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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 Cell Cell 185, 5040-5058.e19 (2022)

Bhatia, H.S.# ; Brunner, A.D.# ; Öztürk, F.# ; Kapoor, S. ; Rong, Z. ; Mai, H. ; Thielert, M. ; Ali, M. ; Al-Maskari, R. ; Paetzold, J.C. ; Kofler, F. ; Todorov, M.I. ; Molbay, M. ; Kolabas, Z.I. ; Negwer, M. ; Höher, L. ; Steinke, H. ; Dima, A. ; Gupta, B. ; Kaltenecker, D. ; Caliskan, Ö.S. ; Brandt, D. ; Krahmer, N. ; Müller, S. ; Lichtenthaler, S.F. ; Hellal, F. ; Bechmann, I. ; Menze, B. ; Theis, F.J. ; Mann, M.&deg ; Ertürk, A.&deg

Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. Video abstract: [Figure presented]

2022 Scientific Article in Applied Physics Letters Appl. Phys. Lett. 121:213502 (2022)

Robert, H.L. ; Diederichs, B. ; Muller-Caspary, K.

Contribution of multiple plasmon scattering in low-angle electron diffraction investigated by energy-filtered atomically resolved 4D-STEM.

We report the influence of multiple plasmon losses on the dynamical diffraction of high-energy electrons, in a scanning transmission electron microscopy (STEM) study. Using an experimental setup enabling energy-filtered momentum-resolved STEM, it is shown that the successive excitation of up to five plasmons within the imaged material results in a subsequent and significant redistribution of low-angle intensity in diffraction space. An empirical approach, based on the convolution with a Lorentzian kernel, is shown to reliably model this redistribution in dependence of the energy-loss. Our study demonstrates that both the significant impact of inelastic scattering in low-angle diffraction at elevated specimen thickness and a rather straightforward model can be applied to mimic multiple plasmon scattering, which otherwise is currently not within reach for multislice simulations due to computational complexity.

In: (2022 IEEE International Ultrasonics Symposium (IUS), 10-13 October 2022, Venice, Italy). 2022. DOI: 10.1109/IUS54386.2022.9958665

Laine, N. ; Zahnd, G. ; Bernard, O. ; Orkisz, M. ; Liebgott, H.

Generation of realistic simulated B-mode image texture with a GAN.

The intima-media complex of the common carotid artery is considered the sentinel of a silent killer disease called atherosclerosis. Morphological biomarkers such as the intima-media thickness are already exploitable, but dynamic biomarkers, which reflect tissue deformation over the cardiac cycle, remain to be validated. Recent motion estimation methods seek to quantify compression, shear, and elongation coefficients, but their clinical applicability has not yet been well defined, and their actual accuracy is difficult to assess due to the absence of ground truth. This lack of reference also is the main limitation to explore fully supervised deep learning methods that have shown great potential in other applications. With this in mind, we propose a simulation pipeline to produce realistic in silico sequences, by combining a physics-based simulator with a post-processing based on a generative adversarial network.

In: (2022 IEEE International Ultrasonics Symposium (IUS), 10-13 October 2022, Venice, Italy). 2022. DOI: 10.1109/IUS54386.2022.9957590

Laine, N. ; Zahnd, G. ; Liebgott, H. ; Orkisz, M.

Segmenting the carotid-artery wall in ultrasound image sequences with a dual-resolution U-net.

Thickening of intima-media complex in the common carotid artery is a biomarker of atherosclerosis. To automatically measure this thickness, we propose a region-based segmentation method, involving a supervised deep-learning approach based on the dilated U-net architecture, named caroSegDeep. It was trained and evaluated using 5-fold cross-validation on two open-access databases containing a total of 2676 annotated images. Compared with the methods already evaluated on these databases, caroSegDeep established a new benchmark and achieved a mean absolute error twice smaller than the inter-observer variability.

