM cell migration and tumor invasiveness. Hence, our findings identify the combination of HSP90 inhibition and radiotherapy in principle as a promising strategy for GBM treatment whose performance needs to be further optimized by improved inhibitor substances, better formulations and/or administration routes, and fine-tuned treatment sequences.

2021 Scientific Article in Journal of Clinical Oncology - JCO J. Clin. Oncol. 39, 1468-1478 (2021)

Haffner, I. ; Schierle, K. ; Raimúndez, E. ; Geier, B. ; Maier, D. ; Hasenauer, J. ; Luber, B. ; Walch, A.K. ; Kolbe, K. ; Riera Knorrenschild, J. ; Kretzschmar, A. ; Rau, B. ; Fischer von Weikersthal, L. ; Ahlborn, M. ; Siegler, G. ; Fuxius, S. ; Decker, T. ; Wittekind, C. ; Lordick, F.

HER2 expression, test deviations, and their impact on survival in metastatic gastric cancer: Results from the prospective multicenter VARIANZ study.

PURPOSE: Trastuzumab is the only approved targeted drug for first-line treatment of human epidermal growth factor receptor 2-positive (HER2+) metastatic gastric cancer (mGC). However, not all patients respond and most eventually progress. The multicenter VARIANZ study aimed to investigate the background of response and resistance to trastuzumab in mGC. METHODS: Patients receiving medical treatment for mGC were prospectively recruited in 35 German sites and followed for up to 48 months. HER2 status was assessed centrally by immunohistochemistry and chromogenic in situ hybridization. In addition, HER2 gene expression was assessed using qPCR. RESULTS: Five hundred forty-eight patients were enrolled, and 77 had HER2+ mGC by central assessment (14.1%). A high deviation rate of 22.7% between central and local test results was seen. Patients who received trastuzumab for centrally confirmed HER2+ mGC (central HER2+/local HER2+) lived significantly longer as compared with patients who received trastuzumab for local HER2+ but central HER2- mGC (20.5 months, n = 60 v 10.9 months, n = 65; hazard ratio, 0.42; 95% CI, 8.2 to 14.4; P < .001). In the centrally confirmed cohort, significantly more tumor cells stained HER2+ than in the unconfirmed cohort, and the HER2 amplification ratio was significantly higher. A minimum of 40% HER2+ tumor cells and a HER2 amplification ratio of ≥ 3.0 were calculated as optimized thresholds for predicting benefit from trastuzumab. CONCLUSION: Significant discrepancies in HER2 assessment of mGC were found in tumor specimens with intermediate HER2 expression. Borderline HER2 positivity and heterogeneity of HER2 expression should be considered as resistance factors for HER2-targeting treatment of mGC. HER2 thresholds should be reconsidered. Detailed reports with quantification of HER2 expression and amplification levels may improve selection of patients for HER2-directed treatment.

2021 Scientific Article in Advanced healthcare materials Adv. Healthc. Mater. 10:e2002115 (2021)

Liu, N. ; O'Connor, P. ; Gujrati, V. ; Gorpas, D. ; Glasl, S. ; Blutke, A. ; Walch, A.K. ; Kleigrewe, K. ; Sattler, M. ; Plettenburg, O. ; Ntziachristos, V.

Facile synthesis of a croconaine-based nanoformulation for optoacoustic imaging and photothermal therapy.

Near-infrared (NIR) light absorbing theranostic agents can integrate optoacoustic imaging and photothermal therapy for effective personalized precision medicine. However, most of these agents face the challenges of unstable optical properties, material-associated toxicity, and nonbiodegradability, all of which limit their biomedical application. Several croconaine-based organic agents able to overcome some of these limitations have been recently reported, but these suffer from complicated multistep synthesis protocols. Herein, the use of CR760, a croconaine dye with excellent optical properties, is reported for nanoparticle formulation and subsequent optoacoustic imaging and photothermal therapy. Importantly, CR760 can be conveniently prepared in a single step from commercially available materials. Furthermore, CR760 can be covalently attached, via a polyethylene glycol linker, to the αvβ3 integrin ligand c(RGDyC), resulting in self-assembled nanoparticles (NPs) with cancer-targeting capability. Such CR760RGD-NPs exhibit strong NIR absorption, high photostability, high optoacoustic generation efficiency, and active tumor-targeting, making them ideal candidates for optoacoustic imaging. Due to favorable electron transfer, CR760RGD-NPs display a 45.37% photothermal conversion efficiency thereby rendering them additionally useful for photothermal therapy. Targeted tumor elimination, biosafety, and biocompatibility are demonstrated in a 4T1 murine breast tumor model. This work points to the use of CR760RGD-NPs as a promising nanoagent for NIR-based cancer phototheranostics.

2021 Nature Nature 592:E1 (2021)

Ansarullah ; Jain, C. ; Far, F.F. ; Homberg, S. ; Wißmiller, K. ; von Hahn, F. ; Raducanu, A. ; Schirge, S. ; Sterr, M. ; Bilekova, S. ; Siehler, J. ; Wiener, J. ; Oppenländer, L. ; Morshedi, A. ; Bastidas-Ponce, A. ; Collden, G. ; Irmler, M. ; Beckers, J. ; Feuchtinger, A. ; Grzybek, M. ; Ahlbrecht, C. ; Feederle, R. ; Plettenburg, O. ; Müller, T.D. ; Meier, M. ; Tschöp, M.H. ; Coskun, Ü. ; Lickert, H.

Author Correction: Inceptor counteracts insulin signalling in β-cells to control glycaemia.

In this Article, the affiliations for author Ünal Coskun were incorrect. They should be ‘German Center for Diabetes Research (DZD), Neuherberg, Germany’, ‘Paul Langerhans Institute Dresden of Helmholtz Center Munich, Technical University Dresden, Dresden, Germany’ and ‘Institute for Clinical Chemistry and Laboratory Medicine, Faculty of Medicine and University Clinic Carl Gustav Carus, Technical University Dresden, Dresden, Germany’ (affiliations 2, 10 and 14, respectively), and not ‘Department of Microsystems Engineering (IMTEK), University of Freiburg, Freiburg, Germany’ (affiliation 5). The original Article has been corrected online.

2021 Scientific Article in Endocrine-Related Cancer Endocr. Relat. Cancer 28, 213-224 (2021)

Stauffer, E. ; Weber, P. ; Heider, T. ; Dalke, C. ; Blutke, A. ; Walch, A.K. ; Burgstaller, G. ; Brix, N. ; Lauber, K. ; Zitzelsberger, H. ; Unger, K. ; Selmansberger, M.

Transcriptomic landscape of radiation-induced murine thyroid proliferative lesions.

Thyroid carcinoma incidence rates in western societies are among the fastest rising, compared to all malignant tumors over the past two decades. While risk factors such as age and exposure to ionizing radiation are known, early-state carcinogenic processes or pre-lesions are poorly understood or unknown. This study aims at the identification and characterization of early-state radiation-associated neoplastic processes by histologic and transcriptomic analyses of thyroid tissues derived from a mouse model. Comprehensive histological examination of 246 thyroids (164 exposed, 82 non-exposed) was carried out. Proliferative and normal tissues from exposed cases and normal tissue from non-exposed cases were collected by laser-capture microdissection, followed by RNAseq transcriptomic profiling using a low input 3`-library preparation protocol, differential gene expression analysis and functional association by Gene Set Enrichment Analysis. Nine exposed samples exhibited proliferative lesions, while none of the non-exposed samples showed histological abnormalities, indicating an association of ionizing radiation exposure with histological abnormalities. Activated immune response signaling and deregulated metabolic processes were observed in irradiated tissue with normal histology compared to normal tissue from non-exposed samples. Proliferative lesions compared to corresponding normal tissues showed enrichment for mainly proliferation-associated gene sets. Consistently, proliferative lesion samples from exposed mice showed elevated proliferation-associated signaling and deregulated metabolic processes compared to normal samples from non-exposed mice. Our findings suggest that a molecular deregulation may be detectable in histologically normal thyroid tissues and in early proliferative lesions in the frame of multi-step progression from irradiated normal tissue to tumorous lesions.

2021 Scientific Article in Journal of Hepatology J. Hepatol. 75, 74-85 (2021)

Yuan, S. ; Liao, G. ; Zhang, M. ; Zhu, Y. ; Wang, K. ; Xiao, W. ; Jia, C. ; Dong, M. ; Sun, N. ; Walch, A.K. ; Xu, P. ; Zhang, J.&deg ; Deng, Q.&deg ; Hu, R.&deg

Translatomic profiling reveals novel self-restricting virus-host interactions during HBV infection.

BACKGROUND AND AIMS: Hepatitis B Virus remains to be yet unresolved global threat to human health. It remains incompletely understood how HBV self-restricts in host during most adulthood infections, and multi-omics analyses were performed to systematically interrogate into HBV-host interaction and the life cycle of HBV. METHODS: RNA-sequencing and ribosome profiling were conducted with cell-based models for HBV replication and gene expression. The novel translational events or products hereby detected were then characterized, and functionally assessed in both cell and mouse models. Moreover, quasi-species analyses of HBV subpopulations were conducted with patients at immune tolerance or activation phases, using next- or third-generation sequencing. RESULTS: We identified EnhI-SL (Enhancer I-stem loop) as a new cis element in HBV genome, and the mutations disrupting EnhI-SL were found to elevate viral polymerase expression. Furthermore, while re-discovering HpZ/P', a previously under-explored isoform of HBV polymerase, we also identified HBxZ as a novel short isoform of HBX and confirmed their existence and functionally characterized them as potent suppressors for HBV gene expression or genome replication. Mechanistically, HpZ/P' was found to repress HBV gene expression partially through interacting with, and sequestering SUPV3L1. The abundances of the HBV mutants either deficient of HpZ/P' or disrupted in EnhI-SL seemed to be diminished upon the activation of host immune system. Finally, SRSF2, a HBV-down-regulated host protein in RNA spliceosome, was found to promote the splicing of viral pre-genomic RNA and HpZ/P' biogenesis. CONCLUSION: This study has identified multiple viral self-restricting mechanisms in HBV-host interaction. Particularly, SRSF2-HpZ/P' appeared to constitute another negative feedback mechanism in controlling HBV life-cycle. Targeting host splicing machinery might thus represent a yet under-explored strategy to intervene into HBV-host interaction.

2021 Scientific Article in PLoS ONE PLoS ONE 16:e0248594 (2021)

Theobalt, N. ; Hofmann, I. ; Fiedler, S. ; Renner, S. ; Dhom, G. ; Feuchtinger, A. ; Walch, A.K. ; Hrabě de Angelis, M. ; Wolf, E. ; Wanke, R. ; Blutke, A.

Unbiased analysis of obesity related, fat depot specific changes of adipocyte volumes and numbers using light sheet fluorescence microscopy.

In translational obesity research, objective assessment of adipocyte sizes and numbers is essential to characterize histomorphological alterations linked to obesity, and to evaluate the efficacies of experimental medicinal or dietetic interventions. Design-based quantitative stereological techniques based on the analysis of 2D-histological sections provide unbiased estimates of relevant 3D-parameters of adipocyte morphology, but often involve complex and time-consuming tissue processing and analysis steps. Here we report the application of direct 3D light sheet fluorescence microscopy (LSFM) for effective and accurate analysis of adipocyte volumes and numbers in optically cleared adipose tissue samples from a porcine model of diet-induced obesity (DIO). Subcutaneous and visceral adipose tissue samples from DIO-minipigs and lean controls were systematically randomly sampled, optically cleared with 3DISCO (3-dimensional imaging of solvent cleared organs), stained with eosin, and subjected to LSFM for detection of adipocyte cell membrane autofluorescence. Individual adipocytes were unbiasedly sampled in digital 3D reconstructions of the adipose tissue samples, and their individual cell volumes were directly measured by automated digital image analysis. Adipocyte numbers and mean volumes obtained by LSFM analysis did not significantly differ from the corresponding values obtained by unbiased quantitative stereological analysis techniques performed on the same samples, thus proving the applicability of LSFM for efficient analysis of relevant morphological adipocyte parameters. The results of the present study demonstrate an adipose tissue depot specific plasticity of adipocyte growth responses to nutrient oversupply. This was characterized by an exclusively hypertrophic growth of visceral adipocytes, whereas adipocytes in subcutaneous fat tissue depots also displayed a marked (hyperplastic) increase in cell number. LSFM allows for accurate and efficient determination of relevant quantitative morphological adipocyte parameters. The applied stereological methods and LSFM protocols are described in detail and can serve as a guideline for unbiased quantitative morphological analyses of adipocytes in other studies and species.

2021 Scientific Article in Molecular Metabolism Mol. Metab. 45:101147 (2021)

Ruiz Ojeda, F.J. ; Wang, J. ; Bäcker, T. ; Krueger, M. ; Zamani, S. ; Rosowski, S. ; Gruber, T. ; Onogi, Y. ; Feuchtinger, A. ; Schulz, T.J. ; Fässler, R. ; Müller, T.D. ; García-Cáceres, C. ; Meier, M. ; Blüher, M. ; Ussar, S.

Active integrins regulate white adipose tissue insulin sensitivity and brown fat thermogenesis.

Objective: Reorganization of the extracellular matrix is a prerequisite for healthy adipose tissue expansion, whereas fibrosis is a key feature of adipose dysfunction and inflammation. However, very little is known about the direct effects of impaired cell–matrix interaction in adipocyte function and insulin sensitivity. The objective of this study was to determine whether integrin activity can regulate insulin sensitivity in adipocytes and thereby systemic metabolism. Methods: We characterized integrin activity in adipose tissue and its consequences on whole-body metabolism using adipose-selective deletion of β1 integrin (Itgb1adipo-cre) and Kindlin-2 (Kind2adipo-cre) in mice. Results: We demonstrate that integrin signaling regulates white adipocyte insulin action and systemic metabolism. Consequently, loss of adipose integrin activity, similar to loss of adipose insulin receptors, results in a lipodystrophy-like phenotype and systemic insulin resistance. However, brown adipose tissue of Kind2adipo-cre and Itgb1adipo-cre mice is chronically hyperactivated and has increased substrate delivery, reduced endothelial basement membrane thickness, and increased endothelial vesicular transport. Conclusions: Thus, we establish integrin-extracellular matrix interactions as key regulators of white and brown adipose tissue function and whole-body metabolism.

2021 Scientific Article in Nature Nature 590, 326–331 (2021)

Ansarullah ; Jain, C. ; Far, F.F. ; Homberg, S. ; Wissmiller, K. ; Gräfin von Hahn, F. ; Raducanu, A. ; Schirge, S. ; Sterr, M. ; Bilekova, S. ; Siehler, J. ; Wiener, J. ; Oppenländer, L. ; Morshedi, A. ; Bastidas-Ponce, A. ; Collden, G. ; Irmler, M. ; Beckers, J. ; Feuchtinger, A. ; Grzybek, M. ; Ahlbrecht, C. ; Feederle, R. ; Plettenburg, O. ; Müller, T.D. ; Meier, M. ; Tschöp, M.H. ; Coskun, Ü. ; Lickert, H.

Inceptor counteracts insulin signalling in β-cells to control glycaemia.

Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic β-cells causes overt diabetes in mice; thus, therapies that sensitize β-cells to insulin may protect patients with diabetes against β-cell failure1–3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse β-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir−/−) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir−/− mice showed an increase in the activation of INSR–IGF1R in Iir−/− pancreatic tissue, resulting in an increase in the proliferation and mass of β-cells. Similarly, inducible β-cell-specific Iir−/− knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR–IGF1R and increased proliferation of β-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR–IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR–IGF1R in β-cells. Together, our findings show that inceptor shields insulin-producing β-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR–IGF1R sensitization and diabetes therapy.

2021 Scientific Article in Life Science Alliance Life Sci. All. 4:e202000898 (2021)

Bühler, L. ; Maida, A. ; Vogl, E.S. ; Georgiadi, A. ; Takacs, A. ; Kluth, O. ; Schürmann, A. ; Feuchtinger, A. ; von Toerne, C. ; Tsokanos, F.-F. ; Klepac, K. ; Wolff, G. ; Sakurai, M. ; Ekim Üstünel, B. ; Nawroth, P.P. ; Herzig, S.

Lipocalin 13 enhances insulin secretion but is dispensable for systemic metabolic control.

Members of the lipocalin protein family serve as biomarkers for kidney disease and acute phase inflammatory reactions, and are under preclinical development for the diagnosis and therapy of allergies. However, none of the lipocalin family members has made the step into clinical development, mostly due to their complex biological activity and the lack of in-depth mechanistic knowledge. Here, we show that the hepatokine lipocalin 13 (LCN13) triggers glucose-dependent insulin secretion and cell proliferation of primary mouse islets. However, inhibition of endogenous LCN13 expression in lean mice did not alter glucose and lipid homeostasis. Enhanced hepatic secretion of LCN13 in either diet-induced or genetic obesity led to no discernible impact on systemic glucose and lipid metabolism, neither in preventive nor therapeutic setting. Of note, loss or forced LCN13 hepatic secretion did not trigger any compensatory regulation of related lipocalin family members. Together, these data are in stark contrast to the suggested gluco-regulatory and therapeutic role of LCN13 in obesity, and imply complex regulatory steps in LCN13 biology at the organismic level mitigating its principal insulinotropic effects.

2021 Scientific Article in Cell Metabolism Cell Metab. 33, 833-844.e5 (2021)

Zhang, Q. ; Delessa, C.T. ; Augustin, R. ; Bakhti, M. ; Collden, G. ; Drucker, D.J. ; Feuchtinger, A. ; García-Cáceres, C. ; Grandl, G. ; Harger, A. ; Herzig, S. ; Hofmann, S.M. ; Holleman, C.L. ; Jastroch, M. ; Keipert, S. ; Kleinert, M. ; Knerr, P.J. ; Kulaj, K. ; Legutko, B. ; Lickert, H. ; Liu, X. ; Luippold, G. ; Lutter, D. ; Malogajski, E. ; Tarquis Medina, M. ; Mowery, S.A. ; Blutke, A. ; Perez-Tilve, D. ; Salinno, C. ; Sehrer, L. ; DiMarchi, R.D. ; Tschöp, M.H. ; Stemmer, K. ; Finan, B. ; Wolfrum, C. ; Müller, T.D.

The glucose-dependent insulinotropic polypeptide (GIP) regulates body weight and food intake via CNS-GIPR signaling.

Uncertainty exists as to whether the glucose-dependent insulinotropic polypeptide receptor (GIPR) should be activated or inhibited for the treatment of obesity. Gipr was recently demonstrated in hypothalamic feeding centers, but the physiological relevance of CNS Gipr remains unknown. Here we show that HFD-fed CNS-Gipr KO mice and humanized (h)GIPR knockin mice with CNS-hGIPR deletion show decreased body weight and improved glucose metabolism. In DIO mice, acute central and peripheral administration of acyl-GIP increases cFos neuronal activity in hypothalamic feeding centers, and this coincides with decreased body weight and food intake and improved glucose handling. Chronic central and peripheral administration of acyl-GIP lowers body weight and food intake in wild-type mice, but shows blunted/absent efficacy in CNS-Gipr KO mice. Also, the superior metabolic effect of GLP-1/GIP co-agonism relative to GLP-1 is extinguished in CNS-Gipr KO mice. Our data hence establish a key role of CNS Gipr for control of energy metabolism.

2021 Scientific Article in Molecular Oncology Mol. Oncol. 15, 1040-1053 (2021)

Schinke, H.# ; Heider, T.# ; Herkommer, T. ; Simon, F. ; Blancke Soares, A. ; Kranz, G. ; Samaga, D. ; Dajka, L. ; Feuchtinger, A. ; Walch, A.K. ; Valeanu, L. ; Walz, C. ; Kirchner, T. ; Canis, M. ; Baumeister, P. ; Belka, C. ; Maihöfer, C. ; Marschner, S. ; Pflugradt, U. ; Ganswindt, U. ; Hess-Rieger, J. ; Zitzelsberger, H. ; Gires, O.

Digital scoring of EpCAM and slug expression as prognostic markers in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinomas (HNSCCs) have poor clinical outcome owing to therapy resistance and frequent recurrences that are among others attributable to tumor cells in partial epithelial-to-mesenchymal transition (pEMT). We compared side-by-side software-based and visual quantification of immunohistochemistry (IHC) staining of epithelial marker EpCAM and EMT regulator Slug in n = 102 primary HNSCC to assess optimal analysis protocols. IHC scores incorporated expression levels and percentages of positive cells. Digital and visual evaluation of membrane-associated EpCAM yielded correlating scorings, whereas visual evaluation of nuclear Slug resulted in significantly higher overall scores. Multivariable Cox proportional hazard analysis defined the median EpCAM expression levels resulting from visual quantification as an independent prognostic factor of overall survival. Slug expression levels resulting from digital quantification were an independent prognostic factor of recurrence-free survival, locoregional recurrence-free survival, and disease-specific survival. Hence, we propose to use visual assessment for the membrane-associated EpCAM protein, whereas nuclear protein Slug assessment was more accurate following digital measurement.

2021 Scientific Article in Breast Care Breast Care 16, 523-531 (2021)

Napieralski, R. ; Schricker, G. ; Auer, G. ; Aubele, M. ; Perkins, J. ; Magdolen, V. ; Ulm, K. ; Hamann, M. ; Walch, A.K. ; Weichert, W. ; Kiehle, M. ; Weichert, O.G.

PITX2 DNA-methylation: Predictive versus prognostic value for anthracycline-based chemotherapy in triple-negative breast cancer patients.

Background: PITX2 DNA methylation has been shown to predict outcomes in high-risk breast cancer patients after anthracycline-based chemotherapy. To determine its prognostic versus predictive value, the impact of PITX2 DNA methylation on outcomes was studied in an untreated cohort vs. an anthracycline-treated triple-negative breast cancer (TNBC) cohort. Material and Methods: The percent DNA methylation ratio (PMR) of paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time PCR test. Patient samples of routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue and clinical data from 144 TNBC patients of 2 independent cohorts (i.e., 66 untreated patients and 78 patients treated with anthracycline-based chemotherapy) were analyzed. Results: The risk of 5- and 10-year overall survival (OS) increased continuously with rising PITX2 DNA methylation in the anthracycline-treated population, but it increased only slightly during 10-year follow-up time in the untreated patient population. PITX2 DNA methylation with a PMR cutoff of 2 did not show significance for poor vs. good outcomes (OS) in the untreated patient cohort (HR = 1.55; p = 0.259). In contrast, the PITX2 PMR cutoff of 2 identified patients with poor (PMR >2) vs. good (PMR ≤2) outcomes (OS) with statistical significance in the anthracycline-treated cohort (HR = 3.96; p = 0.011). The results in the subgroup of patients who did receive anthracyclines only (no taxanes) confirmed this finding (HR = 5.71; p = 0.014). Conclusion: In this hypothesis-generating study PITX2 DNA methylation demonstrated predominantly predictive value in anthracycline treatment in TNBC patients. The risk of poor outcome (OS) correlates with increasing PITX2 DNA methylation.

2021 Scientific Article in Life Science Alliance Life Sci. All. 4:e202000924 (2021)

Karlina, R.# ; Lutter, D.#&deg ; Miok, V. ; Fischer, D.S. ; Altun, I. ; Schöttl, T. ; Schorpp, K.K. ; Israel, A. ; Cero, C. ; Johnson, J.W. ; Kapser-Fischer, I. ; Böttcher, A. ; Keipert, S. ; Feuchtinger, A. ; Graf, E. ; Strom, T.M. ; Walch, A.K. ; Lickert, H. ; Walzthoeni, T. ; Heinig, M. ; Theis, F.J. ; García-Cáceres, C. ; Cypess, A.M. ; Ussar, S.&deg

Identification and characterization of distinct brown adipocyte subtypes in C57BL/6J mice.

Brown adipose tissue (BAT) plays an important role in the regulation of body weight and glucose homeostasis. Although increasing evidence supports white adipose tissue heterogeneity, little is known about heterogeneity within murine BAT. Recently, UCP1 high and low expressing brown adipocytes were identified, but a developmental origin of these subtypes has not been studied. To obtain more insights into brown preadipocyte heterogeneity, we use single-cell RNA sequencing of the BAT stromal vascular fraction of C57/BL6 mice and characterize brown preadipocyte and adipocyte clonal cell lines. Statistical analysis of gene expression profiles from brown preadipocyte and adipocyte clones identify markers distinguishing brown adipocyte subtypes. We confirm the presence of distinct brown adipocyte populations in vivo using the markers EIF5, TCF25, and BIN1. We also demonstrate that loss of Bin1 enhances UCP1 expression and mitochondrial respiration, suggesting that BIN1 marks dormant brown adipocytes. The existence of multiple brown adipocyte subtypes suggests distinct functional properties of BAT depending on its cellular composition, with potentially distinct functions in thermogenesis and the regulation of whole body energy homeostasis.

2021 Scientific Article in International Journal of Radiation Biology Int. J. Radiat. Biol. 97, 156-169 (2021)

Ung, M.-C. ; Garrett, L. ; Dalke, C. ; Leitner, V. ; Dragosa, D. ; Hladik, D ; Neff, F. ; Wagner, F. ; Zitzelsberger, H. ; Miller, G. ; Hrabě de Angelis, M. ; Rößler, U. ; Vogt Weisenhorn, D.M. ; Wurst, W. ; Graw, J. ; Hölter, S.M.

Dose-dependent long-term effects of a single radiation event on behaviour and glial cells.

Purpose: The increasing use of low-dose ionizing radiation in medicine requires a systematic study of its long-term effects on the brain, behaviour and its possible association with neurodegenerative disease vulnerability. Therefore, we analysed the long-term effects of a single low-dose irradiation exposure at 10 weeks of age compared to medium and higher doses on locomotor, emotion-related and sensorimotor behaviour in mice as well as on hippocampal glial cell populations. Materials and methods: We determined the influence of radiation dose (0, 0.063, 0.125 or 0.5 Gy), time post-irradiation (4, 12 and 18 months p.i.), sex and genotype (wild type versus mice with Ercc2 DNA repair gene point mutation) on behaviour. Results: The high dose (0.5 Gy) had early-onset adverse effects at 4 months p.i. on sensorimotor recruitment and late-onset negative locomotor effects at 12 and 18 months p.i. Notably, the low dose (0.063 Gy) produced no early effects but subtle late-onset (18 months) protective effects on sensorimotor recruitment and exploratory behaviour. Quantification and morphological characterization of the microglial and the astrocytic cells of the dentate gyrus 24 months p.i. indicated heightened immune activity after high dose irradiation (0.125 and 0.5 Gy) while conversely, low dose (0.063 Gy) induced more neuroprotective features. Conclusion: This is one of the first studies demonstrating such long-term and late-onset effects on brain and behaviour after a single radiation event in adulthood.

2021 Scientific Article in European Radiology Eur. Radiol. 31, 4175–4183 (2021)

Burkhardt, R. ; Gora, T. ; Fingerle, A.A. ; Sauter, A.P. ; Meurer, F. ; Umkehrer, S. ; Von Teuffenbach, M. ; Kampfer, S. ; Schilling, D. ; Feuchtinger, A. ; Walch, A.K. ; Rummeny, E. ; Combs, S.E. ; Schmid, T.E. ; Pfeiffer, F. ; Wilkens, J.J. ; Herzen, J.

Early detection of radiation-induced lung damage with X-ray dark-field radiography in mice.

Objective: Assessing the advantage of x-ray dark-field contrast over x-ray transmission contrast in radiography for the detection of developing radiation-induced lung damage in mice. Methods: Two groups of female C57BL/6 mice (irradiated and control) were imaged obtaining both contrasts monthly for 28 weeks post irradiation. Six mice received 20 Gy of irradiation to the entire right lung sparing the left lung. The control group of six mice was not irradiated. A total of 88 radiographs of both contrasts were evaluated for both groups based on average values for two regions of interest, covering (irradiated) right lung and healthy left lung. The ratio of these average values, R, was distinguished between healthy and damaged lungs for both contrasts. The time-point when deviations of R from healthy lung exceeded 3σ was determined and compared among contrasts. The Wilcoxon-Mann-Whitney test was used to test against the null hypothesis that there is no difference between both groups. A selection of 32 radiographs was assessed by radiologists. Sensitivity and specificity were determined in order to compare the diagnostic potential of both contrasts. Inter-reader and intra-reader accuracy were rated with Cohen’s kappa. Results: Radiation-induced morphological changes of lung tissue caused deviations from the control group that were measured on average 10 weeks earlier with x-ray dark-field contrast than with x-ray transmission contrast. Sensitivity, specificity, and accuracy doubled using dark-field radiography. Conclusion: X-ray dark-field radiography detects morphological changes of lung tissue associated with radiation-induced damage earlier than transmission radiography in a pre-clinical mouse model. Key Points: • Significant deviations from healthy lung due to irradiation were measured after 16 weeks with x-ray dark-field radiography (p = 0.004). • Significant deviations occur on average 10 weeks earlier for x-ray dark-field radiography in comparison to x-ray transmission radiography. • Sensitivity and specificity doubled when using x-ray dark-field radiography instead of x-ray transmission radiography.

2021 Scientific Article in Diabetes, Obesity and Metabolism Diabetes Obes. Metab. 23, 195-207 (2021)

Sachs, S. ; Niu, L. ; Geyer, P. ; Jall, S. ; Kleinert, M. ; Feuchtinger, A. ; Stemmer, K. ; Brielmeier, M. ; Finan, B. ; DiMarchi, R.D. ; Tschöp, M.H. ; Wewer Albrechtsen, N. ; Mann, M. ; Müller, T.D.&deg ; Hofmann, S.M.&deg

Plasma proteome profiles treatment efficacy of incretin dual agonism in diet-induced obese female and male mice.

Aims Unimolecular peptides targeting the receptors for glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) (GLP-1/GIP co-agonist) have been shown to outperform each single peptide in the treatment of obesity and cardiometabolic disease in preclinical and clinical trials. By combining physiological treatment endpoints with plasma proteomic profiling (PPP), we aimed to identify biomarkers to advance non-invasive metabolic monitoring of compound treatment success and exploration of ulterior treatment effects on an individual basis.Materials and methods We performed metabolic phenotyping along with PPP in body weight-matched male and female diet-induced obese (DIO) mice treated for 21 days with phosphate-buffered saline, single GIP and GLP-1 mono-agonists, or a GLP-1/GIP co-agonist.Results GLP-1R/GIPR co-agonism improved obesity, glucose intolerance, non-alcoholic fatty liver disease (NAFLD) and dyslipidaemia with superior efficacy in both male and female mice compared with mono-agonist treatments. PPP revealed broader changes of plasma proteins after GLP-1/GIP co-agonist compared with mono-agonist treatments in both sexes, including established and potential novel biomarkers for systemic inflammation, NAFLD and atherosclerosis. Subtle sex-specific differences have been observed in metabolic phenotyping and PPP.Conclusions We herein show that a recently developed unimolecular GLP-1/GIP co-agonist is more efficient in improving metabolic disease than either mono-agonist in both sexes. PPP led to the identification of a sex-independent protein panel with the potential to monitor non-invasively the treatment efficacies on metabolic function of this clinically advancing GLP-1/GIP co-agonist.

2021 Scientific Article in International Journal of Radiation Oncology, Biology, Physics Int. J. Radiat. Oncol. Biol. Phys. 109, 76-83 (2021)

Sammer, M.# ; Dombrowsky, A.# ; Schauer, J. ; Oleksenko, K. ; Bicher, S. ; Schwarz, B. ; Rudigkeit, S. ; Matejka, N. ; Reindl, J. ; Bartzsch, S. ; Blutke, A. ; Feuchtinger, A. ; Combs, S.E. ; Dollinger, G. ; Schmid, T.E.

Normal tissue response of combined temporal and spatial fractionation in proton minibeam radiation therapy.

Purpose: Proton minibeam radiation therapy, a spatial fractionation concept, widens the therapeutic window. By reducing normal tissue toxicities, it allows a temporally fractionated regime with high daily doses. However, an array shift between daily fractions can affect the tissue-sparing effect by decreasing the total peak-to-valley dose ratio. Therefore, combining temporal fractions with spatial fractionation raises questions about the impact of daily applied dose modulations, reirradiation accuracies, and total dose modulations. Methods and Materials: Healthy mouse ear pinnae were irradiated with 4 daily fractions of 30 Gy mean dose, applying proton pencil minibeams (pMB) of Gaussian σ = 222 μm in 3 different schemes: a 16 pMB array with a center-to-center distance of 1.8 mm irradiated the same position in all sessions (FS1) or was shifted by 0.9 mm to never hit the previously irradiated tissue in each session (FS2), or a 64 pMB array with a center-to-center distance of 0.9 mm irradiated the same position in all sessions (FS3), resulting in the same total dose distribution as FS2. Reirradiation positioning and its accuracy were obtained from image guidance using the unique vessel structure of ears. Acute toxicities (swelling, erythema, and desquamation) were evaluated for 153 days after the first fraction. Late toxicities (fibrous tissue, inflammation) were analyzed on day 153. Results: Reirradiation of highly dose-modulated arrays at a positioning accuracy of 110 ± 52 μm induced the least severe acute and late toxicities. A shift of the same array in FS2 led to significantly inducted acute toxicities, a higher otitis score, and a slight increase in fibrous tissue. FS3 led to the strongest increase in acute and late toxicities. Conclusions: The highest normal-tissue sparing is achieved after accurate reirradiation of a highly dose modulated pMB array, although high positioning accuracies are challenging in a clinical environment. Nevertheless, the same integral dose applied in highly dose-modulated fractions is superior to low daily dose-modulated fractions.

2020 Scientific Article in PLoS ONE PLoS ONE 15:e0243462 (2020)

Fiedler, S. ; Wünnemann, H. ; Hofmann, I. ; Theobalt, N. ; Feuchtinger, A. ; Walch, A.K. ; Schwaiger, J. ; Wanke, R. ; Blutke, A.

A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies.

Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium (i.e., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300–2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments.

2020 Scientific Article in Frontiers in cell and developmental biology Front. Cell Dev. Biol. 8:570305 (2020)

Wang, Q.# ; Yang, L.# ; Fan, Y. ; Tang, W. ; Sun, H. ; Xu, Z. ; Zhou, J. ; Zhang, Y. ; Zhu, B.&deg ; Cao, X.&deg

Circ-ZDHHC5 accelerates esophageal squamous cell carcinoma progression in vitro via miR-217/ZEB1 axis.

Circular RNA (circRNA) exhibits a covalently closed circular conformation and is structurally stable. Nevertheless, the precise effects exerted by circRNA in esophageal squamous cell carcinoma (ESCC) remains uncertain. circRNA was ascertained by a human circRNA array study and was confirmed by the quantification of reverse transcriptase polymerase reactions. A luciferase reporter, fluorescence in situ hybridization experiment was exploited to explore the interaction between circ-ZDHHC5 and miR-217. The function of circ-ZDHHC5 was determined by siRNA-mediated knockout of circ-ZDHHC5 in in vitro proliferation, migration, and invasion. circ-ZDHHC5, rather than linear ZDHHC5 mRNA, rose in the tissues of patients with ESCC, plasma, and ESCC cell lines in comparison with normal controls. Knockdown of circ-ZDHHC5 inhibited tumorigenesis in ESCC cells, and the co-transfection of si-circ-ZDHHC5 and miR-217 mimics further enhanced the above effect. Noticeably, the present study showed that circ-ZDHHC5 was an miR-217 sponge that modulated the expression of zinc finger E-box binding homeobox 1 (ZEB1), further facilitating ESCC tumorigenesis. As revealed by this study, circ-ZDHHC5 can act as a new potential circular biomarker for detecting ESCC. It provides a novel perceptivity for the treatment of ESCC suggesting that circ-ZDHHC5 could impact on ESCC progression by sponging miR-217 with ZEB1.

2020 Scientific Article in European Urology Open Science Eu. Urol. Open Sci. 22, 88-96 (2020)

Papathomas, T.# ; Tzortzakakis, A. ; Sun, N.# ; Erlmeier, F. ; Feuchtinger, A. ; Trpkov, K. ; Bazarova, A. ; Arvanitis, A. ; Wang, W. ; Bozoky, B. ; Kokaraki, G. ; Axelsson, R. ; Walch, A.K.

In situ metabolomics expands the spectrum of renal tumours positive on 99mTc-sestamibi single photon emission computed tomography/computed tomography examination.

Background: Definite noninvasive characterisation of renal tumours positive on 99mTc-sestamibi single photon emission computed tomography/computed tomography (SPECT/CT) examination including renal oncocytomas (ROs), hybrid oncocytic chromophobe tumours (HOCTs), and chromophobe renal cell carcinoma (chRCC) is currently not feasible. Objective: To investigate whether combined 99mTc-sestamibi SPECT/CT and in situ metabolomic profiling can accurately characterise renal tumours exhibiting 99mTc-sestamibi uptake. Design, setting, and participants: A tissue microarray analysis of 33 tumour samples from 28 patients was used to investigate whether their in situ metabolomic status correlates with their features on 99mTc-sestamibi SPECT/CT examination. In order to validate emerging data, an independent cohort comprising 117 tumours was subjected to matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI MSI). Outcome measurements and statistical analysis: MALDI MSI data analysis and image generation were facilitated by FlexImaging v. 4.2, while k-means analysis by SCiLS Lab software followed by R-package CARRoT analysis was used for assessing the highest predictive power in the differential of RO versus chRCC. Heatmap-based clustering, sparse partial least-squares discriminant analysis, and volcano plots were created with MetaboAnalyst 3.0. Results and limitations: We identified a discriminatory metabolomic signature for 99mTc-sestamibi SPECT/CT–positive Birt-Hogg-Dubè–associated HOCTs versus other renal oncocytic tumours. Metabolomic differences were also evident between 99mTc-sestamibi–positive and 99mTc-sestamibi–negative chRCCs, prompting additional expert review; two of three 99mTc-sestamibi–positive chRCCs were reclassified as low-grade oncocytic tumours (LOTs). Differences were identified between distal-derived tumours from those of proximal tubule origin, including differences between ROs and chRCCs. Conclusions: The current study expands the spectrum of 99mTc-sestamibi SPECT/CT–positive renal tumours, encompassing ROs, HOCTs, LOTs, and chRCCs, and supports the feasibility of in situ metabolomic profiling in the diagnostics and classification of renal tumours. Patient summary: For preoperative evaluation of solid renal tumours, 99mTc-sestamibi single photon emission computed tomography/computed tomography (SPECT/CT) is a novel examination method. To increase diagnostic accuracy, we propose that 99mTc-sestamibi–positive renal tumours should be biopsied and followed by a combined histometabolomic analysis.

2020 Scientific Article in Hypertension Hypertension 76, 1769-1777 (2020)

Vohra, T. ; Kemter, E. ; Sun, N. ; Dobenecker, B. ; Hinrichs, A. ; Burrello, J. ; Gomez-Sanchez, E.P. ; Gomez-Sanchez, C.E. ; Wang, J. ; Kinker, I.S. ; Teupser, D. ; Fischer, K. ; Schnieke, A. ; Peitzsch, M. ; Eisenhofer, G. ; Walch, A.K. ; Reincke, M. ; Wolf, E. ; Williams, T.A.

Effect of dietary sodium modulation on pig adrenal steroidogenesis and transcriptome profiles.

Primary aldosteronism is a frequent form of endocrine hypertension caused by aldosterone overproduction from the adrenal cortex. Regulation of aldosterone biosynthesis has been studied in rodents despite differences in adrenal physiology with humans. We, therefore, investigated pig adrenal steroidogenesis, morphology, and transcriptome profiles of the zona glomerulosa (zG) and zona fasciculata in response to activation of the renin-angiotensin-aldosterone system by dietary sodium restriction. Six-week-old pigs were fed a low- or high-sodium diet for 14 days (3 pigs per group, 0.4 g sodium/kg feed versus 6.8 g sodium/kg). Plasma aldosterone concentrations displayed a 43-fold increase (P=0.011) after 14 days of sodium restriction (day 14 versus day 0). Low dietary sodium caused a 2-fold increase in thickness of the zG (P<0.001) and an almost 3-fold upregulation of CYP11B (P<0.05) compared with high dietary sodium. Strong immunostaining of the KCNJ5 (G protein-activated inward rectifier potassium channel 4), which is frequently mutated in primary aldosteronism, was demonstrated in the zG. mRNA sequencing transcriptome analysis identified significantly altered expression of genes modulated by the renin-angiotensin-aldosterone system in the zG (n=1172) and zona fasciculata (n=280). These genes included many with a known role in the regulation of aldosterone synthesis and adrenal function. The most highly enriched biological pathways in the zG were related to cholesterol biosynthesis, steroid metabolism, cell cycle, and potassium channels. This study provides mechanistic insights into the physiology and pathophysiology of aldosterone production in a species closely related to humans and shows the suitability of pigs as a translational animal model for human adrenal steroidogenesis.

2020 Review in Frontiers in Oncology Front. Oncol. 10:575037 (2020)

Wu, F. ; Cheng, Y. ; Wu, L. ; Zhang, W. ; Zheng, W. ; Wang, Q.&deg ; Cao, H. ; Pan, X. ; Tang, W.&deg

Emerging landscapes of tumor immunity and metabolism.

The metabolic reprogramming of cancer tissue has higher metabolic activity than surrounding tissues. At the same time, the local infiltration of immunosuppressive cells is also significantly increased, resulting in a significant decrease in tumor immunity. During the progression of cancer cells, immunosuppressive tumor microenvironment is formed around the tumor due to their metabolic reprogramming. In addition, it is the changes in metabolic patterns that make tumor cells resistant to certain drugs, impeding cancer treatment. This article reviews the mechanisms of immune escape caused by metabolic reprogramming, and aims to provide new ideas for clinical tumor immunotherapy combined with metabolic intervention for tumor treatment.

2020 Review in Animal reproduction Anim. Reprod. 17:e20200064 (2020)

Zettler, S. ; Renner, S. ; Kemter, E. ; Hinrichs, A. ; Klymiuk, N. ; Backman, M. ; Riedel, E.O. ; Mueller, C. ; Streckel, E. ; Braun-Reichhart, C. ; Martins, A.S. ; Kurome, M. ; Keßler, B. ; Zakhartchenko, V. ; Flenkenthaler, F. ; Arnold, G.J. ; Fröhlich, T. ; Blum, H. ; Blutke, A. ; Wanke, R. ; Wolf, E.

A decade of experience with genetically tailored pig models for diabetes and metabolic research.

The global prevalence of diabetes mellitus and other metabolic diseases is rapidly increasing. Animal models play pivotal roles in unravelling disease mechanisms and developing and testing therapeutic strategies. Rodents are the most widely used animal models but may have limitations in their resemblance to human disease mechanisms and phenotypes. Findings in rodent models are consequently often difficult to extrapolate to human clinical trials. To overcome this 'translational gap', we and other groups are developing porcine disease models. Pigs share many anatomical and physiological traits with humans and thus hold great promise as translational animal models. Importantly, the toolbox for genetic engineering of pigs is rapidly expanding. Human disease mechanisms and targets can therefore be reproduced in pigs on a molecular level, resulting in precise and predictive porcine (PPP) models. In this short review, we summarize our work on the development of genetically (pre)diabetic pig models and how they have been used to study disease mechanisms and test therapeutic strategies. This includes the generation of reporter pigs for studying beta-cell maturation and physiology. Furthermore, genetically engineered pigs are promising donors of pancreatic islets for xenotransplantation. In summary, genetically tailored pig models have become an important link in the chain of translational diabetes and metabolic research.

2020 Scientific Article in Communications Biology Comm. Biol. 3:628 (2020)

Chhabra, N.F. ; Amarie, O.V. ; Wu, M. ; Amend, A.-L. ; Rubey, M. ; Gradinger, D. ; Irmler, M. ; Beckers, J. ; Rathkolb, B. ; Wolf, E. ; Feuchtinger, A. ; Huypens, P. ; Teperino, R. ; Rozman, J. ; Przemeck, G.K.H. ; Hrabě de Angelis, M.

PAX6 mutation alters circadian rhythm and beta cell function in mice without affecting glucose tolerance.

The transcription factor PAX6 is involved in the development of the eye and pancreatic islets, besides being associated with sleep-wake cycles. Here, we investigated a point mutation in the RED subdomain of PAX6, previously described in a human patient, to present a comprehensive study of a homozygous Pax6 mutation in the context of adult mammalian metabolism and circadian rhythm. Pax6(Leca2) mice lack appropriate retinal structures for light perception and do not display normal daily rhythmic changes in energy metabolism. Despite beta cell dysfunction and decreased insulin secretion, mutant mice have normal glucose tolerance. This is associated with reduced hepatic glucose production possibly due to altered circadian variation in expression of clock and metabolic genes, thereby evading hyperglycemia. Hence, our findings show that while the RED subdomain is important for beta cell functional maturity, the Leca2 mutation impacts peripheral metabolism via loss of circadian rhythm, thus revealing pleiotropic effects of PAX6. Nirav Chhabra et al. characterize adult mice carrying a homozygous mutation in Pax6 that was identified in a patient with foveal hypoplasia. They find that the Pax6 point mutation has pleiotropic effects, including defects in the mouse retinal structures, loss of the optic nerve, changes in energy metabolism and circadian rhythms, and dysregulation of genes expressed in the pancreas.

2020 Scientific Article in International Journal of Molecular Sciences Int. J. Mol. Sci. 21:6832 (2020)

Azimzadeh, O. ; Azizova, T. ; Merl-Pham, J. ; Blutke, A. ; Moseeva, M. ; Zubkova, O. ; Anastasov, N. ; Feuchtinger, A. ; Hauck, S.M. ; Atkinson, M.J. ; Tapio, S.

Chronic occupational exposure to ionizing radiation induces alterations in the structure and metabolism of the heart: A proteomic analysis of human formalin-fixed paraffin-embedded (FFPE) cardiac tissue.

Epidemiological studies on workers employed at the Mayak plutonium enrichment plant have demonstrated an association between external gamma ray exposure and an elevated risk of ischemic heart disease (IHD). In a previous study using fresh-frozen post mortem samples of the cardiac left ventricle of Mayak workers and non-irradiated controls, we observed radiation-induced alterations in the heart proteome, mainly downregulation of mitochondrial and structural proteins. As the control group available at that time was younger than the irradiated group, we could not exclude age as a confounding factor. To address this issue, we have now expanded our study to investigate additional samples using archival formalin-fixed paraffin-embedded (FFPE) tissue. Importantly, the control group studied here is older than the occupationally exposed (>500 mGy) group. Label-free quantitative proteomics analysis showed that proteins involved in the lipid metabolism, sirtuin signaling, mitochondrial function, cytoskeletal organization, and antioxidant defense were the most affected. A histopathological analysis elucidated large foci of fibrotic tissue, myocardial lipomatosis and lymphocytic infiltrations in the irradiated samples. These data highlight the suitability of FFPE material for proteomics analysis. The study confirms the previous results emphasizing the role of adverse metabolic changes in the radiation-associated IHD. Most importantly, it excludes age at the time of death as a confounding factor.

2020 Scientific Article in Analytica Chimica Acta Anal. Chim. Acta 1134, 125-135 (2020)

Neef, S.K. ; Winter, S. ; Hofmann, U. ; Mürdter, T.E. ; Schaeffeler, E. ; Horn, H. ; Buck, A. ; Walch, A.K. ; Hennenlotter, J. ; Ott, G. ; Fend, F. ; Bedke, J. ; Schwab, M. ; Haag, M.

Optimized protocol for metabolomic and lipidomic profiling in formalin-fixed paraffin-embedded kidney tissue by LC-MS.