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 Medical Image Analysis Med. Image Anal. 84:102680 (2022)

Bilic, P. ; Christ, P. ; Li, H.B. ; Vorontsov, E.A. ; Ben-Cohen, A. ; Kaissis, G. ; Szeskin, A. ; Jacobs, C. ; Mamani, G.E.H. ; Chartrand, G. ; Lohöfer, F. ; Holch, J.W. ; Sommer, W. ; Hofmann, F. ; Hostettler, A. ; Lev-Cohain, N. ; Drozdzal, M. ; Amitai, M.M. ; Vivanti, R. ; Sosna, J. ; Ezhov, I. ; Sekuboyina, A. ; Navarro, F. ; Kofler, F. ; Paetzold, J.C. ; Shit, S. ; Hu, X. ; Lipkova, J. ; Rempfler, M. ; Piraud, M. ; Kirschke, J. ; Wiestler, B. ; Zhang, Z. ; Hülsemeyer, C. ; Beetz, M. ; Ettlinger, F. ; Antonelli, M. ; Bae, W. ; Bellver, M. ; Bi, L. ; Chen, H. ; Chlebus, G. ; Dam, E.B. ; Dou, Q. ; Fu, C.W. ; Georgescu, B. ; Giró-I-Nieto, X. ; Gruen, F. ; Han, X. ; Heng, P.A. ; Hesser, J. ; Moltz, J.H. ; Igel, C. ; Isensee, F. ; Jäger, P. ; Jia, F. ; Kaluva, K.C. ; Khened, M. ; Kim, I. ; Kim, J.H. ; Kim, S. ; Kohl, S. ; Konopczynski, T. ; Kori, A. ; Krishnamurthi, G. ; Li, F. ; Li, H. ; Li, J. ; Li, X. ; Lowengrub, J. ; Ma, J. ; Maier-Hein, K. ; Maninis, K.K. ; Meine, H. ; Merhof, D. ; Pai, A. ; Perslev, M. ; Petersen, J. ; Pont-Tuset, J. ; Qi, J. ; Qi, X. ; Rippel, O. ; Roth, K. ; Sarasua, I. ; Schenk, A. ; Shen, Z. ; Torres, J. ; Wachinger, C. ; Wang, C. ; Weninger, L. ; Wu, J. ; Xu, D. ; Yang, X. ; Yu, S.C.H. ; Yuan, Y. ; Yue, M. ; Zhang, L. ; Cardoso, J. ; Bakas, S. ; Braren, R. ; Heinemann, V. ; Pal, C. ; Tang, A. ; Kadoury, S. ; Soler, L. ; Van Ginneken, B. ; Greenspan, H. ; Joskowicz, L. ; Menze, B.

The Liver Tumor Segmentation Benchmark (LiTS).

In this work, we report the set-up and results of the Liver Tumor Segmentation Benchmark (LiTS), which was organized in conjunction with the IEEE International Symposium on Biomedical Imaging (ISBI) 2017 and the International Conferences on Medical Image Computing and Computer-Assisted Intervention (MICCAI) 2017 and 2018. The image dataset is diverse and contains primary and secondary tumors with varied sizes and appearances with various lesion-to-background levels (hyper-/hypo-dense), created in collaboration with seven hospitals and research institutions. Seventy-five submitted liver and liver tumor segmentation algorithms were trained on a set of 131 computed tomography (CT) volumes and were tested on 70 unseen test images acquired from different patients. We found that not a single algorithm performed best for both liver and liver tumors in the three events. The best liver segmentation algorithm achieved a Dice score of 0.963, whereas, for tumor segmentation, the best algorithms achieved Dices scores of 0.674 (ISBI 2017), 0.702 (MICCAI 2017), and 0.739 (MICCAI 2018). Retrospectively, we performed additional analysis on liver tumor detection and revealed that not all top-performing segmentation algorithms worked well for tumor detection. The best liver tumor detection method achieved a lesion-wise recall of 0.458 (ISBI 2017), 0.515 (MICCAI 2017), and 0.554 (MICCAI 2018), indicating the need for further research. LiTS remains an active benchmark and resource for research, e.g., contributing the liver-related segmentation tasks in In addition, both data and online evaluation are accessible via