Formalin-fixed and paraffin-embedded (FFPE) tissue represents a valuable resource to examine cancer metabolic alterations and to identify potential markers of disease. Protocols commonly used for liquid-chromatography mass spectrometry (LC-MS)-based FFPE metabolomics have not been optimized for lipidomic analysis and pre-analytical factors, that potentially affect metabolite levels, were scarcely investigated. We here demonstrate the assessment and optimization of sample preparation procedures for comprehensive metabolomic and lipidomic profiling in FFPE kidney tissue by LC-QTOF-MS. The optimized protocol allows improved monitoring of lipids including ceramides (Cer), glycosphingolipids (GSL) and triglycerides (TAGs) while the profiling capability for small polar molecules is maintained. Further, repeatable sample preparation (CVs < 20%) along with high analytical (CVs < 10%) and inter-day precision (CVs < 20%) is achieved. As proof of concept, we analyzed a set of clear cell renal cell carcinoma (ccRCC) and corresponding non-tumorous FFPE tissue samples, achieving phenotypic distinction. Investigation of the impact of tissue fixation time (6 h, 30 h and 54 h) on FFPE tissue metabolic profiles revealed metabolite class-dependent differences on their detection abundance. Whereas specific lipids (e.g. phosphatidylinositoles, GSLs, saturated fatty acids and saturated lyso-phosphatidytlethanolamines LPED remained largely unaffected (CVs < 20% between groups of fixation time), neutral lipids (e.g. Cer and TAGs) exhibited high variability (CVs > 80%). Strikingly, out of the lipid classes assigned as unaffected, fatty acids 18:0, 16:0 and LPE 18:0 were detectable by high-resolution MALDI-FT-ICR MS imaging in an independent cohort of ccRCC tissues (n = 64) and exhibited significant differences between tumor and non-tumor regions.

2020 Scientific Article in Scientific Reports Sci. Rep. 10:14461 (2020)

Blutke, A. ; Sun, N. ; Xu, Z. ; Buck, A. ; Harrison, L. ; Schriever, S.C. ; Pfluger, P.T. ; Wiles, D. ; Kunzke, T. ; Huber, K. ; Schlegel, J. ; Aichler, M. ; Feuchtinger, A. ; Matiasek, K. ; Hauck, S.M. ; Walch, A.K.

Light sheet fluorescence microscopy guided MALDI-imaging mass spectrometry of cleared tissue samples.

Light sheet fluorescence microscopy (LSFM) of optically cleared biological samples represents a powerful tool to analyze the 3-dimensional morphology of tissues and organs. Multimodal combinations of LSFM with additional analyses of the identical sample help to limit the consumption of restricted specimen and reduce inter-sample variation. Here, we demonstrate the proof-of-concept that LSFM of cleared brain tissue samples can be combined with Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) for detection and quantification of proteins. Samples of freshly dissected murine brain and of archived formalin-fixed paraffin-embedded (FFPE) human brain tissue were cleared (3DISCO). Tissue regions of interest were defined by LSFM and excised, (re)-embedded in paraffin, and sectioned. Mouse sections were coated with sinapinic acid matrix. Human brain sections were pre-digested with trypsin and coated with α-cyano-4-hydroxycinnamic acid matrix. Subsequently, sections were subjected to MALDI-time-of-flight (TOF)-MSI in mass ranges between 0.8 to 4 kDa (human tissue sections), or 2.5–25 kDa (mouse tissue sections) with a lateral resolution of 50 µm. Protein- and peptide-identities corresponding to acquired MALDI-MSI spectra were confirmed by parallel liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis. The spatial abundance- and intensity-patterns of established marker proteins detected by MALDI-MSI were also confirmed by immunohistochemistry.

2020 Scientific Article in American Journal of Cancer Research Am. J. Cancer Res. 10, 1785-1792 (2020)

Geng, X. ; Babayeva, L. ; Walch, A.K. ; Aubele, M. ; Gross, E. ; Kiechle, M. ; Bronger, H. ; Dreyer, T. ; Magdolen, V. ; Dorn, J.

High levels of KLK7 protein expression are related to a favorable prognosis in triple-negative breast cancer patients.

In normal physiology, kallikrein-related peptidase 7 (KLK7), together with other members of the kallikrein-related peptidase family, is mainly involved in skin desquamation and keratinization processes. Moreover, expression of KLK7 was shown in various tumor types to be dysregulated and to correlate to patients' survival time. However, there are contradictory reports in breast cancer whether KLK7 represents an unfavorable or favorable prognostic biomarker. In the present study, we examined the prognostic value of KLK7 protein expression in triple-negative breast cancer (TNBC), determined by immunohistochemistry (IHC). A cohort encompassing 133 TNBC specimens, present on tissue microarrays, was analyzed. For quantification of the staining intensity, an automated digital IHC image analysis algorithm was applied. In both Kaplan-Meier and univariate Cox analyses, elevated KLK7 protein levels were significantly linked with prolonged overall survival (OS). In multivariable Cox analysis, addition of KLK7 immunoreactivity scores to the base model (including the clinical parameters age, tumor size, and nodal status) demonstrated that KLK7 protein expression remained as a statistically significant, independent parameter for prolonged OS. These results strongly indicate that KLK7 is a favorable prognostic biomarker in triple negative breast cancer.

2020 Scientific Article in Angewandte Chemie - Internationale Edition Angew. Chem.-Int. Edit. 132, 17600-17603 (2020)

Ščupáková, K. ; Dewez, F. ; Walch, A.K. ; Heeren, R.M.A. ; Balluff, B.

Morphometric cell classification for single-Cell MALDI-mass spectrometry imaging.

The large-scale and label-free molecular characterization of single cells in their natural tissue habitat remains a major challenge in molecular biology. We present a method that integrates morphometric image analysis to delineate and classify individual cells with their single-cell-specific molecular profiles. This approach provides a new means to study spatial biological processes such as cancer field effects and the relationship between morphometric and molecular features.

2020 Scientific Article in Nature Communications Nat. Commun. 11:3068 (2020)

Fischer, A. ; Koopmans, T. ; Ramesh, P. ; Christ, S. ; Strunz, M. ; Wannemacher, J. ; Aichler, M. ; Feuchtinger, A. ; Walch, A.K. ; Ansari, M. ; Theis, F.J. ; Schorpp, K.K. ; Hadian, K. ; Neumann, P.A. ; Schiller, H. B. ; Rinkevich, Y.

Post-surgical adhesions are triggered by calcium-dependent membrane bridges between mesothelial surfaces.

Surgical adhesions are bands of scar tissues that abnormally conjoin organ surfaces. Adhesions are a major cause of post-operative and dialysis-related complications, yet their patho-mechanism remains elusive, and prevention agents in clinical trials have thus far failed to achieve efficacy. Here, we uncover the adhesion initiation mechanism by coating beads with human mesothelial cells that normally line organ surfaces, and viewing them under adhesion stimuli. We document expansive membrane protrusions from mesothelia that tether beads with massive accompanying adherence forces. Membrane protrusions precede matrix deposition, and can transmit adhesion stimuli to healthy surfaces. We identify cytoskeletal effectors and calcium signaling as molecular triggers that initiate surgical adhesions. A single, localized dose targeting these early germinal events completely prevented adhesions in a preclinical mouse model, and in human assays. Our findings classifies the adhesion pathology as originating from mesothelial membrane bridges and offer a radically new therapeutic approach to treat adhesions.

2020 Scientific Article in ChemBioChem ChemBioChem 21, 2495-2502 (2020)

Niu, Z. ; Sarkar, R. ; Aichler, M. ; Wester, H. ; Yousefi, B.H.&deg ; Reif, B.&deg

Mapping of the binding interface of PET tracer molecules and Alzheimer Disease Aβ fibrils using MAS solid-state NMR spectroscopy.

Positron emission tomography (PET) tracer molecules like thioflavin T specifically recognize amyloid deposition in brain tissue by selective binding to hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The molecular basis of this interaction is, however, not well understood. We have employed magic angle spinning (MAS) solid-state NMR spectroscopy to characterize Aβ-PET tracer complexes at atomic resolution. We established a titration protocol by using bovine serum albumin as a carrier to transfer hydrophobic small molecules to Aβ(1-40) fibrillar aggregates. The same Aβ(1-40) amyloid fibril sample was employed in subsequent titrations to minimize systematic errors that potentially arise from sample preparation. In the experiments, the small molecules 13C-methylated Pittsburgh compound B (PiB) as well as a novel Aβ tracer based on a diarylbithiazole (DABTA) scaffold were employed. Classical 13C-detected as well as proton-detected spectra of protonated and perdeuterated samples with back-substituted protons, respectively, were acquired and analyzed. After titration of the tracers, chemical-shift perturbations were observed in the loop region involving residues Gly25-Lys28 and Ile32-Gly33, thus suggesting that the PET tracer molecules interact with the loop region connecting β-sheets β1 and β2 in Aβ fibrils. We found that titration of the PiB derivatives suppressed fibril polymorphism and stabilized the amyloid fibril structure.

2020 Review in Hormone and Metabolic Research Horm. Metab. Res. 52, 435-447 (2020)

Li, F. ; Feuchtinger, A. ; Walch, A.K. ; Sun, N.

In situ metabolite mass spectrometry imaging: New insights into the adrenal gland.

The adrenal gland integrates catecholamine-producing neuroendocrine cells and steroid-producing cells with mesenchymal origin in a structured manner under one capsule and is a key regulator for vital bioactivity. In addition to adrenal-specific disease, dysregulation of adrenal hormones is associated with systemic effects, leading to undesirable metabolic and cardiovascular consequences. Mass spectrometry imaging (MSI) technique can simultaneously measure a broad range of biomolecules, including metabolites and hormones, which has enabled the study of tissue metabolic and hormone alterations in adrenal and adrenal-related diseases. Furthermore, this technique coupled with labeled immunohistochemistry staining has enabled the study of the pathophysiological adaptation of the adrenal gland under normal and abnormal conditions at different molecular levels. This review discusses the recent applications of in situ MSI in the adrenal gland. For example, the combination of formalin-fixed paraffin-embedded tissue microarray and MSI to tissues from patient cohorts has facilitated the discovery of clinically relevant prognostic biomolecules and generated promising hypotheses for new sights into physiology and pathophysiology of adrenal gland. MSI also has enabled the discovery of clinically significant tissue molecular (i. e., biomarker) and pathway changes in adrenal disease, particularly in adrenal tumors. In addition, MSI has advanced the ability to optimally identify and detect adrenal gland specific molecules. Thus, as a novel analytical methodology, MSI has provided unprecedented capabilities for in situ tissue study.

2020 Scientific Article in Frontiers in Oncology Front. Oncol. 10:494 (2020)

Bergmann, N. ; Delbridge, C. ; Gempt, J. ; Feuchtinger, A. ; Walch, A.K. ; Schirmer, L. ; Bunk, W. ; Aschenbrenner, T. ; Liesche-Starnecker, F. ; Schlegel, J.

The intratumoral heterogeneity reflects the intertumoral subtypes of glioblastoma multiforme: A regional immunohistochemistry analysis.

Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor in adults. Despite extensive therapy the prognosis for GBM patients remains poor and the extraordinary therapy resistance has been attributed to intertumoral heterogeneity of glioblastoma. Different prognostic relevant GBM tumor subtypes have been identified based on their molecular profile. This approach, however, neglects the heterogeneity within individual tumors, that is, the intratumoral heterogeneity. Here, we detected the regional immunoreactivity by immunohistochemistry and immunofluorescence using nine different markers on resected GBM specimens (IDH wildtype, WHO grade IV). We found repetitive expression profiles, that could be classified into clusters. These clusters could then be assigned to five pathophysiologically relevant groups that reflect the previously described subclasses of GBM, including mesenchymal, classical, and proneural subtype. Our data indicate the presence of tumor differentiations and tumor subclasses that occur within individual tumors, and might therefore contribute to develop adapted, individual-based therapies.

2020 Scientific Article in Molecular Oncology Mol. Oncol. 14, 1653-1669 (2020)

Hedegger, K. ; Algül, H. ; Lesina, M. ; Blutke, A. ; Schmid, R.M. ; Schneider, M.R. ; Dahlhoff, M.

Unraveling ERBB network dynamics upon betacellulin signaling in pancreatic ductal adenocarcinoma in mice.

Pancreatic ductal adenocarcinoma (PDAC) will soon belong to the top three cancer killers. The only approved specific PDAC therapy targets the epidermal growth factor receptor (EGFR). Although EGFR is a crucial player in PDAC development, EGFR-based therapy is disappointing. In this study, we evaluated the role of the EGFR ligand betacellulin (BTC) in PDAC. The expression of BTC was investigated in human pancreatic cancer specimen. Then, we generated a BTC knockout mouse model by CRISPR/Cas9 technology and a BTC overexpression model. Both models were crossed with the Ptf1a(Cre/+);KRAS(G12D/+) (KC) mouse model (B-/-KC or BKC, respectively). In addition, EGFR, ERBB2, and ERBB4 were investigated by the pancreas-specific deletion of each receptor using the Cre-loxP system. Tumor initiation and progression were analyzed in all mouse lines, and the underlying molecular biology of PDAC was investigated at different time points. BTC is expressed in human and murine PDAC. B-/-KC mice showed a decelerated PDAC progression, associated with decreased EGFR activation. BKC mice developed severe PDAC with a poor survival rate. The dramatically increased BTC-mediated tumor burden was EGFR-dependent, but also ERBB4 and ERBB2 were involved in PDAC development or progression, as depletion of EGFR, ERBB2, or ERBB4 significantly improved the survival rate of BTC-mediated PDAC. BTC increases PDAC tumor burden dramatically by enhanced RAS activation. EGFR signaling, ERBB2 signaling, and ERBB4 signaling are involved in accelerated PDAC development mediated by BTC indicating that targeting the whole ERBB family, instead of a single receptor, is a promising strategy for the development of future PDAC therapies.

2020 Scientific Article in Molecular Metabolism Mol. Metab. 36:100953 (2020)

Prade, V.M.# ; Kunzke, T.# ; Feuchtinger, A. ; Rohm, M. ; Luber, B. ; Lordick, F. ; Buck, A.&deg ; Walch, A.K.&deg

De novo discovery of metabolic heterogeneity with immunophenotype-guided imaging mass spectrometry.

Background: Imaging mass spectrometry enables in situ label-free detection of thousands of metabolites from intact tissue samples. However, automated steps for multi-omics analyses and interpretation of histological images have not yet been implemented in mass spectrometry data analysis workflows. The characterization of molecular properties within cellular and histological features is done via time-consuming, nonobjective, and irreproducible definitions of regions of interest, which are often accompanied by a loss of spatial resolution due to mass spectra averaging.Methods: We developed a new imaging pipeline called Spatial Correlation Image Analysis (SPACiAL), which is a computational multimodal workflow designed to combine molecular imaging data with multiplex immunohistochemistry (IHC). SPACiAL allows comprehensive and spatially resolved in situ correlation analyses on a cellular resolution. To demonstrate the method, matrix-assisted laser desorption-ionization (MALDI) Fourier-transform ion cyclotron resonance (FTICR) imaging mass spectrometry of metabolites and multiplex IHC staining were performed on the very same tissue section of mouse pancreatic islets and on human gastric cancer tissue specimens. The SPACiAL pipeline was used to perform an automatic, semantic-based, functional tissue annotation of histological and cellular features to identify metabolic profiles. Spatial correlation networks were generated to analyze metabolic heterogeneity associated with cellular features.Results: To demonstrate the new method, the SPACiAL pipeline was used to identify metabolic signatures of alpha and beta cells within islets of Langerhans, which are cell types that are not distinguishable via morphology alone. The semantic-based, functional tissue annotation allows an unprecedented analysis of metabolic heterogeneity via the generation of spatial correlation networks. Additionally, we demonstrated intra- and intertumoral metabolic heterogeneity within HER2/neu-positive and -negative gastric tumor cells.Conclusions: We developed the SPACiAL workflow to provide IHC-guided in situ metabolomics on intact tissue sections. Diminishing the workload by automated recognition of histological and functional features, the pipeline allows comprehensive analyses of metabolic heterogeneity. The multimodality of immunohistochemical staining and extensive molecular information from imaging mass spectrometry has the advantage of increasing both the efficiency and precision for spatially resolved analyses of specific cell types. The SPACiAL method is a stepping stone for the objective analysis of high-throughput, multi-omics data from clinical research and practice that is required for diagnostics, biomarker discovery, or therapy response prediction.

2020 Scientific Article in Gut (eGut) Gut 69, 1939-1951 (2020)

Khaloian, S. ; Rath, E. ; Hammoudi, N. ; Gleisinger, E. ; Blutke, A. ; Giesbertz, P. ; Berger, E. ; Metwaly, A. ; Waldschmitt, N. ; Allez, M. ; Haller, D.

Mitochondrial impairment drives intestinal stem cell transition into dysfunctional Paneth cells predicting Crohn's disease recurrence.

Objective Reduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn's disease (CD). PCs reside in proximity to Lgr5(+) intestinal stem cells (ISC) and mitochondria are critical for ISC-renewal and differentiation. Here, we characterise ISC and PC appearance under inflammatory conditions and describe the role of mitochondrial function for ISC niche-maintenance.Design Ileal tissue samples from patients with CD, mouse models for mitochondrial dysfunction (Hsp60(Delta/Delta ISC)) and CD-like ileitis (TNF Delta ARE), and intestinal organoids were used to characterise PCs and ISCs in relation to mitochondrial function.Results In patients with CD and TNF Delta ARE mice, inflammation correlated with reduced numbers of Lysozyme-positive granules in PCs and decreased Lgr5 expression in crypt regions. Disease-associated changes in PC and ISC appearance persisted in non-inflamed tissue regions of patients with CD and predicted the risk of disease recurrence after surgical resection. ISC-specific deletion of Hsp60 and inhibition of mitochondrial respiration linked mitochondrial function to the aberrant PC phenotype. Consistent with reduced stemness in vivo, crypts from inflamed TNF Delta ARE mice fail to grow into organoids ex vivo. Dichloroacetate-mediated inhibition of glycolysis, forcing cells to shift to mitochondrial respiration, improved ISC niche function and rescued the ability of TNF Delta ARE mice-derived crypts to form organoids.Conclusion We provide evidence that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggers a metabolic imbalance, causing reduced stemness and acquisition of a dysfunctional PC phenotype. Blocking glycolysis might be a novel drug target to antagonise PC dysfunction in the pathogenesis of CD.

2020 Nature metabolism Nat. Metab. 2, 380 (2020)

Sachs, S. ; Bastidas-Ponce, A. ; Tritschler, S. ; Bakhti, M. ; Böttcher, A. ; Sánchez-Garrido, M.A. ; Tarquis Medina, M. ; Kleinert, M. ; Fischer, K. ; Jall, S. ; Harger, A. ; Bader, E. ; Roscioni, S. ; Ussar, S. ; Feuchtinger, A. ; Yesildag, B. ; Neelakandhan, A. ; Jensen, C.B. ; Cornu, M. ; Yang, B. ; Finan, B. ; DiMarchi, R.D. ; Tschöp, M.H. ; Theis, F.J.&deg ; Hofmann, S.M.&deg ; Müller, T.D.&deg ; Lickert, H.&deg

Author Correction: Targeted pharmacological therapy restores β-cell function for diabetes remission (Nature Metabolism, (2020), 2, 2, (192-209), 10.1038/s42255-020-0171-3).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

2020 Scientific Article in Cell Death & Disease Cell Death Dis. 11:192 (2020)

Weigand, I. ; Schreiner, J. ; Röhrig, F. ; Sun, N. ; Landwehr, L.S. ; Urlaub, H. ; Kendl, S. ; Kiseljak-Vassiliades, K. ; Wierman, M.E. ; Angeli, J.P.F. ; Walch, A.K. ; Sbiera, S. ; Fassnacht, M. ; Kroiss, M.

Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction.

Conditions of impaired adrenal function and tissue destruction, such as in Addison's disease, and treatment resistance of adrenocortical carcinoma (ACC) necessitate improved understanding of the pathophysiology of adrenal cell death. Due to relevant oxidative processes in the adrenal cortex, our study investigated the role of ferroptosis, an iron-dependent cell death mechanism and found high adrenocortical expression of glutathione peroxidase 4 (GPX4) and long-chain-fatty-acid CoA ligase 4 (ACSL4) genes, key factors in the initiation of ferroptosis. By applying MALDI mass spectrometry imaging to normal and neoplastic adrenocortical tissue, we detected high abundance of arachidonic and adrenic acid, two long chain polyunsaturated fatty acids which undergo peroxidation during ferroptosis. In three available adrenal cortex cell models (H295R, CU-ACC1 and CU-ACC-2) a high susceptibility to GPX4 inhibition with RSL3 was documented with EC50 values of 5.7 x 10(-8), 8.1 x 10(-7) and 2.1 x 10(-8) M, respectively, while all non-steroidogenic cells were significantly less sensitive. Complete block of GPX4 activity by RSL3 led to ferroptosis which was completely reversed in adrenal cortex cells by inhibition of steroidogenesis with ketoconazole but not by blocking the final step of cortisol synthesis with metyrapone. Mitotane, the only approved drug for ACC did not induce ferroptosis, despite strong induction of lipid peroxidation in ACC cells. Together, this report is the first to demonstrate extraordinary sensitivity of adrenal cortex cells to ferroptosis dependent on their active steroid synthetic pathways. Mitotane does not induce this form of cell death in ACC cells.

2020 Scientific Article in Hypertension Hypertension 75, 634-644 (2020)

Sun, N. ; Meyer, L.S. ; Feuchtinger, A. ; Kunzke, T. ; Knösel, T. ; Reincke, M. ; Walch, A.K. ; Williams, T.A.

Mass spectrometry imaging establishes 2 distinct metabolic phenotypes of aldosterone-producing cell clusters in primary aldosteronism.

Aldosterone-producing adenomas (APAs) are one of the main causes of primary aldosteronism and the most prevalent surgically correctable form of hypertension. Aldosterone-producing cell clusters (APCCs) comprise tight nests of zona glomerulosa cells, strongly positive for CYP11B2 (aldosterone synthase) in immunohistochemistry. APCCs have been suggested as possible precursors of APAs because they frequently carry driver mutations for constitutive aldosterone production, and a few adrenal lesions with histopathologic features of both APCCs and APAs have been identified. Our objective was to investigate the metabolic phenotypes of APCCs (n=27) compared with APAs (n=6) using in situ matrix-assisted laser desorption/ionization mass spectrometry imaging of formalin-fixed paraffin-embedded adrenals from patients with unilateral primary aldosteronism. Specific distribution patterns of metabolites were associated with APCCs and classified 2 separate APCC subgroups (subgroups 1 and 2) indistinguishable by CYP11B2 immunohistochemistry. Metabolic profiles of APCCs in subgroup 1 were tightly clustered and distinct from subgroup 2 and APAs. Multiple APCCs from the same adrenal displayed metabolic profiles of the same subgroup. Metabolites of APCC subgroup 2 were highly similar to the APA group and indicated enhanced metabolic pathways favoring cell proliferation compared with APCC subgroup 1. In conclusion, we demonstrate specific subgroups of APCCs with strikingly divergent distribution patterns of metabolites. One subgroup displays a metabolic phenotype convergent with APAs and may represent the progression of APCCs to APAs.

2020 Scientific Article in Molecular Metabolism Mol. Metab. 36:100978 (2020)

Riedel, E.O. ; Hinrichs, A. ; Kemter, E. ; Dahlhoff, M. ; Backman, M. ; Rathkolb, B. ; Prehn, C. ; Adamski, J. ; Renner, S. ; Blutke, A. ; Hrabě de Angelis, M. ; Bidlingmaier, M. ; Schopohl, J. ; Arnold, G.J. ; Fröhlich, T. ; Wolf, E.

Functional changes of the liver in the absence of growth hormone (GH) action - Proteomic and metabolomic insights from a GH receptor deficient pig model.

Objective: The liver is a central target organ of growth hormone (GH), which stimulates the synthesis of insulin-like growth factor 1 (IGF1) and affects multiple biochemical pathways. A systematic multi-omics analysis of GH effects in the liver has not been performed. GH receptor (GHR) deficiency is a unique model for studying the consequences of lacking GH action. In this study, we used molecular profiling techniques to capture a broad spectrum of these effects in the liver of a clinically relevant large animal model for Laron syndrome.Methods: We performed holistic proteome and targeted metabolome analyses of liver samples from 6-month-old GHR-deficient (GHR-KO) pigs and GHR-expressing controls (four males, four females per group).Results: GHR deficiency resulted in an increased abundance of enzymes involved in amino acid degradation, in the urea cycle, and in the tricarboxylic acid cycle. A decreased ratio of long-chain acylcarnitines to free carnitine suggested reduced activity of carnitine palmitoyltransferase 1A and thus reduced mitochondrial import of fatty acids for beta-oxidation. Increased levels of short-chain acylcarnitines in the liver and in the circulation of GHR-KO pigs may result from impaired beta-oxidation of short-chain fatty acids or from increased degradation of specific amino acids. The concentration of mono-unsaturated glycerophosphocholines was significantly increased in the liver of GHR-KO pigs without morphological signs of steatosis, although the abundances of several proteins functionally linked to non-alcoholic fatty liver disease (fetuin B, retinol binding protein 4, several mitochondrial proteins) were increased. Moreover, GHR-deficient liver samples revealed distinct changes in the methionine and glutathione metabolic pathways, in particular, a significantly increased level of glycine N-methyltransferase and increased levels of total and free glutathione. Several proteins revealed a sex-related abundance difference in the control group but not in the GHR-KO group.Conclusions: Our integrated proteomics/targeted metabolomics study of GHR-deficient and control liver samples from a clinically relevant large animal model identified a spectrum of biological pathways that are significantly altered in the absence of GH action. Moreover, new insights into the role of GH in the sex-related specification of liver functions were provided.

2020 Scientific Article in Scientific Reports Sci. Rep. 10:79 (2020)

Lohöfer, F. ; Buchholz, R. ; Glinzer, A. ; Huber, K. ; Haas, H. ; Kaissis, G. ; Feuchtinger, A. ; Aichler, M. ; Sporns, P.B. ; Höltke, C. ; Stölting, M. ; Schilling, F. ; Botnar, R.M. ; Kimm, M.A. ; Faber, C. ; Walch, A.K. ; Zernecke, A. ; Karst, U. ; Wildgruber, M.

Mass Spectrometry imaging of atherosclerosis-affine Gadofluorine following Magnetic Resonance imaging.

Molecular imaging of atherosclerosis by Magnetic Resonance Imaging (MRI) has been impaired by a lack of validation of the specific substrate responsible for the molecular imaging signal. We therefore aimed to investigate the additive value of mass spectrometry imaging (MSI) of atherosclerosis-affine Gadofluorine P for molecular MRI of atherosclerotic plaques. Atherosclerotic Ldlr(-/-) mice were investigated by high-field MRI (7T) at different time points following injection of atherosclerosis-affine Gadofluorine P as well as at different stages of atherosclerosis formation (4, 8, 16 and 20 weeks of HFD). At each imaging time point mice were immediately sacrificed after imaging and aortas were excised for mass spectrometry imaging: Matrix Assisted Laser Desorption Ionization (MALDI) Imaging and Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS) imaging. Mass spectrometry imaging allowed to visualize the localization and measure the concentration of the MR imaging probe Gadofluorine P in plaque tissue ex vivo with high spatial resolution and thus adds novel and more target specific information to molecular MR imaging of atherosclerosis.

2020 Scientific Article in Radiation Oncology Radiat. Oncol. 15:7 (2020)

Schüttrumpf, L. ; Marschner, S. ; Scheu, K. ; Hess-Rieger, J. ; Rietzler, S. ; Walch, A.K. ; Baumeister, P. ; Kirchner, T. ; Ganswindt, U. ; Zitzelsberger, H. ; Belka, C. ; Maihoefer, C.

Definitive chemoradiotherapy in patients with squamous cell cancers of the head and neck-results from an unselected cohort of the clinical cooperation group "Personalized Radiotherapy in Head and Neck Cancer".

Background Definitive chemoradiotherapy (dCRT) is a standard treatment for patients with locally advanced head and neck cancer. There is a clinical need for a stratification of this prognostically heterogeneous group of tumors in order to optimize treatment of individual patients. We retrospectively reviewed all patients with head and neck squamous cell carcinoma (HNSCC) of the oral cavity, oropharynx, hypopharynx, or larynx, treated with dCRT from 09/2008 until 03/2016 at the Department of Radiation Oncology, LMU Munich. Here we report the clinical results of the cohort which represent the basis for biomarker discovery and molecular genetic research within the framework of a clinical cooperation group. Methods Patient data were collected and analyzed for outcome and treatment failures with regard to previously described and established risk factors. Results We identified 184 patients with a median follow-up of 65 months and a median age of 64 years. Patients received dCRT with a median dose of 70 Gy and simultaneous chemotherapy in 90.2% of cases, mostly mitomycin C / 5-FU in concordance with the ARO 95-06 trial. The actuarial 3-year overall survival (OS), local, locoregional and distant failure rates were 42.7, 29.8, 34.0 and 23.4%, respectively. Human papillomavirus-associated oropharynx cancer (HPVOPC) and smaller gross tumor volume were associated with significantly improved locoregional tumor control rate, disease-free survival (DFS) and OS in multivariate analysis. Additionally, lower hemoglobin levels were significantly associated with impaired DFS und OS in univariate analysis. The extent of lymph node involvement was associated with distant failure, DFS and OS. Moreover, 92 patients (50%) of our cohort have been treated in concordance with the ARO 95-06 study, corroborating the results of this study. Conclusion Our cohort is a large unselected monocentric cohort of HNSCC patients treated with dCRT. Tumor control rates and survival rates compare favorably with the results of previously published reports. The clinical data, together with the available tumor samples from biopsies, will allow translational research based on molecular genetic analyses.

2020 Review in Cell and Tissue Research Cell Tissue Res. 380, 341-378 (2020)

Renner, S. ; Blutke, A. ; Clauss, S. ; Deeg, C.A. ; Kemter, E. ; Merkus, D. ; Wanke, R. ; Wolf, E.

Porcine models for studying complications and organ crosstalk in diabetes mellitus.

The worldwide prevalence of diabetes mellitus and obesity is rapidly increasing not only in adults but also in children and adolescents. Diabetes is associated with macrovascular complications increasing the risk for cardiovascular disease and stroke, as well as microvascular complications leading to diabetic nephropathy, retinopathy and neuropathy. Animal models are essential for studying disease mechanisms and for developing and testing diagnostic procedures and therapeutic strategies. Rodent models are most widely used but have limitations in translational research. Porcine models have the potential to bridge the gap between basic studies and clinical trials in human patients. This article provides an overview of concepts for the development of porcine models for diabetes and obesity research, with a focus on genetically engineered models. Diabetes-associated ocular, cardiovascular and renal alterations observed in diabetic pig models are summarized and their similarities with complications in diabetic patients are discussed. Systematic multi-organ biobanking of porcine models of diabetes and obesity and molecular profiling of representative tissue samples on different levels, e.g., on the transcriptome, proteome, or metabolome level, is proposed as a strategy for discovering tissue-specific pathomechanisms and their molecular key drivers using systems biology tools. This is exemplified by a recent study providing multi-omics insights into functional changes of the liver in a transgenic pig model for insulin-deficient diabetes mellitus. Collectively, these approaches will provide a better understanding of organ crosstalk in diabetes mellitus and eventually reveal new molecular targets for the prevention, early diagnosis and treatment of diabetes mellitus and its associated complications.

2020 Scientific Article in Nature metabolism Nat. Metab. 2, 192-209 (2020)

Sachs, S.# ; Bastidas-Ponce, A.# ; Tritschler, S.# ; Bakhti, M. ; Böttcher, A. ; Sánchez-Garrido, M.A. ; Tarquis Medina, M. ; Kleinert, M. ; Fischer, K. ; Jall, S. ; Harger, A. ; Bader, E. ; Roscioni, S. ; Ussar, S. ; Feuchtinger, A. ; Yesildag, B. ; Neelakandhan, A. ; Jensen, C.B. ; Cornu, M. ; Yang, B. ; Finan, B. ; DiMarchi, R.D. ; Tschöp, M.H. ; Theis, F.J.&deg ; Hofmann, S.M.&deg ; Müller, T.D.&deg ; Lickert, H.&deg

Targeted pharmacological therapy restores β-cell function for diabetes remission.

Dedifferentiation of insulin-secreting β cells in the islets of Langerhans has been proposed to be a major mechanism of β-cell dysfunction. Whether dedifferentiated β cells can be targeted by pharmacological intervention for diabetes remission, and ways in which this could be accomplished, are unknown as yet. Here we report the use of streptozotocin-induced diabetes to study β-cell dedifferentiation in mice. Single-cell RNA sequencing (scRNA-seq) of islets identified markers and pathways associated with β-cell dedifferentiation and dysfunction. Single and combinatorial pharmacology further show that insulin treatment triggers insulin receptor pathway activation in β cells and restores maturation and function for diabetes remission. Additional β-cell selective delivery of oestrogen by Glucagon-like peptide-1 (GLP-1-oestrogen conjugate) decreases daily insulin requirements by 60%, triggers oestrogen-specific activation of the endoplasmic-reticulum-associated protein degradation system, and further increases β-cell survival and regeneration. GLP-1-oestrogen also protects human β cells against cytokine-induced dysfunction. This study not only describes mechanisms of β-cell dedifferentiation and regeneration, but also reveals pharmacological entry points to target dedifferentiated β cells for diabetes remission.

2020 Scientific Article in Growth Hormone and IGF Research Growth Horm. IGF Res. 51, 6-16 (2020)

Hofmann, I. ; Kemter, E. ; Theobalt, N. ; Fiedler, S. ; Bidlingmaier, M. ; Hinrichs, A. ; Aichler, M. ; Burkhardt, K. ; Klymiuk, N. ; Wolf, E. ; Wanke, R. ; Blutke, A.

Linkage between growth retardation and pituitary cell morphology in a dystrophin-deficient pig model of Duchenne muscular dystrophy.

Objective: Human patients with Duchenne muscular dystrophy (DMD) commonly exhibit a short stature, but the pathogenesis of this growth retardation is not completely understood. Due to the suspected involvement of the growth hormone/insulin-like growth factor 1 (GH/IGF1) system, controversial therapeutic approaches have been developed, including both GH- administration, as well as GH-inhibition. In the present study, we examined relevant histomorphological and ultrastructural features of adenohypophyseal GH-producing somatotroph cells in a porcine DMD model.Methods: The numbers and volumes of immunohistochemically labelled somatotroph cells were determined in consecutive semi-thin sections of plastic resin embedded adenohypophyseal tissue samples using unbiased state-of-the-art quantitative stereological analysis methods.Results: DMD pigs displayed a significant growth retardation, accounting for a 55% reduction of body weight, accompanied by a significant 50% reduction of the number of somatotroph cells, as compared to controls. However, the mean volumes of somatotroph cells and the volume of GH-granules per cell were not altered. Western blot analyses of the adenohypophyseal protein samples showed no differences in the relative adenohypophyseal GH-abundance between DMD pigs and controls.Conclusion: The findings of this study do not provide evidence for involvement of somatotroph cells in the pathogenesis of growth retardation of DMD pigs. These results are in contrast with previous findings in other dystrophin-deficient animal models, such as the golden retriever model of Duchenne muscular dystrophy, where increased mean somatotroph cell volumes and elevated volumes of intracellular GH-granules were reported and associated with DMD-related growth retardation. Possible reasons for the differences of somatotroph morphology observed in different DMD models are discussed.

2020 Scientific Article in Journal of Cachexia, Sarcopenia and Muscle J. Cachexia Sarcopenia Muscle 11, 226-240 (2020)

Kunzke, T. ; Buck, A. ; Prade, V.M. ; Feuchtinger, A. ; Prokopchuk, O. ; Martignoni, M.E. ; Heisz, S. ; Hauner, H. ; Janssen, K.P. ; Walch, A.K. ; Aichler, M.

Derangements of amino acids in cachectic skeletal muscle are caused by mitochondrial dysfunction.

Background Cachexia is the direct cause of at least 20% of cancer-associated deaths. Muscle wasting in skeletal muscle results in weakness, immobility, and death secondary to impaired respiratory muscle function. Muscle proteins are massively degraded in cachexia; nevertheless, the molecular mechanisms related to this process are poorly understood. Previous studies have reported conflicting results regarding the amino acid abundances in cachectic skeletal muscle tissues. There is a clear need to identify the molecular processes of muscle metabolism in the context of cachexia, especially how different types of molecules are involved in the muscle wasting process. Methods New in situ -omics techniques were used to produce a more comprehensive picture of amino acid metabolism in cachectic muscles by determining the quantities of amino acids, proteins, and cellular metabolites. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging, we determined the in situ concentrations of amino acids and proteins, as well as energy and other cellular metabolites, in skeletal muscle tissues from genetic mouse cancer models (n = 21) and from patients with cancer (n = 6). Combined results from three individual MALDI mass spectrometry imaging methods were obtained and interpreted. Immunohistochemistry staining for mitochondrial proteins and myosin heavy chain expression, digital image analysis, and transmission electron microscopy complemented the MALDI mass spectrometry imaging results. Results Metabolic derangements in cachectic mouse muscle tissues were detected, with significantly increased quantities of lysine, arginine, proline, and tyrosine (P = 0.0037, P = 0.0048, P = 0.0430, and P = 0.0357, respectively) and significantly reduced quantities of glutamate and aspartate (P = 0.0008 and P = 0.0124). Human skeletal muscle tissues revealed similar tendencies. A majority of altered amino acids were released by the breakdown of proteins involved in oxidative phosphorylation. Decreased energy charge was observed in cachectic muscle tissues (P = 0.0101), which was related to the breakdown of specific proteins. Additionally, expression of the cationic amino acid transporter CAT1 was significantly decreased in the mitochondria of cachectic mouse muscles (P = 0.0133); this decrease may play an important role in the alterations of cationic amino acid metabolism and decreased quantity of glutamate observed in cachexia. Conclusions Our results suggest that mitochondrial dysfunction has a substantial influence on amino acid metabolism in cachectic skeletal muscles, which appears to be triggered by diminished CAT1 expression, as well as the degradation of mitochondrial proteins. These findings provide new insights into the pathobiochemistry of muscle wasting.

2020 Scientific Article in Radiation and Environmental Biophysics Radiat. Environ. Biophys. 59, 111-120 (2020)

Dombrowsky, A. ; Burger, K. ; Porth, A.K. ; Stein, M. ; Dierolf, M. ; Günther, B. ; Achterhold, K. ; Gleich, B. ; Feuchtinger, A. ; Bartzsch, S. ; Beyreuther, E. ; Combs, S.E. ; Pfeiffer, F. ; Wilkens, J.J. ; Schmid, T.E.

A proof of principle experiment for microbeam radiation therapy at the Munich compact light source.

Microbeam radiation therapy (MRT), a preclinical form of spatially fractionated radiotherapy, uses an array of microbeams of hard synchrotron X-ray radiation. Recently, compact synchrotron X-ray sources got more attention as they provide essential prerequisites for the translation of MRT into clinics while overcoming the limited access to synchrotron facilities. At the Munich compact light source (MuCLS), one of these novel compact X-ray facilities, a proof of principle experiment was conducted applying MRT to a xenograft tumor mouse model. First, subcutaneous tumors derived from the established squamous carcinoma cell line FaDu were irradiated at a conventional X-ray tube using broadbeam geometry to determine a suitable dose range for the tumor growth delay. For irradiations at the MuCLS, FaDu tumors were irradiated with broadbeam and microbeam irradiation at integral doses of either 3 Gy or 5 Gy and tumor growth delay was measured. Microbeams had a width of 50 µm and a center-to-center distance of 350 µm with peak doses of either 21 Gy or 35 Gy. A dose rate of up to 5 Gy/min was delivered to the tumor. Both doses and modalities delayed the tumor growth compared to a sham-irradiated tumor. The irradiated area and microbeam pattern were verified by staining of the DNA double-strand break marker γH2AX. This study demonstrates for the first time that MRT can be successfully performed in vivo at compact inverse Compton sources.

2020 Scientific Article in Haematologica - The Hematology Journal Haematologica 105, 937-950 (2020)

Altamura, S.# ; Vegi, N.M.# ; Hoppe, P.S. ; Schroeder, T. ; Aichler, M. ; Walch, A.K. ; Okreglicka, K. ; Hültner, L. ; Schneider, M. ; Ladinig, C. ; Kuklik-Roos, C. ; Mysliwietz, J. ; Janik, D. ; Neff, F. ; Rathkolb, B. ; Hrabě de Angelis, M. ; Buske, C. ; da Silva, A.R. ; Muedder, K. ; Conrad, M. ; Ganz, T. ; Kopf, M. ; Muckenthaler, M.U. ; Bornkamm, G.W.

Glutathione peroxidase 4 and vitamin E control reticulocyte maturation, stress erythropoiesis and iron homeostasis.

Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing . oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the ation of mitochondria but is now known to occur through mitophary Yet, ggenetic ablation of the Alox15 gene in mice failed to provice evidence or this hyppothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullaty erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation.

In: Fundamentals and Applications of Fourier Transform Mass Spectrometry. 2019. 253-279

Kreutzer, L. ; Aichler, M. ; Walch, A.K.

In situ metabolomics in cancer tissue by high-resolution mass spectrometry imaging.

This chapter introduces the in situ investigation on metabolomics in cancer tissues by high-resolution mass spectrometry imaging. Metabolomics is a rapidly increasing field, since the detection of biochemical processes can improve the diagnostic, therapeutic treatment prediction, and prognosis in diseases such as cancer. By analyzing metabolic alterations in cancer tissues, insights into the pathway regulations and the resulting clinical outcome can be obtained and associated. In recent years, especially high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was an emerging technique in the analysis of metabolite molecular data and their spatial distribution in tissues. The main intentions in the combination of metabolomics with MALDI MSI are the discovery of molecular biomarkers and metabolic pathways altered in tumors, as well as therapy response prediction and method development. Therefore, increasing numbers of studies were published recently, investigating the optimization of the MSI methods, overcoming the current difficulties and studying the role of clinical translation.

2019 Scientific Article in Scientific Reports Sci. Rep. 9:19483 (2019)

Baumann, P. ; Schriever, S.C. ; Kullmann, S. ; Zimprich, A. ; Feuchtinger, A. ; Amarie, O.V. ; Peter, A. ; Walch, A.K. ; Gailus-Durner, V. ; Fuchs, H. ; Hrabě de Angelis, M. ; Wurst, W. ; Tschöp, M.H. ; Heni, M. ; Hölter, S.M. ; Pfluger, P.T.

Dusp8 affects hippocampal size and behavior in mice and humans.

Dual-specificity phosphatase 8 (Dusp8) acts as physiological inhibitor for the MAPKs Jnk, Erk and p38 which are involved in regulating multiple CNS processes. While Dusp8 expression levels are high in limbic areas such as the hippocampus, the functional role of Dusp8 in hippocampus morphology, MAPK-signaling, neurogenesis and apoptosis as well as in behavior are still unclear. It is of particular interest whether human carriers of a DUSP8 allelic variant show similar hippocampal alterations to mice. Addressing these questions using Dusp8WT and KO mouse littermates, we found that KOs suffered from mildly impaired spatial learning, increased locomotor activity and elevated anxiety. Cell proliferation, apoptosis and p38 and Jnk phosphorylation were unaffected, but phospho-Erk levels were higher in hippocampi of the KOs. Consistent with a decreased hippocampus size in Dusp8 KO mice, we found reduced volumes of the hippocampal subregions subiculum and CA4 in humans carrying the DUSP8 allelic variant SNP rs2334499:C > T. Overall, aberrations in morphology and behavior in Dusp8 KO mice and a decrease in hippocampal volume of SNP rs2334499:C > T carriers point to a novel, translationally relevant role of Dusp8 in hippocampus function that warrants further studies on the role of Dusp8 within the limbic network.

2019 Scientific Article in International Journal of Molecular Sciences Int. J. Mol. Sci. 20:2103 (2019)

Hladik, D. ; Buratovic, S. ; von Toerne, C. ; Azimzadeh, O. ; Subedi, P. ; Philipp, J. ; Winkler, S. ; Feuchtinger, A. ; Samson, E. ; Hauck, S.M. ; Stenerlöw, B. ; Eriksson, P. ; Atkinson, M.J. ; Tapio, S.

Combined treatment with low-dose ionizing radiation and ketamine induces adverse changes in CA1 neuronal structure in murine hippocampi.

In children, ketamine sedation is often used during radiological procedures. Combined exposure of ketamine and radiation at doses that alone did not affect learning and memory induced permanent cognitive impairment in mice. The aim of this study was to elucidate the mechanism behind this adverse outcome. Neonatal male NMRI mice were administered ketamine (7.5 mg kg(-1)) and irradiated (whole-body, 100 mGy or 200 mGy, Cs-137) one hour after ketamine exposure on postnatal day 10. The control mice were injected with saline and sham-irradiated. The hippocampi were analyzed using label-free proteomics, immunoblotting, and Golgi staining of CA1 neurons six months after treatment. Mice co-exposed to ketamine and low-dose radiation showed alterations in hippocampal proteins related to neuronal shaping and synaptic plasticity. The expression of brain-derived neurotrophic factor, activity-regulated cytoskeleton-associated protein, and postsynaptic density protein 95 were significantly altered only after the combined treatment (100 mGy or 200 mGy combined with ketamine, respectively). Increased numbers of basal dendrites and branching were observed only after the co-exposure, thereby constituting a possible reason for the displayed alterations in behavior. These data suggest that the risk of radiation-induced neurotoxic effects in the pediatric population may be underestimated if based only on the radiation dose.

2019 Scientific Article in Nature Nature 576, 287-292 (2019)

Correa-Gallegos, D.# ; Jiang, D.# ; Christ, S. ; Ramesh, P. ; Ye, H. ; Wannemacher, J. ; Kalgudde Gopal, S. ; Yu, Q. ; Aichler, M. ; Walch, A.K. ; Mirastschijski, U. ; Volz, T. ; Rinkevich, Y.

Patch repair of deep wounds by mobilized fascia.

Mammals form scars to quickly seal wounds and ensure survival by an incompletely understood mechanism(1-5). Here we show that skin scars originate from prefabricated matrix in the subcutaneous fascia. Fate mapping and live imaging revealed that fascia fibroblasts rise to the skin surface after wounding, dragging their surrounding extracellular jelly-like matrix, including embedded blood vessels, macrophages and peripheral nerves, to form the provisional matrix. Genetic ablation of fascia fibroblasts prevented matrix from homing into wounds and resulted in defective scars, whereas placing an impermeable film beneath the skin-preventing fascia fibroblasts from migrating upwards-led to chronic open wounds. Thus, fascia contains a specialized prefabricated kit of sentry fibroblasts, embedded within a movable sealant, that preassemble together diverse cell types and matrix components needed to heal wounds. Our findings suggest that chronic and excessive skin wounds may be attributed to the mobility of the fascia matrix.

2019 Scientific Article in Small Small 15:1904112 (2019)

Yang, L. ; Gradl, R. ; Dierolf, M. ; Möller, W. ; Kutschke, D. ; Feuchtinger, A. ; Hehn, L. ; Donnelley, M. ; Günther, B. ; Achterhold, K. ; Walch, A.K. ; Stöger, T. ; Razansky, D. ; Pfeiffer, F. ; Morgan, K.S. ; Schmid, O.

Multimodal precision imaging of pulmonary nanoparticle delivery in mice: Dynamics of application, spatial distribution, and dosimetry.

Targeted delivery of nanomedicine/nanoparticles (NM/NPs) to the site of disease (e.g., the tumor or lung injury) is of vital importance for improved therapeutic efficacy. Multimodal imaging platforms provide powerful tools for monitoring delivery and tissue distribution of drugs and NM/NPs. This study introduces a preclinical imaging platform combining X-ray (two modes) and fluorescence imaging (three modes) techniques for time-resolved in vivo and spatially resolved ex vivo visualization of mouse lungs during pulmonary NP delivery. Liquid mixtures of iodine (contrast agent for X-ray) and/or (nano)particles (X-ray absorbing and/or fluorescent) are delivered to different regions of the lung via intratracheal instillation, nasal aspiration, and ventilator-assisted aerosol inhalation. It is demonstrated that in vivo propagation-based phase-contrast X-ray imaging elucidates the dynamic process of pulmonary NP delivery, while ex vivo fluorescence imaging (e.g., tissue-cleared light sheet fluorescence microscopy) reveals the quantitative 3D drug/particle distribution throughout the entire lung with cellular resolution. The novel and complementary information from this imaging platform unveils the dynamics and mechanisms of pulmonary NM/NP delivery and deposition for each of the delivery routes, which provides guidance on optimizing pulmonary delivery techniques and novel-designed NM for targeting and efficacy.

2019 Scientific Article in Nature metabolism Nat. Metab. 1, 1009-1026 (2019)

Seitz, S. ; Kwon, Y. ; Hartleben, G. ; Jülg, J. ; Sekar, R. ; Krahmer, N. ; Najafi, B. ; Loft, A. ; Gancheva, S. ; Stemmer, K. ; Feuchtinger, A. ; Hrabě de Angelis, M. ; Müller, T.D. ; Mann, M. ; Blüher, M. ; Roden, M. ; Berriel Diaz, M. ; Behrends, C. ; Gilleron, J. ; Herzig, S. ; Zeigerer, A.

Hepatic Rab24 controls blood glucose homeostasis via improving mitochondrial plasticity.

Non-alcoholic fatty liver disease (NAFLD) represents a key feature of obesity-related type 2 diabetes with increasing prevalence worldwide. To our knowledge, no treatment options are available to date, paving the way for more severe liver damage, including cirrhosis and hepatocellular carcinoma. Here, we show an unexpected function for an intracellular trafficking regulator, the small Rab GTPase Rab24, in mitochondrial fission and activation, which has an immediate impact on hepatic and systemic energy homeostasis. RAB24 is highly upregulated in the livers of obese patients with NAFLD and positively correlates with increased body fat in humans. Liver-selective inhibition of Rab24 increases autophagic flux and mitochondrial connectivity, leading to a strong improvement in hepatic steatosis and a reduction in serum glucose and cholesterol levels in obese mice. Our study highlights a potential therapeutic application of trafficking regulators, such as RAB24, for NAFLD and establishes a conceptual functional connection between intracellular transport and systemic metabolic dysfunction.

2019 Scientific Article in Pathogens Pathogens 8:177 (2019)

Arndt, D. ; Fux, R. ; Blutke, A. ; Schwaiger, J. ; El-Matbouli, M. ; Sutter, G. ; Langenmayer, M.C.

Proliferative kidney disease and proliferative darkening syndrome are linked with brown trout (Salmo trutta fario) mortalities in the pre-alpine Isar River.

For many years, brown trout (Salmo trutta fario) mortalities within the pre-alpine Isar River in Germany were reported by the Bavarian Fisheries Association (Landesfischereiverband Bayern e.V.) and local recreational anglers during August and September. Moribund fish seemed to be affected by proliferative darkening syndrome (PDS). In addition, proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae was discussed. To investigate this phenomenon, the present field study monitored brown trout mortalities by daily river inspection in 2017 and 2018. Moribund brown trout (n = 31) were collected and examined using histology, immunohistochemistry, qPCR, and quantitative stereology. Our investigations identified 29 (93.5%) brown trout affected by PKD. Four brown trout (12.9%) displayed combined hepatic and splenic lesions fitting the pathology of PDS. The piscine orthoreovirus 3, suspected as causative agent of PDS, was not detectable in any of the samples. Quantitative stereological analysis of the kidneys revealed a significant increase of the renal tissue volumes with interstitial inflammation and hematopoietic hyperplasia in PKD-affected fish as compared to healthy brown trout. The identified T. bryosalmonae strain was classified as part of the North American clade by phylogenetical analysis. This study highlights PKD and PDS as contributing factors to recurrent autumnal brown trout mortalities.

2019 Scientific Article in PLoS ONE PLoS ONE 14:e0221454 (2019)

Sammer, M. ; Teiluf, K. ; Girst, S. ; Greubel, C. ; Reindl, J. ; Ilicic, K. ; Walsh, D.W.M. ; Aichler, M. ; Walch, A.K. ; Combs, S.E. ; Wilkens, J.J. ; Dollinger, G. ; Schmid, T.E.

Beam size limit for pencil minibeam radiotherapy determined from side effects in an in-vivo mouse ear model.

Side effects caused by radiation are a limiting factor to the amount of dose that can be applied to a tumor volume. A novel method to reduce side effects in radiotherapy is the use of spatial fractionation, in which a pattern of sub-millimeter beams (minibeams) is applied to spare healthy tissue. In order to determine the skin reactions in dependence of single beam sizes, which are relevant for spatially fractionated radiotherapy approaches, single pencil beams of submillimeter to 6 millimeter size were applied in BALB/c mice ears at a Small Animal Radiation Research Platform (SARRP) with a plateau dose of 60 Gy. Radiation toxicities in the ears were observed for 25 days after irradiation. Severe radiation responses were found for beams >= 3 mm diameter. The larger the beam diameter the stronger the observed reactions. No ear swelling and barely reddening or desquamation were found for the smallest beam sizes (0.5 and 1 mm). The findings were confirmed by histological sections. Sub-millimeter beams are preferred in minibeam therapy to obtain optimized tissue sparing. The gradual increase of radiation toxicity with beam size shows that also larger beams are capable of healthy tissue sparing in spatial fractionation.

2019 Scientific Article in Journal of Cell Science J. Cell Sci. 132:jcs223891 (2019)

Wagner, A. ; Hofmeister, O. ; Rolland, S.G. ; Maiser, A. ; Aasumets, K. ; Schmitt, S. ; Schorpp, K.K. ; Feuchtinger, A. ; Hadian, K. ; Schneider, S. ; Zischka, H. ; Leonhardt, H. ; Conradt, B. ; Gerhold, J.M. ; Wolf, A.

Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and C. elegans.

The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of N3-methylcytosine (m(3)C) in RNA- and single-stranded DNA oligonucleotides, demethylation of N-1-methyladenosine (m1A) in tRNA or formation of 5-formyl cytosine (f(5)C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM-microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in Caenorhabditis elegans, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPRmt) in C. elegans.

2019 Scientific Article in JCI insight JCI insight 4:e130356 (2019)

Murakami, M. ; Rhayem, Y. ; Kunzke, T. ; Sun, N. ; Feuchtinger, A. ; Ludwig, P. ; Strom, T.M. ; Gomez-Sanchez, C. ; Knösel, T. ; Kirchner, T. ; Williams, T.A. ; Reincke, M. ; Walch, A.K. ; Beuschlein, F.

In situ metabolomics of aldosterone-producing adenomas.

Recent genetic examinations and multisteroid profiles have provided the basis for subclassification of aldosterone-producing adenomas (APAs). The objective of the current study was to produce a comprehensive, high-resolution mass spectrometry imaging (MSI) map of APAs in relation to morphometry, immunohistochemical profiles, mutational status, and clinical outcome. The study cohort comprised 136 patients with unilateral primary aldosteronism. Matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI was conducted, and metabolite profiles were analyzed with genotype/phenotype information, including digital image analysis from morphometry and IHC of steroidogenic enzymes. Distinct molecular signatures between KCNJ5- and CACNA1D-mutated APAs with significant differences of 137 metabolites, including metabolites of purine metabolism and steroidogenesis, were observed. Intratumor concentration of 18-oxocortisol and 18-hydroxycortisol were inversely correlated with the staining intensity of CYP11B1. Lower staining intensity of CYP11B1 and higher levels of 18-oxocortisol were associated with a higher probability of complete clinical success after surgery. The present study demonstrates distinct metabolomic profiles of APAs in relation to tumor genotype. In addition, we reveal an inverse correlation between cortisol derivatives and CYP11B1 and the impact of 18-oxocortisol and CYP11B1 on clinical outcome, which provides unprecedented insights into the pathophysiology, clinical features, and steroidogenesis of APAs.

2019 Scientific Article in Clinical Chemistry Clin. Chem. 65, 1276-1286 (2019)

Sun, N.# ; Kunzke, T.# ; Sbiera, S. ; Kircher, S. ; Feuchtinger, A. ; Aichler, M. ; Herterich, S. ; Ronchi, C.L. ; Weigand, I. ; Schlegel, N. ; Waldmann, J. ; Fragoso, M.C.V. ; Whitsett, T.G. ; Gill, A.J. ; Fassnacht, M. ; Walch, A.K. ; Kroiss, M.

Prognostic relevance of steroid sulfation in adrenocortical carcinoma revealed by molecular phenotyping using high resolution mass spectrometry imaging.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare tumor with variable prognosis even within the same tumor stage. Cancer-related sex hormones and their sulfated metabolites in body fluids can be used as tumor markers. The role of steroid sulfation in ACC has not yet been studied. MALDI mass spectrometry imaging (MALDI-MSI) is a novel tool for tissue-based chemical phenotyping.METHODS: We performed phenotyping of formalin-fixed, paraffin-embedded tissue samples from 72 ACC by MALDI-MSI at a metabolomics level.RESULTS: Tumoral steroid hormone metabolites-estradiol sulfate [hazard ratio (HR) 0.26; 95% CI, 0.10-0.69; P = 0.005] and estrone 3-sulfate (HR 0.22; 95% CI, 0.07-0.63; P = 0.003)-were significantly associated with prognosis in Kaplan-Meier analyses and after multivariable adjustment for age, tumor stage, and sex (HR 0.29; 95% CI, 0.11-0.79; P= 0.015 and HR 0.30; 95% CI, 0.10-0.91; P = 0.033, respectively). Expression of sulfotransferase SULT2A1 was associated with prognosis to a similar extent and was validated to be a prognostic factor in two published data sets. We discovered the presence of estradiol-17 beta 3,17-disulfate (E2S2) in a subset of tumors with particularly poor overall survival. Electron microscopy revealed novel membrane-delimited organelles in only these tumors. By applying duster analyses of metabolomic data, 3 sulfation-related phenotypes exhibited specific metabolic features unrelated to steroid metabolism.CONCLUSIONS: MALDI-MSI provides novel insights into the pathophysiology of ACC. Steroid hormone sulfation may be used for prognostication and treatment stratification. Sulfation-related metabolic reprogramming may be of relevance also in conditions beyond the rare ACC and can be directly investigated by the use of MALDI-MSI.

2019 Scientific Article in Frontiers in Endocrinology Front. Endocrin. 10:487 (2019)

Siebert, C. ; Ciato, D. ; Murakami, M. ; Frei-Stuber, L. ; Perez-Rivas, L.G. ; Monteserin-Garcia, J.L. ; Noelting, S. ; Maurer, J. ; Feuchtinger, A. ; Walch, A.K. ; Haak, H.R. ; Bertherat, J. ; Mannelli, M. ; Fassnacht, M. ; Korpershoek, E. ; Reincke, M. ; Stalla, G.K. ; Hantel, C. ; Beuschlein, F.

Heat shock protein 90 as a prognostic marker and therapeutic target for adrenocortical carcinoma.

Background: Adrenocortical carcinoma (ACC) is a rare tumor entity with restricted therapeutic opportunities. HSP90 (Heat Shock Protein 90) chaperone activity is fundamental for cell survival and contributes to different oncogenic signaling pathways. Indeed, agents targeting HSP90 function have shown therapeutic efficacy in several cancer types. We have examined the expression of HSP90 in different adrenal tumors and evaluated the use of HSP90 inhibitors in vitro as possible therapy for ACC.Methods: Immunohistochemical expression of HSP90 isoforms was investigated in different adrenocortical tumors and associated with clinical features. Additionally, a panel of N-terminal (17-allylamino-17-demethoxygeldanamycin (17-AAG), luminespib, and ganetespib) and C-terminal (novobiocin and silibinin) HSP90 inhibitors were tested on various ACC cell lines.Results: Within adrenocortical tumors, ACC samples exhibited the highest expression of HSP90 beta. Within a cohort of ACC patients, HSP90 beta expression levels were inversely correlated with recurrence-free and overall survival. In functional assays, among five different compounds tested luminespib and ganetespib induced a significant decrease in cell viability in single as well as in combined treatments with compounds of the clinically used EDP-M scheme (etoposide, doxorubicin, cisplatin, mitotane). Inhibition of cell viability correlated furthermore with a decrease in proliferation, in cell migration and an increase in apoptosis. Moreover, analysis of cancer pathways indicated a modulation of the ERK1/2-and AKT-pathways by luminespib and ganetespib treatment.Conclusions: Our findings emphasize HSP90 as a marker with prognostic impact and promising target with N-terminal HSP90 inhibitors as drugs with potential therapeutic efficacy toward ACC.

2019 Scientific Article in Disease Models and Mechanisms Dis. Model. Mech. 12:dmm039156 (2019)

Renner, S. ; Martins, A.S. ; Streckel, E. ; Braun-Reichhart, C. ; Backman, M. ; Prehn, C. ; Klymiuk, N. ; Bähr, A. ; Blutke, A. ; Landbrecht-Schessl, C. ; Wünsch, A. ; Kessler, B. ; Kurome, M. ; Hinrichs, A. ; Koopmans, S.J. ; Krebs, S. ; Kemter, E. ; Rathkolb, B. ; Nagashima, H. ; Blum, H. ; Ritzmann, M. ; Wanke, R. ; Aigner, B. ; Adamski, J. ; Hrabě de Angelis, M. ; Wolf, E.

Mild maternal hyperglycemia in INSC93S transgenic pigs causes impaired glucose tolerance and metabolic alterations in neonatal offspring.

Alongside the obesity epidemic, the prevalence of maternal diabetes is rising worldwide, and adverse effects on fetal development and metabolic disturbances in the offspring's later life have been described. To clarify whether metabolic programming effects are due to mild maternal hyperglycemia without confounding obesity, we investigated wild-type offspring of INSC93S transgenic pigs, which are a novel genetically modified large-animal model expressing mutant insulin (INS) C93S in pancreatic beta-cells. This mutation results in impaired glucose tolerance, mild fasting hyperglycemia and insulin resistance during late pregnancy. Compared with offspring from wildtype sows, piglets from hyperglycemic mothers showed impaired glucose tolerance and insulin resistance (homeostatic model assessment of insulin resistance: +3-fold in males; +4.4-fold in females) prior to colostrum uptake. Targeted metabolomics in the fasting and insulin-stimulated state revealed distinct alterations in the plasma metabolic profile of piglets from hyperglycemic mothers. They showed increased levels of acylcarnitines, gluconeogenic precursors such as alanine, phospholipids (in particular lysophosphatidylcholines) and a-aminoadipic acid, a potential biomarker for type 2 diabetes. These observations indicate that mild gestational hyperglycemia can cause impaired glucose tolerance, insulin resistance and associated metabolic alterations in neonatal offspring of a large-animal model born at a developmental maturation status comparable to human babies.

2019 Scientific Article in Scientific Reports Sci. Rep. 9:9925 (2019)

Rehm, S.R.T. ; Smirnova, N.F. ; Morrone, C. ; Götzfried, J. ; Feuchtinger, A. ; Pedersen, J. ; Korkmaz, B. ; Yildirim, A.Ö. ; Jenne, D.

Premedication with a cathepsin C inhibitor alleviates early primary graft dysfunction in mouse recipients after lung transplantation.

Neutrophil serine proteases (NSPs), like proteinase 3 (PR3) and neutrophil elastase (NE) are implicated in ischemia-reperfusion responses after lung transplantation (LTx). Cathepsin C (CatC) acts as the key regulator of NSP maturation during biosynthesis. We hypothesized that CatC inhibitors would reduce vascular breakdown and inflammation during reperfusion in pretreated lung transplant recipients by blocking NSP maturation in the bone marrow. An orthotopic LTx model in mice was used to mimic the induction of an ischemia-reperfusion response after 18 h cold storage of the graft and LTx. Recipient mice were treated subcutaneously with a chemical CatC inhibitor (ICatC) for 10 days prior to LTx. We examined the effect of the ICatC treatment by measuring the gas exchange function of the left lung graft, protein content, neutrophil numbers and NSP activities in the bone marrow 4 h after reperfusion. Pre-operative ICatC treatment of the recipient mice improved early graft function and lead to the disappearance of active NSP protein in the transplanted lung. NSP activities were also substantially reduced in bone marrow neutrophils. Preemptive NSP reduction by CatC inhibition may prove to be a viable and effective approach to reduce immediate ischemia reperfusion responses after LTx.

2019 Scientific Article in ACS Nano ACS Nano 13, 8114-8123 (2019)

Sigmund, F.# ; Pettinger, S.# ; Kube, M. ; Schneider, F. ; Schifferer, M. ; Schneider, S. ; Efremova, M.V. ; Pujol-Martí, J. ; Aichler, M. ; Walch, A.K. ; Misgeld, T. ; Dietz, H. ; Westmeyer, G.G.

Iron-sequestering nanocompartments as multiplexed Electron Microscopy gene reporters.

Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of Quasibacillus thermotolerans (Qt), which in combination with the recently characterized encapsulin system from Myxococcus xanthus (Mx) enables multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with T = 4 icosahedral symmetry and a diameter of similar to 43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM via a deep learning classifier to enable automated multiplexed EM gene reporter imaging.

2019 Scientific Article in Scientific Reports Sci. Rep. 9:8492 (2019)

Schneider, A. ; Kurz, S. ; Manske, K. ; Janas, M.K. ; Heikenwälder, M. ; Misgeld, T. ; Aichler, M. ; Weissmann, S.F. ; Zischka, H. ; Knolle, P. ; Wohlleber, D.

Single organelle analysis to characterize mitochondrial function and crosstalk during viral infection.

Mitochondria are key for cellular metabolism and signalling processes during viral infection. We report a methodology to analyse mitochondrial properties at the single-organelle level during viral infection using a recombinant adenovirus coding for a mitochondrial tracer protein for tagging and detection by multispectral flow cytometry. Resolution at the level of tagged individual mitochondria revealed changes in mitochondrial size, membrane potential and displayed a fragile phenotype during viral infection of cells. Thus, single-organelle and multi-parameter resolution allows to explore altered energy metabolism and antiviral defence by tagged mitochondria selectively in virus-infected cells and will be instrumental to identify viral immune escape and to develop and monitor novel mitochondrial-targeted therapies.

2019 Meeting abstract in Strahlentherapie und Onkologie : Journal of Radiation Oncology, Biology, Physics Strahlenther. Onkol. 195, S24-S25 (2019)

Wintergerst, L. ; Selmansberger, M. ; Maihoefer, C. ; Schuettrumpf, L. ; Walch, A.K. ; Wilke, C. ; Pitea, A. ; Woischke, C. ; Baumeister, P. ; Kirchner, T. ; Belka, C. ; Ganswindt, U. ; Zitzelsberger, H. ; Unger, K. ; Hess-Rieger, J.

A 16q24.3 4-gene mRNA signature predicts outcome in radio(chemo)therapy-treated head and neck squamous cell carcinoma.

2019 Meeting abstract in Strahlentherapie und Onkologie : Journal of Radiation Oncology, Biology, Physics Strahlenther. Onkol. 195, S25-S25 (2019)

Hess-Rieger, J. ; Unger, K. ; Maihoefer, C. ; Schuettrumpf, L. ; Schneider, L. ; Heider, T. ; Weber, P. ; Marschner, S. ; Braselmann, H. ; Kuger, S. ; Pflugradt, U. ; Baumeister, P. ; Walch, A.K. ; Woischke, C. ; Kirchner, T. ; Werner, M. ; Werner, K. ; Baumann, M. ; Budach, V. ; Combs, S.E. ; Debus, J. ; Grosu, A.-L. ; Krause, M. ; Roedel, C. ; Stuschke, M. ; Zips, D. ; Zitzelsberger, H. ; Ganswindt, U. ; Henke, M. ; Belka, C.

A five-microRNA-signature predicts recurrence and survival in HPV-negative HNSCC.

2019 Scientific Article in Veterinary Medicine Austria Wien. Tierärztl. Mschr. 106, 109-115 (2019)

Wiesner, M. ; Glawischnig, W. ; Lutzmann, I. ; Grimm, F. ; Blutke, A.

Autochthonous Taenia crassiceps infection in a ring-tailed lemur (Lemur catta) in the Salzburg Zoo.

Captive primates are susceptible to infection with cestodes of the family of Taeniidae. This report describes the infection of a five-year-old female ring tailed-lemur (Lemur catta) in Salzburg Zoo (Salzburg, Austria) with Taenia crassiceps. Necropsy revealed extensive amounts of organized and free cysts in the thoracic cavity, completely encasing and compressing the lungs and the heart. Infection probably occurred by oral uptake of Taenia crassiceps eggs from faeces of red fox (Vulpes vulpes) within the zoo or in the surrounding park, where the lemurs roam freely. Two foxes shot in the vicinity of the zoo were confirmed to have an intestinal infection with Taenia crassiceps. The simultaneous detection of Taenia crassiceps tapeworms in a natural definite host (fox) and of their metacestodes in an accidental intermediate host provides evidence of an autochthonous infection. Compared to other zoo primates, ring-tailed lemurs (Lemur catta) seem to be highly susceptible to infection with Taenia crassiceps. Therapy and preventive methods are discussed.

2019 Scientific Article in Laboratory Investigation Lab. Invest. 99, 1535-1546 (2019)

Huber, K. ; Kunzke, T. ; Buck, A. ; Langer, R. ; Luber, B. ; Feuchtinger, A. ; Walch, A.K.

Multimodal analysis of formalin-fixed and paraffin-embedded tissue by MALDI imaging and fluorescence in situ hybridization for combined genetic and metabolic analysis.

Multimodal tissue analyses that combine two or more detection technologies provide synergistic value compared to single methods and are employed increasingly in the field of tissue-based diagnostics and research. Here, we report a technical pipeline that describes a combined approach of HER2/CEP17 fluorescence in situ hybridization (FISH) analysis with MALDI imaging on the very same section of formalin-fixed and paraffin-embedded (FFPE) tissue. FFPE biopsies and a tissue microarray of human gastroesophageal adenocarcinoma were analyzed by MALDI imaging. Subsequently, the very same section was hybridized by HER2/CEP17 FISH. We found that tissue morphology of both, the biopsies and the tissue microarray, was unaffected by MALDI imaging and the HER2 and CEP17 FISH signals were analyzable. In comparison with FISH analysis of samples without MALDI imaging, we observed no difference in terms of fluorescence signal intensity and gene copy number. Our combined approach revealed adenosine monophosphate, measured by MALDI imaging, as a prognostic marker. HER2 amplification, which was detected by FISH, is a stratifier between good and poor patient prognosis. By integrating both stratification parameters on the basis of our combined approach, we were able to strikingly improve the prognostic effect. Combining molecules detected by MALDI imaging with the gene copy number detected by HER2/CEP17 FISH, we found a synergistic effect, which enhances patient prognosis. This study shows that our combined approach allows the detection of genetic and metabolic properties from one very same FFPE tissue section, which are specific for HER2 and hence suitable for prognosis. Furthermore, this synergism might be useful for response prediction in tumors.

2019 Scientific Article in Journal of Experimental Medicine J. Exp. Med. 216, 1700-1723 (2019)

von Gamm, M. ; Schaub, A. ; Jones, A. ; Wolf, C. ; Behrens, G. ; Lichti, J. ; Essig, K. ; Macht, A. ; Pircher, J. ; Ehrlich, A. ; Davari, K. ; Chauhan, D. ; Busch, B. ; Wurst, W. ; Feederle, R. ; Feuchtinger, A. ; Tschöp, M.H. ; Friedel, C.C. ; Hauck, S.M. ; Sattler, M. ; Geerlof, A. ; Hornung, V. ; Heissmeyer, V. ; Schulz, C. ; Heikenwalder, M. ; Glasmacher, E.

Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3.

The RNase Regnase-1 is a master RNA regulator in macrophages and T cells that degrades cellular and viral RNA upon NF-κB signaling. The roles of its family members, however, remain largely unknown. Here, we analyzed Regnase-3-deficient mice, which develop hypertrophic lymph nodes. We used various mice with immune cell-specific deletions of Regnase-3 to demonstrate that Regnase-3 acts specifically within myeloid cells. Regnase-3 deficiency systemically increased IFN signaling, which increased the proportion of immature B and innate immune cells, and suppressed follicle and germinal center formation. Expression analysis revealed that Regnase-3 and Regnase-1 share protein degradation pathways. Unlike Regnase-1, Regnase-3 expression is high specifically in macrophages and is transcriptionally controlled by IFN signaling. Although direct targets in macrophages remain unknown, Regnase-3 can bind, degrade, and regulate mRNAs, such as Zc3h12a (Regnase-1), in vitro. These data indicate that Regnase-3, like Regnase-1, is an RNase essential for immune homeostasis but has diverged as key regulator in the IFN pathway in macrophages.

2019 Scientific Article in Molecular Metabolism Mol. Metab. 26, 30-44 (2019)

Backman, M.# ; Flenkenthaler, F.# ; Blutke, A. ; Dahlhoff, M. ; Ländström, E. ; Renner, S. ; Philippou-Massier, J. ; Krebs, S. ; Rathkolb, B. ; Prehn, C. ; Grzybek, M. ; Coskun, Ü. ; Rothe, M. ; Adamski, J. ; Hrabě de Angelis, M. ; Wanke, R. ; Fröhlich, T. ; Arnold, G.J. ; Blum, H. ; Wolf, E.

Multi-omics insights into functional alterations of the liver in insulin-deficient diabetes mellitus.

Objective: The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically.Methods: We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics.Results: Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples.Conclusions: The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets. (C) 2019 The Authors. Published by Elsevier GmbH.

2019 Scientific Article in Cancers Cancers 11:727 (2019)

Dombrowsky, A. ; Schauer, J. ; Sammer, M. ; Blutke, A. ; Walsh, D.W.M. ; Schwarz, B. ; Bartzsch, S. ; Feuchtinger, A. ; Reindl, J. ; Combs, S.E. ; Dollinger, G. ; Schmid, T.E.

Acute skin damage and late radiation-induced fibrosis and inflammation in murine ears after high-dose irradiation.

The use of different scoring systems for radiation-induced toxicity limits comparability between studies. We examined dose-dependent tissue alterations following hypofractionated X-ray irradiation and evaluated their use as scoring criteria. Four dose fractions (0, 5, 10, 20, 30 Gy/fraction) were applied daily to ear pinnae. Acute effects (ear thickness, erythema, desquamation) were monitored for 92 days after fraction 1. Late effects (chronic inflammation, fibrosis) and the presence of transforming growth factor beta 1 (TGF beta 1)-expressing cells were quantified on day 92. The maximum ear thickness displayed a significant positive correlation with fractional dose. Increased ear thickness and erythema occurred simultaneously, followed by desquamation from day 10 onwards. A significant dose-dependency was observed for the severity of erythema, but not for desquamation. After 4 x 20 and 4 x 30 Gy, inflammation was significantly increased on day 92, whereas fibrosis and the abundance of TGF beta 1-expressing cells were only marginally increased after 4 x 30 Gy. Ear thickness significantly correlated with the severity of inflammation and fibrosis on day 92, but not with the number of TGF beta 1-expressing cells. Fibrosis correlated significantly with inflammation and fractional dose. In conclusion, the parameter of ear thickness can be used as an objective, numerical and dose-dependent quantification criterion to characterize the severity of acute toxicity and allow for the prediction of late effects.

2019 Scientific Article in Journal of Inherited Metabolic Disease J. Inherit. Metab. Dis. 42, 839-849 (2019)

Segal, J. ; Mülleder, M. ; Krüger, A. ; Adler, T. ; Scholze-Wittler, M. ; Becker, L. ; Calzada-Wack, J. ; Garrett, L. ; Hölter, S.M. ; Rathkolb, B. ; Rozman, J. ; Rácz, I. ; Fischer, R. ; Busch, D.H. ; Neff, F. ; Klingenspor, M. ; Klopstock, T. ; Grüning, N.M. ; Michel, S. ; Lukaszewska-McGreal, B. ; Voigt, I. ; Hartmann, L. ; Timmermann, B. ; Lehrach, H. ; Wolf, E. ; Wurst, W. ; Gailus-Durner, V. ; Fuchs, H. ; Hrabě de Angelis, M. ; Schrewe, H. ; Yuneva, M. ; Ralser, M.

Low catalytic activity is insufficient to induce disease pathology in triosephosphate isomerase deficiency.

Triosephosphate isomerase (TPI) deficiency is a fatal genetic disorder characterized by hemolytic anemia and neurological dysfunction. Although the enzyme defect in TPI was discovered in the 1960s, the exact etiology of the disease is still debated. Some aspects indicate the disease could be caused by insufficient enzyme activity, whereas other observations indicate it could be a protein misfolding disease with tissue-specific differences in TPI activity. We generated a mouse model in which exchange of a conserved catalytic amino acid residue (isoleucine to valine, Ile170Val) reduces TPI specific activity without affecting the stability of the protein dimer. TPIIle170Val/Ile170Val mice exhibit an approximately 85% reduction in TPI activity consistently across all examined tissues, which is a stronger average, but more consistent, activity decline than observed in patients or symptomatic mouse models that carry structural defect mutant alleles. While monitoring protein expression levels revealed no evidence for protein instability, metabolite quantification indicated that glycolysis is affected by the active site mutation. TPIIle170Val/Ile170Val mice develop normally and show none of the disease symptoms associated with TPI deficiency. Therefore, without the stability defect that affects TPI activity in a tissue-specific manner, a strong decline in TPI catalytic activity is not sufficient to explain the pathological onset of TPI deficiency.

2019 Scientific Article in Cancers Cancers 11:594 (2019)

Bechmann, N. ; Poser, I. ; Seifert, V. ; Greunke, C. ; Ullrich, M. ; Qin, N. ; Walch, A.K. ; Peitzsch, M. ; Robledo, M. ; Pacak, K. ; Pietzsch, J. ; Richter, S. ; Eisenhofer, G.

Impact of extrinsic and intrinsic hypoxia on catecholamine biosynthesis in absence or presence of Hif2 alpha in pheochromocytoma cells.

Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2 alpha (MPC and MTT) or expressing both Hif1 alpha and Hif2 alpha (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hifla- and Hif2 alpha-driven effects we expressed Hif2 alpha in MTT and MPC-mCherry cells (naturally lacking Hif2 alpha). Presence of Hif2 alpha resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1 alpha in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2 alpha under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24-48 h) by HIf1 alpha. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2 alpha-mediated phosphorylation of TH (permanent status).

2019 Scientific Article in Blood Blood 133, 2597-2609 (2019)

Sperling, S.# ; Fiedler, P.# ; Lechner, M. ; Pollithy, A. ; Ehrenberg, S. ; Schiefer, A.I. ; Kenner, L. ; Feuchtinger, A. ; Kühn, R. ; Swinerd, G. ; Schmidt-Supprian, M. ; Strobl, L.J. ; Zimber-Strobl, U.

Chronic CD30 signaling in B cells results in lymphomagenesis by driving the expansion of plasmablasts and B1 cells.

CD30 is expressed on a variety of B-cell lymphomas, such as Hodgkin lymphoma, primary effusion lymphoma, and a diffuse large B-cell lymphoma subgroup. In normal tissues, CD30 is expressed on some activated B and T lymphocytes. However, the physiological function of CD30 signaling and its contribution to the generation of CD30(+) lymphomas are still poorly understood. To gain a better understanding of CD30 signaling in B cells, we studied the expression of CD30 in different murine B-cell populations. We show that B1 cells expressed higher levels of CD30 than B2 cells and that CD30 was upregulated in IRF4(+) plasmablasts (PBs). Furthermore, we generated and analyzed mice expressing a constitutively active CD30 receptor in B lymphocytes. These mice displayed an increase in B1 cells in the peritoneal cavity (PerC) and secondary lymphoid organs as well as increased numbers of plasma cells (PCs). TI-2 immunization resulted in a further expansion of B1 cells and PCs. We provide evidence that the expanded B1 population in the spleen included a fraction of PBs. CD30 signals seemed to enhance PC differentiation by increasing activation of NF-kappa B and promoting higher levels of phosphorylated STAT3 and STAT6 and nuclear IRF4. In addition, chronic CD30 signaling led to B-cell lymphomagenesis in aged mice. These lymphomas were localized in the spleen and PerC and had a B1-like/plasmablastic phenotype. We conclude that our mouse model mirrors chronic B-cell activation with increased numbers of CD30(+) lymphocytes and provides experimental proof that chronic CD30 signaling increases the risk of B-cell lymphomagenesis.

2019 Scientific Article in Diabetes Diabetes 68, 1329-1340 (2019)

Ratner, C. ; He, Z. ; Grunddal, K.V. ; Skov, L.J. ; Hartmann, B. ; Zhang, F. ; Feuchtinger, A. ; Bjerregaard, A. ; Christoffersen, C. ; Tschöp, M.H. ; Finan, B. ; DiMarchi, R.D. ; Leinninger, G.M. ; Williams, K.W. ; Clemmensen, C. ; Holst, B.

Long-acting neurotensin synergizes with liraglutide to reverse obesity through a melanocortin-dependent pathway.

Neurotensin (NT), a gut hormone and neuropeptide, increases in circulation after bariatric surgery in rodents and humans and inhibits food intake in mice. However, its potential to treat obesity and the subsequent metabolic dysfunctions have been difficult to assess owing to its short half-life in vivo. Here, we demonstrate that a long-acting, pegylated analog of the NT peptide (P-NT) reduces food intake, body weight, and adiposity in diet-induced obese mice when administered once daily for 6 days. Strikingly, when P-NT was combined with the glucagon-like peptide 1 mimetic liraglutide, the two peptides syner-gized to reduce food intake and body weight relative to each monotherapy, without inducing a taste aversion. Further, P-NT and liraglutide coadministration improved glycemia and reduced steatohepatitis. Finally, we show that the melanocortin pathway is central for P-NT–induced anorexia and necessary for the full synergistic effect of P-NT and liraglutide combination therapy. Overall, our data suggest that P-NT and liraglutide combination therapy could be an enhanced treatment for obesity with improved tolerability compared with liraglutide monotherapy.

2019 Scientific Article in Nature Communications Nat. Commun. 10:1114 (2019)

Gujrati, V.# ; Prakash, J.# ; Malekzadeh Najafabadi, J. ; Stiel, A.-C. ; Klemm, U. ; Mettenleiter, G. ; Aichler, M. ; Walch, A.K. ; Ntziachristos, V.

Bioengineered bacterial vesicles as biological nano-heaters for optoacoustic imaging.

Advances in genetic engineering have enabled the use of bacterial outer membrane vesicles (OMVs) to deliver vaccines, drugs and immunotherapy agents, as a strategy to circumvent biocompatibility and large-scale production issues associated with synthetic nanomaterials. We investigate bioengineered OMVs for contrast enhancement in optoacoustic (photoacoustic) imaging. We produce OMVs encapsulating biopolymer-melanin (OMVMel) using a bacterial strain expressing a tyrosinase transgene. Our results show that upon near-infrared light irradiation, OMVMel generates strong optoacoustic signals appropriate for imaging applications. In addition, we show that OMVMel builds up intense heat from the absorbed laser energy and mediates photothermal effects both in vitro and in vivo. Using multispectral optoacoustic tomography, we noninvasively monitor the spatio-temporal, tumour-associated OMVMel distribution in vivo. This work points to the use of bioengineered vesicles as potent alternatives to synthetic particles more commonly employed for optoacoustic imaging, with the potential to enable both image enhancement and photothermal applications.

2019 Review in Histochemistry and Cell Biology Histochem. Cell Biol. 151, 201-216 (2019)

Papathomas, T.G. ; Sun, N. ; Chortis, V. ; Taylor, A.E. ; Arlt, W. ; Richter, S. ; Eisenhofer, G. ; Ruiz-Babot, G. ; Guasti, L. ; Walch, A.K.

Novel methods in adrenal research: A metabolomics approach.

Metabolic alterations have implications in a spectrum of tissue functions and disease. Aided by novel molecular biological and computational tools, our understanding of physiological and pathological processes underpinning endocrine and endocrine-related disease has significantly expanded over the last decade. Herein, we focus on novel metabolomics-related methodologies in adrenal research: in situ metabolomics by mass spectrometry imaging, steroid metabolomics by gas and liquid chromatography-mass spectrometry, energy pathway metabologenomics by liquid chromatography-mass spectrometry-based metabolomics of Krebs cycle intermediates, and cellular reprogramming to generate functional steroidogenic cells and hence to modulate the steroid metabolome. All four techniques to assess and/or modulate the metabolome in biological systems provide tremendous opportunities to manage neoplastic and non-neoplastic disease of the adrenal glands in the era of precision medicine. In this context, we discuss emerging clinical applications and/or promising metabolic-driven research towards diagnostic, prognostic, predictive and therapeutic biomarkers in tumours arising from the adrenal gland and extra-adrenal paraganglia as well as modern approaches to delineate and reprogram adrenal metabolism.

2019 Scientific Article in PLoS ONE PLoS ONE 14:e0210998 (2019)

Ogrinc Wagner, A. ; Friedrich, V. ; Barthels, C. ; Marconi, P. ; Blutke, A. ; Brombacher, F. ; Brocker, T.

Strain specific maturation of Dendritic cells and production of IL-1β controls CD40-driven colitis.

Intestinal integrity is maintained by balanced numbers of CD103(+) Dendritic cells (DCs), which generate peripherally induced regulatory T cells (iTregs). We have developed a mouse model where DC-specific constitutive CD40 signals caused a strong reduction of CD103(+) DCs in the lamina propria (LP) and intestinal lymph nodes (LN). As a consequence, also iTregs were strongly reduced and transgenic mice on the C57Bl/6-background (B6) developed fatal colitis. Here we describe that transgenic mice on a pure Balb/c-background (B/c) do not show any pathologies, while transgenic C57Bl/6 x Balb/c (F1) mice develop weak colon inflammation, without fatal colitis. This graded pathology correlated with the effects of CD40-signalling on DCs in each background, with striking loss of CD103(+) DCs in B6, but reduced in F1 and diminished in B/c background. We further show direct correlation of CD103(+) DC-numbers with numbers of iTregs, the frequencies of which behave correspondingly. Striking effects on B6-DCs reflected robust loss of surface MHCII, known to be crucial for iTreg induction. Furthermore, elevated levels of IL-23 together with IL-1, found only in B6 mice, support generation of intestinal IFN-gamma(+) IL-17(+) Th17 cells and IFN-gamma(+) Th1 cells, responsible for onset of disease. Together, this demonstrates a novel aspect of colitis-control, depending on genetic background. Moreover, strain-specific environmental sensing might alter the CD103(+) DC/iTreg-axis to tip intestinal homeostatic balance to pathology.

2019 Scientific Article in Cell Metabolism Cell Metab. 29, 932-949.e4 (2019)

Norheim, F. ; Hasin-Brumshtein, Y. ; Vergnes, L. ; Chella Krishnan, K. ; Pan, C. ; Seldin, M.M. ; Hui, S.T. ; Mehrabian, M. ; Zhou, Z. ; Gupta, S. ; Parks, B.W. ; Walch, A.K. ; Reue, K. ; Hofmann, S.M. ; Arnold, A.P. ; Lusis, A.J.

Gene-by-sex interactions in mitochondrial functions and cardio-metabolic traits.

We studied sex differences in over 50 cardio-metabolic traits in a panel of 100 diverse inbred strains of mice. The results clearly showed that the effects of sex on both clinical phenotypes and gene expression depend on the genetic background. In support of this, genetic loci associated with the traits frequently showed sex specificity. For example, Lyplal1, a gene implicated in human obesity, was shown to underlie a sex-specific locus for diet induced obesity. Global gene expression analyses of tissues across the panel implicated adipose tissue "beiging" and mitochondrial functions in the sex differences. Isolated mitochondria showed gene-bysex interactions in oxidative functions, such that some strains (C57BL/6J) showed similar function between sexes, whereas others (DBA/2J and A/J) showed increased function in females. Reduced adipose mitochondria! function in males as compared to females was associated with increased susceptibility to obesity and insulin resistance. Gonadectomy studies indicated that gonadal hormones acting in a tissue-specific manner were responsible in part for the sex differences.

2019 Scientific Article in Proteomics - Clinical Applications Proteomics Clin. Appl. 13:e1800137 (2019)

Balluff, B. ; Buck, A. ; Martin-Lorenzo, M. ; Dewez, F. ; Langer, R. ; McDonnell, L.A. ; Walch, A.K. ; Heeren, R.M.A.

Integrative clustering in mass spectrometry imaging for enhanced patient stratification.

Scope In biomedical research, mass spectrometry imaging (MSI) can obtain spatially-resolved molecular information from tissue sections. Especially matrix-assisted laser desorption/ionization (MALDI) MSI offers, depending on the type of matrix, the detection of a broad variety of molecules ranging from metabolites to proteins, thereby facilitating the collection of multilevel molecular data. Lately, integrative clustering techniques have been developed that make use of the complementary information of multilevel molecular data in order to better stratify patient cohorts, but which have not yet been applied in the field of MSI. Materials and Methods In this study, the potential of integrative clustering is investigated for multilevel molecular MSI data to subdivide cancer patients into different prognostic groups. Metabolomic and peptidomic data are obtained by MALDI-MSI from a tissue microarray containing material of 46 esophageal cancer patients. The integrative clustering methods Similarity Network Fusion, iCluster, and moCluster are applied and compared to non-integrated clustering. Conclusion The results show that the combination of multilevel molecular data increases the capability of integrative algorithms to detect patient subgroups with different clinical outcome, compared to the single level or concatenated data. This underlines the potential of multilevel molecular data from the same subject using MSI for subsequent integrative clustering.

2019 Scientific Article in ACS Nano ACS Nano 13, 1029-1041 (2019)

Yang, L. ; Feuchtinger, A. ; Möller, W. ; Ding, Y. ; Kutschke, D. ; Möller, G. ; Schittny, J.C. ; Burgstaller, G. ; Hofmann, W. ; Stöger, T. ; Razansky, D. ; Walch, A.K. ; Schmid, O.

Three-dimensional quantitative co-mapping of pulmonary morphology and nanoparticle distribution with cellular resolution in non-dissected murine lungs.

Deciphering biodistribution, biokinetics, and biological effects of nanoparticles (NPs) in entire organs with cellular resolution remains largely elusive due to the lack of effective imaging tools. Here, light sheet fluorescence microscopy in combination with optical tissue clearing was validated for concomitant three-dimensional mapping of lung morphology and NP biodistribution with cellular resolution in nondissected ex viva murine lungs. Tissue autofluorescence allowed for label-free, quantitative morphometry of the entire bronchial tree, acinar structure, and blood vessels. Co-registration of fluorescent NPs with lung morphology revealed significant differences in pulmonary NP distribution depending on the means of application (intratracheal instillation and ventilator-assisted aerosol inhalation under anesthetized conditions). Inhalation exhibited a more homogeneous NP distribution in conducting airways and acini indicated by a central-to-peripheral (C/P) NP deposition ratio of unity (0.98 +/- 0.13) as compared to a 2-fold enhanced central deposition (C/P = 1.98 +/- 0.37) for instillation. After inhalation most NPs were observed in the proximal part of the acini as predicted by computational fluid dynamics simulations. At cellular resolution patchy NP deposition was visualized in bronchioles and acini, but more pronounced for instillation. Excellent linearity of the fluorescence intensity dose response curve allowed for accurate NP dosimetry and revealed ca. 5% of the inhaled aerosol was deposited in the lungs. This single-modality imaging technique allows for quantitative co-registration of tissue architecture and NP biodistribution, which could accelerate elucidation of NP biokinetics and bioactivity within intact tissues, facilitating both nanotoxicology studies and the development of nanomedicines.

2019 Scientific Article in Gastroenterology Gastroenterology 156, 203-217.e20 (2019)

Görgülü, K. ; Diakopoulos, K.N. ; Ai, J. ; Schoeps, B. ; Kabacaoglu, D. ; Karpathaki, A.F. ; Ciecielski, K.J. ; Kaya-Aksoy, E. ; Ruess, D.A. ; Berninger, A. ; Kowalska, M. ; Stevanovic, M. ; Wörmann, S.M. ; Wartmann, T. ; Zhao, Y. ; Halangk, W. ; Voronina, S. ; Tepikin, A. ; Schlitter, A.M. ; Steiger, K. ; Artati, A. ; Adamski, J. ; Aichler, M. ; Walch, A.K. ; Jastroch, M. ; Hartleben, G. ; Mantzoros, C.S. ; Weichert, W. ; Schmid, R.M. ; Herzig, S. ; Krüger, A. ; Sainz, B. ; Lesina, M. ; Algül, H.

Levels of the autophagy-related 5 protein affect progression and metastasis of pancreatic tumors in mice.

BACKGROUND AND AIMS: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. METHODS: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5; Kras) or heterozygous disruption of Atg5 (A5(+/-); Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. RESULTS: A5(+/-); Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5; Kras mice did not develop any tumors. Cultured A5(+/-); Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2thorn oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5(+/-); Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5(+/-); Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. CONCLUSIONS: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.

2019 Scientific Article in International Journal of Obesity Int. J. Obes. 43, 1305-1318 (2019)

Harrison, L. ; Schriever, S.C. ; Feuchtinger, A. ; Kyriakou, E. ; Baumann, P. ; Pfuhlmann, K. ; Messias, A.C. ; Walch, A.K. ; Tschöp, M.H. ; Pfluger, P.T.

Fluorescent blood-brain barrier tracing shows intact leptin transport in obese mice.

Background/objectives Individuals carrying loss-of-function gene mutations for the adipocyte hormone leptin are morbidly obese, but respond favorably to replacement therapy. Recombinant leptin is however largely ineffective for the vast majority of obese individuals due to leptin resistance. One theory underlying leptin resistance is impaired leptin transport across the blood-brain-barrier (BBB). Here, we aim to gain new insights into the mechanisms of leptin BBB transport, and its role in leptin resistance.Methods We developed a novel tool for visualizing leptin transport using infrared fluorescently labeled leptin, combined with tissue clearing and light-sheet fluorescence microscopy. We corroborated these data using western blotting.Results Using 3D whole brain imaging, we display comparable leptin accumulation in circumventricular organs of lean and obese mice, predominantly in the choroid plexus (CP). Protein quantification revealed comparable leptin levels in micro-dissected mediobasal hypothalami (MBH) of lean and obese mice (p = 0.99). We further found increased leptin receptor expression in the CP (p = 0.025, p = 0.0002) and a trend toward elevated leptin protein levels in the MBH (p = 0.17, p = 0.078) of obese mice undergoing weight loss interventions by calorie restriction or exendin-4 treatment.Conclusions Overall, our findings suggest a crucial role for the CP in controlling the transport of leptin into the cerebrospinal fluid and from there to target areas such as the MBH, potentially mediated via the leptin receptor. Similar leptin levels in circumventricular organs and the MBH of lean and obese mice further suggest intact leptin BBB transport in leptin resistant mice.

2019 Scientific Article in Clinical Cancer Research Clin. Cancer Res. 25, 1505-1516 (2019)

Hess-Rieger, J.# ; Unger, K.# ; Maihoefer, C. ; Schüttrumpf, L. ; Wintergerst, L. ; Heider, T. ; Weber, P. ; Marschner, S. ; Braselmann, H. ; Samaga, D. ; Kuger, S. ; Pflugradt, U. ; Baumeister, P. ; Walch, A.K. ; Woischke, C. ; Kirchner, T. ; Werner, M. ; Werner, K. ; Baumann, M. ; Budach, V. ; Combs, S.E. ; Debus, J. ; Grosu, A.-L. ; Krause, M. ; Linge, A. ; Rödel, C. ; Stuschke, M. ; Zips, D. ; Zitzelsberger, H. ; Ganswindt, U. ; Henke, M. ; Belka, C.

A five-microRNA signature predicts survival and disease control of patients with head and neck cancer negative for HPV-infection.