2022 Scientific Article in Small Small 19:e2205318 (2022)

Lin, Y. ; Wilk, U. ; Pöhmerer, J. ; Hörterer, E. ; Höhn, M. ; Luo, X. ; Mai, H. ; Wagner, E.&deg ; Lächelt, U.&deg

Folate receptor-mediated delivery of Cas9 RNP for enhanced immune checkpoint disruption in cancer cells.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system offers great opportunities for the treatment of numerous diseases by precise modification of the genome. The functional unit of the system is represented by Cas9/sgRNA ribonucleoproteins (RNP), which mediate sequence-specific cleavage of DNA. For therapeutic applications, efficient and cell-specific transport into target cells is essential. Here, Cas9 RNP nanocarriers are described, which are based on lipid-modified oligoamino amides and folic acid (FolA)-PEG to realize receptor-mediated uptake and gene editing in cancer cells. In vitro studies confirm strongly enhanced potency of receptor-mediated delivery, and the nanocarriers enable efficient knockout of GFP and two immune checkpoint genes, PD-L1 and PVR, at low nanomolar concentrations. Compared with non-targeted nanoparticles, FolA-modified nanocarriers achieve substantially higher gene editing including dual PD-L1/PVR gene disruption after injection into CT26 tumors in vivo. In the syngeneic mouse model, dual disruption of PD-L1 and PVR leads to CD8+ T cell recruitment and distinct CT26 tumor growth inhibition, clearly superior to the individual knockouts alone. The reported Cas9 RNP nanocarriers represent a versatile platform for potent and receptor-specific gene editing. In addition, the study demonstrates a promising strategy for cancer immunotherapy by permanent and combined immune checkpoint disruption.

2022 Scientific Article in BioSpektrum BioSpektrum 28, 711-715 (2022)

Göllner, S. ; Mishra, K. ; Stiel, A.-C.

Schallschalter: Photoschaltbare Reporter und Sensoren in der Optoakustik.

Optoacoustic imaging offers a unique combination of observation volume and achievable resolution in vivo. However, the use of transgene labeling agents has been impractical because their signal is low compared to the background of the tissue. Thus, detection was limited to large numbers of cells. We tackle this problem by using switchable agents — switching creates a signal modulation which allows to separate the labeled cells from the constant background — making it virtually invisible.

2022 Scientific Article in Frontiers in Oncology Front. Oncol. 12:925542 (2022)

Schauer, J. ; Wieser, H.P. ; Huang, Y. ; Ruser, H. ; Lascaud, J. ; Würl, M. ; Chmyrov, A. ; Vidal, M. ; Herault, J. ; Ntziachristos, V. ; Assmann, W. ; Parodi, K. ; Dollinger, G.

Proton beam range verification by means of ionoacoustic measurements at clinically relevant doses using a correlation-based evaluation.