Purpose: Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is associated with unfavorable prognosis, while independent prognostic markers remain to be defined.Experimental Design: We retrospectively performed miRNA expression profiling. Patients were operated for locally advanced HPV-negative HNSCC and had received radiochemotherapy in eight different hospitals (DKTK-ROG; n = 85). Selection fulfilled comparable demographic, treatment, and follow-up characteristics. Findings were validated in an independent single-center patient sample (LMU-KKG; n = 77). A prognostic miRNA signature was developed for freedom from recurrence and tested for other endpoints. Recursivepartitioning analysis was performed on the miRNA signature, tumor and nodal stage, and extracapsular nodal spread. Technical validation used qRT-PCR. An miRNA-mRNA target network was generated and analyzed.Results: For DKTK-ROG and LMU-KKG patients, the median follow-up was 5.1 and 5.3 years, and the 5-year freedom from recurrence rate was 63.5% and 75.3%, respectively. A five-miRNA signature (hsa-let-7g-3p, hsamiR- 6508-5p, hsa-miR-210-5p, hsa-miR-4306, and hsa-miR-7161-3p) predicted freedom from recurrence in DKTK-ROG [hazard ratio (HR) 4.42; 95% confidence interval (CI), 1.98-9.88, P < 0.001], which was confirmed in LMU-KKG (HR 4.24; 95% CI, 1.40-12.81, P = 0.005). The signature also predicted overall survival (HR 3.03; 95% CI, 1.50-6.12, P = 0.001), recurrence-free survival (HR 3.16; 95% CI, 1.65-6.04, P < 0.001), and disease-specific survival (HR 5.12; 95% CI, 1.88-13.92, P < 0.001), all confirmed in LMU-KKG data. Adjustment for relevant covariates maintained the miRNA signature predicting all endpoints. Recursive- partitioning analysis of both samples combined classified patients into low (n = 17), low-intermediate (n = 80), high-intermediate (n = 48), or high risk (n = 17) for recurrence (P < 0.001).Conclusions: The five-miRNA signature is a strong and independent prognostic factor for disease recurrence and survival of patients with HPV-negative HNSCC.

2018 Nature Communications Nat. Commun. 9:4975 (2018)

Clemmensen, C. ; Jall, S. ; Kleinert, M. ; Quarta, C. ; Gruber, T. ; Reber, J. ; Sachs, S. ; Fischer, K. ; Feuchtinger, A. ; Karlas, A. ; Simonds, S.E. ; Grandl, G. ; Loher, D. ; Sanchez-Quant, E. ; Keipert, S. ; Jastroch, M. ; Hofmann, S.M. ; Nascimento, E.B.M. ; Schrauwen, P. ; Ntziachristos, V. ; Cowley, M.A. ; Finan, B. ; Müller, T.D. ; Tschöp, M.H.

Coordinated targeting of cold and nicotinic receptors synergistically improves obesity and type 2 diabetes (vol 9, 4304, 2018).

In the original PDF version of this article, affiliation 1, 'Institute for Diabetes and Obesity, Helmholtz Diabetes Center (HDC), Helmholtz Zentrum Muenchen & German Center for Diabetes Research (DZD), Neuherberg, Germany', was incorrectly given as 'Institute of Diabetes and Regeneration Research, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health (GmbH), Neuherberg, Germany '. This has now been corrected in the PDF version of the article; the HTML version was correct at the time of publication.

2018 Scientific Article in Nature Communications Nat. Commun. 9:4304 (2018)

Clemmensen, C. ; Jall, S. ; Kleinert, M. ; Quarta, C. ; Gruber, T. ; Reber, J. ; Sachs, S. ; Fischer, K. ; Feuchtinger, A. ; Karlas, A. ; Simonds, S.E. ; Grandl, G. ; Loher, D. ; Sanchez-Quant, E. ; Keipert, S. ; Jastroch, M. ; Hofmann, S.M. ; Nascimento, E.B.M. ; Schrauwen, P. ; Ntziachristos, V. ; Cowley, M.A. ; Finan, B.&deg ; Müller, T.D.&deg ; Tschöp, M.H.&deg

Coordinated targeting of cold and nicotinic receptors synergistically improves obesity and type 2 diabetes.

Pharmacological stimulation of brown adipose tissue (BAT) thermogenesis to increase energy expenditure is progressively being pursued as a viable anti-obesity strategy. Here, we report that pharmacological activation of the cold receptor transient receptor potential cation channel subfamily M member 8 (TRPM8) with agonist icilin mimics the metabolic benefits of cold exposure. In diet-induced obese (DIO) mice, treatment with icilin enhances energy expenditure, and decreases body weight, without affecting food intake. To further potentiate the thermogenic action profile of icilin and add complementary anorexigenic mechanisms, we set out to identify pharmacological partners next to icilin. To that end, we specifically targeted nicotinic acetylcholine receptor (nAChR) subtype alpha3beta4 (α3β4), which we had recognized as a potential regulator of energy homeostasis and glucose metabolism. Combinatorial targeting of TRPM8 and nAChR α3β4 by icilin and dimethylphenylpiperazinium (DMPP) orchestrates synergistic anorexic and thermogenic pathways to reverse diet-induced obesity, dyslipidemia, and glucose intolerance in DIO mice.

2018 Scientific Article in Molecular Oncology Mol. Oncol., DOI: 10.1002/1878-0261.12388 (2018)

Wintergerst, L. ; Selmansberger, M. ; Maihoefer, C. ; Schüttrumpf, L. ; Walch, A.K. ; Wilke, C. ; Pitea, A. ; Woischke, C. ; Baumeister, P. ; Kirchner, T. ; Belka, C. ; Ganswindt, U. ; Zitzelsberger, H. ; Unger, K. ; Hess-Rieger, J.

A prognostic mRNA expression signature of four 16q24.3 genes in radio(chemo)therapy-treated head and neck squamous cell carcinoma (HNSCC).

Previously, we have shown that copy number gain of the chromosomal band 16q24.3 is associated with impaired clinical outcome of radiotherapy-treated head and neck squamous cell carcinoma (HNSCC) patients. We set out to identify a prognostic mRNA signature from genes located on 16q24.3 in radio(chemo)therapy-treated HNSCC patients of the TCGA (The Cancer Genome Atlas, n = 99) cohort. We applied stepwise forward selection using expression data of 41 16q24.3 genes. The resulting optimal Cox-proportional hazards regression model included the genes APRT, CENPBD1, CHMP1A, and GALNS. Afterward, the prognostic value of the classifier was confirmed in an independent cohort of HNSCC patients treated by adjuvant radio(chemo)therapy (LMU-KKG cohort). The signature significantly differentiated high- and low-risk patients with regard to overall survival (HR = 2.01, 95% CI 1.10–3.70; P = 0.02125), recurrence-free survival (HR = 1.84, 95% CI 1.01–3.34; P = 0.04206), and locoregional recurrence-free survival (HR = 1.87, 95% CI 1.03–3.40; P = 0.03641). The functional impact of the four signature genes was investigated after reconstruction of a gene association network from transcriptome data of the TCGA HNSCC cohort using a partial correlation approach. Subsequent pathway enrichment analysis of the network neighborhood (first and second) of the signature genes suggests involvement of HNSCC-associated signaling pathways such as apoptosis, cell cycle, cell adhesion, EGFR, JAK-STAT, and mTOR. Furthermore, a detailed analysis of the first neighborhood revealed a cluster of co-expressed genes located on chromosome 16q, substantiating the impact of 16q24.3 alterations in poor clinical outcome of HNSCC. The reported gene expression signature represents a prognostic marker in HNSCC patients following postoperative radio(chemo)therapy.

2018 Scientific Article in Molecular Metabolism Mol. Metab., DOI: 10.1016/j.molmet.2018.07.002 (2018)

Suwandhi, L. ; Hausmann, S. ; Braun, A. ; Gruber, T. ; Heinzmann, S.S. ; Gálvez, E.J.C. ; Buck, A. ; Legutko, B. ; Israel, A. ; Feuchtinger, A. ; Haythorne, E. ; Staiger, H. ; Heni, M. ; Häring, H.-U. ; Schmitt-Kopplin, P. ; Walch, A.K. ; Cáceres, C.G. ; Tschöp, M.H. ; Rutter, G.A. ; Strowig, T. ; Elsner, M. ; Ussar, S.

Chronic d-serine supplementation impairs insulin secretion.

OBJECTIVE: The metabolic role of d-serine, a non-proteinogenic NMDA receptor co-agonist, is poorly understood. Conversely, inhibition of pancreatic NMDA receptors as well as loss of the d-serine producing enzyme serine racemase have been shown to modulate insulin secretion. Thus, we aim to study the impact of chronic and acute d-serine supplementation on insulin secretion and other parameters of glucose homeostasis. METHODS: We apply MALDI FT-ICR mass spectrometry imaging, NMR based metabolomics, 16s rRNA gene sequencing of gut microbiota in combination with a detailed physiological characterization to unravel the metabolic action of d-serine in mice acutely and chronically treated with 1% d-serine in drinking water in combination with either chow or high fat diet feeding. Moreover, we identify SNPs in SRR, the enzyme converting L-to d-serine and two subunits of the NMDA receptor to associate with insulin secretion in humans, based on the analysis of 2760 non-diabetic Caucasian individuals. RESULTS: We show that chronic elevation of d-serine results in reduced high fat diet intake. In addition, d-serine leads to diet-independent hyperglycemia due to blunted insulin secretion from pancreatic beta cells. Inhibition of alpha 2-adrenergic receptors rapidly restores glycemia and glucose tolerance in d-serine supplemented mice. Moreover, we show that single nucleotide polymorphisms (SNPs) in SRR as well as in individual NMDAR subunits are associated with insulin secretion in humans. CONCLUSION: Thus, we identify a novel role of d-serine in regulating systemic glucose metabolism through modulating insulin secretion.

2018 Scientific Article in EMBO Journal, The EMBO J.:e94813 (2018)

Jain, M.#&deg ; Mann, T.D.# ; Stulić, M.# ; Rao, S.P. ; Kirsch, A. ; Pullirsch, D. ; Strobl, X. ; Rath, C. ; Reissig, L. ; Moreth, K. ; Klein-Rodewald, T. ; Bekeredjian, R. ; Gailus-Durner, V. ; Fuchs, H. ; Hrabě de Angelis, M. ; Pablik, E. ; Cimatti, L. ; Martin, D. ; Zinnanti, J. ; Graier, W.F. ; Sibilia, M. ; Frank, S. ; Levanon, E.Y. ; Jantsch, M.F.&deg

RNA editing of Filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure.

Epitranscriptomic events such as adenosine-to-inosine (A-to-I) RNA editing by ADAR can recode mRNAs to translate novel proteins. Editing of the mRNA that encodes actin crosslinking protein Filamin A (FLNA) mediates a Q-to-R transition in the interactive C-terminal region. While FLNA editing is conserved among vertebrates, its physiological function remains unclear. Here, we show that cardiovascular tissues in humans and mice show massive editing and that FLNA RNA is the most prominent substrate. Patient-derived RNA-Seq data demonstrate a significant drop in FLNA editing associated with cardiovascular diseases. Using mice with only impaired FLNA editing, we observed increased vascular contraction and diastolic hypertension accompanied by increased myosin light chain phosphorylation, arterial remodeling, and left ventricular wall thickening, which eventually causes cardiac remodeling and reduced systolic output. These results demonstrate a causal relationship between RNA editing and the development of cardiovascular disease indicating that a single epitranscriptomic RNA modification can maintain cardiovascular health.

2018 Scientific Article in Biochemical and Biophysical Research Communications Biochem. Biophys. Res. Commun. 503, 2770-2777 (2018)

Clemen, C.S. ; Winter, L. ; Strucksberg, K.H. ; Berwanger, C. ; Türk, M. ; Kornblum, C. ; Florin, A. ; Aguilar-Pimentel, J.A. ; Amarie, O.V. ; Becker, L. ; Garrett, L. ; Hans, W. ; Moreth, K. ; Neff, F. ; Pingen, L. ; Rathkolb, B. ; Rácz, I. ; Rozman, J. ; Treise, I. ; Fuchs, H. ; Gailus-Durner, V. ; Hrabě de Angelis, M. ; Vorgerd, M. ; Eichinger, L. ; Schröder, R.

The heterozygous R155C VCP mutation: Toxic in humans! Harmless in mice?

Heterozygous missense mutations in the human VCP gene cause inclusion body myopathy associated with Paget disease of bone and fronto-temporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). The exact molecular mechanisms by which VCP mutations cause disease manifestation in different tissues are incompletely understood. In the present study, we report the comprehensive analysis of a newly generated R155C VCP knock-in mouse model, which expresses the ortholog of the second most frequently occurring human pathogenic VCP mutation. Heterozygous R155C VCP knock-in mice showed decreased plasma lactate, serum albumin and total protein concentrations, platelet numbers, and liver to body weight ratios, and increased oxygen consumption and CD8+/Ly6C + T-cell fractions, but none of the typical human IBMPFD or ALS pathologies. Breeding of heterozygous mice did not yield in the generation of homozygous R155C VCP knock-in animals. Immunoblotting showed identical total VCP protein levels in human IBMPFD and murine R155C VCP knock-in tissues as compared to wild-type controls. However, while in human IBMPFD skeletal muscle tissue 70% of the total VCP mRNA was derived from the mutant allele, in R155C VCP knock-in mice only 5% and 7% mutant mRNA were detected in skeletal muscle and brain tissue, respectively. The lack of any obvious IBMPFD or ALS pathology could thus be a consequence of the very low expression of mutant VCP. We conclude that the increased and decreased fractions of the R155C mutant VCP mRNA in man and mice, respectively, are due to missense mutation-induced, divergent alterations in the biological half-life of the human and murine mutant mRNAs. Furthermore, our work suggests that therapy approaches lowering the expression of the mutant VCP mRNA below a critical threshold may ameliorate the intrinsic disease pathology.

2018 Scientific Article in Science Science 361:eaar7191 (2018)

International Wheat Genome Sequencing Consortium (Eversole, K.&deg ; Feuillet, C. ; Keller, B. ; Rogers, J. ; Stein, N.&deg ; Appels, R.&deg) ; Pozniak, C.J. ; Choulet, F. ; Distelfeld, A. ; Poland, J. ; Ronen, G ; Sharpe, A.G. ; Pozniak, C. ; Barad, O. ; Baruch, K. ; Choulet, F. ; Keeble-Gagnère, G. ; Mascher, M. ; Ben-Zvi, G. ; Josselin, A.A. ; Himmelbach, A. ; Keeble-Gagnère, G. ; Balfourier, F. ; Gutierrez-Gonzalez, J. ; Hayden, M. ; Josselin, A.A. ; Koh, C. ; Muehlbauer, G. ; Pasam, R.K. ; Paux, E. ; Rigault, P. ; Tibbits, J. ; Tiwari, V. ; Choulet, F. ; Keeble-Gagnère, G. ; Spannagl, M. ; Lang, D. ; Gundlach, H. ; Haberer, G. ; Mayer, K.F.X. ; Ormanbekova, D. ; Paux, E. ; Prade, V.M. ; Šimková, H. ; Wicker, T. ; Swarbreck, D. ; Rimbert, H. ; Guilhot, N. ; Kaithakottil, G. ; Keilwagen, J. ; Leroy, P. ; Lux, T. ; Twardziok, S.O. ; Venturini, L. ; Juhász, A ; Abrouk, M ; Fischer, I.P. ; Uauy, C. ; Borrill, P. ; Ramirez-Gonzalez, R.H. ; Arnaud, D. ; Chalabi, S. ; Chalhoub, B. ; Cory, A. ; Datla, R. ; Davey, M.W. ; Hayden, M. ; Jacobs, J. ; Robinson, S.J. ; Steuernagel, B. ; Tibbits, J. ; Tiwari, V. ; van Ex, F. ; Wulff, B.B.H. ; Robinson, S.J. ; Cory, A. ; Benhamed, M. ; Paux, E. ; Bendahmane, A. ; Concia, L. ; Latrasse, D. ; Rogers, J. ; Alaux, M. ; Bartoš, J. ; Bellec, A. ; Berges, H. ; Doležel, J. ; Frenkel, Z ; Gill, B. ; Korol, A. ; Letellier, T. ; Olsen, O.A. ; Singh, K. ; Valárik, M. ; van der Vossen, E. ; Vautrin, S. ; Weining, S. ; Korol, A. ; Frenkel, Z. ; Fahima, T. ; Glikson, V. ; Raats, D. ; Rogers, J. ; Tiwari, V. ; Gill, B. ; Paux, E. ; Číhalíková, J. ; Toegelová, H. ; Vrána, J. ; Sourdille, P. ; Darrier, B. ; Barabaschi, D. ; Cattivelli, L ; Hernandez, P. ; Galvez, S. ; Budak, H. ; Jones, J.D.G. ; Witek, K. ; Yu, G. ; Small, I. ; Melonek, J. ; Zhou, R. ; Juhász, A. ; Belova, T. ; Olsen, O.A. ; Kanyuka, K. ; King, R. ; Nilsen, K. ; Walkowiak, S. ; Cuthbert, R. ; Datla, R. ; Knox, R. ; Wiebe, K. ; Xiang, D. ; Rohde, A. ; Golds, T. ; Čížková, J. ; Akpinar, B.A. ; Biyiklioglu, S. ; Muehlbauer, G. ; Gao, L. ; Gutierrez-Gonzalez, J. ; N'Daiye, A. ; Číhalíková, J. ; Kubaláková, M. ; Šafář, J. ; Berges, H. ; Bellec, A. ; Vautrin, S. ; Alaux, M. ; Alfama, F. ; Adam-Blondon, A.F. ; Flores, R. ; Guerche, C. ; Letellier, T. ; Loaec, M. ; Quesneville, H. ; Condie, J. ; Ens, J. ; Koh, C. ; Maclachlan, R. ; Tan, Y. ; Paux, E. ; Alberti, A. ; Aury, J.M. ; Barbe, V. ; Couloux, A. ; Cruaud, C. ; Labadie, K. ; Mangenot, S. ; Wincker, P. ; Gill, B. ; Kaur, G. ; Luo, M. ; Sehgal, S. ; Singh, K. ; Chhuneja, P. ; Gupta, O.P. ; Jindal, S. ; Kaur, P. ; Malik, P. ; Sharma, P. ; Yadav, B. ; Singh, N.K. ; Khurana, J. ; Chaudhary, C. ; Khurana, P. ; Kumar, V. ; Mahato, A. ; Mathur, S. ; Sevanthi, A. ; Sharma, N. ; Tomar, R.S. ; Jacobs, J. ; Alaux, M. ; Bellec, A. ; Berges, H. ; Frenkel, Z. ; Gill, B. ; Korol, A. ; van der Vossen, E. ; Vautrin, S. ; Kaur, G. ; Luo, M. ; Sehgal, S. ; Bartoš, J. ; Holušová, K. ; Plíhal, O. ; Clark, M.D. ; Heavens, D. ; Kettleborough, G. ; Wright, J. ; Valárik, M. ; Abrouk, M. ; Balcárková, B. ; Holušová, K. ; Hu, Y. ; Luo, M. ; Salina, E. ; Ravin, N. ; Skryabin, K. ; Beletsky, A. ; Kadnikov, V. ; Mardanov, A. ; Nesterov, M. ; Rakitin, A. ; Sergeeva, E. ; Handa, H. ; Kanamori, H. ; Katagiri, S. ; Kobayashi, F. ; Nasuda, S. ; Tanaka, T. ; Wu, J. ; Hayden, M. ; Rigault, P. ; Belova, T. ; Cattonaro, F. ; Jiumeng, M. ; Kugler, K.G. ; Pfeifer, M. ; Sandve, S. ; Xun, X. ; Zhan, B. ; Abrouk, M. ; Batley, J. ; Bayer, P.E. ; Edwards, D. ; Hayashi, S. ; Tulpová, Z. ; Visendi, P. ; Weining, S. ; Cui, L. ; Du, X. ; Feng, K. ; Nie, X. ; Tong, W.G. ; Wang, L. ; Borrill, P. ; Galvez, S. ; Lux, T. ; Ramirez-Gonzalez, R.H. ; Venturini, L. ; Borrill, P ; Hernandez, P ; Kanyuka, K ; Paux, E.

Shifting the limits in wheat research and breeding using a fully annotated reference genome.

An annotated reference sequence representing the hexaploid bread wheat genome in 21 pseudomolecules has been analyzed to identify the distribution and genomic context of coding and noncoding elements across the A, B, and D subgenomes. With an estimated coverage of 94% of the genome and containing 107,891 high-confidence gene models, this assembly enabled the discovery of tissue-and developmental stage-related coexpression networks by providing a transcriptome atlas representing major stages of wheat development. Dynamics of complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. This community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding.

2018 Scientific Article in European Respiratory Journal Eur. Respir. J. 52:1702314 (2018)

Sun, N.# ; Fernandez, I.E.# ; Wei, M. ; Witting, M. ; Aichler, M. ; Feuchtinger, A. ; Burgstaller, G. ; Verleden, S.E. ; Schmitt-Kopplin, P. ; Eickelberg, O. ; Walch, A.K.

Pharmacometabolic response to pirfenidone in pulmonary fibrosis detected by MALDI-FTICR-MSI.

Idiopathic pulmonary fibrosis (IPF) is a fatal condition that reduces life expectancy and shows a limited response to available therapies. Pirfenidone has been approved for treatment of IPF, but little is known about the distinct metabolic changes that occur in the lung upon pirfenidone administration.Here, we performed a proof-of-concept study using high-resolution quantitative matrix-assisted laser desorption/ionisation Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI) to simultaneously detect, visualise and quantify in situ endogenous and exogenous metabolites in lungs of mice subjected to experimental fibrosis and human patients with IPF, and to assess the effect of pirfenidone treatment on metabolite levels.Metabolic pathway analysis and endogenous metabolite quantification revealed that pirfenidone treatment restores redox imbalance and glycolysis in IPF tissues, and downregulates ascorbate and aldarate metabolism, thereby likely contributing to in situ modulation of collagen processing. As such, we detected specific alterations in metabolite pathways in fibrosis and, importantly, metabolic recalibration following pirfenidone treatment.Together, these results highlight the suitability of high-resolution MALDI-FTICR-MSI for deciphering the therapeutic effects of pirfenidone and provide a preliminary analysis of the metabolic changes that occur during pirfenidone treatment in vivo These data may therefore contribute to improvement of currently available therapies for IPF.

2018 Scientific Article in American Journal of Respiratory and Critical Care Medicine Am. J. Respir. Crit. Care Med. 198, 1527-1538 (2018)

Martin-Medina, A. ; Lehmann, M. ; Burgy, O. ; Hermann, S. ; Baarsma, H.A. ; Wagner, D.E. ; De Santis, M. ; Ciolek, F. ; Hofer, T.P. ; Frankenberger, M. ; Aichler, M. ; Lindner, M. ; Gesierich, W. ; Guenther, A. ; Walch, A.K. ; Coughlan, C. ; Wolters, P.  ; Lee, J.S. ; Behr, J. ; Königshoff, M.

Increased extracellular vesicles mediate WNT5A signaling in idiopathic pulmonary fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo) fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored.Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF.Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies.Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-beta in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A.Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.

2018 Scientific Article in Analytical and Bioanalytical Chemistry Anal. Bioanal. Chem. 410, 5969–5980 (2018)

Buck, A. ; Heijs, B. ; Beine, B. ; Schepers, J. ; Cassese, A. ; Heeren, R.M.A. ; McDonnell, L.A. ; Henkel, C. ; Walch, A.K. ; Balluff, B.

Round robin study of formalin-fixed paraffin-embedded tissues in mass spectrometry imaging.

Mass spectrometry imaging (MSI) has provided many results with translational character, which still have to be proven robust in large patient cohorts and across different centers. Although formalin-fixed paraffin-embedded (FFPE) specimens are most common in clinical practice, no MSI multicenter study has been reported for FFPE samples. Here, we report the results of the first round robin MSI study on FFPE tissues with the goal to investigate the consequences of inter-and intracenter technical variation on masking biological effects. A total of four centers were involved with similar MSI instrumentation and sample preparation equipment. A FFPE multi-organ tissue microarray containing eight different types of tissue was analyzed on a peptide and metabolite level, which enabled investigating different molecular and biological differences. Statistical analyses revealed that peptide intercenter variation was significantly lower and metabolite intercenter variation was significantly higher than the respective intracenter variations. When looking at relative univariate effects of mass signals with statistical discriminatory power, the metabolite data was more reproducible across centers compared to the peptide data. With respect to absolute effects (cross-center common intensity scale), multivariate classifiers were able to reach on average > 90% accuracy for peptides and > 80% for metabolites if trained with sufficient amount of cross-center data. Overall, our study showed that MSI data from FFPE samples could be reproduced to a high degree across centers. While metabolite data exhibited more reproducibility with respect to relative effects, peptide data-based classifiers were more directly transferable between centers and therefore more robust than expected.

2018 Scientific Article in Radiation Oncology Radiat. Oncol. 13:123 (2018)

Maihoefer, C. ; Schüttrumpf, L. ; Macht, C. ; Pflugradt, U. ; Hess-Rieger, J. ; Schneider, L. ; Woischke, C. ; Walch, A.K. ; Baumeister, P. ; Kirchner, T. ; Zitzelsberger, H. ; Belka, C. ; Ganswindt, U.

Postoperative (chemo) radiation in patients with squamous cell cancers of the head and neck - clinical results from the cohort of the clinical cooperation group "Personalized Radiotherapy in Head and Neck Cancer".

Background: Postoperative (chemo) radiation improves tumor control and survival in high-risk patients with head and neck squamous cell carcinoma based on established risk factors. The clinical cooperation group "Personalized Radiotherapy in Head and Neck Cancer" focuses on the identification and validation of new biomarkers, which are aimed at eventually stratifying and personalizing the therapy concept. Hence, we reviewed all patients with head and neck squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx, treated with postoperative (chemo) radiation from 06/2008 until 06/2015 at the Department of Radiation Oncology in the University Hospital, LMU Munich. Here we report the clinical results of the cohort, laying the foundation for further research within the framework of a clinical cooperation group.Methods: Patient data were retrospectively (until 2013) and prospectively (from 2013) collected and analyzed for outcome and treatment failures with regard to previously described and established risk factors.Results: We identified 302 patients (median follow-up 45 months, average age 60.7 years), having received postoperative (chemo) radiation (median 64 Gy). Chemotherapy was added in 58% of cases, mostly Cisplatin/5-Fluorouracil in concordance with the ARO 96-3 study. The 3-year overall survival, local, locoregional and distant failure estimates were 70.5, 9.7, 12.2 and 13.5%, respectively. Human papillomavirus-associated oropharyngeal cancer was associated with a significant improved overall survival, locoregional, distant and overall tumor control rates in multivariate analysis. Additionally, in multivariate analysis, for local failure, resection status and perineural invasion, for locoregional and distant failure extracapsular extension and for overall survival the presence of nodal disease were significant adverse factors. Moreover, 138 patients have been treated in concordance with the ARO 96-3 protocol, corroborating the results of this study.Conclusions: Our cohort represents a large unselected cohort of patients with head and neck squamous cell carcinoma treated with postoperative (chemo) radiation. Tumor control rates and survival rates are consistent with the results of previously reported data.

2018 Scientific Article in BMC Evolutionary Biology BMC Evol. Biol. 18:96 (2018)

Bräuer, K. ; Brockers, K. ; Moneer, J. ; Feuchtinger, A. ; Wollscheid-Lengeling, E. ; Lengeling, A. ; Wolf, A.

Phylogenetic and genomic analyses of the ribosomal oxygenases Riox1 (No66) and Riox2 (Mina53) provide new insights into their evolution.

Background Translation of specific mRNAs can be highly regulated in different cells, tissues or under pathological conditions. Ribosome heterogeneity can originate from variable expression or post-translational modifications of ribosomal proteins. The ribosomal oxygenases RIOX1 (NO66) and RIOX2 (MINA53) modify ribosomal proteins by histidine hydroxylation. A similar mechanism is present in prokaryotes. Thus, ribosome hydroxylation may be a well-conserved regulatory mechanism with implications in disease and development. However, little is known about the evolutionary history of Riox1 and Riox2 genes and their encoded proteins across eukaryotic taxa. Results In this study, we have analysed Riox1 and Riox2 orthologous genes from 49 metazoen species and have constructed phylogenomic trees for both genes. Our genomic and phylogenetic analyses revealed that Arthropoda, Annelida, Nematoda and Mollusca lack the Riox2 gene, although in the early phylum Cnidaria both genes, Riox1 and Riox2, are present and expressed. Riox1 is an intronless single-exon-gene in several species, including humans. In contrast to Riox2, Riox1 is ubiquitously present throughout the animal kingdom suggesting that Riox1 is the phylogenetically older gene from which Riox2 has evolved. Both proteins have maintained a unique protein architecture with conservation of active sites within the JmjC domains, a dimerization domain, and a winged-helix domain. In addition, Riox1 proteins possess a unique N-terminal extension domain. Immunofluorescence analyses in Hela cells and in Hydra vulgaris identified a nucleolar localisation signal within the extended N-terminal domain of human RIOX1 and an altered subnuclear localisation for the Hydra Riox2. Conclusions Conserved active site residues and uniform protein domain architecture suggest a consistent enzymatic activity within the Riox orthologs throughout evolution. However, differences in genomic architecture, like single exon genes and alterations in subnuclear localisation, as described for Hydra, point towards adaption mechanisms that may correlate with taxa- or species-specific requirements. The diversification of Riox1/Riox2 gene structures throughout evolution suggest that functional requirements in expression of protein isoforms and/or subcellular localisation of proteins may have evolved by adaptation to lifestyle.  

2018 Scientific Article in Neural regeneration research Neural Regen. Res. 13, 854-861 (2018)

Saller, M.M. ; Huettl, R.E. ; Mayer, J.M. ; Feuchtinger, A. ; Krug, C. ; Holzbach, T. ; Volkmer, E.

Validation of a novel animal model for sciatic nerve repair with an adipose-derived stem cell loaded fibrin conduit.

Flatworms of the species Schmidtea mediterranea are immortal-adult animals contain a large pool of pluripotent stem cells that continuously differentiate into all adult cell types. Therefore, single-cell transcriptome profiling of adult animals should reveal mature and progenitor cells. By combining perturbation experiments, gene expression analysis, a computational method that predicts future cell states from transcriptional changes, and a lineage reconstruction method, we placed all major cell types onto a single lineage tree that connects all cells to a single stem cell compartment. We characterized gene expression changes during differentiation and discovered cell types important for regeneration. Our results demonstrate the importance of single-cell transcriptome analysis for mapping and reconstructing fundamental processes of developmental and regenerative biology at high resolution.

2018 Scientific Article in Heliyon Heliyon 4:e00606 (2018)

Lohöfer, F. ; Hoffmann, L. ; Buchholz, R. ; Huber, K. ; Glinzer, A. ; Kosanke, K. ; Feuchtinger, A. ; Aichler, M. ; Feuerecker, B. ; Kaissis, G. ; Rummeny, E.J. ; Höltke, C. ; Faber, C. ; Schilling, F. ; Botnar, R.M. ; Walch, A.K. ; Karst, U. ; Wildgruber, M.

Molecular imaging of myocardial infarction with Gadofluorine P – A combined magnetic resonance and mass spectrometry imaging approach.

Background: Molecular MRI is becoming increasingly important for preclinical research. Validation of targeted gadolinium probes in tissue however has been cumbersome up to now. Novel methodology to assess gadolinium distribution in tissue after in vivo application is therefore needed. Purpose: To establish combined Magnetic Resonance Imaging (MRI) and Mass Spectrometry Imaging (MSI) for improved detection and quantification of Gadofluorine P deposition in scar formation and myocardial remodeling. Materials and methods: Animal studies were performed according to institutionally approved protocols. Myocardial infarction was induced by permanent ligation of the left ascending artery (LAD) in C57BL/6J mice. MRI was performed at 7T at 1 week and 6 weeks after myocardial infarction. Gadofluorine P was used for dynamic T1mapping of extracellular matrix synthesis during myocardial healing and compared to Gd-DTPA. After in vivo imaging contrast agent concentration as well as distribution in tissue were validated and quantified by spatially resolved Matrix-Assisted Laser Desorption Ionization (MALDI) MSI and Laser Ablation – Inductively Coupled Plasma – Mass Spectrometry (LA-ICP-MS) imaging. Results: Both Gadofluorine P enhancement as well as local tissue content in the myocardial scar were highest at 15 minutes post injection. R1values increased from 1 to 6 weeks after MI (1.62 s−1vs 2.68 s−1, p = 0.059) paralleled by an increase in Gadofluorine P concentration in the infarct from 0.019 mM at 1 week to 0.028 mM at 6 weeks (p = 0.048), whereas Gd-DTPA enhancement showed no differences (3.95 s−1vs 3.47 s−1, p = 0.701). MALDI-MSI results were corroborated by elemental LA-ICP-MS of Gadolinium in healthy and infarcted myocardium. Histology confirmed increased extracellular matrix synthesis at 6 weeks compared to 1 week. Conclusion: Adding quantitative MSI to MR imaging enables a quantitative validation of Gadofluorine P distribution in the heart after MI for molecular imaging.

2018 Scientific Article in Chemical Communications Chem. Commun. 54, 5426-5429 (2018)

Rodriguez Camargo, D.C.&deg ; Garg, D. ; Buday, K. ; Frankó, A. ; Rodriguez Camargo, A. ; Schmidt, F. ; Cox, S.J. ; Suladze, S. ; Haslbeck, M. ; Mideksa, Y.G. ; Gemmecker, G. ; Aichler, M. ; Mettenleiter, G. ; Schulz, M. ; Walch, A.K. ; Hrabě de Angelis, M. ; Feige, M.J. ; Sierra, C.A. ; Conrad, M. ; Tripsianes, K. ; Ramamoorthy, A. ; Reif, B.&deg

hIAPP forms toxic oligomers in plasma.

In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in β-sheet rich oligomers with increased β-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.

2018 Scientific Article in PLoS Biology PLoS Biol. 16:e2005019 (2018)

André, V. ; Gau, C. ; Scheideler, A. ; Aguilar-Pimentel, J.A. ; Amarie, O.V. ; Becker, L. ; Garrett, L. ; Hans, W. ; Hölter, S.M. ; Janik, D. ; Moreth, K. ; Neff, F. ; Östereicher, M.A. ; Rácz, I. ; Rathkolb, B. ; Rozman, J. ; Bekeredjian, R. ; Graw, J. ; Klingenspor, M. ; Klopstock, T. ; Ollert, M. ; Schmidt-Weber, C.B. ; Wolf, E. ; Wurst, W. ; Gailus-Durner, V. ; Brielmeier, M. ; Fuchs, H. ; Hrabě de Angelis, M.

Laboratory mouse housing conditions can be improved using common environmental enrichment without compromising data.

Animal welfare requires the adequate housing of animals to ensure health and well-being. The application of environmental enrichment is a way to improve the well-being of laboratory animals. However, it is important to know whether these enrichment items can be incorporated in experimental mouse husbandry without creating a divide between past and future experimental results. Previous small-scale studies have been inconsistent throughout the literature, and it is not yet completely understood whether and how enrichment might endanger comparability of results of scientific experiments. Here, we measured the effect on means and variability of 164 physiological parameters in 3 conditions: with nesting material with or without a shelter, comparing these 2 conditions to a “barren” regime without any enrichments. We studied a total of 360 mice from each of 2 mouse strains (C57BL/6NTac and DBA/2NCrl) and both sexes for each of the 3 conditions. Our study indicates that enrichment affects the mean values of some of the 164 parameters with no consistent effects on variability. However, the influence of enrichment appears negligible compared to the effects of other influencing factors. Therefore, nesting material and shelters may be used to improve animal welfare without impairment of experimental outcome or loss of comparability to previous data collected under barren housing conditions.

2018 Scientific Article in International Journal of Cancer Int. J. Cancer 143, 1505-1515 (2018)

Wilke, C.# ; Braselmann, H.# ; Hess-Rieger, J. ; Klymenko, S.V. ; Chumak, V.V. ; Zakhartseva, L.M. ; Bakhanova, E.V. ; Walch, A.K. ; Selmansberger, M. ; Samaga, D. ; Weber, P. ; Schneider, L. ; Fend, F. ; Bösmüller, H.C. ; Zitzelsberger, H. ; Unger, K.

A genomic copy number signature predicts radiation exposure in post-Chernobyl breast cancer.

Breast cancer is the second leading cause of cancer death among women worldwide and besides life style, age and genetic risk factors, exposure to ionizing radiation is known to increase the risk for breast cancer. Further, DNA copy number alterations (CNAs), which can result from radiation-induced double-strand breaks, are frequently occurring in breast cancer cells. We set out to identify a signature of CNAs discriminating breast cancers from radiation-exposed and non-exposed female patients. We analyzed resected breast cancer tissues from 68 exposed female Chernobyl clean-up workers and evacuees and 68 matched non-exposed control patients for CNAs by array comparative genomic hybridization analysis (aCGH). Using a stepwise forward-backward selection approach a non-complex CNA signature, that is, less than ten features, was identified in the training data set, which could be subsequently validated in the validation data set (p value <0.05). The signature consisted of nine copy number regions located on chromosomal bands 7q11.22-11.23, 7q21.3, 16q24.3, 17q21.31, 20p11.23-11.21, 1p21.1, 2q35, 2q35, 6p22.2. The signature was independent of any clinical characteristics of the patients. In all, we identified a CNA signature that has the potential to allow identification of radiation-associated breast cancer at the individual level.

2018 Scientific Article in Scientific Reports Sci. Rep. 8:5975 (2018)

Treise, I. ; Huber, E.M. ; Klein-Rodewald, T. ; Heinemeyer, W. ; Grassmann, S.A. ; Basler, M. ; Adler, T. ; Rathkolb, B. ; Helming, L. ; Andres, C. ; Klaften, M. ; Landbrecht, C. ; Wieland, T. ; Strom, T.M. ; McCoy, K.D. ; Macpherson, A.J. ; Wolf, E. ; Groettrup, M. ; Ollert, M. ; Neff, F. ; Gailus-Durner, V. ; Fuchs, H. ; Hrabě de Angelis, M. ; Groll, M.&deg ; Busch, D.H.&deg

Defective immuno- and thymoproteasome assembly causes severe immunodeficiency.

By N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated the mutant mouse line TUB6 that is characterised by severe combined immunodeficiency (SCID) and systemic sterile autoinflammation in homozygotes, and a selective T cell defect in heterozygotes. The causative missense point mutation results in the single amino acid exchange G170W in multicatalytic endopeptidase complex subunit-1 (MECL-1), the β2i-subunit of the immuno- and thymoproteasome. Yeast mutagenesis and crystallographic data suggest that the severe TUB6-phenotype compared to the MECL-1 knockout mouse is caused by structural changes in the C-terminal appendage of β2i that prevent the biogenesis of immuno- and thymoproteasomes. Proteasomes are essential for cell survival, and defective proteasome assembly causes selective death of cells expressing the mutant MECL-1, leading to the severe immunological phenotype. In contrast to the immunosubunits β1i (LMP2) and β5i (LMP7), mutations in the gene encoding MECL-1 have not yet been assigned to human disorders. The TUB6 mutant mouse line exemplifies the involvement of MECL-1 in immunopathogenesis and provides the first mouse model for primary immuno- and thymoproteasome-associated immunodeficiency that may also be relevant in humans.

2018 Scientific Article in Proceedings of the National Academy of Sciences of the United States of America Proc. Natl. Acad. Sci. U.S.A. 115, E2348-E2357 (2018)

Xie, K. ; Ryan, D.P. ; Pearson, B.L. ; Henzel, K.S. ; Neff, F. ; Vidal, R.O. ; Hennion, M. ; Lehmann, I. ; Schleif, M. ; Schröder, S. ; Adler, T. ; Rathkolb, B. ; Rozman, J. ; Schütz, A.L. ; Prehn, C. ; Mickael, M.E. ; Weiergräber, M. ; Adamski, J. ; Busch, D.H. ; Ehninger, G. ; Matynia, A. ; Jackson, W.S. ; Wolf, E. ; Fuchs, H. ; Gailus-Durner, V. ; Bonn, S. ; Hrabě de Angelis, M. ; Ehninger, D.

Epigenetic alterations in longevity regulators, reduced life span, and exacerbated aging-related pathology in old father offspring mice.

Advanced age is not only a major risk factor for a range of disorders within an aging individual but may also enhance susceptibility for disease in the next generation. In humans, advanced paternal age has been associated with increased risk for a number of diseases. Experiments in rodent models have provided initial evidence that paternal age can influence behavioral traits in offspring animals, but the overall scope and extent of paternal age effects on health and disease across the life span remain underexplored. Here, we report that old father offspring mice showed a reduced life span and an exacerbated development of aging traits compared with young father offspring mice. Genome-wide epigenetic analyses of sperm from aging males and old father offspring tissue identified differentially methylated promoters, enriched for genes involved in the regulation of evolutionarily conserved longevity pathways. Gene expression analyses, biochemical experiments, and functional studies revealed evidence for an overactive mTORC1 signaling pathway in old father offspring mice. Pharmacological mTOR inhibition during the course of normal aging ameliorated many of the aging traits that were exacerbated in old father offspring mice. These findings raise the possibility that inherited alterations in longevity pathways contribute to intergenerational effects of aging in old father offspring mice.

2018 Scientific Article in Scientific Reports Sci. Rep. 8:1116 (2018)

Frankó, A.&deg ; Rodriguez Camargo, D.C. ; Böddrich, A. ; Garg, D. ; Rodriguez Camargo, A. ; Rathkolb, B. ; Janik, D. ; Aichler, M. ; Feuchtinger, A. ; Neff, F. ; Fuchs, H. ; Wanker, E.E. ; Reif, B. ; Häring, H.-U. ; Peter, A. ; Hrabě de Angelis, M.&deg

Epigallocatechin gallate (EGCG) reduces the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) transgenic mice.

The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application.

2018 Scientific Article in Endocrinology Endocrinology 159, 1511-1524 (2018)

Sun, N. ; Wu, Y. ; Nanba, K. ; Sbiera, S. ; Kircher, S. ; Kunzke, T. ; Aichler, M. ; Berezowska, S. ; Reibetanz, J. ; Rainey, W.E. ; Fassnacht, M. ; Walch, A.K. ; Kroiss, M.

High resolution tissue mass spectrometry imaging reveals a refined functional anatomy of the human adult adrenal gland.

It is undeniably one of the greatest findings in biology that (with some very minor exceptions) every cell in the body possesses the whole genetic information needed to generate a complete individual. Today, this concept has been so thoroughly assimilated that we struggle to still see how surprising this finding actually was: all cellular phenotypes naturally occurring in one person are generated from genetic uniformity, and thus are per definition epigenetic. Transcriptional mechanisms are clearly critical for developing and protecting cell identities, because a mis-expression of few or even single genes can efficiently induce inappropriate cellular programmes. However, how transcriptional activities are molecularly controlled and which of the many known epigenomic features have causal roles remains unclear. Today, clarification of this issue is more pressing than ever because profiling efforts and epigenome-wide association studies (EWAS) continuously provide comprehensive datasets depicting epigenomic differences between tissues and disease states. In this commentary, we propagate the idea of a widespread follow-up use of epigenome editing technology in EWAS studies. This would enable them to address the questions of which features, where in the genome, and which circumstances are essential to shape development and trigger disease states.

2018 Scientific Article in Radiation and Environmental Biophysics Radiat. Environ. Biophys. 57, 99-113 (2018)

Dalke, C. ; Neff, F. ; Bains, S.K. ; Bright, S. ; Lord, D.J. ; Reitmeir, P. ; Rößler, U. ; Samaga, D. ; Unger, K. ; Braselmann, H. ; Wagner, F. ; Greiter, M. ; Gomolka, M. ; Hornhardt, S. ; Kunze, S. ; Kempf, S.J. ; Garrett, L. ; Hölter, S.M. ; Wurst, W. ; Rosemann, M. ; Azimzadeh, O. ; Tapio, S. ; Aubele, M. ; Theis, F.J. ; Hoeschen, C. ; Slijepcevic, P. ; Kadhim, M. ; Atkinson, M.J. ; Zitzelsberger, H. ; Kulka, U. ; Graw, J.

Lifetime study in mice after acute low-dose ionizing radiation: A multifactorial study with special focus on cataract risk.

Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.

2018 Scientific Article in International Journal of Oncology Int. J. Oncol. 52, 755-767 (2018)

Absmaier, M. ; Napieralski, R. ; Schuster, T. ; Aubele, M. ; Walch, A.K. ; Magdolen, V. ; Dorn, J. ; Gross, E. ; Harbeck, N. ; Noske, A. ; Kiechle, M. ; Schmitt, M.

PITX2 DNA-methylation predicts response to anthracycline-based adjuvant chemotherapy in triple-negative breast cancer patients.

Triple-negative breast cancer (TNBC) constitutes a heterogeneous breast cancer subgroup with poor prognosis; survival rates are likely to be lower with TNBC compared to other breast cancer subgroups. For this disease, systemic adjuvant chemotherapy regimens often yield suboptimal clinical results. To improve treatment regimens in TNBC, identification of molecular biomarkers may help to select patients for individualized adjuvant therapy. Evidence has accumulated that determination of the methylation status of the PITX2 gene provides a predictive value in various breast cancer subgroups, either treated with endocrine-based therapy or anthracycline-containing chemotherapy. To further explore the validity of this novel predictive candidate biomarker, in the present exploratory retrospective study, determination of the PITX2 DNA-methylation status was assessed for non-metastatic TNBC patients treated with adjuvant anthracycline-based chemotherapy by molecular analysis of breast cancer tissues. The PITX2 DNA-methylation status was determined in fresh-frozen tumor tissue specimens (n=56) by methylation-specific qRT-PCR (qMSP) and the data related to disease-free and overall survival, applying an optimized DNA-methylation score of 6.35%. For non-metastatic TNBC patients treated with adjuvant systemic anthracycline-based chemotherapy, a low PITX2 DNA-methylation status (<6.35) defines TNBC patients with poor disease-free and overall survival. Univariate and multivariate analyses demonstrate the statistically independent predictive value of PITX2 DNA-methylation. For non-metastatic TNBC patients, selective determination of the PITX2 DNA-methylation status may serve as a cancer biomarker for predicting response to anthracycline-based adjuvant chemotherapy. The assay based on methylation of the PIXT2 gene can be applied to frozen and routinely available formalin-fixed, paraffin-embedded (FFPE) breast cancer tumor tissues that will not only define those TNBC patients who may benefit from anthracycline-based chemotherapy but also those who should be spared the necessity of such potentially toxic treatment. Such patients should be allocated to alternative treatment options.

2018 Scientific Article in Cell Cell 172, 409–422.e21 (2018)

Ingold, I. ; Berndt, C. ; Schmitt, S. ; Doll, S. ; Poschmann, G. ; Buday, K. ; Roveri, A. ; Peng, X. ; Porto Freitas, F. ; Seibt, T. ; Mehr, L. ; Aichler, M. ; Walch, A.K. ; Lamp, D. ; Jastroch, M. ; Miyamoto, S. ; Wurst, W. ; Ursini, F. ; Arnér, E.S.J. ; Fradejas-Villar, N. ; Schweizer, U. ; Zischka, H. ; Friedmann Angeli, J.P.F. ; Conrad, M.

Selenium utilization by GPX4 is required to prevent hydroperoxide-induced ferroptosis.

Selenoproteins are rare proteins among all kingdoms of life containing the 21 st amino acid, selenocysteine. Selenocysteine resembles cysteine, differing only by the substitution of selenium for sulfur. Yet the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins also exist as cysteine-containing homologs. Here, we demonstrate that selenolate-based catalysis of the essential mammalian selenoprotein GPX4 is unexpectedly dispensable for normal embryogenesis. Yet the survival of a specific type of interneurons emerges to exclusively depend on selenocysteine-containing GPX4, thereby preventing fatal epileptic seizures. Mechanistically, selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation as cells expressing a cysteine variant are highly sensitive toward peroxide-induced ferroptosis. Remarkably, concomitant deletion of all selenoproteins in Gpx4 cys/cys cells revealed that selenoproteins are dispensable for cell viability provided partial GPX4 activity is retained. Conclusively, 200 years after its discovery, a specific and indispensable role for selenium is provided. The trace element selenium protects a critical population of interneurons from ferroptotic cell death.

2018 Scientific Article in Endocrine-Related Cancer Endocr. Relat. Cancer 25, 145-162 (2018)

Molatore, S. ; Kügler, A. ; Irmler, M. ; Wiedemann, T. ; Neff, F. ; Feuchtinger, A. ; Beckers, J. ; Robledo, M. ; Roncaroli, F. ; Pellegata, N.S.

Characterization of neuroendocrine tumors in heterozygous mutant MENX rats: A novel model of invasive medullary thyroid carcinoma.