Purpose: The Bragg peak located at the end of the ion beam range is one of the main advantages of ion beam therapy compared to X-Ray radiotherapy. However, verifying the exact position of the Bragg peak within the patient online is a major challenge. The goal of this work was to achieve submillimeter proton beam range verification for pulsed proton beams of an energy of up to 220 MeV using ionoacoustics for a clinically relevant dose deposition of typically 2 Gy per fraction by i) using optimal proton beam characteristics for ionoacoustic signal generation and ii) improved signal detection by correlating the signal with simulated filter templates. Methods: A water tank was irradiated with a preclinical 20 MeV proton beam using different pulse durations ranging from 50 ns up to 1 μs in order to maximise the signal-to-noise ratio (SNR) of ionoacoustic signals. The ionoacoustic signals were measured using a piezo-electric ultrasound transducer in the MHz frequency range. The signals were filtered using a cross correlation-based signal processing algorithm utilizing simulated templates, which enhances the SNR of the recorded signals. The range of the protons is evaluated by extracting the time of flight (ToF) of the ionoacoustic signals and compared to simulations from a Monte Carlo dose engine (FLUKA). Results: Optimised SNR of 28.0 ± 10.6 is obtained at a beam current of 4.5 μA and a pulse duration of 130 ns at a total peak dose deposition of 0.5 Gy. Evaluated ranges coincide with Monte Carlo simulations better than 0.1 mm at an absolute range of 4.21 mm. Higher beam energies require longer proton pulse durations for optimised signal generation. Using the correlation-based post-processing filter a SNR of 17.8 ± 5.5 is obtained for 220 MeV protons at a total peak dose deposition of 1.3 Gy. For this clinically relevant dose deposition and proton beam energy, submillimeter range verification was achieved at an absolute range of 303 mm in water. Conclusion: Optimal proton pulse durations ensure an ideal trade-off between maximising the ionoacoustic amplitude and minimising dose deposition. In combination with a correlation-based post-processing evaluation algorithm, a reasonable SNR can be achieved at low dose levels putting clinical applications for online proton or ion beam range verification into reach.

2022 Nature Nature 612:E7 (2022)