Rats affected by the MENX syndrome spontaneously develop multiple neuroendocrine tumors (NETs) including adrenal, pituitary and thyroid gland neoplasms. MENX was initially reported to be inherited as a recessive trait and affected rats were found to be homozygous for the predisposing Cdkn1b mutation encoding p27. We here report that heterozygous MENX-mutant rats (p27+/mut) develop the same spectrum of NETs seen in the homozygous (p27mut/mut) animals but with slower progression. Consequently, p27+/mut rats have a significantly shorter lifespan compared with their wild-type (p27+/+) littermates. In the tumors of p27+/mut rats, the wild-type Cdkn1b allele is neither lost nor silenced, implying that p27 is haploinsufficient for tumor suppression in this model. Transcriptome profiling of rat adrenal (pheochromocytoma) and pituitary tumors having different p27 dosages revealed a tissue-specific, dose-dependent effect of p27 on gene expression. In p27+/mut rats, thyroid neoplasms progress to invasive and metastatic medullary thyroid carcinomas (MTCs) accompanied by increased calcitonin levels, as in humans. Comparison of expression signatures of late-stage vs early-stage MTCs from p27+/mut rats identified genes potentially involved in tumor aggressiveness. The expression of a subset of these genes was evaluated in human MTCs and found to be associated with aggressive RET-M918T-positive tumors. Altogether, p27 haploinsufficiency in MENX rats uncovered a novel, representative model of invasive and metastatic MTC exploitable for translational studies of this often aggressive and incurable cancer.

2018 Scientific Article in Laboratory Investigation Lab. Invest. 98, 141-149 (2018)

Aichler, M. ; Kunzke, T. ; Buck, A. ; Sun, N. ; Ackermann, M. ; Jonigk, D. ; Gaumann, A. ; Walch, A.K.

Molecular similarities and differences from human pulmonary fibrosis and corresponding mouse model: MALDI imaging mass spectrometry in comparative medicine.

© 2018 USCAP, Inc All rights reserved. Animal models can reproduce some model-specific aspects of human diseases, but some animal models translate poorly or fail to translate to the corresponding human disease. Here, we develop a strategy to systematically compare human and mouse tissues, and conduct a proof-of-concept experiment to identify molecular similarities and differences using patients with idiopathic pulmonary fibrosis and a bleomycin-induced fibrosis mouse model. Our novel approach employs high-throughput tissue microarrays (TMAs) of humans and mice, high-resolution matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance-mass spectrometry imaging (MALDI-FT-ICR-MSI) to spatially resolve mass spectra at the level of specific metabolites, and hierarchical clustering and pathway enrichment analysis to identify functionally similar/different molecular patterns and pathways in pathological lesions of humans and mice. We identified a large number of common molecules (n=1366) and fewer exclusive molecules in humans (n=83) and mice (n=54). Among the common molecules, the 'ascorbate and aldarate metabolism' pathway had the highest similarity in human and mouse lesions. This proof-of-concept study demonstrates that our novel strategy employing a reliable and easy-to-perform experimental design accurately identifies pathways and factors that can be directly compared between animal models and human diseases.

2018 Scientific Article in International Journal of Cancer Int. J. Cancer 142, 573-583 (2018)

Wilke, C. ; Hess-Rieger, J. ; Klymenko, S.V. ; Chumak, V.V. ; Zakhartseva, L.M. ; Bakhanova, E.V. ; Feuchtinger, A. ; Walch, A.K. ; Selmansberger, M. ; Braselmann, H. ; Schneider, L. ; Pitea, A. ; Steinhilber, J. ; Fend, F. ; Bösmüller, H.C. ; Zitzelsberger, H. ; Unger, K.

Expression of miRNA-26b-5p and its target TRPS1 is associated with radiation exposure in post-Chernobyl breast cancer.

Ionising radiation is a well-recognised risk factor for the development of breast cancer, however, it is unknown whether radiation-specific molecular oncogenic mechanisms exist. We investigated post-Chernobyl breast cancers from radiation-exposed female clean-up workers and non-exposed controls for molecular changes. Radiation-associated alterations identified in the discovery cohort (n=38) were subsequently validated in a second cohort (n=39). Increased expression of hsa-miR-26b-5p was associated with radiation exposure in both of the cohorts. Moreover, downregulation of the TRPS1 protein, which is a transcriptional target of hsa-miR-26b-5p was associated with radiation exposure. Since TRPS1 overexpression is common in sporadic breast cancer its observed downregulation in radiation-associated breast cancer warrants clarification of the specific functional role of TRPS1 in the radiation context. For this purpose, the impact of TRPS1 on the transcriptome was characterised in two radiation-transformed breast cell culture models after siRNA-knockdown. Deregulated genes upon TRPS1 knockdown were associated with DNA-repair, cell cycle, mitosis, cell migration, angiogenesis and EMT pathways. Furthermore, we identified the interaction partners of TRPS1 from the transcriptomic correlation networks derived from gene expression data on radiation-transformed breast cell culture models and sporadic breast cancer tissues provided by the TCGA database. The genes correlating with TRPS1 in the radiation-transformed breast cell lines were primarily linked to DNA damage response and chromosome segregation, whilst the transcriptional interaction partners in the sporadic breast cancers were mostly associated with apoptosis. Thus, upregulation of hsa-miR-26b-5p and downregulation of TRPS1 in radiation-associated breast cancer tissue samples suggests these molecules representing radiation markers in breast cancer.

2017 Scientific Article in Plant Journal, The Plant J. 93, 502-514 (2017)

Prade, V.M. ; Gundlach, H. ; Twardziok, S.O. ; Chapman, B. ; Tan, C.S. ; Langridge, P. ; Schulman, A.H. ; Stein, N. ; Waugh, R. ; Zhang, G. ; Platzer, M. ; Li, C. ; Spannagl, M. ; Mayer, K.F.X.

The pseudogenes of barley.

Pseudogenes have a reputation of being 'evolutionary relics' or 'junk DNA'. While they are well characterized in mammals, studies in more complex plant genomes have so far been hampered by the absence of reference genome sequences. Barley is one of the economically most important cereals and has a genome size of 5.1 Gb. With the first high-quality genome reference assembly available for a Triticeae crop, we conducted a whole-genome assessment of pseudogenes on the barley genome. We identified, characterized and classified 89 440 gene fragments and pseudogenes scattered along the chromosomes, with occasional hotspots and higher densities at the chromosome ends. Full-length pseudogenes (11 015) have preferentially retained their exon-intron structure. Retrotransposition of processed mRNAs only plays a marginal role in their creation. However, the distribution of retroposed pseudogenes reflects the Rabl configuration of barley chromosomes and thus hints at founding mechanisms. While parent genes related to the defense-response were found to be under-represented in cultivated barley, we detected several defense-related pseudogenes in wild barley accessions. The percentage of transcriptionally active pseudogenes is 7.2%, and these may potentially adopt new regulatory roles. The barley genome is rich in pseudogenes and small gene fragments mainly located towards chromosome tips or as tandemly repeated units. Our results indicate non-random duplication and pseudogenization preferences and improve our understanding of the dynamics of gene birth and death in large plant genomes and the mechanisms that lead to evolutionary innovations.

2017 Scientific Article in Science Science 358:eaan4368 (2017)

Klaeger, S. ; Heinzlmeir, S. ; Wilhelm, M. ; Polzer, H. ; Vick, B. ; Koenig, P.A. ; Reinecke, M. ; Ruprecht, B. ; Petzoldt, S. ; Meng, C. ; Zecha, J. ; Reiter, K. ; Qiao, H. ; Helm, D. ; Koch, H. ; Schoof, M. ; Canevari, G. ; Casale, E. ; Depaolini, S.R. ; Feuchtinger, A. ; Wu, Z. ; Schmidt, T. ; Rueckert, L. ; Becker, W. ; Huenges, J. ; Garz, A.K. ; Gohlke, B.O. ; Zolg, D.P. ; Kayser, G. ; Vooder, T. ; Preissner, R. ; Hahne, H. ; Tõnisson, N. ; Kramer, K. ; Götze, K. ; Bassermann, F. ; Schlegl, J. ; Ehrlich, H.C. ; Aiche, S. ; Walch, A.K. ; Greif, P.A. ; Schneider, S. ; Felder, E.R. ; Ruland, J. ; Médard, G. ; Jeremias, I. ; Spiekermann, K. ; Kuster, B.

The target landscape of clinical kinase drugs.

Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.

2017 Scientific Article in eLife eLife 6:e31226 (2017)

Rodriguez Camargo, D.C. ; Korshavn, K.J. ; Jussupow, A. ; Raltchev, K. ; Goricanec, D. ; Fleisch, M. ; Sarkar, R. ; Xue, K. ; Aichler, M. ; Mettenleiter, G. ; Walch, A.K. ; Camilloni, C. ; Hagn, F. ; Reif, B. ; Ramamoorthy, A.

Stabilization and structural analysis of a membrane-associated hIAPP aggregation intermediate.

Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique b-strand structure distinct from the conventional amyloid b-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.

2017 Scientific Article in Rapid Communications in Mass Spectrometry Rapid Commun. Mass Spectrom. 32, 159-166 (2017)

Desbenoit, N. ; Walch, A.K. ; Spengler, B. ; Brunelle, A. ; Römpp, A.

Correlative mass spectrometry imaging, applying TOF-SIMS and AP-MALDI to a single tissue section.

RATIONALE: Mass spectrometry imaging (MSI) is a powerful tool for mapping the surface of a sample. Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) and Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization (AP-MALDI) offer complementary capabilities. Here, we present a workflow to apply both techniques to a single tissue section and combine the resulting data on the example of human colon cancer tissue. METHODS: Following cryo-sectioning, images were acquired using the high spatial resolution (1 μm pixel size) provided by TOF-SIMS. The same section was then coated with a para-nitroaniline matrix and images were acquired using AP-MALDI coupled to an Orbitrap mass spectrometer, offering high mass resolution, high mass accuracy and MS/MS capabilities. Datasets provided by both mass spectrometers were converted into the open and vendor-independent imzML file format and processed with the open-source software MSiReader. RESULTS: The TOF-SIMS and AP-MALDI-MS mass spectra show strong signals of fatty acids, cholesterol, phosphatidylcholine and sphingomyelin. We showed a high correlation between the fatty acid ions detected with TOF-SIMS in negative ion mode and the phosphatidylcholine ions detected with AP-MALDI in positive ion mode using a similar setting for visualization. Histological staining on the same section allowed the identification of the anatomical structures and their correlation with the ion images. CONCLUSIONS: This multimodal approach using two MSI platforms shows an excellent complementarity for the localization and identification of lipids. The spatial resolution of both systems is at or close to cellular dimensions and thus spatial correlation can only be obtained if the same tissue section is analyzed sequentitially. imzML-based data processing allows a real correlation of the imaging datasets provided by these two technologies and opens the way for a more complete molecular view of the anatomical structures of biological tissues.

2017 Scientific Article in OncoTarget Oncotarget 8, 78917-78929 (2017)

Jacobs, L. ; Habringer, S. ; Slawska, J. ; Huber, K. ; Hauf, E. ; Li, Z. ; Refaeli, Y. ; Schwaiger, M. ; Rudelius, M. ; Walch, A.K. ; Keller, U.

Functional imaging in combination with mutation status aids prediction of response to inhibiting B-cell receptor signaling in lymphoma.

Aberrant B-cell receptor (BCR) signaling is known to contribute to malignant transformation. Two small molecule inhibitors targeting BCR pathway signaling include ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, and idelalisib, a specific Phosphatidylinositol-4,5-bisphosphate 3-kinase delta (PI3Kd) inhibitor, both of which have been approved for use in haematological malignancies. Despite the identification of various diffuse large B-cell lymphoma (DLBCL) subtypes, mutation status alone is not sufficient to predict patient response and therapeutic resistance can arise. Herein we apply early molecular imaging across alternative activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL subtypes to investigate the effects of BCR pathway inhibition. Treatment with both inhibitors adversely affected cell growth and viability. These effects were partially predictable based upon mutation status. Accordingly, very early 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography ( 18 F-FDG-PET) and 3'-deoxy-3'[18F]-fluorothymidine positron emission tomography ( 18 F-FLT-PET) reported tumour regression and reductions in tumour metabolism and proliferation upon treatment. Furthermore, matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) identified alterations in the proteome of a model of ABC DLBCL upon treatment with ibrutinib or idelalisib. In conclusion we demonstrate that very early molecular imaging adds predictive value in addition to mutational status of DLBCL that may be useful in directing patient therapy.

2017 Scientific Article in Biochimica et Biophysica Acta Biochim. Biophys. Acta 1862, 51-60 (2017)

Urban, C. ; Buck, A. ; Siveke, J.T. ; Lordick, F. ; Luber, B. ; Walch, A.K.&deg ; Aichler, M.&deg

PAXgene fixation enables comprehensive metabolomic and proteomic analyses of tissue specimens by MALDI MSI.

An alcohol-based non-crosslinking tissue fixative, PAXgene Tissue System, has been proposed as alternative fixation method to formalin, providing superior and morphological preservation. To date, metabolites have not been assessed in PAXgene-fixed tissues. The study focuses on a comparison between PAXgene and standard formalin fixation for metabolomic analysis by MALDI mass spectrometry imaging. Therefore, fifty-six samples from seven mice organs were fixed with PAXgene (PFPE) or formalin (FFPE), embedded in paraffin, and processed to a tissue microarray. PAXgene was able to spatially preserve metabolites in organs achieving an overlap of common metabolites ranging from 34 to 78% with FFPE. Highly similar signal intensities and visualization of molecules demonstrated negligible differences for metabolite imaging on PFPE compared to FFPE tissues. In addition, we performed proteomic analysis of intact proteins and peptides derived from enzymatic digestion. An overlap of 33 to 58% was found between FFPE and PFPE tissue samples in peptide analysis with a higher number of PFPE-specific peaks. Analysis of intact proteins achieved an overlap in the range of 0 to 28% owing to the poor detectability of cross-linked proteins in formalin-fixed tissues. Furthermore, metabolite and peptide profiles obtained from PFPE tissues were able to correctly classify organs independent of the fixation method, whereas a distinction of organs by protein profiles was only achieved by PAXgene fixation. Finally, we applied MALDI MSI to human biopsies by sequentially analyzing metabolites and peptides within the same tissue section. Concerning prospective studies, PAXgene can be used as an alternative fixative for multi-omic tissue analysis.

2017 Scientific Article in Journal of Pathology, The J. Pathol. 243, 481-495 (2017)

Fichter, C.D. ; Przypadlo, C.M. ; Buck, A. ; Herbener, N. ; Riedel, B. ; Schäfer, L. ; Nakagawa, H. ; Walch, A.K. ; Reinheckel, T. ; Werner, M. ; Lassmann, S.

A new model system identifies EGFR/HER2 and HER2/HER3 heterodimers as potent inducers of oesophageal epithelial cell invasion.

Oesophageal squamous cell carcinomas and oesophageal adenocarcinomas display distinct patterns of ErbB expression and dimers. The functional effects of specific ErbB homo- or heterodimers on oesophageal (cancer) cell behaviour, particularly invasion of early carcinogenesis remains unknown. Here, a new cellular model system for controlled activation of EGFR or HER2 and EGFR/HER2 or HER2/HER3 homo- and heterodimers was studied in non-neoplastic squamous oesophageal epithelial Het-1A cells. EGFR, HER2 and HER3 intracellular domains (ICDs) were fused to dimerization domains (DmrA / DmrA and DmrC), and transduced into Het-1A cells lacking ErbB expression. Dimerization of EGFR, HER2 or EGFR/HER2, HER2/HER3 ICDs was induced by synthetic ligands (A/A or A/C dimerizers). This was accompanied by phosphorylation of the respective EGFR, HER2 and HER3 ICDs and activation of distinct down-stream signalling pathways, such as PLCγ1, Akt, STAT and Src family kinases. Phenotypically, ErbB homo- and heterodimers caused cell rounding and non-apoptotic blebbing in EGFR/HER2 and HER2/HER3 heterodimer cells. In a Transwell assay, cell migration velocity was elevated in HER2-dimer as compared to empty vector cells. In addition, HER2-dimer cells showed in increased cell invasion, reaching significance for induced HER2/HER3 heterodimers (p=0.015). Importantly, in three-dimensional organotypic cultures, empty vector cells grew as a superficial cell layer, resembling oesophageal squamous epithelium. In contrast, induced HER2-dimer cells (HER2 homodimers) were highly invasive into the matrix and formed cell clusters. This was associated with partial loss of CK7 (when HER2 homodimers were modelled) and p63 (when EGFR/HER2 heterodimers were modelled), which suggests a change or loss of squamous cell differentiation. Controlled activation of specific EGFR, HER2 and HER3 homo- and heterodimers caused oesophageal squamous epithelial cell migration and/or invasion, especially in a three dimensional microenvironment, thereby functionally identifying ErbB homo- and heterodimers as important drivers of oesophageal carcinogenesis.

2017 Scientific Article in Journal of Biomedical Science J. Biomed. Sci. 24:57 (2017)

Kumar, S. ; Rathkolb, B. ; Sabrautzki, S. ; Krebs, S. ; Kemter, E. ; Becker, L. ; Beckers, J. ; Bekeredjian, R. ; Brommage, R. ; Calzada-Wack, J. ; Garrett, L. ; Hölter, S.M. ; Horsch, M. ; Klingenspor, M. ; Klopstock, T. ; Moreth, K. ; Neff, F. ; Rozman, J. ; Fuchs, H. ; Gailus-Durner, V. ; Hrabě de Angelis, M. ; Wolf, E. ; Aigner, B.

Standardized, systemic phenotypic analysis reveals kidney dysfunction as main alteration of Kctd1I27N mutant mice.

BACKGROUND: Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically dominant mutant line HST014 was established and further analyzed. METHODS: Analysis of the causative mutation as well as the standardized, systemic phenotypic analysis of the mutant line was carried out. RESULTS: The causative mutation was detected in the potassium channel tetramerization domain containing 1 (Kctd1) gene which leads to the amino acid exchange Kctd1 (I27N) thereby affecting the functional BTB domain of the protein. This line is the first mouse model harboring a Kctd1 mutation. Kctd1 (I27N) homozygous mutant mice die perinatally. Standardized, systemic phenotypic analysis of Kctd1 (I27N) heterozygous mutants was carried out in the German Mouse Clinic (GMC). Systematic morphological investigation of the external physical appearance did not detect the specific alterations that are described in KCTD1 mutant human patients affected by the scalp-ear-nipple (SEN) syndrome. The main pathological phenotype of the Kctd1 (I27N) heterozygous mutant mice consists of kidney dysfunction and secondary effects thereof, without gross additional primary alterations in the other phenotypic parameters analyzed. Genome-wide transcriptome profiling analysis at the age of 4 months revealed about 100 differentially expressed genes (DEGs) in kidneys of Kctd1 (I27N) heterozygous mutants as compared to wild-type controls. CONCLUSIONS: In summary, the main alteration of the Kctd1 (I27N) heterozygous mutants consists in kidney dysfunction. Additional analyses in 9-21 week-old heterozygous mutants revealed only few minor effects.

2017 Scientific Article in Proceedings of SPIE Proc. SPIE 10411:104110F (2017)

Koch, M. ; de Jong, J.S. ; Glatz, J. ; Symvoulidis, P. ; Lamberts, L.E. ; Adams, A.L.L. ; Kranendonk, M.E.G. ; van Scheltinga, A.T. ; Aichler, M. ; Jansen, L. ; de Vries, J. ; Lub-de Hooge, M. ; Schröder, C.P. ; Jorritsma-Smit, A. ; Linssen, M.D. ; de Boer, E. ; van der Vegt, B. ; Nagengast, W.B. ; Elias, S.G. ; Oliveira, S. ; Witkamp, A.J. ; Mali, W.P.Th.M. ; van der Wall, E. ; Garcia-Allende, P. ; van Diest, P.J. ; de Vries, E.G. ; Walch, A.K. ; van Dam, G.M. ; Ntziachristos, V.

Fluorescently labeled bevacizumab in human breast cancer: Defining the classification threshold.

In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging.

2017 Scientific Article in American Journal of Pathology, The Am. J. Pathol. 187, 2570-2589 (2017)

Schlecht, A. ; Leimbeck, S.V. ; Jägle, H. ; Feuchtinger, A. ; Tamm, E.R. ; Braunger, B.M.

Deletion of endothelial TGF-β signaling leads to choroidal neovascularization.

The molecular pathogenesis of choroidal neovascularization (CNV), an angiogenic process that critically contributes to vision loss in age-related macular degeneration (AMD) is unclear. Here we analyzed the role of transforming growth factor (TGF)-β signaling for CNV formation by generating a series of mutant mouse models with induced conditional deletion of TGF-β signaling in the entire eye, the retinal pigment epithelium (RPE) or the vascular endothelium. Deletion of TGF-β signaling in the eye caused CNV, irrespectively if it was ablated in newborn or three-week-old mice. Areas of CNV showed photoreceptor degeneration, multilayered RPE, basal lamina deposits and accumulations of monocytes/macrophages. The changes progressed leading to marked structural and functional alterations of the retina. While the specific deletion of TGF-β signaling in the RPE caused no obvious changes, specific deletion in vascular endothelial cells caused CNV and a phenotype quite similar to that observed after the deletion in the entire eye. We conclude that impairment of TGF-β signaling in the vascular endothelium of the eye is sufficient to trigger CNV formation. Our findings highlight the importance of TGF-β signaling as key player in the development of ocular neovascularization and indicate a fundamental role of TGF-β signaling in the pathogenesis of AMD.

2017 Scientific Article in Biological Chemistry Biol. Chem. 398, 1151-1164 (2017)

Yang, F. ; Aubele, M. ; Walch, A.K. ; Gross, E. ; Napieralski, R. ; Zhao, S. ; Ahmed, N. ; Kiechle, M. ; Reuning, U. ; Dorn, J. ; Sweep, F.C. ; Magdolen, V. ; Schmitt, M.

Tissue kallikrein-related peptidase 4 (KLK4), a novel biomarker in triple-negative breast cancer.

Triple-negative breast cancer (TNBC), lacking the steroid hormone receptors ER and PR and the oncoprotein HER2, is characterized by its aggressive pattern and insensitivity to endocrine and HER2-directed therapy. Human kallikrein-related peptidases KLK1-15 provide a rich source of serine protease-type biomarkers associated with tumor growth and cancer progression for a variety of malignant diseases. In this study, recombinant KLK4 protein was generated and affinity-purified KLK4-directed polyclonal antibody pAb587 established to allow localization of KLK4 protein expression in tumor cell lines and archived formalin-fixed, paraffin-embedded TNBC tumor tissue specimens. For this, KLK4 protein expression was assessed by immunohistochemistry in primary tumor tissue sections (tissue microarrays) of 188 TNBC patients, mainly treated with anthracycline- or CMF-based polychemotherapy. KLK4 protein is localized in the cytoplasm of tumor and stroma cells. In this patient cohort, elevated stroma cell KLK4 expression, but not tumor cell KLK4 expression, is predictive for poor disease-free survival by univariate analysis (hazard ratio: 2.26, p=0.001) and multivariable analysis (hazard ratio: 2.12, p<0.01). Likewise, univariate analysis revealed a trend for statistical significance of elevated KLK4 stroma cell expression for overall survival of TNBC patients as well.

2017 Scientific Article in OncoTarget Oncotarget 8, 68012-68025 (2017)

Kunzke, T. ; Balluff, B. ; Feuchtinger, A. ; Buck, A. ; Langer, R. ; Luber, B. ; Lordick, F. ; Zitzelsberger, H. ; Aichler, M. ; Walch, A.K.

Native glycan fragments detected by MALDI-FT-ICR mass spectrometry imaging impact gastric cancer biology and patient outcome.

Glycosylation in cancer is a highly dynamic process that has a significant impact on tumor biology. Further, the attachment of aberrant glycan forms is already considered a hallmark of the disease state. Mass spectrometry has become a prominent approach to analyzing glycoconjugates. Specifically, matrix-assisted laser desorption/ionisation -mass spectrometric imaging (MALDI-MSI) is a powerful technique that combines mass spectrometry with histology and enables the spatially resolved and label-free detection of glycans. The most common approach to the analysis of glycans is the use of mass spectrometry adjunct to PNGase F digestion and other chemical reactions. In the current study, we perform the analysis of formalin-fixed, paraffin-embedded (FFPE) tissues for natively occurring bioactive glycan fragments without prior digestion or chemical reactions using MALDI-FT-ICR-MSI. We examined 106 primary resected gastric cancer patient tissues in a tissue microarray and correlated native-occurring fragments with clinical endpoints, therapeutic targets such as epidermal growth factor receptor (EGFR) and HER2/neu expressions and the proliferation marker MIB1. The detection of a glycosaminoglycan fragment in tumor stroma regions was determined to be an independent prognostic factor for gastric cancer patients. Native glycan fragments were significantly linked to the expression of EGFR, HER2/neu and MIB1. In conclusion, we are the first to report the in situ detection of native-occurring bioactive glycan fragments in FFPE tissues that influence patient outcomes. These findings highlight the significance of glycan fragments in gastric cancer tumor biology and patient outcome.

2017 Meeting abstract in Haematologica - The Hematology Journal Haematologica 102, 333-333 (2017)

Altamura, S. ; Vegi, N. ; Hueltner, L. ; Schneider, M. ; Höppe, P. ; Schroeder, T. ; Canli, Ö. ; Greten, F.R. ; Aichler, M. ; Walch, A.K. ; Neff, F. ; Janik, D. ; Kuklik-Roos, C. ; Ladinig, C. ; Mysliwietz, J. ; Rathkolb, B. ; Buske, C. ; Conrad, M. ; Muckenthaler, M.U. ; Bornkamm, G.W.

Lack of the ferroptosis inhibitor GPX4 in erythroid cells causes a block in reticulocyte maturation and a hypoxic signature with impaired hepcidin regulation.

2017 Scientific Article in Nature Communications Nat. Commun. 8:15126 (2017)

Düwel, S. ; Hundshammer, C. ; Gersch, M. ; Feuerecker, B. ; Steiger, K. ; Buck, A. ; Walch, A.K. ; Haase, A. ; Glaser, S.J. ; Schwaiger, M. ; Schilling, F.

Imaging of pH in vivo using hyperpolarized 13C-labelled zymonic acid.

Natural pH regulatory mechanisms can be overruled during several pathologies such as cancer, inflammation and ischaemia, leading to local pH changes in the human body. Here we demonstrate that (13)C-labelled zymonic acid (ZA) can be used as hyperpolarized magnetic resonance pH imaging sensor. ZA is synthesized from [1-(13)C]pyruvic acid and its (13)C resonance frequencies shift up to 3.0 p.p.m. per pH unit in the physiological pH range. The long lifetime of the hyperpolarized signal enhancement enables monitoring of pH, independent of concentration, temperature, ionic strength and protein concentration. We show in vivo pH maps within rat kidneys and subcutaneously inoculated tumours derived from a mammary adenocarcinoma cell line and characterize ZA as non-toxic compound predominantly present in the extracellular space. We suggest that ZA represents a reliable and non-invasive extracellular imaging sensor to localize and quantify pH, with the potential to improve understanding, diagnosis and therapy of diseases characterized by aberrant acid-base balance.

2017 Scientific Article in Journal of Trace Elements in Medicine and Biology J. Trace Elem. Med. Biol. 44, 71-75 (2017)

Hachmöller, O. ; Aichler, M. ; Schwamborn, K. ; Lutz, L. ; Werner, M. ; Sperling, M. ; Walch, A.K. ; Karst, U.

Investigating the influence of standard staining procedures on the copper distribution and concentration in Wilson's disease liver samples by laser ablation-inductively coupled plasma-mass spectrometry.

The influence of rhodanine and haematoxylin and eosin (HE) staining on the copper distribution and concentration in liver needle biopsy samples originating from patients with Wilson's disease (WD), a rare autosomal recessive inherited disorder of the copper metabolism, is investigated. In contemporary diagnostic of WD, rhodanine staining is used for histopathology, since rhodanine and copper are forming a red to orange-red complex, which can be recognized in the liver tissue using a microscope. In this paper, a laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method is applied for the analysis of eight different WD liver samples. Apart from a spatially resolved elemental detection as qualitative information, this LA-ICP-MS method offers also quantitative information by external calibration with matrix-matched gelatine standards. The sample set of this work included an unstained and a rhodanine stained section of each WD liver sample. While unstained sections of WD liver samples showed very distinct structures of the copper distribution with high copper concentrations, rhodanine stained sections revealed a blurred copper distribution with significant decreased concentrations in a range from 20 to more than 90%. This implies a copper removal from the liver tissue by complexation during the rhodanine staining. In contrast to this, a further HE stained sample of one WD liver sample did not show a significant decrease in the copper concentration and influence on the copper distribution in comparison to the unstained section. Therefore, HE staining can be combined with the analysis by means of LA-ICP-MS in two successive steps from one thin section of a biopsy specimen. This allows further information to be gained on the elemental distribution by LA-ICP-MS additional to results obtained by histological staining.

2017 Scientific Article in Cell Metabolism Cell Metab. 25, 1334-1347.e4 (2017)

Aichler, M. ; Borgmann, D.M. ; Krumsiek, J. ; Buck, A. ; MacDonald, P.E. ; Fox, J.E.M. ; Lyon, J. ; Light, P.E. ; Keipert, S. ; Jastroch, M. ; Feuchtinger, A. ; Müller, N.S. ; Sun, N. ; Palmer, A. ; Alexandrov, T. ; Hrabě de Angelis, M. ; Neschen, S. ; Tschöp, M.H. ; Walch, A.K.

N-acyl taurines and acylcarnitines cause an imbalance in insulin synthesis and secretion provoking β cell dysfunction in type 2 diabetes.

The processes contributing to β cell dysfunction in type 2 diabetes (T2D) are uncertain, largely because it is difficult to access β cells in their intact immediate environment. We examined the pathophysiology of β cells under T2D progression directly in pancreatic tissues. We used MALDI imaging of Langerhans islets (LHIs) within mouse tissues or from human tissues to generate in situ-omics data, which we supported with in vitro experiments. Molecular interaction networks provided information on functional pathways and molecules. We found that stearoylcarnitine accumulated in β cells, leading to arrest of insulin synthesis and energy deficiency via excessive β-oxidation and depletion of TCA cycle and oxidative phosphorylation metabolites. Acetylcarnitine and an accumulation of N-acyl taurines, a group not previously detected in β cells, provoked insulin secretion. Thus, β cell dysfunction results from enhanced insulin secretion combined with an arrest of insulin synthesis.

2017 Scientific Article in Scientific Reports Sci. Rep. 7:2397 (2017)

Kaklamanos, A. ; Rozman, J. ; Roulis, M. ; Karagianni, N. ; Armaka, M. ; Wu, M. ; Brachthäuser, L. ; Calzada-Wack, J. ; Horsch, M. ; Beckers, J. ; Rathkolb, B. ; Adler, T. ; Neff, F. ; Wolf, E. ; Gailus-Durner, V. ; Fuchs, H. ; Hrabě de Angelis, M. ; Kollias, G.

Extensive phenotypic characterization of a new transgenic mouse reveals pleiotropic perturbations in physiology due to mesenchymal hGH minigene expression.

The human growth hormone (hGH) minigene used for transgene stabilization in mice has been recently identified to be locally expressed in the tissues where transgenes are active and associated with phenotypic alterations. Here we extend these findings by analyzing the effect of the hGH minigene in TgC6hp55 transgenic mice which express the human TNFR1 under the control of the mesenchymal cell-specific CollagenVI promoter. These mice displayed a fully penetrant phenotype characterized by growth enhancement accompanied by perturbations in metabolic, skeletal, histological and other physiological parameters. Notably, this phenotype was independent of TNF-TNFR1 signaling since the genetic ablation of either Tnf or Tradd did not rescue the phenotype. Further analyses showed that the hGH minigene was expressed in several tissues, also leading to increased hGH protein levels in the serum. Pharmacological blockade of GH signaling prevented the development of the phenotype. Our results indicate that the unplanned expression of the hGH minigene in CollagenVI expressing mesenchymal cells can lead through local and/or systemic mechanisms to enhanced somatic growth followed by a plethora of primary and/or secondary effects such as hyperphagia, hypermetabolism, disturbed glucose homeostasis, altered hematological parameters, increased bone formation and lipid accumulation in metabolically critical tissues.

2017 Scientific Article in Molecular Psychiatry Mol. Psychiatry 23, 1345-1355 (2017)

Ryan, D.P. ; Henzel, K.S. ; Pearson, B.L. ; Siwek, M.E. ; Papazoglou, A. ; Guo, L. ; Paesler, K. ; Yu, M. ; Müller, R. ; Xie, K. ; Schröder, S. ; Becker, L. ; Garrett, L. ; Hölter, S.M. ; Neff, F. ; Rácz, I. ; Rathkolb, B. ; Rozman, J. ; Ehninger, G. ; Klingenspor, M. ; Klopstock, T. ; Wolf, E. ; Wurst, W. ; Zimmer, A.D. ; Fuchs, H. ; Gailus-Durner, V. ; Hrabě de Angelis, M. ; Sidiropoulou, K. ; Weiergräber, M. ; Zhou, Y. ; Ehninger, D.

A paternal methyl donor-rich diet altered cognitive and neural functions in offspring mice.

Dietary intake of methyl donors, such as folic acid and methionine, shows considerable intra-individual variation in human populations. While it is recognized that maternal departures from the optimum of dietary methyl donor intake can increase the risk for mental health issues and neurological disorders in offspring, it has not been explored whether paternal dietary methyl donor intake influences behavioral and cognitive functions in the next generation. Here, we report that elevated paternal dietary methyl donor intake in a mouse model, transiently applied prior to mating, resulted in offspring animals (methyl donor-rich diet (MD) F1 mice) with deficits in hippocampus-dependent learning and memory, impaired hippocampal synaptic plasticity and reduced hippocampal theta oscillations. Gene expression analyses revealed altered expression of the methionine adenosyltransferase Mat2a and BK channel subunit Kcnmb2, which was associated with changes in Kcnmb2 promoter methylation in MD F1 mice. Hippocampal overexpression of Kcnmb2 in MD F1 mice ameliorated altered spatial learning and memory, supporting a role of this BK channel subunit in the MD F1 behavioral phenotype. Behavioral and gene expression changes did not extend into the F2 offspring generation. Together, our data indicate that paternal dietary factors influence cognitive and neural functions in the offspring generation.Molecular Psychiatry advance online publication, 4 April 2017; doi:10.1038/mp.2017.53.

2017 Scientific Article in International Journal of Cancer Int. J. Cancer 141, 816-828 (2017)

Smida, J.&deg ; Xu, H. ; Zhang, Y. ; Baumhoer, D. ; Ribi, S. ; Kovac, M. ; von Luettichau, I. ; Bielack, S. ; O'Leary, V.B. ; Leib-Mösch, C. ; Frishman, D.&deg ; Nathrath, M.

Genome-wide analysis of somatic copy number alterations and chromosomal breakages in osteosarcoma.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. It is characterized by highly complex karyotypes with structural and numerical chromosomal alterations. The observed OS-specific characteristics in localization and frequencies of chromosomal breakages strongly implicate a specific set of responsible driver genes or a specific mechanism of fragility induction. In this study, a comprehensive assessment of somatic copy number alterations (SCNAs) was performed in 160 OS samples using whole-genome CytoScan High Density arrays (Affymetrix, Santa Clara, CA). Genes or regions frequently targeted by SCNAs were identified. Breakage analysis revealed OS specific unstable regions in which well-known OS tumor suppressor genes, including TP53, RB1, WWOX, DLG2, and LSAMP are located. Certain genomic features, such as transposable elements and non-B DNA-forming motifs were found to be significantly enriched in the vicinity of chromosomal breakage sites. A complex breakage pattern - chromothripsis - has been suggested as a widespread phenomenon in OS. It was further demonstrated that hyperploidy and in particular chromothripsis were strongly correlated with OS patient clinical outcome. The revealed OS-specific fragility pattern provides novel clues for understanding the biology of osteosarcoma.

2017 Scientific Article in Molecular Metabolism Mol. Metab. 6, 440-446 (2017)

Jall, S. ; Sachs, S. ; Clemmensen, C. ; Finan, B. ; Neff, F. ; DiMarchi, R.D. ; Tschöp, M.H. ; Müller, T.D.&deg ; Hofmann, S.M.&deg

Monomeric GLP-1/GIP/glucagon triagonism corrects obesity, hepatosteatosis, and dyslipidemia in female mice.

Objective: Obesity is a major health threat that affects men and women equally. Despite this fact, weight-loss potential of pharmacotherapies is typically first evaluated in male mouse models of diet-induced obesity (DIO). To address this disparity we herein determined whether a monomeric peptide with agonism at the receptors for glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon is equally efficient in correcting DIO, dyslipidemia, and glucose metabolism in DIO female mice as it has been previously established for DIO male mice. Methods: Female C57BL/6J mice and a cohort of fatmass-matched C57BL/6J male mice were treated for 27 days via subcutaneous injections with either the GLP-1/GIP/glucagon triagonist or PBS. A second cohort of C57BL/6J male mice was included to match the females in the duration of the high-fat, high-sugar diet (HFD) exposure. Results: Our results show that GLP-1/GIP/glucagon triple agonism inhibits food intake and decreases body weight and body fat mass with comparable potency in male and female mice that have been matched for body fat mass. Treatment improved dyslipidemia in both sexes and reversed diet-induced steatohepatitis to a larger extent in female mice compared to male mice. Conclusions: We herein show that a recently developed unimolecular peptide triagonist is equally efficient in both sexes, suggesting that this polypharmaceutical strategy might be a relevant alternative to bariatric surgery for the treatment of obesity and related metabolic disorders.

2017 Scientific Article in JCI insight JCI insight 2:93136 (2017)

Arlt, W. ; Lang, K. ; Sitch, A.J. ; Dietz, A.S. ; Rhayem, Y. ; Bancos, I. ; Feuchtinger, A. ; Chortis, V. ; Gilligan, L.C. ; Ludwig, P. ; Riester, A. ; Asbach, E. ; Hughes, B.A. ; O'Neil, D.M. ; Bidlingmaier, M. ; Tomlinson, J.W. ; Hassan-Smith, Z.K. ; Rees, D.A. ; Adolf, C. ; Hahner, S. ; Quinkler, M. ; Dekkers, T. ; Deinum, J. ; Biehl, M. ; Keevil, B.G. ; Shackleton, C.H.L. ; Deeks, J.J. ; Walch, A.K. ; Beuschlein, F.&deg ; Reincke, M.&deg

Steroid metabolome analysis reveals prevalent glucocorticoid excess in primary aldosteronism.

BACKGROUND: Adrenal aldosterone excess is the most common cause of secondary hypertension and is associated with increased cardiovascular morbidity. However, adverse metabolic risk in primary aldosteronism extends beyond hypertension, with increased rates of insulin resistance, type 2 diabetes, and osteoporosis, which cannot be easily explained by aldosterone excess. METHODS: We performed mass spectrometry-based analysis of a 24-hour urine steroid metabolome in 174 newly diagnosed patients with primary aldosteronism (103 unilateral adenomas, 71 bilateral adrenal hyperplasias) in comparison to 162 healthy controls, 56 patients with endocrine inactive adrenal adenoma, 104 patients with mild subclinical, and 47 with clinically overt adrenal cortisol excess. We also analyzed the expression of cortisol-producing CYP11B1 and aldosterone-producing CYP11B2 enzymes in adenoma tissue from 57 patients with aldosterone-producing adenoma, employing immunohistochemistry with digital image analysis. RESULTS: Primary aldosteronism patients had significantly increased cortisol and total glucocorticoid metabolite excretion (all P < 0.001), only exceeded by glucocorticoid output in patients with clinically overt adrenal Cushing syndrome. Several surrogate parameters of metabolic risk correlated significantly with glucocorticoid but not mineralocorticoid output. Intratumoral CYP11B1 expression was significantly associated with the corresponding in vivo glucocorticoid excretion. Unilateral adrenalectomy resolved both mineralocorticoid and glucocorticoid excess. Postoperative evidence of adrenal insufficiency was found in 13 (29%) of 45 consecutively tested patients. CONCLUSION: Our data indicate that glucocorticoid cosecretion is frequently found in primary aldosteronism and contributes to associated metabolic risk. Mineralocorticoid receptor antagonist therapy alone may not be sufficient to counteract adverse metabolic risk in medically treated patients with primary aldosteronism. FUNDING: Medical Research Council UK, Wellcome Trust, European Commission.

2017 Scientific Article in Molecular Oncology Mol. Oncol. 11, 1288-1301 (2017)

von Heyking, K. ; Calzada-Wack, J. ; Göllner, S. ; Neff, F. ; Schmidt, O. ; Hensel, T. ; Schirmer, D. ; Fasan, A. ; Esposito, I. ; Müller-Tidow, C. ; Sorensen, P.H.B. ; Burdach, S. ; Richter, G.H.

The endochondral bone protein CHM1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma.

Ewing sarcomas (ES) are highly malignant, osteolytic bone or soft tissue tumors, which are characterized by EWS-ETS translocations and early metastasis to lung and bone. In this study, we investigated the role of the BRICHOS chaperone domain-containing endochondral bone protein chondromodulin I (CHM1) in ES pathogenesis. CHM1 is significantly over-expressed in ES, and chromosome immunoprecipitation (ChIP) data demonstrate CHM1 to be directly bound by an EWS-ETS translocation, EWS-FLI1. Using RNA interference we observed that CHM1 promoted chondrogenic differentiation capacity of ES cells but decreased the expression of osteolytic genes such as HIF1A, IL6, JAG1 and VEGF. This was in line with the induction of the number of tartrate-resistant acid phosphatase (TRAP(+) ) stained osteoclasts in an orthotopic model of local tumor growth after CHM1 knock down, indicating that CHM1-mediated inhibition of osteomimicry might play a role in homing, colonization and invasion into bone tissues. We further demonstrate that CHM1 enhanced the invasive potential of ES cells in vitro. This invasiveness was in part mediated via CHM1-regulated MMP9 expression and correlated with the observation that, in an xenograft mouse model, CHM1 was essential for the establishment of lung metastases. This finding is in line with the observed increase in CHM1 expression in patient specimens with ES lung metastases. Our results suggest that CHM1 seems to have pleiotropic functions in ES, that need to be further investigated, but appears to be essential for the invasive and metastatic capacities of ES.

2017 Scientific Article in American Journal of Respiratory Cell and Molecular Biology Am. J. Respir. Cell Mol. Biol. 57, 77-90 (2017)

Knüppel, L. ; Ishikawa, Y. ; Aichler, M. ; Heinzelmann, K. ; Hatz, R. ; Behr, J. ; Walch, A.K. ; Bächinger, H.P. ; Eickelberg, O. ; Staab-Weijnitz, C.A.

A novel antifibrotic mechanism of nintedanib and pirfenidone: Inhibition of collagen fibril assembly.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix, in particular collagens. Two IPF therapeutics, nintedanib and pirfenidone, decelerate lung function decline, but their underlying mechanisms of action are poorly understood. In this study we sought to analyze their effects on collagen synthesis and maturation at important regulatory levels. METHODS: Primary human fibroblasts from IPF patients and healthy donors were treated with nintedanib (0.01-1.0µM) or pirfenidone (0.1-1.0mM) in absence or presence of TGF-β1. Effects on collagen, fibronectin, FKBP10, HSP47 expression and collagen I and III secretion were analyzed by qPCR and Western Blot. Appearance of collagen fibrils was monitored by scanning electron microscopy (SEM) and kinetics of collagen fibril assembly was assessed in a light scattering approach. RESULTS: In IPF fibroblasts, nintedanib reduced the expression of collagen I, V, fibronectin and FKBP10 and attenuated secretion of collagen I and III. Pirfenidone also downregulated collagen V, but otherwise showed fewer and less pronounced effects. By and large, effects were similar in donor fibroblasts. For both drugs, electron microscopy of IPF fibroblast cultures revealed fewer and thinner collagen fibrils compared with untreated controls. Finally, both drugs dose-dependently delayed fibril formation of purified collagen I. CONCLUSIONS: Both drugs act on important regulatory levels in collagen synthesis and processing. Nintedanib was more effective in downregulating profibrotic gene expression and collagen secretion. Importantly, both drugs inhibited collagen I fibril formation and caused reduction and an altered appearance of collagen fibril bundles, representing a completely novel mechanism of action for both drugs.

2017 Scientific Article in Cancer Immunology, Immunotherapy Cancer Immunol. Immunother. 66, 777-786 (2017)

Dislich, B. ; Stein, A. ; Seiler, C.A. ; Kröll, D. ; Berezowska, S. ; Zlobec, I. ; Galvan, J.A. ; Slotta-Huspenina, J. ; Walch, A.K. ; Langer, R.

Expression patterns of programmed death-ligand 1 in esophageal adenocarcinomas: Comparison between primary tumors and metastases.

Expression analysis of programmed death-ligand 1 (PD-L1) may be helpful in guiding clinical decisions for immune checkpoint inhibition therapy, but testing by immunohistochemistry may be hampered by heterogeneous staining patterns within tumors and expression changes during metastatic course. PD-L1 expression (clone SP142) was investigated in esophageal adenocarcinomas using tissue microarrays (TMA) from 112 primary resected tumors, preoperative biopsies and full slide sections from a subset of these cases (n = 24), corresponding lymph node (n = 55) and distant metastases (n = 17). PD-L1 expression was scored as 0.1-1, >1, >5, >50% positive membranous staining of tumor cells and any positive staining of tumor-associated inflammatory infiltrates and/or stroma cells. There was a significant correlation with overall PD-L1 expression between the full slide sections and the TMA (p = 0.001), but not with the corresponding biopsies. PD-L1 expression in tumor cells >1% was detected in 8.0% of cases (9/112) and 51.8% of cases (58/112) in tumor-associated inflammatory infiltrates and/or stroma cells of primary tumors. Epithelial expression in metastases was found in 5.6% of cases (4/72) and immune cell expression in 18.1% of cases (13/72), but did not correlate with the expression pattern in the primary tumor. Overall PD-L1 expression in the primary tumor did not influence survival. However, PD-L1 expression was correlated with the number of CD3(+) tumor-infiltrating lymphocytes in the tumor center, and a combinational score of PD-L1 status/CD3(+) tumor-infiltrating lymphocytes was correlated with patients' overall survival.

2017 Scientific Article in PLoS ONE PLoS ONE 12:e0172788 (2017)

Echtler, K. ; Konrad, I. ; Lorenz, M. ; Schneider, S. ; Hofmaier, S. ; Plenagl, F. ; Stark, K. ; Czermak, T. ; Tirniceriu, A. ; Eichhorn, M.E. ; Walch, A.K. ; Enders, G. ; Massberg, S. ; Schulz, C.

Platelet GPIIb supports initial pulmonary retention but inhibits subsequent proliferation of melanoma cells during hematogenic metastasis.

Platelets modulate the process of cancer metastasis. However, current knowledge on the direct interaction of platelets and tumor cells is mostly based on findings obtained in vitro. We addressed the role of the platelet fibrinogen receptor glycoprotein IIb (integrin αIIb) for experimental melanoma metastasis in vivo. Highly metastatic B16-D5 melanoma cells were injected intravenously into GPIIb-deficient (GPIIb-/-) or wildtype (WT) mice. Acute accumulation of tumor cells in the pulmonary vasculature was assessed in real-time by confocal videofluorescence microscopy. Arrest of tumor cells was dramatically reduced in GPIIb-/- mice as compared to WT. Importantly, we found that mainly multicellular aggregates accumulated in the pulmonary circulation of WT, instead B16-D5 aggregates were significantly smaller in GPIIb-/- mice. While pulmonary arrest of melanoma was clearly dependent on GPIIb in this early phase of metastasis, we also addressed tumor progression 10 days after injection. Inversely, and unexpectedly, we found that melanoma metastasis was now increased in GPIIb-/- mice. In contrast, GPIIb did not regulate local melanoma proliferation in a subcutaneous tumor model. Our data suggest that the platelet fibrinogen receptor has a differential role in the modulation of hematogenic melanoma metastasis. While platelets clearly support early steps in pulmonary metastasis via GPIIb-dependent formation of platelet-tumor-aggregates, at a later stage its absence is associated with an accelerated development of melanoma metastases.