Mishra, A. ; Malik, R. ; Hachiya, T. ; Jürgenson, T. ; Namba, S. ; Posner, D.C. ; Kamanu, F.K. ; Koido, M. ; Le Grand, Q. ; Shi, M. ; He, Y. ; Georgakis, M.K. ; Caro, I. ; Krebs, K. ; Liaw, Y.C. ; Vaura, F.C. ; Lin, K. ; Winsvold, B.S. ; Srinivasasainagendra, V. ; Parodi, L. ; Bae, H.J. ; Chauhan, G. ; Chong, M.R. ; Tomppo, L. ; Akinyemi, R. ; Roshchupkin, G.V. ; Habib, N. ; Jee, Y.H. ; Thomassen, J.Q. ; Abedi, V. ; Cárcel-Márquez, J. ; Nygaard, M. ; Leonard, H.L. ; Yang, C. ; Yonova-Doing, E. ; Knol, M.J. ; Lewis, A.J. ; Judy, R.L. ; Ago, T. ; Amouyel, P. ; Armstrong, N.D. ; Bakker, M.K. ; Bartz, T.M. ; Bennett, D.A. ; Bis, J.C. ; Bordes, C. ; Børte, S. ; Cain, A. ; Ridker, P.M. ; Cho, K. ; Chen, Z. ; Cruchaga, C. ; Cole, J.W. ; de Jager, P.L. ; de Cid, R. ; Endres, M. ; Ferreira, L.E. ; Geerlings, M.I. ; Gasca, N.C. ; Gudnason, V. ; Hata, J. ; He, J. ; Heath, A.K. ; Ho, Y.L. ; Havulinna, A.S. ; Hopewell, J.C. ; Hyacinth, H.I. ; Jacob, M.A. ; Jeon, C.E. ; Jern, C. ; Kamouchi, M. ; Keene, K.L. ; Kitazono, T. ; Kittner, S.J. ; Konuma, T. ; Kumar, A. ; Lacaze, P. ; Launer, L.J. ; Lee, K.J.D. ; Lepik, K. ; Li, J. ; Li, L. ; Manichaikul, A. ; Markus, H.S. ; Marston, N.A. ; Meitinger, T. ; Mitchell, B.D. ; Montellano, F.A. ; Morisaki, T. ; Mosley, T.H. ; Nalls, M.A. ; Nordestgaard, B.G. ; O'Donnell, M.J. ; Onland-Moret, N.C. ; Ovbiagele, B. ; Peters, A. ; Psaty, B.M. ; Rich, S.S. ; Rosand, J. ; Sabatine, M.S. ; Sacco, R.L. ; Saleheen, D. ; Sandset, E.C. ; Salomaa, V. ; Sargurupremraj, M. ; Sasaki, M. ; Satizabal, C.L. ; Schmidt, C.O. ; Shimizu, A. ; Smith, N.L. ; Sloane, K.L. ; Sutoh, Y. ; Sun, Y.V. ; Tanno, K. ; Tiedt, S. ; Tatlisumak, T. ; Torres-Aguila, N.P. ; Tiwari, H.K. ; Trégouët, D.A. ; Trompet, S. ; Tuladhar, A.M. ; Tybjærg-Hansen, A. ; van Vugt, M. ; Vibo, R. ; Verma, S.S. ; Wiggins, K.L. ; Wennberg, P. ; Woo, D. ; Wilson, P.W.F. ; Xu, H. ; Yang, Q. ; Yoon, K. ; Millwood, I.Y. ; Gieger, C. ; Ninomiya, T. ; Grabe, H.J. ; Jukema, J.W. ; Rissanen, I.L. ; Strbian, D. ; Kim, Y.J. ; Chen, P.H. ; Mayerhofer, E. ; Howson, J.M.M. ; Irvin, M.R. ; Adams, H.H. ; Wassertheil-Smoller, S. ; Christensen, K. ; Ikram, M.A. ; Rundek, T. ; Worrall, B.B. ; Lathrop, G.M. ; Riaz, M. ; Simonsick, E.M. ; Kõrv, J. ; França, P.H.C. ; Zand, R. ; Prasad, K. ; Frikke-Schmidt, R. ; de Leeuw, F.E. ; Liman, T. ; Haeusler, K.G. ; Ruigrok, Y.M. ; Heuschmann, P.U. ; Longstreth, W.T. Jr. ; Jung, K.J. ; Bastarache, L. ; Paré, G. ; Damrauer, S.M. ; Chasman, D.I. ; Rotter, J.I. ; Anderson, C.D. ; Zwart, J.A. ; Niiranen, T.J. ; Fornage, M. ; Liaw, Y.P. ; Seshadri, S. ; Fernandez-Cadenas, I. ; Walters, R.G. ; Ruff, C.T. ; Owolabi, M.O. ; Huffman, J.E. ; Milani, L. ; Kamatani, Y. ; Dichgans, M.&deg ; Debette, S.&deg