2017 Scientific Article in Scientific Reports Sci. Rep. 7:44041 (2017)

Rodriguez Camargo, D.C. ; Tripsianes, K. ; Buday, K. ; Frankó, A. ; Göbl, C. ; Hartlmüller, C. ; Sarkar, R. ; Aichler, M. ; Mettenleiter, G. ; Schulz, M. ; Böddrich, A. ; Erck, C. ; Martens, H. ; Walch, A.K. ; Madl, T. ; Wanker, E.E. ; Conrad, M. ; Hrabě de Angelis, M. ; Reif, B.

The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.

2017 Scientific Article in Molecular Metabolism Mol. Metab. 6, 256-266 (2017)

Frankó, A.&deg ; Neschen, S. ; Rozman, J. ; Rathkolb, B. ; Aichler, M. ; Feuchtinger, A. ; Brachthäuser, L. ; Neff, F. ; Kovarova, M. ; Wolf, E. ; Fuchs, H. ; Häring, H.-U. ; Peter, A. ; Hrabě de Angelis, M.&deg

Bezafibrate ameliorates diabetes via reduced steatosis and improved hepatic insulin sensitivity in diabetic TallyHo mice.

Objective Recently, we have shown that Bezafibrate (BEZ), the pan-PPAR (peroxisome proliferator-activated receptor) activator, ameliorated diabetes in insulin deficient streptozotocin treated diabetic mice. In order to study whether BEZ can also improve glucose metabolism in a mouse model for fatty liver and type 2 diabetes, the drug was applied to TallyHo mice. Methods TallyHo mice were divided into an early (ED) and late (LD) diabetes progression group and both groups were treated with 0.5% BEZ (BEZ group) or standard diet (SD group) for 8 weeks. We analyzed plasma parameters, pancreatic beta-cell morphology, and mass as well as glucose metabolism of the BEZ-treated and control mice. Furthermore, liver fat content and composition as well as hepatic gluconeogenesis and mitochondrial mass were determined. Results Plasma lipid and glucose levels were markedly reduced upon BEZ treatment, which was accompanied by elevated insulin sensitivity index as well as glucose tolerance, respectively. BEZ increased islet area in the pancreas. Furthermore, BEZ treatment improved energy expenditure and metabolic flexibility. In the liver, BEZ ameliorated steatosis, modified lipid composition and increased mitochondrial mass, which was accompanied by reduced hepatic gluconeogenesis. Conclusions Our data showed that BEZ ameliorates diabetes probably via reduced steatosis, enhanced hepatic mitochondrial mass, improved metabolic flexibility and elevated hepatic insulin sensitivity in TallyHo mice, suggesting that BEZ treatment could be beneficial for patients with NAFLD and impaired glucose metabolism.

2017 Scientific Article in Disease Models and Mechanisms Dis. Model. Mech. 10, 163-171 (2017)

Szibor, M.# ; Dhandapani, P.K.# ; Dufour, E. ; Holmström, K.M. ; Zhuang, Y. ; Salwig, I. ; Wittig, I. ; Heidler, J. ; Gizatullina, Z. ; Gainutdinov, T. ; German Mouse Clinic Consortium (Fuchs, H. ; Gailus-Durner, V. ; Hrabě de Angelis, M. ; Aguilar-Pimentel, J.A. ; Schmidt-Weber, C.B. ; Becker, L. ; Adler, T. ; Treise, I. ; Horsch, M. ; Beckers, J. ; Moreth, K. ; Garrett, L. ; Hölter, S.M. ; Zimprich, A. ; Wurst, W. ; Hans, W. ; Amarie, O.V. ; Graw, J. ; Rozman, J. ; Calzada-Wack, J. ; Da Silva-Buttkus, P. ; Neff, F. ; Rácz, I. ; Rathkolb, B. ; Östereicher, M.A. ; Steinkamp, R. ; Lengger, C. ; Maier, H. ; Stöger, C. ; Leuchtenberger, S.) ; Nandania, J. ; Velagapudi,V. ; Wietelmann, A. ; Rustin, P. ; Gellerich, F.N. ; Jacobs, H.T. ; Braun, T.

Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology.

Plants and many lower organisms, but not mammals, express alternative oxidases (AOX) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell-lines, Drosophila disease models and, most recently, in the mouse, where multiple, lentivector-AOX transgenes conferred substantial expression in specific tissues. Here we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello, whilst animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.

2017 Scientific Article in Advances in Cancer Research Adv. Cancer Res. 134, 117-132 (2017)

Buck, A. ; Aichler, M. ; Huber, K. ; Walch, A.K.

In situ metabolomics in cancer by mass spectrometry imaging.

Metabolomics is a rapidly evolving and a promising research field with the expectation to improve diagnosis, therapeutic treatment prediction, and prognosis of particular diseases. Among all techniques used to assess the metabolome in biological systems, mass spectrometry imaging is the method of choice to qualitatively and quantitatively analyze metabolite distribution in tissues with a high spatial resolution, thus providing molecular data in relation to cancer histopathology. The technique is ideally suited to study tissues molecular content and is able to provide molecular biomarkers or specific mass signatures which can be used in classification or the prognostic evaluation of tumors. Recently, it was shown that FFPE tissue samples are also suitable for metabolic analyses. This progress in methodology allows access to a highly valuable resource of tissues believed to widen and strengthen metabolic discovery-driven studies.

2017 Scientific Article in Neoplasia : An International Journal for Oncology Research Neoplasia 19, 8-16 (2017)

Ma, X. ; van Phi, V. ; Kimm, M.A. ; Prakash, J. ; Kessler, H. ; Kosanke, K. ; Feuchtinger, A. ; Aichler, M. ; Gupta, A. ; Rummeny, E.J. ; Eisenblätter, M. ; Siveke, J. ; Walch, A.K. ; Braren, R. ; Ntziachristos, V. ; Wildgruber, M.

Integrin-targeted hybrid fluorescence molecular tomography/X-ray computed tomography for imaging tumor progression and early response in non-small cell lung cancer.

Integrins play an important role in tumor progression, invasion and metastasis. Therefore we aimed to evaluate a preclinical imaging approach applying ανβ3 integrin targeted hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT) for monitoring tumor progression as well as early therapy response in a syngeneic murine Non-Small Cell Lung Cancer (NSCLC) model. Lewis Lung Carcinomas were grown orthotopically in C57BL/6 J mice and imaged in-vivo using a ανβ3 targeted near-infrared fluorescence (NIRF) probe. ανβ3-targeted FMT-XCT was able to track tumor progression. Cilengitide was able to substantially block the binding of the NIRF probe and suppress the imaging signal. Additionally mice were treated with an established chemotherapy regimen of Cisplatin and Bevacizumab or with a novel MEK inhibitor (Refametinib) for 2 weeks. While μCT revealed only a moderate slowdown of tumor growth, ανβ3 dependent signal decreased significantly compared to non-treated mice already at one week post treatment. ανβ3 targeted imaging might therefore become a promising tool for assessment of early therapy response in the future.

2017 Scientific Article in Biological Chemistry Biol. Chem. 398, 765-773 (2017)

Zhao, S. ; Dorn, J. ; Napieralski, R. ; Walch, A.K. ; Diersch, S. ; Kotzsch, M. ; Ahmed, N. ; Hooper, J. ; Kiechle, M. ; Schmitt, M. ; Magdolen, V.

Plasmin(Ogen) serves as a favorable biomarker for prediction of survival in advanced high-grade serous ovarian cancer.

n serous ovarian cancer, the clinical relevance of tumor cell-expressed plasmin(ogen) (PLG) has not yet been evaluated. Due to its proteolytic activity, plasmin supports tumorigenesis, however, angiostatin(-like) fragments, derived from PLG, can also function as potent antitumorigenic factors. In the present study, we assessed PLG protein expression in 103 cases of advanced high-grade serous ovarian cancer (FIGO III/IV) by immunohistochemistry. In 70/103 cases, positive staining of tumor cells was observed. In univariate Cox regression analysis, PLG staining was positively associated with prolonged overall survival (OS) (hazard ratio [HR] = 0.59, P = 0.026) of the patients. In multivariable analysis, PLG, together with residual tumor mass, remained a statistically significant independent prognostic marker (HR = 0.49, P = 0.009). In another small patient cohort (n=29), we assessed mRNA expression levels of PLG by quantitative PCR. Here, elevated PLG mRNA levels were also significantly associated with prolonged OS of patients (Kaplan-Meier analysis; P = 0.001). This finding was validated by in silico analysis of a microarray data set (n = 398) from The Cancer Genome Atlas (Kaplan-Meier analysis; P = 0.031). In summary, these data indicate that elevated PLG expression represents a favorable prognostic biomarker in advanced (FIGO III/IV) high-grade serous ovarian cancer.

2017 Scientific Article in Cancer Research Cancer Res. 77, 623-631 (2017)

Koch, M. ; de Jong, J.S. ; Glatz, J. ; Symvoulidis, P. ; Lamberts, L.E. ; Adams, A.L.L. ; Kranendonk, M.E.G. ; Terwisscha van Scheltinga, A.G. ; Aichler, M. ; Jansen, L. ; de Vries, J. ; Lub-de, Hoog, M.N. ; Schröder, C.P. ; Jorritsma-Smit, A. ; Linssen, M.D. ; de Boer, E. ; van der Vegt, B. ; Nagengast, W.B. ; Elisas,S.G. ; Oliveira, S. ; Witkamp, A.J. ; Mali, W.P.Th.M. ; van der Wall, E. ; Gracia-Allende, P.B. ; van Diest, P.J. ; de Vries, E.G. ; Walch, A.K. ; van Dam, G.M. ; Ntziachristos, V.

Threshold analysis and biodistribution of fluorescently labeled bevacizumab in human breast cancer.

In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and non-malignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multi-scale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labelled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging.

2017 Scientific Article in Nature Chemical Biology Nat. Chem. Biol. 13, 91-98 (2017)

Doll, S. ; Proneth, B. ; Tyurina, A.A. ; Panzilius, E. ; Kobayashi, S. ; Ingold, I. ; Irmler, M. ; Beckers, J. ; Aichler, M. ; Walch, A.K. ; Prokisch, H. ; Trümbach, D. ; Mao, G. ; Qu, F. ; Bayir, H. ; Füllekrug, J. ; Scheel, C. ; Wurst, W. ; Schick, J. ; Kagan, V.E. ; Friedmann Angeli, J.P.F.&deg ; Conrad, M.&deg

ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.

© 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate ferroptosis are needed. We applied two independent approaches—a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines—to uncover acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential component for ferroptosis execution. Specifically, Gpx4–Acsl4 double-knockout cells showed marked resistance to ferroptosis. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 was preferentially expressed in a panel of basal-like breast cancer cell lines and predicted their sensitivity to ferroptosis. Pharmacological targeting of ACSL4 with thiazolidinediones, a class of antidiabetic compound, ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases.

2016 Scientific Article in Nature medicine Nat. Med. 22, 1428-1438 (2016)

Eisenberg, T.# ; Abdellatif, M.# ; Schroeder, S. ; Primessnig, U. ; Stekovic, S. ; Pendl, T. ; Harger, A. ; Schipke, J. ; Zimmermann, A. ; Schmidt, A. ; Tong, M. ; Ruckenstuhl, C. ; Dammbrueck, C. ; Gross, A.S. ; Herbst, V. ; Magnes, C. ; Trausinger, G. ; Narath, S. ; Meinitzer, A. ; Hu, Z. ; Kirsch, A. ; Eller, K. ; Carmona-Gutierrez, D. ; Büttner, S. ; Pietrocola, F. ; Knittelfelder, O. ; Schrepfer, E. ; Rockenfeller, P. ; Simonini, C. ; Rahn, A. ; Horsch, M. ; Moreth, K. ; Beckers, J. ; Fuchs, H. ; Gailus-Durner, V. ; Neff, F. ; Janik, D. ; Rathkolb, B. ; Rozman, J. ; Hrabě de Angelis, M. ; Moustafa, T. ; Haemmerle, G. ; Mayr, M. ; Willeit, P. ; von Frieling-Salewsky, M. ; Pieske, B. ; Scorrano, L. ; Pieber, T.R. ; Pechlaner, R. ; Willeit, J. ; Sigrist, S.J. ; Linke, W.A. ; Mühlfeld, C. ; Sadoshima, J. ; Dengjel, J. ; Kiechl, S. ; Kroemer, G.&deg ; Sedej, S.&deg ; Madeo, F.&deg

Cardioprotection and lifespan extension by the natural polyamine spermidine.

Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.

2016 Scientific Article in Genes Genomes Genetics G3 Genes Genomes Genetics G3 6, 4035-4046 (2016)

Fuchs, H. ; Sabrautzki, S. ; Przemeck, G.K.H. ; Leuchtenberger, S. ; Lorenz-Depiereux, B. ; Becker, L. ; Rathkolb, B. ; Horsch, M. ; Garrett, L. ; Östereicher, M.A. ; Hans, W. ; Abe, K. ; Sagawa, N. ; Rozman, J. ; Vargas Panesso, I.L. ; Sandholzer, M. ; Lisse, T.S. ; Adler, T. ; Aguilar-Pimentel, J.A. ; Calzada-Wack, J. ; Ehrhard, N. ; Elvert, R. ; Gau, C. ; Hölter, S.M. ; Micklich, K. ; Moreth, K. ; Prehn, C. ; Puk, O. ; Rácz, I. ; Stöger, C. ; Vernaleken, A. ; Michel, D. ; Diener, S. ; Wieland, T. ; Adamski, J. ; Bekeredjian, R. ; Lengger, C. ; Maier, H. ; Neff, F. ; Ollert, M. ; Stöger, T. ; Yildirim, A.Ö. ; Strom, T.M. ; Zimmer, A. ; Wolf, E. ; Wurst, W. ; Klopstock, T. ; Beckers, J. ; Gailus-Durner, V. ; Hrabě de Angelis, M.

The first Scube3 mutant mouse line with pleiotropic phenotypic alterations.

The vertebrate Scube (Signal peptide, CUB and EGF-like domain-containing protein) family consists of three independent members Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K) that clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to understand SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3. In addition, alterations in energy metabolism, behavior and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.

2016 Scientific Article in Proceedings of the National Academy of Sciences of the United States of America Proc. Natl. Acad. Sci. U.S.A. 113, 12244-12249 (2016)

Abdelmoula, W.M. ; Balluff, B. ; Englert, S. ; Dijkstra, J. ; Reinders, M.J.T. ; Walch, A.K. ; McDonnell, L.A. ; Lelieveldt, B.P.F.

Data-driven identification of prognostic tumor subpopulations using spatially mapped t-SNE of mass spectrometry imaging data.

The identification of tumor subpopulations that adversely affect patient outcomes is essential for a more targeted investigation into how tumors develop detrimental phenotypes, as well as for personalized therapy. Mass spectrometry imaging has demonstrated the ability to uncover molecular intratumor heterogeneity. The challenge has been to conduct an objective analysis of the resulting data to identify those tumor subpopulations that affect patient outcome. Here we introduce spatially mapped t-distributed stochastic neighbor embedding (t-SNE), a nonlinear visualization of the data that is able to better resolve the biomolecular intratumor heterogeneity. In an unbiased manner, t-SNE can uncover tumor subpopulations that are statistically linked to patient survival in gastric cancer and metastasis status in primary tumors of breast cancer.

2016 Scientific Article in BMC Cancer BMC Cancer 16:811 (2016)

Gross, E. ; van Tinteren, H. ; Li, Z. ; Raab, S. ; Meul, C. ; Avril, S. ; Laddach, N. ; Aubele, M. ; Propping, C. ; Gkazepis, A. ; Schmitt, M. ; Meindl, A. ; Nederlof, P.M. ; Kiechle, M. ; Lips, E.H.

Identification of BRCA1-like triple-negative breast cancers by quantitative multiplex-ligation-dependent probe amplification (MLPA) analysis of BRCA1-associated chromosomal regions: A validation study.

BACKGROUND: Triple-negative breast cancer (TNBC) with a BRCA1-like molecular signature has been demonstrated to remarkably respond to platinum-based chemotherapy and might be suited for a future treatment with poly(ADP-ribose)polymerase (PARP) inhibitors. In order to rapidly assess this signature we have previously developed a multiplex-ligation-dependent probe amplification (MLPA)-based assay. Here we present an independent validation of this assay to confirm its important clinical impact. METHODS: One-hundred-forty-four TNBC tumor specimens were analysed by the MLPA-based "BRCA1-like" test. Classification into BRCA1-like vs. non-BRCA1-like samples was performed by our formerly established nearest shrunken centroids classifier. Data were subsequently compared with the BRCA1-mutation/methylation status of the samples. T-lymphocyte infiltration and expression of the main target of PARP inhibitors, PARP1, were assessed on a subset of samples by immunohistochemistry. Data acquisition and interpretation was performed in a blinded manner. RESULTS: In the studied TNBC cohort, 63 out of 144 (44 %) tumors were classified into the BRCA1-like category. Among these, the MLPA test correctly predicted 15 out of 18 (83 %) samples with a pathogenic BRCA1-mutation and 20 of 22 (91 %) samples exhibiting BRCA1-promoter methylation. Five false-negative samples were observed. We identified high lymphocyte infiltration as one possible basis for misclassification. However, two falsely classified BRCA1-mutated tumors were also characterized by rather non-BRCA1-associated histopathological features such as borderline ER expression. The BRCA1-like vs. non-BRCA1-like signature was specifically enriched in high-grade (G3) cancers (90 % vs. 58 %, p = 0.0004) and was also frequent in tumors with strong (3+) nuclear PARP1 expression (37 % vs. 16 %; p = 0.087). CONCLUSIONS: This validation study confirmed the good performance of the initial MLPA assay which might thus serve as a valuable tool to select patients for platinum-based chemotherapy regimens. Moreover, frequent PARP1 upregulation in BRCA1-like tumors may also point to susceptibility to treatment with PARP inhibitors. Limitations are the requirement of high tumor content and high-quality DNA.

2016 Scientific Article in European journal of microbiology and immunology Eur. J. Micr. Imm. 6, 186-196 (2016)

Wiedemann, T. ; Hofbaur, S. ; Loell, E. ; Rieder, G.

A C-Terminal coiled-coil region of CagL is responsible for Helicobacter Pylori-induced Il-8 expression.

Interleukin-8 (IL-8) is a potent neutrophil-activating chemokine which triggers the infiltration and migration of neutrophils into areas of bacterial infection. Helicobacter pylori-infected patient studies as well as animal models have revealed that H. pylori type I strains carrying an intact cytotoxin-associated gene pathogenicity island (cag-PAI) with a functional type IV secretion system (T4SS) induce IL-8 expression and secretion in gastric mucosa. This gastric mucosal IL-8 expression correlates with severe histological changes due to H. pylori infection. In the present study, we explored a new recognition pattern on the bacterial adhesion protein CagL inducing IL-8 expression in H. pylori-infected host cells. To analyze the secreted IL-8 concentration, we performed IL-8 enzyme-linked immunosorbent assay (ELISA). To investigate the H. pylori-induced IL-8 expression on the transcriptional level, we transiently transfected gastric epithelial cells (AGS) with a human IL-8 luciferase reporter construct. The results of this study demonstrate that specifically the C-terminal coiled-coil region of the H. pylori CagL protein, a protein described to be located on the tip of the T4SS-pilus, is responsible for several in vitro observations: 1) H. pylori-induced IL-8 secretion via the transforming growth factor (TGF)-α activated epidermal growth factor-receptor (EGF-R) signaling pathway; 2) H. pylori-induced elongation of the cells, a typical CagA-induced phenotype; and 3) the bridging of the T4SS to its human target cells. This novel bacterial-host recognition sequence allows a new insight into how H. pylori induces the inflammatory response in gastric epithelial cells and facilitates the development of precancerous conditions.

2016 Scientific Article in Brain and behavior Brain Behav. 6:e00578 (2016)

Schenck, T.L. ; Lin, S. ; Stewart, J.K. ; Koban, K.C. ; Aichler, M. ; Rezaeian, F. ; Giunta, R.E.

Sensory reanimation of the hand by transfer of the superficial branch of the radial nerve to the median and ulnar nerve.

Background: It remains a surgical challenge to treat high-grade nerve injuries of the upper extremity. Extra-anatomic reconstructions through the transfer of peripheral nerves have gained clinical importance over the past decades. This contribution outlines the anatomic and histomorphometric basis for the transfer of the superficial branch of the radial nerve (SBRN) to the median nerve (MN) and the superficial branch of the ulnar nerve (SBUN). Methods: The SBRN, MN, and SBUN were identified in 15 specimens and the nerve transfer performed. A favorable site for coaptation was chosen and its location described using relevant anatomical landmarks. Histomorphometric characteristics of donor and target were compared to evaluate the chances of a clinical success. Results: A suitable location for dissecting the SBRN was identified prior to its first bifurcation. Coaptations were possible near the pronator quadratus muscle, approximately 22 cm distal to the lateral epicondyle of the humerus. The MN and SBUN had to be dissected interfasciculary over 82 ± 5.7 mm and 49 ± 5.5 mm, respectively. Histomorphometric analysis revealed sufficient donor-to-recipient axon ratios for both transfers and identified the SBRN as a suitable donor with high axon density. Conclusion: Our anatomic and histomorphometric results indicate that the SBRN is a suitable donor for the MN and SBUN at wrist level. The measurements show feasibility of this procedure and shall help in planning this sensory nerve transfer. High axon density in the SBRN identifies it or its branches an ideal candidate for sensory reanimation of fingers and thumbs.

2016 Scientific Article in PLoS ONE PLoS ONE 11:e0164298 (2016)

Wittmann, A. ; Grimm, M. ; Scherthan, H. ; Horsch, M. ; Beckers, J. ; Fuchs, H. ; Gailus-Durner, V. ; Hrabě de Angelis, M. ; Ford, S.J. ; Burton, N.C. ; Razansky, D. ; Trümbach, D. ; Aichler, M. ; Walch, A.K. ; Calzada-Wack, J. ; Neff, F. ; Wurst, W. ; Hartmann, T. ; Floß, T.

Sphingomyelin synthase 1 is essential for male fertility in mice.

Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility.

2016 Scientific Article in OncoTarget Oncotarget 7, 71817-71832 (2016)

Kempf, S.J. ; Janik, D. ; Barjaktarovic, Z. ; Braga-Tanaka III, I. ; Tanaka, S. ; Neff, F. ; Saran, A. ; Larsen, M.R. ; Tapio, S.

Chronic low-dose-rate ionising radiation affects the hippocampal phosphoproteome in the ApoE-/- Alzheimer's mouse model.

Accruing data indicate that radiation-induced consequences resemble pathologies of neurodegenerative diseases such as Alzheimer´s. The aim of this study was to elucidate the effect on hippocampus of chronic low-dose-rate radiation exposure (1 mGy/day or 20 mGy/day) given over 300 days with cumulative doses of 0.3 Gy and 6.0 Gy, respectively. ApoE deficient mutant C57Bl/6 mouse was used as an Alzheimer´s model. Using mass spectrometry, a marked alteration in the phosphoproteome was found at both dose rates. The radiation-induced changes in the phosphoproteome were associated with the control of synaptic plasticity, calcium-dependent signalling and brain metabolism. An inhibition of CREB signalling was found at both dose rates whereas Rac1-Cofilin signalling was found activated only at the lower dose rate. Similarly, the reduction in the number of activated microglia in the molecular layer of hippocampus that paralleled with reduced levels of TNFα expression and lipid peroxidation was significant only at the lower dose rate. Adult neurogenesis, investigated by Ki67, GFAP and NeuN staining, and cell death (activated caspase-3) were not influenced at any dose or dose rate. This study shows that several molecular targets induced by chronic low-dose-rate radiation overlap with those of Alzheimer´s pathology. It may suggest that ionising radiation functions as a contributing risk factor to this neurodegenerative disease.

2016 Review in Histochemistry and Cell Biology Histochem. Cell Biol. 146, 1-26 (2016)

Feuchtinger, A. ; Walch, A.K. ; Dobosz, M.

Deep tissue imaging: A review from a preclinical cancer research perspective.

This review delves into the rapidly evolving field of deep tissue imaging at cellular resolution, reviewing popular tissue clearing and staining methods in combination with light-sheet fluorescence microscopy (LSFM) including quantification and three-dimensional visualization tools, the field of applications and perspective, particularly with the focus on preclinical cancer research and drug development. The LSFM technique presented here allows an extremely fast optical sectioning for three-dimensional reconstruction of centimeter-sized tissue samples at cellular resolution. However, optical clearing methods are required to receive optical transparent tissue. Application of either tissue autofluorescence, in vivo fluorescence labeling, endogenous fluorescence or ex vivo whole-mount immunolabeling enables three-dimensional in situ visualization of morphological and functional features of unsectioned whole-mount tissue samples. This powerful and innovative imaging technique opens up a new dimension of tissue analysis providing detailed and comprehensive insights into biology. It enables the investigation of normal and pathological tissue features and disease progression and allows precise monitoring of potential therapeutic interventions in intact biological tissue on a cellular level.

2016 Scientific Article in Cell Cell 167, 843-857.e14 (2016)

Finan, B. ; Clemmensen, C. ; Zhu, Z. ; Stemmer, K. ; Gauthier, K. ; Müller, L. ; de Angelis, M. ; Moreth, K. ; Neff, F. ; Perez-Tilve, D. ; Fischer, K. ; Lutter, D. ; Sánchez-Garrido, M.A. ; Liu, P. ; Tuckermann, J.P. ; Malehmir, M. ; Healy, M.E. ; Weber, A. ; Heikenwälder, M. ; Jastroch, M. ; Kleinert, M. ; Jall, S. ; Brandt, S. ; Flamant, F. ; Schramm, K.-W. ; Biebermann, H. ; Döring, Y. ; Weber, C. ; Habegger, K.M. ; Keuper, M. ; Gelfanov, V. ; Liu, F. ; Köhrle, J. ; Rozman, J. ; Fuchs, H. ; Gailus-Durner, V. ; Hrabě de Angelis, M. ; Hofmann, S.M. ; Yang, B. ; Tschöp, M.H. ; DiMarchi, R. ; Müller, T.D.

Chemical hybridization of glucagon and thyroid hormone optimizes therapeutic Impact for metabolic disease.

Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.

2016 Scientific Article in Gut (eGut) Gut 67, 146-156 (2016)

Kong, B. ; Bruns, P. ; Behler, N.A. ; Chang, L. ; Schlitter, A.M. ; Cao, J. ; Gewies, A. ; Ruland, J. ; Fritzsche, S. ; Valkovskaya, N. ; Jian, Z. ; Regel, I. ; Raulefs, S. ; Irmler, M. ; Beckers, J. ; Friess, H. ; Erkan, M. ; Müller, N.S. ; Roth, S. ; Hackert, T. ; Esposito, I. ; Theis, F.J. ; Kleeff, J. ; Michalski, C.W.

Dynamic landscape of pancreatic carcinogenesis reveals early molecular networks of malignancy.

OBJECTIVE: The initial steps of pancreatic regeneration versus carcinogenesis are insufficiently understood. Although a combination of oncogenic Kras and inflammation has been shown to induce malignancy, molecular networks of early carcinogenesis remain poorly defined. DESIGN: We compared early events during inflammation, regeneration and carcinogenesis on histological and transcriptional levels with a high temporal resolution using a well-established mouse model of pancreatitis and of inflammation-accelerated Kras(G12D)-driven pancreatic ductal adenocarcinoma. Quantitative expression data were analysed and extensively modelled in silico. RESULTS: We defined three distinctive phases-termed inflammation, regeneration and refinement-following induction of moderate acute pancreatitis in wild-type mice. These corresponded to different waves of proliferation of mesenchymal, progenitor-like and acinar cells. Pancreas regeneration required a coordinated transition of proliferation between progenitor-like and acinar cells. In mice harbouring an oncogenic Kras mutation and challenged with pancreatitis, there was an extended inflammatory phase and a parallel, continuous proliferation of mesenchymal, progenitor-like and acinar cells. Analysis of high-resolution transcriptional data from wild-type animals revealed that organ regeneration relied on a complex interaction of a gene network that normally governs acinar cell homeostasis, exocrine specification and intercellular signalling. In mice with oncogenic Kras, a specific carcinogenic signature was found, which was preserved in full-blown mouse pancreas cancer. CONCLUSIONS: These data define a transcriptional signature of early pancreatic carcinogenesis and a molecular network driving formation of preneoplastic lesions, which allows for more targeted biomarker development in order to detect cancer earlier in patients with pancreatitis.

2016 Scientific Article in BMC Cancer BMC Cancer 16:615 (2016)

Huber, M. ; Mall, R. ; Braselmann, H. ; Feuchtinger, A. ; Molatore, S. ; Lindner, K. ; Walch, A.K. ; Gross, E. ; Schmitt, M. ; Falkenberg, N. ; Aubele, M.

uPAR enhances malignant potential of triple-negative breast cancer by directly interacting with uPA and IGF1R.

BACKGROUND: Due to lack of a targeted therapy for the triple-negative breast cancer (TNBC) patients, it is important to explore this aggressive breast cancer type in more detail and to establish novel therapeutic approaches. TNBC is defined negative for the protein expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). One prominent feature of this cancer type is the frequent overexpression of major components of the urokinase-type plasminogen activator system (uPAS) including uPA, its receptor uPAR and the inhibitor PAI-1, which may be valuable as therapeutic targets. METHODS: Direct interactions of uPAR with interactors were demonstrated by immunoprecipitations and proximity ligation assays. For stable knockdowns of target proteins, lentiviral vectors were used and the effects were analysed by immunoblottings and using in vitro cell viability, migration and invasion assays. Immunohistochemical and statistical analyses of biomarkers and clinical parameters were conducted in a TNBC cohort (n = 174). RESULTS: Direct tumour-promoting interactions of uPAR with uPA and the insulin-like growth factor receptor 1 (IGF1R) were shown in TNBC cells and these interactions were significantly reduced (p = 0.001) when uPAR was downregulated. The combined knockdown of uPAR and uPA or IGF1R additively and significantly reduced cell viability, migration and invasion of the model cell lines. In TNBC tissue, the complexes formed by uPAR with uPA or with IGF1R significantly correlated with the histological grade (p = 0.0019) as well as with cathepsin B and D (p ≤ 0.0001) that are implicated in cell invasion and metastasis. CONCLUSIONS: Our outcomes show that not only overexpressed biomarkers promote tumourigenesis, but rather their interactions further potentiate tumour progression. This study emphasises the potential of combined approaches targeting uPAR and its interactors with regard to an improved therapy of TNBC.

2016 Scientific Article in Blood Blood 128, 2435-2449 (2016)

Stark, K. ; Philippi, V. ; Stockhausen, S. ; Busse, J. ; Antonelli, A. ; Miller, M. ; Schubert, I. ; Hoseinpour, P. ; Chandraratne, S. ; von Brühl, M.L. ; Gärtner, F. ; Lorenz, M. ; Agresti, A. ; Coletti, R. ; Antoine, D.J. ; Heermann, R. ; Jung, K. ; Reese, S. ; Laitinen, I. ; Schwaiger, M. ; Walch, A.K. ; Sperandio, M. ; Nawroth, P.P. ; Reinhardt, C. ; Jäckel, S. ; Bianchi, M.E. ; Massberg, S.

Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice.

Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. While sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new anti-inflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior we identified blood-derived high-mobility group box 1 protein (HMGB1) - a prototypical mediator of sterile inflammation - to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1(-/-) chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through RAGE and TLR2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis.

2016 Scientific Article in Neoplasia : An International Journal for Oncology Research Neoplasia 18, 459-467 (2016)

Chekkoury, A.# ; Nunes, A.# ; Gateau, J. ; Symvoulidis, P. ; Feuchtinger, A. ; Bézière, N. ; Ovsepian, S.V. ; Walch, A.K. ; Ntziachristos, V.

High-resolution multispectral optoacoustic tomography of the vascularization and constitutive hypoxemia of cancerous tumors.

Diversity of the design and alignment of illumination and ultrasonic transducers empower the fine scalability and versatility of optoacoustic imaging. In this study, we implement an innovative high-resolution optoacoustic mesoscopy for imaging the vasculature and tissue oxygenation within subcutaneous and orthotopic cancerous implants of mice in vivo through acquisition of tomographic projections over 180° at a central frequency of 24 MHz. High-resolution volumetric imaging was combined with multispectral functional measurements to resolve the exquisite inner structure and vascularization of the entire tumor mass using endogenous and exogenous optoacoustic contrast. Evidence is presented for constitutive hypoxemia within the carcinogenic tissue through analysis of the hemoglobin absorption spectra and distribution. Morphometric readouts obtained with optoacoustic mesoscopy have been verified with high-resolution ultramicroscopic studies. The findings described herein greatly extend the applications of optoacoustic mesoscopy toward structural and multispectral functional measurements of the vascularization and hemodynamics within solid tumors in vivo and are of major relevance to basic and preclinical oncological studies in small animal models.

2016 Scientific Article in Journal of Molecular and Cellular Cardiology J. Mol. Cell. Cardiol. 99, 57-64 (2016)

Regn, M. ; Laggerbauer, B. ; Jentzsch, C. ; Ramanujam, D. ; Ahles, A. ; Sichler, S. ; Calzada-Wack, J. ; Koenen, R. ; Braun, A. ; Nieswandt, B. ; Engelhardt, S.

Peptidase inhibitor 16 is a membrane-tethered regulator of chemerin processing in the myocardium.

A key response of the myocardium to stress is the secretion of factors with paracrine or endocrine function. Intriguing in this respect is peptidase inhibitor 16 (PI16), a member of the CAP family of proteins which we found to be highly upregulated in cardiac disease. Up to this point, the mechanism of action and physiological function of PI16 remained elusive. Here, we show that PI16 is predominantly expressed by cardiac fibroblasts, which expose PI16 to the interstitium via a glycophosphatidylinositol-(GPI) membrane anchor. Based on a reported genetic association of PI16 and plasma levels of the chemokine chemerin, we investigated whether PI16 regulates post-translational processing of its precursor pro-chemerin. PI16-deficient mice were engineered and found to generate higher levels of processed chemerin than wildtype mice. Purified recombinant PI16 efficiently inhibited cathepsin K, a chemerin-activating protease, in vitro. Moreover, we show that conditioned medium from PI16-overexpressing cells impairs the activation of pro-chemerin. Together, our data indicate that PI16 suppresses chemerin activation in the myocardium and suggest that this circuit may be part of the cardiac stress response.

2016 Scientific Article in Nature Protocols Nat. Protoc. 11, 1428-1443 (2016)

Ly, A.# ; Buck, A.# ; Balluff, B. ; Sun, N. ; Gorzolka, K. ; Feuchtinger, A. ; Janssen, K.P. ; Kuppen, P.J. ; van de Velde, C.J. ; Weirich, G. ; Erlmeier, F. ; Langer, R. ; Aubele, M. ; Zitzelsberger, H. ; McDonnell, L.A. ; Aichler, M. ; Walch, A.K.

High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue.

Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected ∼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.

2016 Scientific Article in Journal of Nuclear Medicine J. Nucl. Med. 57, 1971-1977 (2016)

Mendler, C.T. ; Feuchtinger, A. ; Heid, I. ; Aichler, M. ; D'Alessandria, C. ; Pirsig, S. ; Blechert, B. ; Wester, H.J. ; Braren, R. ; Walch, A.K. ; Skerra, A. ; Schwaiger, M.

Tumor uptake of anti-CD20 Fabs depends on tumor perfusion.

Antibodies have become an established treatment modality in cancer therapy during the last decade. However, these treatments often suffer from insufficient and heterogeneous response despite validated antigen or target receptor expression in the tumor. In fact, therapeutic success depends both on the presence and accessibility of the tumor antigen by the antibody. In search of a suitable preclinical animal model to evaluate the mechanisms of tumor heterogeneity and hemodynamics, we characterized two exemplary non-Hodgkin lymphoma subtypes with comparable CD20 expression and metabolism, SUDHL-4 and Granta, using multimodal imaging techniques. METHODS: To investigate in vivo biodistribution, two differently modified αCD20 antigen-binding fragments (Fab), prepared (i) by PASylation and (ii) by fusion with an albumin-binding domain (ABD), were radiolabeled with (125)I and intravenously injected into immunocompromised mice bearing corresponding xenografts. RESULTS: Validation with (18)F-FDG revealed similar distribution of vital tumor tissue 1 h p.i. However, large differences in tumor uptake were observed when applying the CD20-specific radiotracers (125)I-Fab-ABD and (125)I-Fab-PAS200 with 12.3 and 2.4 % ID/g, respectively, for Granta in comparison with 3.5 and 0.75 % ID/g, respectively, for SUDHL-4 xenografts 24 h p.i. 3D light-sheet fluorescence microscopy with Cy5-Fab-PAS200 confirmed better tracer extravasation in the Granta tumors. Moreover, dynamic contrast enhanced MRI imaging revealed significantly reduced tumor perfusion in the SUHDL-4 xenografts. CONCLUSION: Tracer uptake was highly dependent on local tumor perfusion as well as Fab permeation in the SUDHL-4 and Granta tumors. Thus, the SUDHL-4 xenograft offers an excellent model system to investigate the influence of therapies affecting tumor angiogenesis.

2016 Scientific Article in Nature Nature 535, 430-434 (2016)

Bader, E.# ; Migliorini, A.# ; Gegg, M. ; Moruzzi, N. ; Gerdes, J.M. ; Roscioni, S. ; Bakhti, M. ; Brandl, E. ; Irmler, M. ; Beckers, J. ; Aichler, M. ; Feuchtinger, A. ; Leitzinger, C. ; Zischka, H. ; Wang-Sattler, R. ; Jastroch, M. ; Tschöp, M.H. ; Machicao, F. ; Staiger, H. ; Häring, H.-U. ; Chmelova, H. ; Chouinard, J.A. ; Oskolkov, N. ; Korsgren, O. ; Speier, S. ; Lickert, H.

Identification of proliferative and mature β-cells in the islets of Langerhans.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of β-cells. Pancreatic β-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene, acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature β-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger β-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for β-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional β-cell heterogeneity and induce β-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional β-cell mass in diabetic patients.

2016 Scientific Article in OncoTarget Oncotarget 7, 41767-41780 (2016)

von Heyking, K. ; Roth, L.C. ; Ertl, M. ; Schmidt, O. ; Calzada-Wack, J. ; Neff, F. ; Lawlor, E.R. ; Burdach, S. ; Richter, G.H.

The posterior HOXD locus: Its contribution to phenotype and malignancy of Ewing sarcoma.

Microarray analysis revealed genes of the posterior HOXD locus normally involved in bone formation to be over-expressed in primary Ewing sarcoma (ES). The expression of posterior HOXD genes was not influenced via ES pathognomonic EWS/ETS translocations. However, knock down of the dickkopf WNT signaling pathway inhibitor 2 (DKK2) resulted in a significant suppression of HOXD10, HOXD11 and HOXD13 while over-expression of DKK2 and stimulation with factors of the WNT signaling pathway such as WNT3a, WNT5a or WNT11 increased their expression. RNA interference demonstrated that individual HOXD genes promoted chondrogenic differentiation potential, and enhanced expression of the bone-associated gene RUNX2. Furthermore, HOXD genes increased the level of the osteoblast- and osteoclast-specific genes, osteocalcin (BGLAP) and platelet-derived growth factor beta polypeptide (PDGFB), and may further regulate endochondral bone development via induction of parathyroid hormone-like hormone (PTHLH). Additionally, HOXD11 and HOXD13 promoted contact independent growth of ES, while in vitro invasiveness of ES lines was enhanced by all 3 HOXD genes investigated and seemed mediated via matrix metallopeptidase 1 (MMP1). Consequently, knock down of HOXD11 or HOXD13 significantly suppressed lung metastasis in a xeno-transplant model in immune deficient mice, providing overall evidence that posterior HOXD genes promote clonogenicity and metastatic potential of ES.

2016 Scientific Article in Cancer Research Cancer Res. 76, 4113-4123 (2016)

Mall, S. ; Yusufi, N. ; Wagner, R. ; Klar, R. ; Bianchi, H. ; Steiger, K. ; Straub, M. ; Audehm, S. ; Laitinen, I. ; Aichler, M. ; Peschel, C. ; Ziegler, S. ; Mustafa, M. ; Schwaiger, M. ; D'Alessandria, C. ; Krackhardt, A.M.

Immuno-PET imaging of engineered human T cells in tumors.

Sensitive in vivo imaging technologies applicable to the clinical setting are still lacking for adoptive T-cell-based immunotherapies, an important gap to fill if mechanisms of tumor rejection or escape are to be understood. Here, we propose a highly sensitive imaging technology to track human TCR-transgenic T cells in vivo by directly targeting the murinized constant TCR beta domain (TCRmu) with a zirconium-89 ((89)Zr)-labeled anti-TCRmu-F(ab')2 fragment. Binding of the labeled or unlabeled F(ab')2 fragment did not impair functionality of transgenic T cells in vitro and in vivo Using a murine xenograft model of human myeloid sarcoma, we monitored by Immuno-PET imaging human central memory T cells (TCM), which were transgenic for a myeloid peroxidase (MPO)-specific TCR. Diverse T-cell distribution patterns were detected by PET/CT imaging, depending on the tumor size and rejection phase. Results were confirmed by IHC and semiquantitative evaluation of T-cell infiltration within the tumor corresponding to the PET/CT images. Overall, these findings offer a preclinical proof of concept for an imaging approach that is readily tractable for clinical translation.

2016 Scientific Article in Journal of Clinical Investigation J. Clin. Invest. 126, 2721-2735 (2016)

Lichtmannegger, J.# ; Leitzinger, C.# ; Wimmer, R. ; Schmitt, S. ; Schulz, S. ; Kabiri, Y. ; Eberhagen, C. ; Rieder, T. ; Janik, D. ; Neff, F. ; Straub, B.K. ; Schirmacher, P. ; DiSpirito, A.A. ; Bandow, N. ; Baral, B.S. ; Flatley, A. ; Kremmer, E. ; Denk, G.U. ; Reiter, F.P. ; Hohenester, S. ; Eckardt-Schupp, F. ; Dencher, N.A. ; Adamski, J. ; Sauer, V. ; Niemietz, C. ; Schmidt, H.H. ; Merle, U. ; Gotthardt, D.N. ; Kroemer, G. ; Weiss, K.H. ; Zischka, H.

Methanobactin reverses acute liver failure in a rat model of Wilson disease.

In Wilson disease (WD), functional loss of ATPase copper-transporting β (ATP7B) impairs biliary copper excretion, leading to excessive copper accumulation in the liver and fulminant hepatitis. Current US Food and Drug Administration- and European Medicines Agency-approved pharmacological treatments usually fail to restore copper homeostasis in patients with WD who have progressed to acute liver failure, leaving liver transplantation as the only viable treatment option. Here, we investigated the therapeutic utility of methanobactin (MB), a peptide produced by Methylosinus trichosporium OB3b, which has an exceptionally high affinity for copper. We demonstrated that ATP7B-deficient rats recapitulate WD-associated phenotypes, including hepatic copper accumulation, liver damage, and mitochondrial impairment. Short-term treatment of these rats with MB efficiently reversed mitochondrial impairment and liver damage in the acute stages of liver copper accumulation compared with that seen in untreated ATP7B-deficient rats. This beneficial effect was associated with depletion of copper from hepatocyte mitochondria. Moreover, MB treatment prevented hepatocyte death, subsequent liver failure, and death in the rodent model. These results suggest that MB has potential as a therapeutic agent for the treatment of acute WD.

2016 Scientific Article in OncoTarget Oncotarget 7, 44062-44075 (2016)

Huber, M. ; Falkenberg, N. ; Hauck, S.M. ; Priller, M. ; Braselmann, H. ; Feuchtinger, A. ; Walch, A.K. ; Schmitt, M. ; Aubele, M.

Cyr61 and YB-1 are novel interacting partners of uPAR and elevate the malignancy of triple-negative breast cancer.

The triple-negative breast cancer (TNBC) is a very aggressive tumor type often occurring in young women and is associated with a bad prognosis for the patients. TNBC lacks established targets for breast cancer therapy, such as the estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). Therefore, novel therapeutic targets and strategies are needed for an improved treatment of this breast cancer subtype. TNBC and respective cell lines often overexpress proteins of the urokinase plasminogen activator system (uPAS) including uPA, its receptor uPAR and inhibitor PAI-1, which together with co-factors contribute to the malignancy of TNBC. Here, two novel interacting partners of uPAR, the cysteine-rich angiogenic inducer 61 (Cyr61) and the Y-box-binding protein 1 (YB-1) were identified and their differential expression demonstrated in TNBC cells as well as in tumors. In the TNBC cohort, both interactors significantly correlated with expression levels of cathepsin B, c-Met and the tumor grade. In addition, expression levels of Cyr61 significantly correlated with cathepsin D (p=0.03), insulin receptor (p≤0.001), insulin-like growth factor receptor 1 (IGF1R, p=0.015) and also with YB-1 (p=0.0004) levels. The interactions of uPAR with Cyr61 significantly correlated with expression levels of tumor-promoting biomarkers including plasminogen (p=0.0014), cathepsin B (p=0.032), c-Met (p=0.0192) as well as with the tumor grade (p=0.02). In multivariate survival analysis, YB-1 showed independent prognostic value (p=0.01). As the novel interacting partners, also together with uPAR, contribute to tumor progression and metastasis, both may be potential therapeutic targets in breast cancer.

2016 Scientific Article in Diabetes Diabetes 65, 2540-2552 (2016)

Frankó, A. ; Huypens, P. ; Neschen, S. ; Irmler, M. ; Rozman, J. ; Rathkolb, B. ; Neff, F. ; Prehn, C. ; Dubois, G. ; Baumann, M. ; Massinger, R. ; Gradinger, D. ; Przemeck, G.K.H. ; Repp, B. ; Aichler, M. ; Feuchtinger, A. ; Schommers, P. ; Stöhr, O. ; Sanchez-Lasheras, C. ; Adamski, J. ; Peter, A. ; Prokisch, H. ; Beckers, J. ; Walch, A.K. ; Fuchs, H. ; Wolf, E. ; Schubert, M. ; Wiesner, R.J. ; Hrabě de Angelis, M.

Bezafibrate improves insulin sensitivity and metabolic flexibility in STZ-treated diabetic mice.

Bezafibrate (BEZ), a pan activator of peroxisome proliferator-activated receptors (PPARs), has been generally used to treat hyperlipidemia for decades. Clinical trials with type 2 diabetes patients indicated that BEZ also has beneficial effects on glucose metabolism, although the underlying mechanisms of these effects remain elusive. Even less is known about a potential role for BEZ in treating type 1 diabetes. Here we show that BEZ markedly improves hyperglycemia and glucose and insulin tolerance in streptozotocin (STZ)-treated mice, an insulin-deficient mouse model of type 1 diabetes. BEZ treatment of STZ mice significantly suppressed the hepatic expression of genes that are annotated in inflammatory processes, whereas the expression of PPAR and insulin target gene transcripts was increased. Furthermore, BEZ-treated mice also exhibited improved metabolic flexibility as well as an enhanced mitochondrial mass and function in the liver. Finally, we show that the number of pancreatic islets and the area of insulin positive cells tended to be higher in BEZ-treated mice. Our data suggest that BEZ may improve impaired glucose metabolism by augmenting hepatic mitochondrial performance, suppressing hepatic inflammatory pathways, and improving insulin sensitivity and metabolic flexibility. Thus, BEZ treatment might also be useful for patients with impaired glucose tolerance or diabetes.

2016 Scientific Article in Stroke Stroke 47, 1864-1871 (2016)

Boeckh-Behrens, T. ; Kleine, J.F. ; Zimmer, C. ; Neff, F. ; Scheipl, F. ; Pelisek, J. ; Schirmer, L. ; Nguyen, K.H. ; Karatas, D. ; Poppert, H.

Thrombus histology suggests cardioembolic cause in cryptogenic stroke.

BACKGROUND AND PURPOSE: Ischemic stroke of undetermined cause is a major health issue because of its high frequency and clinical relevance. Histopathologic analysis of human thrombi, retrieved from stroke patients with large-vessel occlusion during mechanical thrombectomy, may provide information about underlying pathologies. This study examines the relationship between stroke causes and histological clot composition to identify specific patterns that might help to distinguish causes of cryptogenic stroke. METHODS: Thrombi of 145 consecutive stroke patients with large-vessel occlusion were collected during intracranial mechanical recanalization. The hematoxylin and eosin-stained specimens were quantitatively analyzed in terms of the relative fractions of the main constituents (red and white blood cells and fibrin/platelets). These data, along with additional clinical and interventional parameters, were compared for different stroke subtypes, as defined by the international Trial of Org 10172 in Acute Stroke Treatment criteria. RESULTS: The composition of thrombi from cardioembolic and noncardioembolic stroke patients differed significantly for all main thrombus components. Cardioembolic thrombi had higher proportions of fibrin/platelets (P=0.009), less erythrocytes (P=0.003), and more leucocytes (P=0.035) than noncardioembolic thrombi. Cryptogenic strokes showed strong overlap with cardioembolic strokes but not with noncardioembolic strokes, in terms of both thrombus histology and interventional and clinical outcome parameters. CONCLUSIONS: Quantitative evaluation of thrombus composition may help to distinguish between different stroke causes. Our findings support the notion that the majority of cryptogenic strokes are cardioembolic.

2016 Nature Communications Nat. Commun. 7:11835 (2016)

Eden, M. ; Meder, B. ; Völkers, M. ; Poomvanicha, M. ; Domes, K. ; Branchereau, M. ; Marck, P. ; Will, R. ; Bernt, A. ; Rangrez, A. ; Busch, M. ; German Mouse Clinic Consortium (Adler, T. ; Aguilar-Pimentel, J.A. ; Becker, L. ; Beckers, J. ; Busch, D.H. ; Calzada-Wack, J. ; Fuchs, H. ; Gailus-Durner, V. ; Garrett, L. ; Graw, J. ; Götz, A. ; Hans, W. ; Hölter, S.M. ; Horsch, M. ; Klein-Rodewald, T. ; Lengger, C. ; Leuchtenberger, S. ; Maier, H. ; Neff, F. ; Ollert, M. ; Prehn, C. ; Puk, O. ; Rácz, I. ; Rathkolb, B. ; Rozman, J. ; Schrewe, A. ; Schulz, H. ; Stöger, C. ; Tost, M. ; Wurst, W.) ; Hrabě de Angelis, M. ; Heymes, C. ; Rottbauer, W. ; Most, P. ; Hofmann, F. ; Frey, N.

Erratum: Myoscape controls cardiac calcium cycling and contractility via regulation of L-type calcium channel surface expression.

2016 Scientific Article in Analytical Chemistry Anal. Chem. 88, 5871-5878 (2016)

Cassese, A. ; Ellis, S. ; Ogrinc Potočnik, N. ; Burgermeister, E. ; Ebert, M. ; Walch, A.K. ; van den Maagdenberg, A.M. ; McDonnell, L.A. ; Heeren, R.M. ; Balluff, B.

Spatial autocorrelation in mass spectrometry imaging.

Mass spectrometry imaging (MSI) is a powerful molecular imaging technique. In microprobe MSI, images are created through a grid-wise interrogation of individual spots by mass spectrometry across a surface. Classical statistical tests for within-sample comparisons fail as close-by measurement spots violate the assumption of independence of these tests, which can lead to an increased false-discovery rate. For spatial data this effect is referred to as spatial autocorrelation. In this study we investigated spatial autocorrelation in three different matrix-assisted laser desorption/ionization MSI datasets. These datasets cover different molecular classes (metabolites/drugs, lipids, and proteins) and different spatial resolutions ranging from 20 µm to 100 µm. Significant spatial autocorrelation was detected in all three datasets and found to increase with decreasing pixel size. To enable statistical testing for differences in mass signal intensities between regions of interest within MSI datasets, we propose the use of Conditional Autoregressive (CAR) models. We show that by accounting for spatial autocorrelation, discovery rates (i.e. the ratio between the features identified and the total number of features) could be reduced between 21% and 69%. The reliability of this approach was validated by control mass signals based on prior knowledge. In light of the advent of larger MSI datasets based on either an increased spatial resolution or 3D datasets, accounting for effects due to spatial autocorrelation becomes even more indispensable. Here we propose a generic and easily applicable workflow to enable within-sample statistical comparisons.

2016 Scientific Article in PLoS ONE PLoS ONE 11:e0152996 (2016)

Côme, C. ; Cvrljevic, A. ; Khan, M.M. ; Treise, I. ; Adler, T. ; Aguilar-Pimentel, J.A. ; Au-Yeung, B. ; Sittig, E. ; Laajala, T.D. ; Chen, Y. ; Oeder, S. ; Calzada-Wack, J. ; Horsch, M. ; Aittokallio, T. ; Busch, D.H. ; Ollert, M. ; Neff, F. ; Beckers, J. ; Gailus-Durner, V. ; Fuchs, H. ; Hrabě de Angelis, M. ; Chen, Z. ; Lahesmaa, R. ; Westermarck, J.

CIP2A promotes T-cell activation and immune response to Listeria monocytogenes infection.

The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2AHOZ) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2AHOZ mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4+ T-cells and CD8+ effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2AHOZ as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects.

2016 Scientific Article in Nature Communications Nat. Commun. 7:11317 (2016)

Eden, M. ; Meder, B. ; Völkers, M. ; Poomvanicha, M. ; Domes, K. ; Branchereau, M. ; Marck, P. ; Will, R. ; Bernt, A. ; Rangrez, A. ; Busch, M. ; German Mouse Clinic Consortium (Adler, T. ; Aguilar-Pimentel, J.A. ; Becker, L. ; Beckers, J. ; Busch, D.H. ; Calzada-Wack, J. ; Fuchs, H. ; Gailus-Durner, V. ; Garrett, L. ; Graw, J. ; Götz, A. ; Hans, W. ; Hölter, S.M. ; Horsch, M. ; Klein-Rodewald, T. ; Lengger, C. ; Leuchtenberger, S. ; Maier, H. ; Neff, F. ; Ollert, M. ; Prehn, C. ; Puk, O. ; Rácz, I. ; Rathkolb, B. ; Rozman, J. ; Schrewe, A. ; Schulz, H. ; Stöger, C. ; Tost, M. ; Wurst, W. ; Frey, N.) ; Hrabě de Angelis, M. ; Heymes, C. ; Rottbauer, W. ; Most, P. ; Hofmann, F.

Myoscape controls cardiac calcium cycling and contractility via regulation of L-type calcium channel surface expression.

Calcium signalling plays a critical role in the pathogenesis of heart failure. Here we describe a cardiac protein named Myoscape/FAM40B/STRIP2, which directly interacts with the L-type calcium channel. Knockdown of Myoscape in cardiomyocytes decreases calcium transients associated with smaller Ca(2+) amplitudes and a lower diastolic Ca(2+) content. Likewise, L-type calcium channel currents are significantly diminished on Myoscape ablation, and downregulation of Myoscape significantly reduces contractility of cardiomyocytes. Conversely, overexpression of Myoscape increases global Ca(2+) transients and enhances L-type Ca(2+) channel currents, and is sufficient to restore decreased currents in failing cardiomyocytes. In vivo, both Myoscape-depleted morphant zebrafish and Myoscape knockout (KO) mice display impairment of cardiac function progressing to advanced heart failure. Mechanistically, Myoscape-deficient mice show reduced L-type Ca(2+)currents, cell capacity and calcium current densities as a result of diminished LTCC surface expression. Finally, Myoscape expression is reduced in hearts from patients suffering of terminal heart failure, implying a role in human disease.

2016 Scientific Article in Analytical Chemistry Anal. Chem. 88, 5281-5289 (2016)

Buck, A. ; Balluff, B. ; Voss, A. ; Langer, R. ; Zitzelsberger, H. ; Aichler, M. ; Walch, A.K.

How suitable is MALDI-TOF for metabolite imaging from clinical formalin-fixed and paraffin-embedded tissue samples in comparison to MALDI-FT-ICR mass spectrometry?

In research and clinical settings formalin-fixed and paraffin-embedded (FFPE) tissue specimens are collected routinely and therefore this material constitutes a highly valuable source to gather insight in metabolic changes of diseases. Among mass spectrometry techniques to examine the molecular content of FFPE tissue mass spectrometry imaging (MSI) is the most appropriate when morphological and histological features shall be related to metabolic information. Currently, high resolution mass spectrometers are widely used for metabolomics studies. However, with regards to matrix-assisted laser desorption/ionization (MALDI) MSI no study has so far addressed the necessity of instrumental mass resolving power in terms of clinical diagnosis and prognosis using archived FFPE tissue. For this matter we performed for the first time a comprehensive comparison between a high mass resolution Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer and a time-of-flight (TOF) instrument with lower mass resolving power. Spectra analysis revealed that about one third of the detected peaks remained unresolved by MALDI-TOF which led to a three to five time lower number of m/z features compared to FT-ICR measurements. Overlaid peak information and background noise in TOF images made a precise assignment of molecular attributes to morphological features more difficult and limited classification approaches. This clearly demonstrates the need for high mass resolution capabilities for metabolite imaging. Nevertheless, MALDI-TOF allowed reproducing and verifying individual markers identified previously by MALDI-FT-ICR MSI. The systematic comparison gives rise to synergistically combine the different MSI platforms for high-throughput discovery and validation of biomarkers.

2016 Scientific Article in American Journal of Cancer Research Am. J. Cancer Res. 6, 61-70 (2016)

Dorn, J. ; Yassouridis, A. ; Walch, A.K. ; Diamandis, E.P. ; Schmitt, M. ; Kiechle, M. ; Wang, P. ; Drecoll, E. ; Schmalfeldt, B. ; Loessner, D. ; Kotzsch, M. ; Magdolen, V.

Assessment of kallikrein-related peptidase 5 (KLK5) protein expression in tumor tissue of advanced ovarian cancer patients by immunohistochemistry and ELISA: Correlation with clinical outcome.

Members of the human kallikrein-related peptidase (KLK) family, including KLK5, have been reported to play an important role in ovarian cancer progression. In the present study, we assessed KLK5 protein expression in ovarian cancer tissues by immunohistochemistry (IHC) and ELISA, and analyzed its association with clinicopathologic parameters and disease outcome in 95 patients with advanced ovarian cancer FIGO stage III/IV. KLK5 immunoexpression was evaluated in ovarian cancer tissue microarrays by IHC using a manual semiquantitative scoring system. KLK5 antigen levels were determined in ovarian cancer tumour tissue extracts by ELISA. KLK5 protein is expressed in ovarian cancer tissue by stromal and tumor cells. Mean KLK5 immunoscore values in tumor cells (KLK5-Tc; 5.7, range 0 to 12) were higher compared to stromal cells (KLK5-Sc; 1.2, range 0 to 9) but the correlation between KLK5-Tc and KLK5-Sc was rather low (rs = 0.34, P < 0.05). No significant associations of clinicopathological parameters with KLK5-Tc, KLK5-Sc, the combined overall score KLK5-Tc+Sc, or ELISA (KLK5-E) expression values were determined, except for KLK5-E protein expression with advanced age and high nuclear grade (G3). In univariate Cox regression analysis, elevated expression levels of KLK5-Sc are significantly linked with both prolonged overall survival (OS) (hazard ratio [HR] = 0.6, P = 0.046) and progression-free survival (PFS) (HR = 0.54, P = 0.032) of advanced ovarian cancer patients. KLK5-Tc and KLK5-Tc+Sc scores as well as the KLK5-E values were not associated with patients' outcome. In multivariable analysis, KLK5-Sc expression was found to be statistically significant for PFS. Patients with elevated KLK5-Sc had a two-fold lower risk of disease recurrence (HR = 0.53, P = 0.037) as compared to patients with low KLK5-Sc. For KLK5-Sc and OS, a trend towards statistical significance was observed (HR = 0.62, P = 0.077). These results indicate that KLK5 overexpression by stromal cells (KLK5-Sc) may be a positive modulator lowering aggressiveness of ovarian cancer.

2016 Scientific Article in Journal of Trace Elements in Medicine and Biology J. Trace Elem. Med. Biol. 35, 97-102 (2016)

Hachmöller, O. ; Aichler, M. ; Schwamborn, K. ; Lutz, L. ; Werner, M. ; Sperling, M. ; Walch, A.K. ; Karst, U.

Element bioimaging of liver needle biopsy specimens from patients with Wilson's disease by laser ablation-inductively coupled plasma-mass spectrometry.

A laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method is developed and applied for the analysis of paraffin-embedded liver needle biopsy specimens of patients with Wilson's disease (WD), a rare autosomal recessive disorder of the copper metabolism causing various hepatic, neurological and psychiatric symptoms due to a copper accumulation in the liver and the central nervous system. The sample set includes two WD liver samples and one negative control sample. The imaging analysis was performed with a spatial resolution of 10 μm. Besides copper, iron was monitored because an elevated iron concentration in the liver is known for WD. In addition to this, both elements were quantified using an external calibration based on matrix-matched gelatine standards. The presented method offers low limits of detection of 1 and 5 μg/g for copper and iron, respectively. The high detection power and good spatial resolution allow the analysis of small needle biopsy specimen using this method. The two analyzed WD samples can be well differentiated from the control sample due to their inhomogeneous copper distribution and high copper concentrations of up to 1200μg/g. Interestingly, the WD samples show an inverse correlation of regions with elevated copper concentrations and regions with high iron concentrations.

2016 Scientific Article in Metallomics Metallomics 8, 648-653 (2016)

Hachmöller, O. ; Buzanich, A.G. ; Aichler, M. ; Radtke, M. ; Dietrich, D. ; Schwamborn, K. ; Lutz, L. ; Werner, M. ; Sperling, M. ; Walch, A.K. ; Karst, U.

Elemental bioimaging and speciation analysis for the investigation of Wilson's disease using μXRF and XANES.

A liver biopsy specimen from a Wilson's disease (WD) patient was analyzed by means of micro-X-ray fluorescence (μXRF) spectroscopy to determine the elemental distribution. First, bench-top μXRF was utilized for a coarse scan of the sample under laboratory conditions. The resulting distribution maps of copper and iron enabled the determination of a region of interest (ROI) for further analysis. In order to obtain more detailed elemental information, this ROI was analyzed by synchrotron radiation (SR)-based μXRF with a beam size of 4 μm offering a resolution at the cellular level. Distribution maps of additional elements to copper and iron like zinc and manganese were obtained due to a higher sensitivity of SR-μXRF. In addition to this, X-ray absorption near edge structure spectroscopy (XANES) was performed to identify the oxidation states of copper in WD. This speciation analysis indicated a mixture of copper(i) and copper(ii) within the WD liver tissue.

2016 Scientific Article in Journal of Proteome Research J. Proteome Res. 15, 1350-1359 (2016)

Ly, A.# ; Merl-Pham, J.# ; Priller, M. ; Gruhn, F. ; Senninger, N. ; Ueffing, M. ; Hauck, S.M.

Proteomic profiling suggests central role of STAT signaling during retinal degeneration in the rd10 mouse model.

The rd10 mouse is a model of retinitis pigmentosa characterized by the dysfunction of a rod-photoreceptor-specific phosphodiesterase. Compared to the rd1 mouse, retinal degeneration in the rd10 mouse begins later in age with a milder phenotype, making it ideal for investigating cell death and neuroprotective mechanisms. Alterations in the rd10 retina proteome at pre-, peak-, and post-degenerative time points were examined using a modified high-recovery filter-aided sample preparation (FASP) method in combination with label-free quantitative mass spectrometry, generating a proteomic dataset on almost 3000 proteins. Our data confirmed a period of protein expression similar to age-matched wild-type mice pre-degeneration, with decreases in proteins associated with phototransduction and increases in signaling proteins at peak- and post-degenerative stages. 57 proteins were differentially expressed in the rd10 retinae during peak-degeneration compared to wild-type mice after stringent FDR correction (q<0.05). Network analysis separated these proteins into a cluster of downregulated photoreceptor proteins, and one of upregulated signaling proteins centered around GFAP, STAT3, and STAT1. This is the first study to identify alterations in STAT1 in the rd10 mouse, which were confirmed with gene expression and immunoblotting experiments underpinning the efficacy of our approach. This unique proteomic dataset on protein dynamics during retinal degeneration could serve as an information source for vision research in the future.

2016 Scientific Article in Molecular Cancer Therapeutics Mol. Cancer Ther. 15, 1145-1152 (2016)

Grüner, B.M. ; Winkelmann, I. ; Feuchtinger, A. ; Sun, N. ; Balluff, B. ; Teichmann, N. ; Herner, A. ; Kalideris, E. ; Steiger, K. ; Braren, R. ; Aichler, M. ; Esposito, I. ; Schmid, R.M. ; Walch, A.K. ; Siveke, J.T.

Modeling therapy response and spatial tissue distribution of erlotinib in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is likely the most aggressive and therapy-resistant of all cancers. Aim of this study was to investigate the emerging technology of matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) as a powerful tool to study drug delivery and spatial tissue distribution in PDAC. We utilized an established genetically engineered mouse model of spontaneous PDAC to examine the distribution of the small molecule inhibitor erlotinib in healthy pancreas and PDAC. MALDI IMS was utilized on sections of single-dose or long-term-treated mice to measure drug tissue distribution. Histological and statistical analyses were performed to correlate morphology, drug distribution and survival. We found that erlotinib levels were significantly lower in PDAC compared to healthy tissue (p = 0.0078). Survival of long-term-treated mice did not correlate with overall levels of erlotinib or with overall histological tumor grade but both with the percentage of atypical glands in the cancer (p = 0.021, rs = 0.59) and the level of erlotinib in those atypical glands (p = 0.019, rs = 0.60). The results of this pilot study present MALDI IMS as a reliable technology to study drug delivery and spatial distribution compounds in a preclinical setting and supports drug imaging-based translational approaches.

2016 Scientific Article in Expert Review of Proteomics Expert Rev. Proteomics 13, 275-284 (2016)

Lahiri, S. ; Sun, N. ; Buck, A. ; Imhof, A. ; Walch, A.K.

MALDI imaging mass spectrometry as a novel tool for detecting histone modifications in clinical tissue samples.

Histone post-translational modifications (PTMs), histone variants and enzymes responsible for the incorporation or the removal of the PTMs are being increasingly associated with human disease. Combinations of histone PTMs and the specific incorporation of variants contribute to the establishment of cellular identity and hence are potential markers that could be exploited in disease diagnostics and prognostics and therapy response prediction. Due to the scarcity of suitable antibodies and the pre-requirement of tissue homogenization for more advanced analytical techniques, comprehensive information regarding the spatial distribution of these factors at the tissue level has been lacking. MALDI imaging mass spectrometry provides an ideal platform to measure histone PTMs and variants from tissues while maintaining the information about their spatial distribution. Discussed in this review are the relevance of histones in the context of human disease and the contribution of MALDI imaging mass spectrometry in measuring histones in situ.

2016 Scientific Article in Cancer Medicine Cancer Med. 5, 703-710 (2016)

Falkenberg, N.# ; Höfig, I.# ; Rosemann, M. ; Szumielewski, J. ; Richter, S. ; Schorpp, K.K. ; Hadian, K. ; Aubele, M. ; Atkinson, M.J. ; Anastasov, N.

Three-dimensional microtissues essentially contribute to preclinical validations of therapeutic targets in breast cancer.

A 3D microtissues using T47D and JIMT-1 cells were generated to analyze tissue-like response of breast cancer cells after combined human epidermal growth factor receptor 2 (HER2)-targeted treatment and radiation. Following lentiviral knockdown of HER2, we compared growth rate alterations using 2D monolayers, 3D microtissues, and mouse xenografts. Additionally, to model combined therapeutic strategies, we treated HER2-depleted T47D cells and 3D microtissues using trastuzumab (anti-HER2 antibody) in combination with irradiation. Comparison of HER2 knockdown with corresponding controls revealed growth impairment due to HER2 knockdown in T47D 2D monolayers, 3D microtissues, and xenografts (after 2, 12, and ≥40 days, respectively). In contrast, HER2 knockdown was less effective in inhibiting growth of trastuzumab-resistant JIMT-1 cells in vitro and in vivo. Combined administration of trastuzumab and radiation treatment was also analyzed using T47D 3D microtissues. Administration of both, radiation (5 Gy) and trastuzumab, significantly enhanced the growth inhibiting effect in 3D microtissues. To improve the predictive power of potential drugs-as single agents or in combination-here, we show that regarding tumor growth analyses, 3D microtissues are highly comparable to outcomes derived from xenografts. Considering increased limitations for animal experiments on the one hand and strong need of novel drugs on the other hand, it is indispensable to include highly reproducible 3D microtissue platform in preclinical analyses to validate more accurately the capacity of future drug-combined radiotherapy.

2016 Scientific Article in International Journal of Radiation Oncology, Biology, Physics Int. J. Radiat. Oncol. Biol. Phys. 95, 234-241 (2016)

Girst, S. ; Greubel, C. ; Reindl, J. ; Siebenwirth, C. ; Zlobinskaya, O. ; Walsh, D.W. ; Ilicic, K. ; Aichler, M. ; Walch, A.K. ; Wilkens, J.J. ; Multhoff, G. ; Dollinger, G. ; Schmid, T.E.

Proton minibeam radiation therapy reduces side effects in an in vivo mouse ear model.

PURPOSE: Proton minibeam radiation therapy is a novel approach to minimize normal tissue damage in the entrance channel by spatial fractionation while keeping tumor control through a homogeneous tumor dose using beam widening with an increasing track length. In the present study, the dose distributions for homogeneous broad beam and minibeam irradiation sessions were simulated. Also, in an animal study, acute normal tissue side effects of proton minibeam irradiation were compared with homogeneous irradiation in a tumor-free mouse ear model to account for the complex effects on the immune system and vasculature in an in vivo normal tissue model. METHODS AND MATERIALS: At the ion microprobe SNAKE, 20-MeV protons were administered to the central part (7.2 × 7.2 mm(2)) of the ear of BALB/c mice, using either a homogeneous field with a dose of 60 Gy or 16 minibeams with a nominal 6000 Gy (4 × 4 minibeams, size 0.18 × 0.18 mm(2), with a distance of 1.8 mm). The same average dose was used over the irradiated area. RESULTS: No ear swelling or other skin reactions were observed at any point after minibeam irradiation. In contrast, significant ear swelling (up to fourfold), erythema, and desquamation developed in homogeneously irradiated ears 3 to 4 weeks after irradiation. Hair loss and the disappearance of sebaceous glands were only detected in the homogeneously irradiated fields. CONCLUSIONS: These results show that proton minibeam radiation therapy results in reduced adverse effects compared with conventional homogeneous broad-beam irradiation and, therefore, might have the potential to decrease the incidence of side effects resulting from clinical proton and/or heavy ion therapy.

2016 Scientific Article in Histochemistry and Cell Biology Histochem. Cell Biol. 145, 201-211 (2016)

Sun, N. ; Fernandez, I.E. ; Wei, M. ; Wu, Y. ; Aichler, M. ; Eickelberg, O.&deg ; Walch, A.K.&deg

Pharmacokinetic and pharmacometabolomic study of pirfenidone in normal mouse tissues using high mass resolution MALDI-FTICR-mass spectrometry imaging.

Given the importance of pirfenidone as the first worldwide-approved drug for idiopathic pulmonary fibrosis treatment, its pharmacodynamic properties and the metabolic response to pirfenidone treatment have not been fully elucidated. The aim of the present study was to get molecular insights of pirfenidone-related pharmacometabolomic response using MALDI-FTICR-MSI. Quantitative MALDI-FTICR-MSI was carried out for determining the pharmacokinetic properties of pirfenidone and its related metabolites 5-hydroxymethyl pirfenidone and 5-carboxy pirfenidone in lung, liver and kidney. To monitor the effect of pirfenidone administration on endogenous cell metabolism, additional in situ endogenous metabolite imaging was performed in lung tissue sections. While pirfenidone is highly abundant and delocalized across the whole micro-regions of lung, kidney and liver, 5-hydroxymethyl pirfenidone and 5-carboxy pirfenidone demonstrate heterogeneous distribution patterns in lung and kidney. In situ endogenous metabolite imaging study of lung tissue indicates no significant effects of pirfenidone on metabolic pathways. Remarkably, we found 129 discriminative m/z values which represent clear differences between control and treated lungs, the majority of which are currently unknown. PCA analysis and heatmap view can accurately distinguish control and treated groups. This is the first pharmacokinetic study to investigate the tissue distribution of orally administered pirfenidone and its related metabolites simultaneously in organs without labeling. The combination of pharmametabolome with histological features provides detailed mapping of drug effects on metabolism as response of heathy lung tissue to pirfenidone treatment.

2016 Scientific Article in OncoTarget Oncotarget 7, 1451-1463 (2016)

Hensel, T. ; Giorgi, C. ; Schmidt, O. ; Calzada-Wack, J. ; Neff, F. ; Buch, T. ; Niggli, F.K. ; Schäfer, B.W. ; Burdach, S. ; Richter, G.H.

Targeting the EWS-ETS transcriptional program by BET bromodomain inhibition in Ewing sarcoma.

Ewing sarcomas (ES) are highly malignant bone or soft tissue tumors. Genetically, ES are defined by balanced chromosomal EWS/ETS translocations, which give rise to chimeric proteins (EWS-ETS) that generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome. By use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in a dose dependent manner. This was further enhanced by co-treatment with an inhibitor of the PI3K pathway. Microarray analysis further revealed JQ1 treatment to block a typical ES associated expression program. The effect on this expression program was mimicked by RNA interference with BRD3 or BRD4 expression, indicating that the EWS-FLI1 mediated expression profile is at least in part mediated via such epigenetic readers. Consequently, contact dependent and independent proliferation of different ES lines was strongly inhibited. Mechanistically, treatment of ES resulted in a partial arrest of the cell cycle as well as induction of apoptosis. Tumor development was suppressed dose dependently in a xeno-transplant model in immune deficient mice, overall indicating that ES may be susceptible to treatment with epigenetic inhibitors blocking BET bromodomain activity and the associated pathognomonic EWS-ETS transcriptional program.

2016 Scientific Article in Proteomics Proteomics 16, 437-447 (2016)

Lahiri, S. ; Sun, N. ; Solis-Mezarino, V. ; Fedisch, A. ; Ninkovic, J. ; Feuchtinger, A. ; Götz, M. ; Walch, A.K. ; Imhof, A.

In situ detection of histone variants and modifications in mouse brain using imaging mass spectrometry.

Histone posttranslational modifications and histone variants control the epigenetic regulation of gene expression and affect a wide variety of biological processes. A complex pattern of such modifications and variants defines the identity of cells within complex organ systems and can therefore be used to characterize cells at a molecular level. However, their detection and identification in situ has been limited so far due to lack of specificity, selectivity and availability of anti-histone antibodies. Here, we describe a novel MALDI imaging mass spectrometry (MALDI-IMS) based workflow, which enables us to detect and characterize histones by their intact mass and their correlation with cytological properties of the tissue using novel statistical and image analysis tools. The workflow allows us to characterize the in situ distribution of the major histone variants and their modification in the mouse brain. This new analysis tool is particularly useful for the investigation of expression patterns of the linker histone H1 variants for which suitable antibodies are so far not available.

2016 Scientific Article in Clinical Neuroradiology Clin. Neuroradiol. 26, 189-197 (2016)

Boeckh-Behrens, T. ; Schubert, M.I. ; Forschler, A. ; Prothmann, S. ; Kreiser, K. ; Zimmer, C. ; Riegger, J. ; Bauer, J. ; Neff, F. ; Kehl, V. ; Pelisek, J. ; Schirmer, L. ; Mehr, M. ; Poppert, H.

The impact of histological clot composition in embolic stroke.

Purpose: Thrombus composition has been suggested to have a decisive impact on the outcome of patients treated by mechanical thrombectomy because of embolic stroke. The recent development of stent retrievers allows collection and, hence, histopathological analysis of fresh thrombus material. Against this background, the aim of this prospective study was to assess the impact of thrombus composition on mechanical recanalization, clinical outcome and stroke etiology. Methods: Thirty-four patients suffering from acute ischemic stroke due to occlusion of the distal internal carotid artery/carotid-T, anterior cerebral artery, or middle cerebral arteries were mechanically recanalized, and thrombus material was obtained. Histological thrombus composition was compared with imaging, clinical, and neurointerventional data. Results: The main findings were that a higher percentage of white blood cells (WBCs) in the thrombus was associated with (i) cardioembolic etiology, (ii) extended mechanical recanalization time, and (iii) less favorable recanalization (Thrombolysis in Cerebral Infarction score) and clinical outcome (National Institute of Health Stroke Scale). Conclusion: Our results suggest that thrombi with a high WBC fraction are related to more organized thrombi of cardioembolic origin associated with less favorable recanalization and clinical outcome in acute ischemic anterior circulation stroke. WBC-mediated immunological and coagulatory processes may play a key role in thrombus formation and pathogenesis of stroke warranting further investigation.

München, Technische Universität, Fakultät für Medizin, Diss., 2015, 158 S.

Leinhäuser, I.

The bone morphogenetic protein 7 (BMP7) plays a pro-tumorigenic role in pheochromocytoma.

This thesis showed that in most of human pheochromocytomas BMP7, a secreted growth factor of the TGF-beta superfamily, is overexpressed and this positively correlates with features suggestive of higher malignant potential. BMP7 promotes proliferation, migration, and invasion of pheochromocytoma cells by signaling through the PI3K/AKT/mTOR pathway which in turn causes the upregulation of integrin beta 1 expression. The blockage of active Bmp7 pathway efficiently reduced the proliferation and induced apoptosis of pheochromocytoma cells, thereby representing a novel therapeutic strategy for treating pheochromocytomas.

2015 Scientific Article in Mammalian Genome Mamm. Genome 27, 17-28 (2015)

Bönisch, C. ; Irmler, M. ; Brachthäuser, L. ; Neff, F. ; Bamberger, M.T. ; Marschall, S. ; Hrabě de Angelis, M. ; Beckers, J.

Dexamethasone treatment alters insulin, leptin, and adiponectin levels in male mice as observed in DIO but does not lead to alterations of metabolic phenotypes in the offspring.

Epigenetic inheritance (EI) of metabolic phenotypes via the paternal lineage has been shown in rodent models of diet-induced obesity (DIO). However, the factors involved in soma-to-germline information transfer remain elusive. Here, we address the role of alterations in insulin, leptin, and adiponectin levels for EI of metabolic phenotypes by treating C57BL/6NTac male mice (F0) with the synthetic glucocorticoid dexamethasone and generating offspring (F1) either by in vitro fertilization or by natural fecundation. Dexamethasone treatment slightly alters F0 body composition by increasing fat mass and decreasing lean mass, and significantly improves glucose tolerance. Moreover, it increases insulin and leptin levels and reduces adiponectin levels in F0 fathers as observed in mouse models of DIO. However, these paternal changes of metabolic hormones do not alter metabolic parameters, such as body weight, body composition and glucose homeostasis in male and female F1 mice even when these are challenged with a high-fat diet. Accordingly, sperm transcriptomes are not altered by dexamethasone treatment. Our results suggest that neither increased glucocorticoid, insulin, and leptin levels, nor decreased adiponectin levels in fathers are sufficient to confer soma-to-germline information transfer in EI of obesity via the paternal lineage.

2015 Scientific Article in Nature Communications Nat. Commun. 6:8940 (2015)

Kovac, M. ; Blattmann, C. ; Ribi, S. ; Smida, J. ; Müller, N.S. ; Engert, F. ; Castro-Giner, F. ; Weischenfeldt, J. ; Kovacova, M. ; Krieg, A. ; Andreou, D. ; Tunn, P.U. ; Dürr, H.R. ; Rechl, H.P. ; Schaser, K.D. ; Melcher, I. ; Burdach, S. ; Kulozik, A. ; Specht, K. ; Heinimann, K. ; Fulda, S. ; Bielack, S. ; Jundt, G. ; Tomlinson, I. ; Korbel, J.O. ; Nathrath, M. ; Baumhoer, D.

Exome sequencing of osteosarcoma reveals mutation signatures reminiscent of BRCA deficiency.

Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which has historically complicated driver gene discovery. Here we sequence exomes of 31 tumours and decipher their evolutionary landscape by inferring clonality of the individual mutation events. Exome findings are interpreted in the context of mutation and SNP array data from a replication set of 92 tumours. We identify 14 genes as the main drivers, of which some were formerly unknown in the context of osteosarcoma. None of the drivers is clearly responsible for the majority of tumours and even TP53 mutations are frequently mapped into subclones. However, >80% of osteosarcomas exhibit a specific combination of single-base substitutions, LOH, or large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours. Our findings imply that multiple oncogenic pathways drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits, which could be therapeutically exploited.

2015 Scientific Article in Endocrine Endocrine 51, 236-244 (2015)

Naves, L.A. ; Daly, A.F. ; Dias, L.A. ; Yuan, B. ; Zakir, J.C. ; Barra, G.B. ; Palmeira, L. ; Villa, C. ; Trivellin, G. ; Júnior, A.J. ; Neto, F.F. ; Liu, P.P. ; Pellegata, N.S. ; Stratakis, C.A. ; Lupski, J.R. ; Beckers, A.

Aggressive tumor growth and clinical evolution in a patient with X-linked acro-gigantism syndrome.

X-linked acro-gigantism (X-LAG) syndrome is a newly described disease caused by microduplications on chromosome Xq26.3 leading to copy number gain of GPR101. We describe the clinical progress of a sporadic male X-LAG syndrome patient with an Xq26.3 microduplication, highlighting the aggressive natural history of pituitary tumor growth in the absence of treatment. The patient first presented elsewhere aged 5 years 8 months with a history of excessive growth for >2 years. His height was 163 cm, his weight was 36 kg, and he had markedly elevated GH and IGF-1. MRI showed a non-invasive sellar mass measuring 32.5 × 23.9 × 29.1 mm. Treatment was declined and the family was lost to follow-up. At the age of 10 years and 7 months, he presented again with headaches, seizures, and visual disturbance. His height had increased to 197 cm. MRI showed an invasive mass measuring 56.2 × 58.1 × 45.0 mm, with compression of optic chiasma, bilateral cavernous sinus invasion, and hydrocephalus. His thyrotrope, corticotrope, and gonadotrope axes were deficient. Surgery, somatostatin analogs, and cabergoline did not control vertical growth and pegvisomant was added, although vertical growth continues (currently 207 cm at 11 years 7 months of age). X-LAG syndrome is a new genomic disorder in which early-onset pituitary tumorigenesis can lead to marked overgrowth and gigantism. This case illustrates the aggressive nature of tumor evolution and the challenging clinical management in X-LAG syndrome.

München, Technische Universität, Fakultät für Medizin, Diss., 2015, 107 S.

Upheber, S.

Die Integrin αvβ3-vermittelte Migration humaner Ovarialkarzinomzellen als Funktion des Metastasierungssuppressors KAI1 (CD82) und seiner Splice-Variante sowie deren Interaktion mit dem Zell/Zell-Adhäsionsmolekül E-Cadherin.

Das Tetraspanin KAI1 wurde in verschiedenen soliden Tumoren als Metastasierungssuppressor identifiziert. Darüber hinaus wurde eine Splice-Variante (KAI1-Splice) beschrieben, deren Expression jedoch mit einer schlechten Prognose von Tumorpatienten assoziiert ist. In der vorliegenden Arbeit wurde die Funktion von KAI1 im Vergleich zu KAI1-Splice in kultivierten humanen Ovarialkarzinomzellen untersucht. In vitro-Zellmigrationsstudien zeigten, dass KAI1 die Integrin alphavbeta3-vermittelte Zellmigration erniedrigte, während KAI1-Splice sich motilitätssteigernd auswirkte. Außerdem war die Interaktion von KAI1-Splice mit dem Zell/Zell-Adhäsionsmolekül E-Cadherin vermindert.

2015 Scientific Article in Diabetes Diabetes 65, 406-420 (2015)

Wiedemann, T. ; Bielohuby, M. ; Müller, T.D. ; Bidlingmaier, M. ; Pellegata, N.S.

Obesity in MENX rats is accompanied by high circulating levels of ghrelin and improved insulin sensitivity.

Ghrelin, the natural ligand of the growth hormone secretagogue receptor (GHS-R1a), is mainly secreted from the stomach and regulates food intake and energy homeostasis. p27 regulates cell cycle progression in many cell types. Here, we report that rats affected by the multiple endocrine neoplasia syndrome MENX, caused by a p27 mutation, develop pancreatic islet hyperplasia containing elevated numbers of ghrelin-producing epsilon cells. The metabolic phenotype of MENX-affected rats featured high endogenous acylated and unacylated plasma ghrelin levels. Supporting increased ghrelin action, MENX rats show increased food intake, enhanced body fat mass, and elevated plasma levels of triglycerides and cholesterol. Ghrelin effect on food intake was confirmed by treating MENX rats with a GHS-R1a antagonist. At 7,5 months, MENX-affected rats show decreased mRNA levels of hypothalamic GHS-R1a, neuropeptide Y (NPY), and agouti-related protein (AgRP), suggesting that prolonged hyperghrelinemia may lead to decreased ghrelin efficacy. In line with ghrelin's proposed role in glucose metabolism, we find decreased glucose-stimulated insulin secretion (GSIS) in MENX rats while insulin sensitivity is improved. In summary, we provide a novel, non-transgenic rat model with high endogenous ghrelin plasma levels and interestingly, improved glucose tolerance. This model might aid in identifying new therapeutic approaches for obesity and obesity-related diseases including type-2 diabetes.

2015 Scientific Article in Journal of Proteome Research J. Proteome Res. 14, 4674-4686 (2015)

Kempf, S.J. ; Sepe, S. ; von Toerne, C. ; Janik, D. ; Neff, F. ; Hauck, S.M. ; Atkinson, M.J. ; Mastroberardino, P.G. ; Tapio, S.

Neonatal irradiation leads to persistent proteome alterations involved in synaptic plasticity in the mouse hippocampus and cortex.

Recent epidemiological data indicate that radiation doses as low as those used in computer tomography may result in long-term neurocognitive side effects. The aim of this study was to elucidate long-term molecular alterations related to memory formation in the brain after low and moderate doses of gamma radiation. Female C57BL/6J mice were irradiated on postnatal day 10 with total body doses of 0.1 Gy, 0.5 Gy or 2.0 Gy; the control group was sham-irradiated. The proteome analysis of hippocampus, cortex and synaptosomes isolated from these brain regions indicated changes in ephrin-related, RhoGDI and axonal guidance signalling.. Immunoblotting and miRNA-quantification demonstrated an imbalance in the synapse morphology-related Rac1-Cofilin pathway and long-term potentiation-related CREB signalling. Proteome profiling also showed impaired oxidative phosphorylation, especially in the synaptic mitochondria. This was accompanied by an early (4 weeks) reduction of mitochondrial respiration capacity in the hippocampus. Although the respiratory capacity was restored by 24 weeks, the number of deregulated mitochondrial complex proteins was increased at this time. All observed changes were significant at doses of 0.5 Gy and 2.0 Gy but not at 0.1 Gy. This study strongly suggests that ionising radiation at the neonatal state triggers persistent proteomic alterations associated with synaptic impairment.

2015 Scientific Article in Biomedical Optics Express Biomed. Opt. Express 6, 3134-3148 (2015)

Chekkoury, A. ; Gateau, J. ; Driessen, W.H.P. ; Symvoulidis, P. ; Bézière, N. ; Feuchtinger, A. ; Walch, A.K. ; Ntziachristos, V.

Optical mesoscopy without the scatter: Broadband multispectral optoacoustic mesoscopy.

Optical mesoscopy extends the capabilities of biological visualization beyond the limited penetration depth achieved by microscopy. However, imaging of opaque organisms or tissues larger than a few hundred micrometers requires invasive tissue sectioning or chemical treatment of the specimen for clearing photon scattering, an invasive process that is regardless limited with depth. We developed previously unreported broadband optoacoustic mesoscopy as a tomographic modality to enable imaging of optical contrast through several millimeters of tissue, without the need for chemical treatment of tissues. We show that the unique combination of three-dimensional projections over a broad 500 kHz-40 MHz frequency range combined with multi-wavelength illumination is necessary to render broadband multispectral optoacoustic mesoscopy (2B-MSOM) superior to previous optical or optoacoustic mesoscopy implementations.

2015 Scientific Article in Cell Metabolism Cell Metab. 22, 838-850 (2015)

Pfluger, P.T. ; Kabra, D.G. ; Aichler, M. ; Schriever, S.C. ; Pfuhlmann, K. ; Casquero García, V. ; Lehti, M. ; Weber, J. ; Kutschke, M. ; Rozman, J. ; Elrod, J.W. ; Hevener, A.L. ; Feuchtinger, A. ; Hrabě de Angelis, M. ; Walch, A.K. ; Rollmann, S.M. ; Aronow, B.J. ; Müller, T.D. ; Perez-Tilve, D. ; Jastroch, M. ; de Luca, M. ; Molkentin, J.D. ; Tschöp, M.H.

Calcineurin links mitochondrial elongation with energy metabolism.

Canonical protein phosphatase 3/calcineurin signaling is central to numerous physiological processes. Here we provide evidence that calcineurin plays a pivotal role in controlling systemic energy and body weight homeostasis. Knockdown of calcineurin in Drosophila melanogaster led to a decrease in body weight and energy stores, and increased energy expenditure. In mice, global deficiency of catalytic subunit Ppp3cb, and tissue-specific ablation of regulatory subunit Ppp3r1 from skeletal muscle, but not adipose tissue or liver, led to protection from high-fat-diet-induced obesity and comorbid sequelæ. Ser637 hyperphosphorylation of dynamin-related protein 1 (Drp1) in skeletal muscle of calcineurin-deficient mice was associated with mitochondrial elongation into power-cable-shaped filaments and increased mitochondrial respiration, but also with attenuated exercise performance. Our data suggest that calcineurin acts as highly conserved pivot for the adaptive metabolic responses to environmental changes such as high-fat, high-sugar diets or exercise.

2015 Scientific Article in OncoTarget Oncotarget 6, 39111-39126 (2015)

Leinhäuser, I. ; Richter, A. ; Lee, M.S. ; Höfig, I. ; Anastasov, N. ; Fend, F. ; Ercolino, T. ; Mannelli, M. ; Gimenez-Roqueplo, A.P. ; Robledo, M. ; de Krijger, R.R. ; Beuschlein, F. ; Atkinson, M.J. ; Pellegata, N.S.

Oncogenic features of the Bone Morphogenic Protein 7 (BMP7) in pheochromocytoma.

BMP7 is a growth factor playing pro- or anti-oncogenic roles in cancer in a cell type-dependent manner. We previously reported that the BMP7 gene is overexpressed in pheochromocytomas (PCCs) developing in MENX-affected rats and human patients. Here, analyzing a large cohort of PCC patients, we found that 72% of cases showed elevated levels of the BMP7 protein. To elucidate the role of BMP7 in PCC, we modulated its levels in PCC cell lines (overexpression in PC12, knockdown in MPC and MTT cells) and conducted functional assays. Active BMP signaling promoted cell proliferation, migration, and invasion, and sustained survival of MENX rat primary PCC cells. In PCC, BMP7 signals through the PI3K/AKT/mTOR pathway and causes integrin β1 up-regulation. Silencing integrin β1 in PC12 cells suppressed BMP7-mediated oncogenic features. Treatment of MTT cells with DMH1, a novel BMP antagonist, suppressed proliferation and migration. To verify the clinical applicability of our findings, we evaluated a dual PI3K/mTOR inhibitor (NVP-BEZ235) in MENX-affected rats in vivo. PCCs treated with NVP-BEZ235 had decreased proliferation and integrin β1 levels, and higher apoptosis. Altogether, BMP7 activates pro-oncogenic pathways in PCC. Downstream effectors of BMP7-mediated signaling may represent novel targets for treating progressive/inoperable PCC, still orphan of effective therapy.

2015 Scientific Article in Journal of Visualized Experiments J. Vis. Exp. 102:e52907 (2015)

Ratschiller, T. ; Deutsch, M.A. ; Calzada-Wack, J. ; Neff, F. ; Roesch, C. ; Guenzinger, R. ; Lange, R. ; Krane, M.

Heterotopic cervical heart transplantation in mice.

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient's common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

2015 Scientific Article in PLoS Genetics PLoS Genet. 11:e1005423 (2015)

Lagouge, M. ; Mourier, A. ; Lee, H.J. ; Spåhr, H. ; Wai, T. ; Kukat, C. ; Silva Ramos, E. ; Motori, E. ; Busch, J.D. ; Siira, S. ; German Mouse Clinic Consortium (Larsson, N.G.&deg ; Aguilar-Pimentel, J.A. ; Amarie, O.V. ; Becker, L. ; Beckers, J. ; Brachthäuser, L. ; Calzada-Wack, J. ; Eickelberg, O. ; Gailus-Durner, V. ; Garrett, L. ; Graw, J. ; Hans, W. ; Hölter, S.M. ; Horsch, M. ; Hrabě de Angelis, M. ; Janik, D. ; Klein-Rodewald, T. ; Lengger, C. ; Leuchtenberger, S. ; Maier, H. ; Moreth, K. ; Neff, F. ; Östereicher, M.A. ; Puk, O. ; Rácz, I. ; Rathkolb, B. ; Rozman, J. ; Steinkamp, R. ; Stöger, C. ; Stöger, T. ; Vernaleken, A. ; Wurst, W. ; Yildirim, A.Ö. ; Zimprich, A.) ; Kremmer, E. ; Filipovska, A.&deg

SLIRP regulates the rate of mitochondrial protein synthesis and protects LRPPRC from degradation.

We have studied the in vivo role of SLIRP in regulation of mitochondrial DNA (mtDNA) gene expression and show here that it stabilizes its interacting partner protein LRPPRC by protecting it from degradation. Although SLIRP is completely dependent on LRPPRC for its stability, reduced levels of LRPPRC persist in the absence of SLIRP in vivo. Surprisingly, Slirp knockout mice are apparently healthy and only display a minor weight loss, despite a 50-70% reduction in the steady-state levels of mtDNA-encoded mRNAs. In contrast to LRPPRC, SLIRP is dispensable for polyadenylation of mtDNA-encoded mRNAs. Instead, deep RNA sequencing (RNAseq) of mitochondrial ribosomal fractions and additional molecular analyses show that SLIRP is required for proper association of mRNAs to the mitochondrial ribosome and efficient translation. Our findings thus establish distinct functions for SLIRP and LRPPRC within the LRPPRC-SLIRP complex, with a novel role for SLIRP in mitochondrial translation. Very surprisingly, our results also demonstrate that mammalian mitochondria have a great excess of transcripts under basal physiological conditions in vivo.