Publisher Correction: Stroke genetics informs drug discovery and risk prediction across ancestries.

2022 Scientific Article in Frontiers in Medicine Front. Med. 9:992993 (2022)

Bartos, L.M. ; Kirchleitner, S.V. ; Blobner, J. ; Wind, K. ; Kunze, L.H. ; Holzgreve, A. ; Gold, L. ; Zatcepin, A. ; Kolabas, Z.I. ; Ulukaya,S. ; Weidner, L. ; Quach, S. ; Messerer, D. ; Bartenstein, P. ; Tonn, J.C. ; Riemenschneider, M.J. ; Ziegler, S. ; von Baumgarten, L. ; Albert, N.L. ; Brendel, M.

18 kDa translocator protein positron emission tomography facilitates early and robust tumor detection in the immunocompetent SB28 glioblastoma mouse model.

Introduction: The 18 kDa translocator protein (TSPO) receives growing interest as a biomarker in glioblastoma. Mouse models can serve as an important tool for the investigation of biomarkers in glioblastoma, but several glioblastoma models indicated only low TSPO-PET signals in contrast to high TSPO-PET signals of human glioblastoma. Thus, we aimed to investigate TSPO-PET imaging in the syngeneic immunocompetent SB28 mouse model, which is thought to closely represent the tumor microenvironment (TME) of human glioblastoma. Methods: Dynamic TSPO-PET/CT imaging was performed for 60 min after injection of 13.6 ± 4.2 MBq [18F]GE-180. Contrast enhanced CT (ceCT) was acquired prior to PET and served for assessment of tumor volumes and attenuation correction. SB28 and sham mice were imaged at an early (week-1; n = 6 SB28, n = 6 sham) and a late time-point (week-3; n = 8 SB28, n = 9 sham) after inoculation. Standard of truth ex vivo tumor volumes were obtained for SB28 mice at the late time-point. Tracer kinetics were analyzed for the lesion site and the carotid arteries to establish an image derived input function (IDIF). TSPO-PET and ceCT lesion volumes were compared with ex vivo volumes by calculation of root-mean-square-errors (RMSE). Volumes of distribution (VTmax/mean) in the lesion were calculated using carotid IDIF and standardized uptake values (SUVmax/mean) were obtained for a 40–60 min time frame. Results: Higher uptake rate constants (K1) were observed for week-1 SB28 tumor lesions when compared to week-3 SB28 tumor lesions. Highest agreement between TSPO-PET lesion volumes and ex vivo tumor volumes was achieved with a 50% maximum threshold (RMSE-VT: 39.7%; RMSE-SUV: 34.4%), similar to the agreement of ceCT tumor volumes (RMSE: 30.1%). Lesions of SB28 mice had higher PET signal when compared to sham mice at week-1 (VTmax 6.6 ± 2.9 vs. 3.9 ± 0.8, p = 0.035; SUVmax 2.3 ± 0.5 vs. 1.2 ± 0.1, p < 0.001) and PET signals remained at a similar level at week-3 (VTmax 5.0 ± 1.6 vs. 2.7 ± 0.8, p = 0.029; SUVmax 1.9 ± 0.5 vs. 1.2 ± 0.2, p = 0.0012). VTmax correlated with SUVmax (R2 = 0.532, p < 0.001). Conclusion: TSPO-PET imaging of immunocompetent SB28 mice facilitates early detection of tumor signals over sham lesions. SB28 tumors mirror high TSPO-PET signals of human glioblastoma and could serve as a valuable translational model to study TSPO as an imaging biomarker.

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 Review in Biosensors Biosensors 12:901 (2022)

Dimaridis, I. ; Sridharan, P. ; Ntziachristos, V. ; Karlas, A. ; Hadjileontiadis, L.J.

Image quality improvement techniques and assessment adequacy in clinical optoacoustic imaging: A systematic review.

Optoacoustic imaging relies on the detection of optically induced acoustic waves to offer new possibilities in morphological and functional imaging. As the modality matures towards clinical application, research efforts aim to address multifactorial limitations that negatively impact the resulting image quality. In an endeavor to obtain a clear view on the limitations and their effects, as well as the status of this progressive refinement process, we conduct an extensive search for optoacoustic image quality improvement approaches that have been evaluated with humans in vivo, thus focusing on clinically relevant outcomes. We query six databases (PubMed, Scopus, Web of Science, IEEE Xplore, ACM Digital Library, and Google Scholar) for articles published from 1 January 2010 to 31 October 2021, and identify 45 relevant research works through a systematic screening process. We review the identified approaches, describing their primary objectives, targeted limitations, and key technical implementation details. Moreover, considering comprehensive and objective quality assessment as an essential prerequisite for the adoption of such approaches in clinical practice, we subject 36 of the 45 papers to a further in-depth analysis of the reported quality evaluation procedures, and elicit a set of criteria with the intent to capture key evaluation aspects. Through a comparative criteria-wise rating process, we seek research efforts that exhibit excellence in quality assessment of their proposed methods, and discuss features that distinguish them from works with similar objectives. Additionally, informed by the rating results, we highlight areas with improvement potential, and extract recommendations for designing quality assessment pipelines capable of providing rich evidence.

2022 Scientific Article in Nature Cell Biology Nat. Cell Biol. 24, 1666-1676 (2022)

Truong, D.J.J.# ; Armbrust, N.# ; Geilenkeuser, J. ; Lederer, E.-M. ; Santl, T. ; Beyer, M. ; Ittermann, S. ; Steinmaßl, E. ; Dyka, M. ; Raffl, G. ; Phlairaharn, T. ; Greisle, T. ; Živanić, M. ; Grosch, M. ; Drukker, M. ; Westmeyer, G.G.

Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs.

Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 (IL2) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest.

2022 Scientific Article in Journal of Biomedical Optics J. Biomed. Opt. 27:106004 (2022)

Madasamy, A. ; Gujrati, V. ; Ntziachristos, V. ; Prakash, J.

Deep learning methods hold promise for light fluence compensation in three-dimensional optoacoustic imaging.

SIGNIFICANCE: Quantitative optoacoustic imaging (QOAI) continues to be a challenge due to the influence of nonlinear optical fluence distribution, which distorts the optoacoustic image representation. Nonlinear optical fluence correction in OA imaging is highly ill-posed, leading to the inaccurate recovery of optical absorption maps. This work aims to recover the optical absorption maps using deep learning (DL) approach by correcting for the fluence effect. AIM: Different DL models were compared and investigated to enable optical absorption coefficient recovery at a particular wavelength in a nonhomogeneous foreground and background medium. APPROACH: Data-driven models were trained with two-dimensional (2D) Blood vessel and three-dimensional (3D) numerical breast phantom with highly heterogeneous/realistic structures to correct for the nonlinear optical fluence distribution. The trained DL models such as U-Net, Fully Dense (FD) U-Net, Y-Net, FD Y-Net, Deep residual U-Net (Deep ResU-Net), and generative adversarial network (GAN) were tested to evaluate the performance of optical absorption coefficient recovery (or fluence compensation) with in-silico and in-vivo datasets. RESULTS: The results indicated that FD U-Net-based deconvolution improves by about 10% over reconstructed optoacoustic images in terms of peak-signal-to-noise ratio. Further, it was observed that DL models can indeed highlight deep-seated structures with higher contrast due to fluence compensation. Importantly, the DL models were found to be about 17 times faster than solving diffusion equation for fluence correction. CONCLUSIONS: The DL methods were able to compensate for nonlinear optical fluence distribution more effectively and improve the optoacoustic image quality.

2022 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 41, 3182-3193 (2022)

Dehner, C. ; Olefir, I. ; Basak, K. ; Jüstel, D.&deg ; Ntziachristos, V.&deg

Deep-learning-based electrical noise removal enables high spectral optoacoustic contrast in deep tissue.

Image contrast in multispectral optoacoustic tomography (MSOT) can be severely reduced by electrical noise and interference in the acquired optoacoustic signals. Previously employed signal processing techniques have proven insufficient to remove the effects of electrical noise because they typically rely on simplified models and fail to capture complex characteristics of signal and noise. Moreover, they often involve time-consuming processing steps that are unsuited for real-time imaging applications. In this work, we develop and demonstrate a discriminative deep learning approach to separate electrical noise from optoacoustic signals prior to image reconstruction. The proposed deep learning algorithm is based on two key features. First, it learns spatiotemporal correlations in both noise and signal by using the entire optoacoustic sinogram as input. Second, it employs training on a large dataset of experimentally acquired pure noise and synthetic optoacoustic signals. We validated the ability of the trained model to accurately remove electrical noise on synthetic data and on optoacoustic images of a phantom and the human breast. We demonstrate significant enhancements of morphological and spectral optoacoustic images reaching 19% higher blood vessel contrast and localized spectral contrast at depths of more than 2 cm for images acquired in vivo. We discuss how the proposed denoising framework is applicable to clinical multispectral optoacoustic tomography and suitable for real-time operation.

2022 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 41, 3373-3384 (2022)

Longo, A. ; Jüstel, D. ; Ntziachristos, V.

Disentangling the frequency content in optoacoustics.