2015 Scientific Article in Nature Genetics Nat. Genet. 47, 969-978 (2015)

Hrabě de Angelis, M.# ; Nicholson, G.# ; Selloum, M.# ; White, J.K.# ; Morgan, H.# ; Ramirez-Solis, R.# ; Sorg, T.# ; Wells, S.# ; Fuchs, H.# ; Fray, M.# ; Adams, D.J. ; Adams, N.C. ; Adler, T. ; Aguilar-Pimentel, J.A. ; Ali-Hadji, D. ; Amann, G. ; André, P. ; Atkins, S. ; Auburtin, A. ; Ayadi, A. ; Becker, J. ; Becker, L. ; Bedu, E. ; Bekeredjian, R. ; Birling, M.C. ; Blake, A. ; Bottomley, J. ; Bowl, M.R. ; Brault, V. ; Busch, D.H. ; Bussell, J.N. ; Calzada-Wack, J. ; Cater, H. ; Champy, M.F. ; Charles, P. ; Chevalier, C. ; Chiani, F. ; Codner, G.F. ; Combe, R. ; Cox, R.D. ; Dalloneau, E. ; Dierich, A. ; di Fenza, A. ; Doe, B. ; Duchon, A. ; Eickelberg, O. ; Esapa, C.T. ; Fertak, L.E. ; Feigel, T. ; Emelyanova, I. ; Estabel, J. ; Favor, J. ; Flenniken, A.M. ; Gambadoro, A. ; Garrett, L. ; Gates, H. ; Gerdin, A.K. ; Gkoutos, G.V. ; Greenaway, S. ; Glasl, L. ; Goetz, P. ; da Cruz, I.G. ; Götz, A. ; Graw, J. ; Guimond, A. ; Hans, W. ; Hicks, G. ; Hölter, S.M. ; Höfler, H. ; Hancock, J.M. ; Hoehndorf, R. ; Hough, T. ; Houghton, R. ; Hurt, A. ; Ivandic, B. ; Jacobs, H. ; Jacquot, S. ; Jones, N. ; Karp, N.A. ; Katus, H.A. ; Kitchen, S. ; Klein-Rodewald, T. ; Klingenspor, M. ; Klopstock, T. ; Lalanne, V. ; Leblanc, S. ; Lengger, C. ; le Marchand, E. ; Ludwig, T. ; Lux, A. ; McKerlie, C. ; Maier, H. ; Mandel, J.L. ; Marschall, S. ; Mark, M. ; Melvin, D.G. ; Meziane, H. ; Micklich, K. ; Mittelhauser, C. ; Monassier, L. ; Moulaert, D. ; Müller, S. ; Naton, B. ; Neff, F. ; Nolan, P.M. ; Nutter, L.M. ; Ollert, M. ; Pavlovic, G. ; Pellegata, N.S. ; Peter, E. ; Petit-Demoulière, B. ; Pickard, A. ; Podrini, C. ; Potter, P. ; Pouilly, L. ; Puk, O. ; Richardson, D. ; Rousseau, S. ; Quintanilla-Fend, L. ; Quwailid, M.M. ; Rácz, I. ; Rathkolb, B. ; Riet, F. ; Rossant, J. ; Roux, M. ; Rozman, J. ; Ryder, E. ; Salisbury, J. ; Santos, L. ; Schäble, K.-H. ; Schiller, E. ; Schrewe, A. ; Schulz, H. ; Steinkamp, R. ; Simon, M. ; Stewart, M. ; Stöger, C. ; Stöger, T. ; Sun, M. ; Sunter, D. ; Teboul, L. ; Tilly, I. ; Tocchini-Valentini, G.P. ; Tost, M. ; Treise, I. ; Vasseur, L. ; Velot, E. ; Vogt Weisenhorn, D.M. ; Wagner, C. ; Walling, A. ; Wattenhofer-Donze, M. ; Weber, B. ; Wendling, O. ; Westerberg, H. ; Willershäuser, M. ; Wolf, E. ; Wolter, A. ; Wood, J. ; Wurst, W. ; Yildirim, A.Ö. ; Zeh, R. ; Zimmer, A. ; Zimprich, A. ; Holmes, C.# ; Steel, K.P.# ; Herault, Y.# ; Gailus-Durner, V.# ; Mallon, A.M.# ; Brown, S.D.#

Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.

2015 Scientific Article in Journal of the American Heart Association J. Am. Heart Assoc. 4:e002153 (2015)

Kiermayer, C. ; Northrup, E. ; Schrewe, A. ; Walch, A.K. ; Hrabě de Angelis, M. ; Schoensiegel, F. ; Zischka, H. ; Prehn, C. ; Adamski, J. ; Bekeredjian, R. ; Ivandic, B. ; Kupatt, C. ; Brielmeier, M.

Heart-specific knockout of the mitochondrial Thioredoxin reductase (Txnrd2) induces metabolic and contractile dysfunction in the aging myocardium.

BACKGROUND: Ubiquitous deletion of thioredoxin reductase 2 (Txnrd2) in mice is embryonically lethal and associated with abnormal heart development, while constitutive, heart-specific Txnrd2 inactivation leads to dilated cardiomyopathy and perinatal death. The significance of Txnrd2 in aging cardiomyocytes, however, has not yet been examined. METHODS AND RESULTS: The tamoxifen-inducible heart-specific αMHC-MerCreMer transgene was used to inactivate loxP-flanked Txnrd2 alleles in adult mice. Hearts and isolated mitochondria from aged knockout mice were morphologically and functionally analyzed. Echocardiography revealed a significant increase in left ventricular end-systolic diameters in knockouts. Fractional shortening and ejection fraction were decreased compared with controls. Ultrastructural analysis of cardiomyocytes of aged mice showed mitochondrial degeneration and accumulation of autophagic bodies. A dysregulated autophagic activity was supported by higher levels of lysosome-associated membrane protein 1 (LAMP1), microtubule-associated protein 1A/1B-light chain 3-I (LC3-I), and p62 in knockout hearts. Isolated Txnrd2-deficient mitochondria used less oxygen and tended to produce more reactive oxygen species. Chronic hypoxia inducible factor 1, α subunit stabilization and altered transcriptional and metabolic signatures indicated that energy metabolism is deregulated. CONCLUSIONS: These results imply a novel role of Txnrd2 in sustaining heart function during aging and suggest that Txnrd2 may be a modifier of heart failure.

2015 Scientific Article in Molecular and Cellular Endocrinology Mol. Cell. Endocrinol. 421, 49-59 (2015)

Wiedemann, T. ; Pellegata, N.S.

Animal models of multiple endocrine neoplasia.

Multiple endocrine neoplasia (MEN) syndromes are autosomal dominant diseases with high penetrance characterized by proliferative lesions (usually hyperplasia or adenoma) arising in at least two endocrine tissues. Four different MEN syndromes have been so far identified: MEN type 1 (MEN1), MEN2A (also referred to as MEN2), MEN2B (or MEN3) and MEN4, which have slightly varying tumor spectra and are caused by mutations in different genes. MEN1 associates with loss-of-function mutations in the MEN1 gene encoding the tumor suppressor menin. The MEN2A and MEN2B syndromes are due to activating mutations in the proto-oncogene RET (Rearranged in Transfection) and are characterized by different phenotypic features of the affected patients. MEN4 was the most recent addition to the family of the MEN syndromes. It was discovered less than 10 years ago thanks to studies of a rat strain that spontaneously develops multiple endocrine tumors (named MENX). These studies identified an inactivating mutation in the Cdkn1b gene, encoding the putative tumor suppressor p27, as the causative mutation of the rat syndrome. Subsequently, germline mutations in the human ortholog CDKN1B were also found in a subset of patients with a MEN-like phenotype and this led to the identification of MEN4. Small animal models have been instrumental in understanding important biochemical, physiological and pathological processes of cancer onset and spread in intact living organisms. Moreover, they have provided us with insight into gene function(s) and molecular mechanisms of disease progression. We here review the currently available animal models of MEN syndromes and their impact on the elucidation of the pathophysiology of these diseases, with a special focus on the rat MENX syndrome that we have been characterizing.

2015 Scientific Article in Molecular Cancer Therapeutics Mol. Cancer Ther. 14, 1877-1883 (2015)

Deppisch, N. ; Ruf, P. ; Eissler, N. ; Neff, F. ; Buhmann, R. ; Lindhofer, H. ; Mocikat, R.

Efficacy and tolerability of a GD2-directed trifunctional bispecific antibody in a preclinical model: Subcutaneous administration is superior to intravenous delivery.

Trifunctional bispecific antibodies (trAb) are novel anticancer drugs that recruit and activate different types of immune effector cells at the targeted tumor. Thus, tumor cells are effectively eliminated and a long-lasting tumor-specific T-cell memory is induced. The trAb Ektomab is directed against human CD3 on T cells and the tumor-associated ganglioside GD2, which is an attractive target for immunotherapy of melanoma in humans. To optimize clinical applicability, we studied different application routes with respect to therapeutic efficacy and tolerability by using the surrogate trAb Surek (anti-GD2 x anti-murine CD3) and a murine melanoma engineered to express GD2. We show that subcutaneous injection of the trAb is superior to the intravenous delivery pathway, which is the standard application route for therapeutic antibodies. Despite lower plasma levels after subcutaneous administration, the same tumor-protective potential was observed in vivo compared to intravenous administration of Surek. However, subcutaneously delivered Surek showed better tolerability. This could be explained by a continuous release of the antibody leading to constant plasma levels and a delayed induction of proinflammatory cytokines. Importantly, the induction of counter-regulatory mechanisms was reduced after subcutaneous application. These findings are relevant for the clinical application of trifunctional bispecific antibodies and possibly also other immunoglobulin constructs.

2015 Scientific Article in PLoS ONE PLoS ONE 10:e0129058 (2015)

Maugg, D. ; Rothenaigner, I. ; Schorpp, K.K. ; Potukuchi, H.K. ; Korsching, E. ; Baumhoer, D. ; Hadian, K. ; Smida, J. ; Nathrath, M.

New small molecules targeting apoptosis and cell viability in osteosarcoma.

Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS.

2015 Scientific Article in Molecular Metabolism Mol. Metab. 4, 537-542 (2015)

Keipert, S. ; Kutschke, M. ; Lamp, D. ; Brachthäuser, L. ; Neff, F. ; Meyer, C.W. ; Oelkrug, R. ; Kharitonenkov, A. ; Jastroch, M.

Genetic disruption of uncoupling protein 1 in mice renders brown adipose tissue a significant source of FGF21 secretion.

Objective: Circulating fibroblast growth factor 21 (FGF21) is an important auto- and endocrine player with beneficial metabolic effects on obesity and diabetes. In humans, thermogenic brown adipose tissue (BAT) was recently suggested as a source of FGF21 secretion during cold exposure. Here, we aim to clarify the role of UCP1 and ambient temperature in the regulation of FGF21 in mice. Methods: Wildtype (WT) and UCP1-knockout (UCP1 KO) mice, the latter being devoid of BAT-derived non-shivering thermogenesis, were exposed to different housing temperatures. Plasma metabolites and FGF21 levels were determined, gene expression was analyzed by qPCR, and tissue histology was performed with adipose tissue. Results: At thermoneutrality, FGF21 gene expression and serum levels were not different between WT and UCP1 KO mice. Cold exposure led to highly increased FGF21 serum levels in UCP1 KO mice, which were reflected in increased FGF21 gene expression in adipose tissues but not in liver and skeletal muscle. Exvivo secretion assays revealed FGF21 release only from BAT, progressively increasing with decreasing ambient temperatures. In association with increased FGF21 serum levels in the UCP1 KO mouse, typical FGF21-related serum metabolites and inguinal white adipose tissue morphology and thermogenic gene expression were altered. Conclusions: Here we show that the genetic ablation of UCP1 increases FGF21 gene expression in adipose tissue. The removal of adaptive nonshivering thermogenesis renders BAT a significant source of endogenous FGF21 under thermal stress. Thus, the thermogenic competence of BAT is not a requirement for FGF21 secretion. Notably, high endogenous FGF21 levels in UCP1-deficient models and subjects may confound pharmacological FGF21 treatments.

München, Technische Universität, Fakultät für Chemie, Diss., 2015, 113 S.

Berchtold, S.

Epithelial-stromal interaction in pancreatic cancer: The role of collagen type V.

The main focus of this work was to unravel the functional role of collagen type V, which is mainly produced by pancreatic stellate cells, in the epithelial-stromal interaction in pancreatic cancer. To this aim, different in vitro and in vivo approaches were used to investigate the influence of collagen type V and of its downstream signaling pathway on pancreatic cancer cells as well as on endothelial and stromal cells. It could be shown that collagen type V promotes the aggressive phenotype of pancreatic cancer, starting from the early phases of pancreatic carcinogenesis until the metastatic disease.

2015 Scientific Article in Clinical Cancer Research Clin. Cancer Res. 21, 4440-4450 (2015)

Groß, C.# ; Steiger, K.# ; Sayyed, S.# ; Heid, I. ; Feuchtinger, A. ; Walch, A.K. ; Hess-Rieger, J. ; Unger, K. ; Zitzelsberger, H. ; Settles, M. ; Schlitter, A.M. ; Dworniczak, J. ; Altomonte, J. ; Ebert, O. ; Schwaiger, M. ; Rummeny, E.J. ; Steingötter, A. ; Esposito, I. ; Braren, R.

Model matters: Differences in orthotopic rat hepatocellular carcinoma physiology determine therapy response to sorafenib.

PURPOSE: Preclinical model systems should faithfully reflect the complexity of the human pathology. In hepatocellular carcinoma (HCC), the tumor vasculature is of particular interest in diagnosis and therapy. By comparing two commonly applied preclinical model systems, diethylnitrosamine induced (DEN) and orthotopically implanted (McA) rat HCC, we aimed to measure tumor biology non-invasively and identify differences between the models. EXPERIMENTAL DESIGN: DEN and McA tumor development was monitored by magnetic resonance imaging (MRI) and positron emission tomography (PET). A slice-based correlation of imaging and histopathology was performed. Array CGH analyses were applied to determine genetic heterogeneity. Therapy response to sorafenib was tested in DEN and McA tumors. RESULTS: Histologically and biochemically confirmed liver damage resulted in increased 18F-fluordeoxyglucose (FDG) PET uptake and perfusion in DEN animals only. DEN tumors exhibited G1-3 grading compared to uniform G3 grading of McA tumors. Array comparative genomic hybridization revealed a highly variable chromosomal aberration pattern in DEN tumors. Heterogeneity of DEN tumors was reflected in more variable imaging parameter values. DEN tumors exhibited lower mean growth rates and FDG uptake and higher diffusion and perfusion values compared to McA tumors. To test the significance of these differences, the multikinase inhibitor sorafenib was administered, resulting in reduced volume growth kinetics and perfusion in the DEN group only. CONCLUSION: This work depicts the feasibility and importance of in depth preclinical tumor model characterization and suggests the DEN model as a promising model system of multifocal nodular HCC in future therapy studies.

2015 Scientific Article in Plant Physiology Plant Physiol. 168, 859-870 (2015)

Velikova, V.B. ; Müller, C. ; Ghirardo, A. ; Rock, T. ; Aichler, M. ; Walch, A.K. ; Schmitt-Kopplin, P. ; Schnitzler, J.-P.

Knocking down of isoprene emission modifies the lipid matrix of thylakoid membranes and influences the chloroplast ultrastructure in poplar.

Isoprene is a small lipophilic molecule with important functions in plant protection against abiotic stresses by improving membrane structure and scavenging reactive oxygen species. Here, we studied the lipid composition of thylakoid membranes and chloroplast ultrastructure in isoprene emitting (IE) and non-isoprene emitting (NE) poplars. We demonstrated that the total amount of mono- (MGDG), di-galactosyldiacylglycerols (DGDG), phospholipids (PL), and fatty acids is reduced in chloroplasts when isoprene biosynthesis is blocked. A significantly lower amount of unsaturated fatty acids, particularly linolenic acid (18:3) in NE chloroplasts was associated with the reduced fluidity of thylakoid membranes, which in turn negatively affects PSII photochemical efficiency (ΦPSII). The low ΦPSII in NE plants was negatively correlated with non-photochemical quenching (NPQ) and the energy-dependent (qE) component of NPQ. Transmission electron microscopy (TEM) revealed alterations in the chloroplast ultrastructure in NE compared with IE plants. NE chloroplasts were more rounded and contained less grana stacks and longer stroma thylakoids, more plastoglobules, and larger associative zones between chloroplasts and mitochondria. These results strongly support the idea that in isoprene-emitting species, the function of this molecule is closely associated with the structural organization and functioning of plastidic membranes.

2015 Scientific Article in Journal of Pathology, The J. Pathol. 237, 123–132 (2015)

Buck, A.# ; Ly, A.# ; Balluff, B. ; Sun, N. ; Gorzolka, K. ; Feuchtinger, A. ; Janssen, K.P. ; Kuppen, P.J. ; van de Velde, C.J. ; Weirich, G. ; Erlmeier, F. ; Langer, R. ; Aubele, M. ; Zitzelsberger, H. ; Aichler, M. ; Walch, A.K.

High-resolution MALDI-FT-ICR MS imaging for the analysis of metabolites from formalin-fixed paraffin-embedded clinical tissue samples.

We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed paraffin-embedded (FFPE) human tissue samples. Using high-resolution Matrix-Assisted Laser Desorption/Ionization Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry Imaging (MALDI-FT-ICR MSI), we conducted a proof of principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients, and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues.

2015 Scientific Article in Frontiers in Oncology Front. Oncol. 5:94 (2015)

Bauer, L. ; Takacs, A. ; Slotta-Huspenina, J. ; Langer, R. ; Becker, K. ; Novotny, A. ; Ott, K. ; Walch, A.K. ; Hapfelmeier, A. ; Keller, G.

Clinical significance of NOTCH1 and NOTCH2 expression in gastric carcinomas: An immunohistochemical study.

BACKGROUND: NOTCH signaling can exert oncogenic or tumor suppressive functions and can contribute to chemotherapy resistance in cancer. In this study, we aimed to clarify the clinicopathological significance and the prognostic and predictive value of NOTCH1 and NOTCH2 expression in gastric cancer (GC). METHODS: NOTCH1 and NOTCH2 expression was determined immunohistochemically in 142 primarily resected GCs using tissue microarrays and in 84 pretherapeutic biopsies from patients treated by neoadjuvant chemotherapy. The results were correlated with survival, response to therapy, and clinico-pathological features. RESULTS: Primarily resected patients with NOTCH1-negative tumors demonstrated worse survival. High NOTCH1 expression was associated with early-stage tumors and with significantly increased survival in this subgroup. Higher NOTCH2 expression was associated with early-stage and intestinal-type tumors and with better survival in the subgroup of intestinal-type tumors. In pretherapeutic biopsies, higher NOTCH1 and NOTCH2 expression was more frequent in non-responding patients, but these differences were statistically not significant. CONCLUSION: Our findings suggested that, in particular, NOTCH1 expression indicated good prognosis in GC. The close relationship of high NOTCH1 and NOTCH2 expression with early tumor stages may indicate a tumor-suppressive role of NOTCH signaling in GC. The role of NOTCH1 and NOTCH2 in neoadjuvantly treated GC is limited.

2015 Scientific Article in Radiation Protection Dosimetry Radiat. Prot. Dosim. 166, 320-323 (2015)

Schöllnberger, H. ; Ozasa, K. ; Neff, F. ; Kaiser, J.C.

Cardiovascular disease mortality of a-bomb survivors and the healthy survivor selection effect.

The latest A-bomb survivor data for cardiovascular diseases are analysed to investigate whether in the first years after the bombings the baseline rates of proximal survivors were markedly different compared with those of the distal survivors. This phenomenon relates to a healthy survivor selection effect. This question is important for the decision whether to include or exclude the early years of follow-up when analysing the biological effects from acute low and high dose exposures following the nuclear weapons explosions in Hiroshima and Nagasaki. The present study shows that for cerebrovascular diseases and heart diseases the baseline rates are not significantly different in the first two decades of follow-up. Thus, for these two detrimental health outcomes, there is no need to exclude distal survivors and the first decades of follow-up time when investigating the shapes of the related dose-responses.

2015 Scientific Article in Histochemistry and Cell Biology Histochem. Cell Biol. 144, 147-156 (2015)

Erlmeier, F. ; Feuchtinger, A. ; Borgmann, D.M. ; Rudelius, M. ; Autenrieth, M. ; Walch, A.K. ; Weirich, G.

Supremacy of modern morphometry in typing renal oncocytoma and malignant look-alikes.

In the era of tumour type-specific therapies, the correct typing of renal tumours is of prime importance. As immunotyping and genotyping approaches are laborious and fall short of standardization, we used whole-scale computer-assisted morphometry instead. Three different types of renal tumours with different prognoses and therapies, notoriously prone to mistyping, were analysed . The sample of 335 tumours included clear cell renal cell carcinoma, chromophobe renal cell carcinoma and renal oncocytoma. The sample was analysed using H&E stains of tissue microarrrays in combination with an image-scanning software. Nuclear and cytoplasmic features were registered with the aid of computer-assisted morphometry. Features included shape, texture, colour and colour intensity for different cell compartments, e.g. nuclei and cytoplasm. The software passed several training steps for final validation. Using morphometry, we were able to classify the three renal tumour types correctly, with a 100 % specificity compared to the WHO typing. Nuclear features dominated the typing of chromophobe renal cell carcinoma, whereas cytoplasmic features were the leading classificators for renal oncocytoma. The grading of clear cell renal cell carcinoma attained a specificity of 80 %. In conclusion, modern morphometry may serve as a tool for typing renal epithelial tumours and additionally draws the attention to future nuclear research in chromophobe renal cell carcinoma.

2015 Scientific Article in Journal of Biological Chemistry, The J. Biol. Chem. 290, 14668-14678 (2015)

Ingold, I. ; Aichler, M. ; Yefremova, E. ; Roveri, A. ; Buday, K. ; Doll, S. ; Tasdemir, A. ; Hoffard, N. ; Wurst, W. ; Walch, A.K. ; Ursini, F. ; Friedmann Angeli, J.P.F. ; Conrad, M.

Expression of a catalytically inactive mutant form of Glutathione peroxidase 4 (Gpx4) confers a dominant-negative effect in male fertility.

The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 (mGpx4) was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with targeted mutation of the active site selenocysteine (Sec) of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4-/- embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breedings and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoan midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mGpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared to Sec at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Since the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and being a structural protein, tightly controlled expression of functional Gpx4 emerges being key for full male fertility.

2015 Scientific Article in GigaScience GigaScience 4:20 (2015)

Oetjen, J. ; Veselkov, K. ; Watrous, J. ; McKenzie, J.S. ; Becker, M. ; Hauberg-Lotte, L. ; Kobarg, J.H. ; Strittmatter, N. ; Mróz, A.K. ; Hoffmann, F. ; Trede, D. ; Palmer, A. ; Schiffler, S. ; Steinhorst, K. ; Aichler, M. ; Goldin, R. ; Guntinas-Lichius, O. ; von Eggeling, F. ; Thiele, H. ; Maedler, K. ; Walch, A.K. ; Maass, P. ; Dorrestein, P.C. ; Takats, Z. ; Alexandrov, T.

Benchmark datasets for 3D MALDI- and DESI-imaging mass spectrometry.

BACKGROUND: Three-dimensional (3D) imaging mass spectrometry (MS) is an analytical chemistry technique for the 3D molecular analysis of a tissue specimen, entire organ, or microbial colonies on an agar plate. 3D-imaging MS has unique advantages over existing 3D imaging techniques, offers novel perspectives for understanding the spatial organization of biological processes, and has growing potential to be introduced into routine use in both biology and medicine. Owing to the sheer quantity of data generated, the visualization, analysis, and interpretation of 3D imaging MS data remain a significant challenge. Bioinformatics research in this field is hampered by the lack of publicly available benchmark datasets needed to evaluate and compare algorithms. FINDINGS: High-quality 3D imaging MS datasets from different biological systems at several labs were acquired, supplied with overview images and scripts demonstrating how to read them, and deposited into MetaboLights, an open repository for metabolomics data. 3D imaging MS data were collected from five samples using two types of 3D imaging MS. 3D matrix-assisted laser desorption/ionization imaging (MALDI) MS data were collected from murine pancreas, murine kidney, human oral squamous cell carcinoma, and interacting microbial colonies cultured in Petri dishes. 3D desorption electrospray ionization (DESI) imaging MS data were collected from a human colorectal adenocarcinoma. CONCLUSIONS: With the aim to stimulate computational research in the field of computational 3D imaging MS, selected high-quality 3D imaging MS datasets are provided that could be used by algorithm developers as benchmark datasets.

2015 Scientific Article in Theranostics Theranostics 5, 667-685 (2015)

Almstätter, I. ; Mykhaylyk, O. ; Settles, M. ; Altomonte, J. ; Aichler, M. ; Walch, A.K. ; Rummeny, E.J. ; Ebert, O. ; Plank, C. ; Braren, R.

Characterization of magnetic viral complexes for targeted delivery in oncology.

Oncolytic viruses are promising new agents in cancer therapy. Success of tumor lysis is often hampered by low intra-tumoral titers due to a strong anti-viral host immune response and insufficient tumor targeting. Previous work on the co-assembly of oncolytic virus particles (VPs) with magnetic nanoparticles (MNPs) was shown to provide shielding from inactivating immune response and improve targeting by external field gradients. In addition, MNPs are detected by magnet resonance imaging (MRI) enabling non-invasive therapy monitoring. In this study two selected core-shell type iron oxide MNPs were assembled with adenovirus (Ad) or vesicular stomatitis virus (VSV). The selected MNPs were characterized by high r2 and r2 (*) relaxivities and thus could be quantified non-invasively by 1.5 and 3.0 tesla MRI with a detection limit below 0.001 mM iron in tissue-mimicking phantoms. Assembly and cell internalization of MNP-VP complexes resulted in 81 - 97 % reduction of r2 and 35 - 82 % increase of r2 (*) compared to free MNPs. The relaxivity changes could be attributed to the clusterization of particles and complexes shown by transmission electron microscopy (TEM). In a proof-of-principle study the non-invasive detection of MNP-VPs by MRI was shown in vivo in an orthotopic rat hepatocellular carcinoma model. In conclusion, MNP assembly and compartmentalization have a major impact on relaxivities, therefore calibration measurements are required for the correct quantification in biodistribution studies. Furthermore, our study provides first evidence of the in vivo applicability of selected MNP-VPs in cancer therapy.

2015 Scientific Article in Journal of Nuclear Medicine J. Nucl. Med. 56, 839-846 (2015)

van Berkel, A. ; Upendrao, J. ; Lenders, J. ; Pellegata, N.S. ; Kusters, B. ; Piscaer, I. ; Hermus, A.R. ; Plantinga, T.S. ; Langenhuijsen, H. ; Vriens, D. ; Janssen, M. ; Gotthardt, M. ; Timmers, H.

Semi-quantitative 123I-metaiodobenzylguanidine scintigraphy to distinguish pheochromocytoma and paraganglioma from physiological adrenal uptake and its correlation with genotype-dependent expression of catecholamine transporters.

(123)I-metaiodobenzylguanidine ((123)I-MIBG) scintigraphy plays an important role in the diagnostic evaluation of patients with pheochromocytoma and paraganglioma (PPGL). MIBG targets cell membrane and vesicular catecholamine transporters of chromaffin cells and facilitates localization of the primary tumor and metastatic lesions. Its specificity for the diagnosis of adrenomedullary chromaffin cell tumors can be jeopardized by physiological uptake by the normal adrenal medulla. The aim of this study was to distinguish between PPGLs and normal adrenal glands by evaluating semi-quantitative (123)I-MIBG uptake and to examine genotype-specific differences in correlation with expression of catecholamine transporter systems. METHODS: Sixty-two PPGLs collected from 57 patients with hereditary mutations in SDHA (n = 1), SDHB (n = 2), SDHD (n = 4), VHL (n = 2), RET (n = 12), NF1 (n = 2), MAX (n = 1) and with sporadic PPGLs (n = 33) were investigated. Pre-operative planar and single-photon emission computed tomographic (SPECT) images were semi-quantitatively analyzed using uptake measurements. Tumor-to-liver (T/L) and normal-adrenal-to-liver (NA/L) ratios were calculated and correlated with clinical characteristics including genotype, tumor size and plasma metanephrines concentrations. The expression of norepinephrine transporter (NET) and vesicular monoamine transporter (VMAT-1) was evaluated immunohistochemically in paraffin-embedded tumor tissues. RESULTS: Mean T/L ratios of PPGL lesions were significantly higher than NA/L ratios (p<0.001). Cut-off values to distinguish between physiological and pathological adrenal uptake were established at 0.7 (100% sensitivity, 10.3% specificity) and 4.3 (100% specificity, 66.1% sensitivity). No statistically significant differences in (123)I-MIBG uptake were found across PPGLs of different genotypes. Mean NET expression in hereditary cluster 2 (RET, NF1, MAX) and apparently sporadic tumors was significantly higher than for hereditary cluster 1 (SDHx, VHL) PPGLs (P = 0.011 and P = 0.006, respectively). Mean VMAT-1 expression in hereditary cluster 1 PPGLs was significantly higher than for cluster 2 tumors (P = 0.010). (123)I-MIBG uptake significantly correlated with maximum tumor diameter (P = 0.002). MIBG uptake, however, did not correlate with either NET or VMAT-1 expression. CONCLUSION: Liver normalized semi-quantitative (123)I-MIBG uptake may be helpful to distinguish between pheochromocytoma and physiological adrenal uptake. Genotype-specific differences in expression of NET and VMAT-1 do not translate into differences in (123)I-MIBG uptake.

2015 Scientific Article in Frontiers in Oncology Front. Oncol. 5:73 (2015)

Genitsch, V. ; Novotny, A. ; Seiler, C.A. ; Kröll, D. ; Walch, A.K. ; Langer, R.

Epstein-Barr virus in gastro-esophageal adenocarcinomas - single center experiences in the context of current literature.

Epstein-Barr virus (EBV)-associated gastric carcinomas (GC) represent a distinct and well-recognized subtype of gastric cancer with a prevalence of around 10% of all GC. In contrast, EBV has not been reported to play a major role in esophageal adenocarcinomas (EAC) and adenocarcinomas of the gastro-esophageal junction (GEJ). We report our experiences on EBV in collections of gastro-esophageal adenocarcinomas from two surgical centers and discuss the current state of research in this field. Tumor samples from 465 primary resected gastro-esophageal adenocarcinomas (118 EAC, 73 GEJ, and 274 GC) were investigated. Presence of EBV was determined by EBV-encoded small RNAs (EBER) in situ hybridization. Results were correlated with pathologic parameters (UICC pTNM category, Her2 status, tumor grading) and survival. EBER positivity was observed in 14 cases. None of the EAC were positive for EBER. In contrast, we observed EBER positivity in 2/73 adenocarcinomas of the GEJ (2.7%) and 12/274 GC (4.4%). These were of intestinal type (seven cases) or unclassifiable (six cases), while only one case was of diffuse type according to the Lauren classification. No association between EBV and pT, pN, or tumor grading was found, neither was there a correlation with clinical outcome. None of the EBER positive cases were Her2 positive. In conclusion, EBV does not seem to play a role in the carcinogenesis of EAC. Moreover, adenocarcinomas of the GEJ show lower rates of EBV positivity compared to GC. Our data only partially correlate with previous reports from the literature. This highlights the need for further research on this distinct entity. Recent reports, however, have identified specific epigenetic and genetic alterations in EBV-associated GC, which might lead to a distinct treatment approach for this specific subtype of GC in the future.

2015 Scientific Article in PLoS ONE PLoS ONE 10:e0123082 (2015)

Poos, K.# ; Smida, J.# ; Maugg, D. ; Eckstein, G.N. ; Baumhoer, D. ; Nathrath, M. ; Korsching, E.

Genomic heterogeneity of osteosarcoma - shift from single candidates to functional modules.

Osteosarcoma (OS), a bone tumor, exhibit a complex karyotype. On the genomic level a highly variable degree of alterations in nearly all chromosomal regions and between individual tumors is observable. This hampers the identification of common drivers in OS biology. To identify the common molecular mechanisms involved in the maintenance of OS, we follow the hypothesis that all the copy number-associated differences between the patients are intercepted on the level of the functional modules. The implementation is based on a network approach utilizing copy number associated genes in OS, paired expression data and protein interaction data. The resulting functional modules of tightly connected genes were interpreted regarding their biological functions in OS and their potential prognostic significance. We identified an osteosarcoma network assembling well-known and lesser-known candidates. The derived network shows a significant connectivity and modularity suggesting that the genes affected by the heterogeneous genetic alterations share the same biological context. The network modules participate in several critical aspects of cancer biology like DNA damage response, cell growth, and cell motility which is in line with the hypothesis of specifically deregulated but functional modules in cancer. Further, we could deduce genes with possible prognostic significance in OS for further investigation (e.g. EZR, CDKN2A, MAP3K5). Several of those module genes were located on chromosome 6q. The given systems biological approach provides evidence that heterogeneity on the genomic and expression level is ordered by the biological system on the level of the functional modules. Different genomic aberrations are pointing to the same cellular network vicinity to form vital, but already neoplastically altered, functional modules maintaining OS. This observation, exemplarily now shown for OS, has been under discussion already for a longer time, but often in a hypothetical manner, and can here be exemplified for OS.

2015 Scientific Article in Laboratory Investigation Lab. Invest. 95, 422-431 (2015)

Aichler, M. ; Walch, A.K.

MALDI Imaging mass spectrometry: Current frontiers and perspectives in pathology research and practice.

MALDI Imaging mass spectrometry has entered the field of tissue-based research by providing unique advantages for analyzing tissue specimen in an unprecedented detail. A broad spectrum of analytes ranging from proteins, peptides, protein modification over small molecules, drugs and their metabolites as well as pharmaceutical components, endogenous cell metabolites, lipids, and other analytes are made accessible by this in situ technique in tissue. Some of them were even not accessible in tissues within the histological context before. Thereby, the great advantage of MALDI Imaging is the correlation of molecular information with traditional histology by keeping the spatial localization information of the analytes after mass spectrometric measurement. This method is label-free and allows multiplex analysis of hundreds to thousands of molecules in the very same tissue section simultaneously. Imaging mass spectrometry brings a new quality of molecular data and links the expert discipline of pathology and deep molecular mass spectrometric analysis to tissue-based research. This review will focus on state-of-the-art of MALDI Imaging mass spectrometry, its recent applications by analyzing tissue specimen and the contributions in understanding the biology of disease as well as its perspectives for pathology research and practice.

2015 Scientific Article in Journal of Proteome Research J. Proteome Res. 14, 1203-1219 (2015)

Azimzadeh, O. ; Sievert, W. ; Sarioglu, H. ; Merl-Pham, J. ; Yentrapalli, R. ; Bakshi, M.V. ; Janik, D. ; Ueffing, M. ; Atkinson, M.J. ; Multhoff, G. ; Tapio, S.

Integrative proteomics and targeted transcriptomics analyses in cardiac endothelial cells unravel mechanisms of long-term radiation-induced vascular dysfunction.

Epidemiological data from radiotherapy patients show the damaging effect of ionizing radiation on heart and vasculature. The endothelium is the main target of radiation damage and contributes essentially to the development of cardiac injury. However, the molecular mechanisms behind the radiation-induced endothelial dysfunction are not fully understood. In the present study, 10-week-old C57Bl/6 mice received local X-ray heart doses of 8 or 16 Gy and were sacrificed after 16 weeks; the controls were sham-irradiated. The cardiac microvascular endothelial cells were isolated from the heart tissue using streptavidin-CD31-coated microbeads. The cells were lysed and proteins were labeled with duplex isotope-coded protein label methodology for quantification. All samples were analyzed by LC–ESI–MS/MS and Proteome Discoverer software. The proteomics data were further studied by bioinformatics tools and validated by targeted transcriptomics, immunoblotting, immunohistochemistry, and serum profiling. Radiation-induced endothelial dysfunction was characterized by impaired energy metabolism and perturbation of the insulin/IGF-PI3K-Akt signaling pathway. The data also strongly suggested premature endothelial senescence, increased oxidative stress, decreased NO availability, and enhanced inflammation as main causes of radiation-induced long-term vascular dysfunction. Detailed data on molecular mechanisms of radiation-induced vascular injury as compiled here are essential in developing radiotherapy strategies that minimize cardiovascular complications.

2015 Scientific Article in OncoTarget Oncotarget 6, 7727-7740 (2015)

Ribi, S.# ; Baumhoer, D.# ; Lee, K.# ; Edison ; Teo, A.S. ; Madan, B. ; Zhang, K. ; Kohlmann, W.K. ; Yao, F. ; Lee, W.H. ; Hoi, Q. ; Cai, S.W. ; Woo, X.Y. ; Tan, P.L. ; Jundt, G. ; Smida, J. ; Nathrath, M. ; Sung, W.K. ; Schiffman, J.D. ; Virshup, D.M.&deg ; Hillmer, A.M.&deg

TP53 intron 1 hotspot rearrangements are specific to sporadic osteosarcoma and can cause Li-Fraumeni syndrome.

Somatic mutations of TP53 are among the most common in cancer and germline mutations of TP53 (usually missense) can cause Li-Fraumeni syndrome (LFS). Recently, recurrent genomic rearrangements in intron 1 of TP53 have been described in osteosarcoma (OS), a highly malignant neoplasm of bone belonging to the spectrum of LFS tumors. Using whole-genome sequencing of OS, we found features of TP53 intron 1 rearrangements suggesting a unique mechanism correlated with transcription. Screening of 288 OS and 1,090 tumors of other types revealed evidence for TP53 rearrangements in 46 (16%) OS, while none were detected in other tumor types, indicating this rearrangement to be highly specific to OS. We revisited a four-generation LFS family where no TP53 mutation had been identified and found a 445 kb inversion spanning from the TP53 intron 1 towards the centromere. The inversion segregated with tumors in the LFS family. Cancers in this family had loss of heterozygosity, retaining the rearranged allele and resulting in TP53 expression loss. In conclusion, intron 1 rearrangements cause p53-driven malignancies by both germline and somatic mechanisms and provide an important mechanism of TP53 inactivation in LFS, which might in part explain the diagnostic gap of formerly classified "TP53 wild-type" LFS.

2015 Scientific Article in Antioxidants & Redox Signaling Antioxid. Redox Signal. 22, 938-950 (2015)

Hellfritsch, J. ; Kirsch, J. ; Schneider, M. ; Fluege, T. ; Wortmann, M. ; Frijhoff, J. ; Dagnell, M. ; Fey, T. ; Esposito, I. ; Kölle, P. ; Pogoda, K. ; Ingold, I. ; Friedmann Angeli, J.P.F. ; Kuhlencordt, P. ; Östman, A. ; Pohl, U. ; Beck, H.&deg ; Conrad, M.&deg

Knockout of mitochondrial thioredoxin reductase stabilizes prolyl hydroxylase 2 and inhibits tumor growth and tumor-derived angiogenesis.

Aims: Mitochondrial thioredoxin reductase (Txnrd2) is a central player in the control of mitochondrial H2O2 abundance by serving as a direct electron donor to the thioredoxin-peroxiredoxin axis. In the present study we investigated the impact of targeted disruption of Txnrd2 on tumor growth. Results: Tumor cells with a Txnrd2-deficiency failed to activate HIF-1α signaling; it rather caused PHD2 accumulation, HIF-1α degradation and decreased VEGF levels, ultimately leading to reduced tumor growth and tumor vascularization. Increased c-Jun NH2-terminal Kinase (JNK) activation proved to be the molecular link between the loss of Txnrd2, an altered mitochondrial redox balance with compensatory upregulation of glutaredoxin-2, and elevated PHD2 expression. Innovation: Our data provide compelling evidence for a yet unrecognized mitochondrial Txnrd-driven, regulatory mechanism that ultimately prevents cellular HIF-1α accumulation. In addition, simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems was used as an efficient therapeutic approach in hindering tumor growth. Conclusion: The present work demonstrates an unexpected regulatory link between mitochondrial Txnrd and the JNK-PHD2-HIF-1α axis which highlights how the loss of Txnrd2 and the resulting altered mitochondrial redox balance impairs tumor growth as well as tumor-related angiogenesis. Furthermore, it opens a new avenue for a therapeutic approach to hinder tumor growth by the simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems.

2015 Scientific Article in Clinical Cancer Research Clin. Cancer Res. 21, 3204-3215 (2015)

Lee, M.S. ; Wiedemann, T. ; Gross, C. ; Leinhäuser, I. ; Roncaroli, F. ; Braren, R. ; Pellegata, N.S.

Targeting PI3K/mTOR signaling displays potent antitumor efficacy against nonfunctioning pituitary adenomas.

PURPOSE: Novel therapeutic approaches are needed to improve the postoperative management of residual nonfunctioning pituitary adenomas (NFPAs), given their high relapse rate. Here, we evaluated the antitumor efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in the only available model of spontaneous NFPAs (MENX rats). EXPERIMENTAL DESIGN: Organotypic cultures of rat primary NFPAs were incubated with NVP-BEZ235 and assessed for cell viability, proliferation, apoptosis, PI3K/mTOR inhibition. NVP-BEZ235, or placebo, was administered to MENX rats and tumor response was monitored non-invasively by diffusion weighted-magnetic resonance imaging (DW-MRI). Following treatment, tumor tissues were investigated for cell proliferation, apoptosis, PI3K/mTOR inhibition. Genes mediating the cytotoxic activity of NVP-BEZ235 were identified by gene expression profiling. Among them, Defb1, encoding beta-defensin 1, was further studied for its role in pituitary cells and in human pancreatic neuroendocrine tumor (NET) cells. RESULTS: NVP-BEZ235 showed anti-proliferative and pro-cell death activities against NFPAs both in vitro and in vivo, and the response to the drug correlated with inhibition of the PI3K pathway. DW-MRI identified early functional changes (decreased cellularity) in the adenomas before their size was affected and emerged as a useful modality to assess therapy response. The cytotoxic effect of PI3K/mTOR blockade in NFPA was mediated by several genes, including Defb1. NVP-BEZ235 treatment induced Defb1 expression in NFPAs in vitro and in vivo, and in pancreatic NET cells. High Defb1 levels sensitized NET cells to PI3K/mTOR inhibition. CONCLUSIONS: Our findings provide rationale for clinical investigation of PI3K/mTOR inhibition in NFPAs and identify novel effectors of PI3K-mediated neuroendocrine cell survival.

2015 Scientific Article in Methods in Molecular Biology Methods Mol. Biol. 1295, 87-97 (2015)

Schmitt, S. ; Eberhagen, C. ; Weber, S. ; Aichler, M. ; Zischka, H.

Isolation of mitochondria from cultured cells and liver tissue biopsies for molecular and biochemical analyses.

We recently reported a new method to isolate functionally intact mitochondria from cell culture and small tissue samples (Schmitt et al., Anal Biochem 443(1):66-74, 2013). This method comprises a semi-automated cell rupture, termed pump controlled cell rupture system (PCC), which can be precisely adjusted to the specific cellular source of isolation and which can be tightly controlled (Schmitt et al., Anal Biochem 443(1):66-74, 2013). Here we provide a detailed hands-on protocol of this PCC method which results in an efficient cell breakage but preserving the mitochondrial integrity. Upon subsequent purification steps, the obtained mitochondrial fraction meets the quality and purity required for molecular analyses, e.g. proteomic comparisons, as well as for biochemical analyses, e.g. determination of diverse enzymatic activities.

2015 Scientific Article in Methods in Molecular Biology Methods Mol. Biol. 1295, 75-86 (2015)

Schulz, S. ; Lichtmannegger, J. ; Schmitt, S. ; Leitzinger, C. ; Eberhagen, C. ; Einer, C. ; Kerth, J. ; Aichler, M. ; Zischka, H.

A protocol for the parallel isolation of intact mitochondria from rat liver, kidney, heart, and brain.

Mitochondria are key organelles for cellular energy production and cell death decisions. Consequently, a plethora of conditions which are toxic to cells are known to directly attack these organelles. However, mitochondria originating from different tissues differ in their sensitivity to toxic insults. Thus, in order to predict the potential organ-specific toxicity of a given drug or pathological condition at the mitochondrial level, test settings are needed that directly compare the responses and vulnerabilities of mitochondria from different organs. As a prerequisite for such test strategies, we provide here a robust, prompt, and easy-to-follow step-by-step protocol to simultaneously isolate functional and intact mitochondria from rat liver, kidney, heart, and brain. This isolation procedure ensures mitochondrial preparations of comparable purity and reproducible quantities which can be subsequently analyzed for organ-specific mitochondrial toxicity.

2015 Scientific Article in Theranostics Theranostics 5, 618-630 (2015)

Wester, H.J. ; Keller, U. ; Schottelius, M. ; Beer, A. ; Philipp-Abbrederis, K. ; Hoffmann, F. ; Simecek, J. ; Gerngross, C. ; Lassmann, M. ; Herrmann, K. ; Pellegata, N.S. ; Rudelius, M. ; Kessler, H. ; Schwaiger, M.

Disclosing the CXCR4 expression in lymphoproliferative diseases by targeted molecular imaging.

Chemokine ligand-receptor interactions play a pivotal role in cell attraction and cellular trafficking, both in normal tissue homeostasis and in disease. In cancer, chemokine receptor-4 (CXCR4) expression is an adverse prognostic factor. Early clinical studies suggest that targeting CXCR4 with suitable high-affinity antagonists might be a novel means for therapy. In addition to the preclinical evaluation of [68Ga]Pentixafor in mice bearing human lymphoma xenografts as an exemplary CXCR4-expressing tumor entity, we report on the first clinical applications of [68Ga]Pentixafor-Positron Emission Tomography as a powerful method for CXCR4 imaging in cancer patients. [68Ga]Pentixafor binds with high affinity and selectivity to human CXCR4 and exhibits a favorable dosimetry. [68Ga]Pentixafor-PET provides images with excellent specificity and contrast. This non-invasive imaging technology for quantitative assessment of CXCR4 expression allows to further elucidate the role of CXCR4/CXCL12 ligand interaction in the pathogenesis and treatment of cancer, cardiovascular diseases and autoimmune and inflammatory disorders.

2015 Scientific Article in Endocrine-Related Cancer Endocr. Relat. Cancer 22, 353-367 (2015)

Beckers, A.# ; Lodish, M.# ; Giampaolo, T. ; Rostomyan, L. ; Lee, M.S. ; Faucz, F.R. ; Yuan, B. ; Choong, C. ; Caberg, J.H. ; Verrua, E. ; Naves, L.A. ; Cheetham, T. ; Young, J. ; Lysy, P. ; Petrossians, P. ; Cotterill, A. ; Shah, N. ; Metzger, D. ; Castermans, E. ; Ambrosio, M.R. ; Villa, C. ; Strebkova, N. ; Mazerkina, N. ; Gaillard, S. ; Barcelos Barra, G. ; Casulari, L.A. ; Neggers, S. ; Salvatori, R. ; Jaffrain-Rea, M.L. ; Zacharin, M. ; Lecumberri Santamaria, B. ; Zacharieva, S. ; Lim, E.M. ; Mantovani, G. ; Zatelli, M.C. ; Collins, M.T. ; Bonneville, J.F. ; Quezado, M.M. ; Chittiboina, P. ; Oldfield, E. ; Bours, V. ; Liu, P. ; de Herder, W.W. ; Pellegata, N.S. ; Lupski, J.R. ; Daly, A.F. ; Stratakis, C.A.

X-Linked Acrogigantism (X-LAG) syndrome: Clinical profile and therapeutic responses.

X-linked acro-gigantism (X-LAG) is a new syndrome of pituitary gigantism, caused by microduplications on chromosome Xq26.3, encompassing the gene GPR101, which is highly upregulated in pituitary tumors. We conducted this study to explore the clinical, radiological and hormonal phenotype and responses to therapy in patients with X-LAG syndrome. The study included 18 patients (13 sporadic) with X-LAG and a microduplication in chromosome Xq26.3. All sporadic cases had unique duplications and the inheritance pattern in 2 families was dominant with all Xq26.3 duplication carriers being affected. Patients began to grow rapidly as early as 2-3 months of age (median 12 months). At diagnosis (median delay 27 months), patients had a median height and weight SDS score of >+3.9 SDS. Apart from the increased overall body size, the children had acromegalic symptoms including acral enlargement and facial coarsening. More than a third of cases had increased appetite. Patients had marked hypersecretion of GH/IGF-1 and prolactin, usually due to a pituitary macroadenoma or hyperplasia. Primary neurosurgical control was achieved with extensive anterior pituitary resection but postoperative hypopituitarism was frequent. Control with somatostatin analogs was not readily achieved despite moderate to high somatostatin receptor subtype-2 expression in tumor tissue. Postoperative adjuvant pegvisomant achieved control of IGF-1 all 5 cases in which it was employed. X-LAG is a new infant-onset gigantism syndrome that has a severe clinical phenotype leading to challenging disease management.

2015 Scientific Article in Circulation Circulation 131, 1191-1201 (2015)

Kessler, T.# ; Zhang, L.# ; Liu, Z. ; Yin, X. ; Huang, Y. ; Wang, Y. ; Fu, Y. ; Mayr, M. ; Ge, Q. ;