Signals acquired by optoacoustic tomography systems have broadband frequency content that encodes information about structures on different physical scales. Concurrent processing and rendering of such broadband signals may result in images with poor contrast and fidelity due to a bias towards low frequency contributions from larger structures. This problem cannot be addressed by filtering different frequency bands and reconstructing them individually, as this procedure leads to artefacts due to its incompatibility with the entangled frequency content of signals generated by structures of different sizes. Here we introduce frequency-band model-based (fbMB) reconstruction to separate frequency-band-specific optoacoustic image components during image formation, thereby enabling structures of all sizes to be rendered with high fidelity. In order to disentangle the overlapping frequency content of image components, fbMB uses soft priors to achieve an optimal trade-off between localization of the components in frequency bands and their structural integrity. We demonstrate that fbMB produces optoacoustic images with improved contrast and fidelity, which reveal anatomical structures in in vivo images of mice in unprecedented detail. These enhancements further improve the accuracy of spectral unmixing in small vasculature. By offering a precise treatment of the frequency components of optoacoustic signals, fbMB improves the quality, accuracy, and quantification of optoacoustic images and provides a method of choice for optoacoustic reconstructions.

2022 Scientific Article in Acta Neuropathologica Communications Acta Neuropathol. Commun. 10:129 (2022)

Ozen, I. ; Mai, H. ; de Maio, A. ; Ruscher, K. ; Michalettos, G. ; Clausen, F. ; Gottschalk, M. ; Ansar, S. ; Arkan, S. ; Ertürk, A. ; Marklund, N.

Purkinje cell vulnerability induced by diffuse traumatic brain injury is linked to disruption of long-range neuronal circuits.

Cerebellar dysfunction is commonly observed following traumatic brain injury (TBI). While direct impact to the cerebellum by TBI is rare, cerebellar pathology may be caused by indirect injury via cortico-cerebellar pathways. To address the hypothesis that degeneration of Purkinje cells (PCs), which constitute the sole output from the cerebellum, is linked to long-range axonal injury and demyelination, we used the central fluid percussion injury (cFPI) model of widespread traumatic axonal injury in mice. Compared to controls, TBI resulted in early PC loss accompanied by alterations in the size of pinceau synapses and levels of non-phosphorylated neurofilament in PCs. A combination of vDISCO tissue clearing technique and immunohistochemistry for vesicular glutamate transporter type 2 show that diffuse TBI decreased mossy and climbing fiber synapses on PCs. At 2 days post-injury, numerous axonal varicosities were found in the cerebellum supported by fractional anisotropy measurements using 9.4 T MRI. The disruption and demyelination of the cortico-cerebellar circuits was associated with poor performance of brain-injured mice in the beam-walk test. Despite a lack of direct input from the injury site to the cerebellum, these findings argue for novel long-range mechanisms causing Purkinje cell injury that likely contribute to cerebellar dysfunction after TBI.

2022 Review in European heart journal - cardiovascular imaging Eur. Heart J. Cardiovasc. Imaging 24, e1-e16 (2022)

Seguchi, M. ; Aytekin, A. ; Lenz, T. ; Nicol, P. ; Klosterman, G.R. ; Beele, A. ; Sabic, E. ; Utsch, L. ; Alyaqoob, A. ; Gorpas, D. ; Ntziachristos, V. ; Jaffer, F.A. ; Rauschendorfer, P. ; Joner, M.

Intravascular molecular imaging: Translating pathophysiology of atherosclerosis into human disease conditions.

Progression of atherosclerotic plaque in coronary arteries is characterized by complex cellular and non-cellular molecular interactions. Within recent years, atherosclerosis has been recognized as inflammation-driven disease condition, where progressive stages are characterized by morphological changes in plaque composition but also relevant molecular processes resulting in increased plaque vulnerability. While existing intravascular imaging modalities are able to resolve key morphological features during plaque progression, they lack capability to characterize the molecular profile of advanced atherosclerotic plaque. Because hybrid imaging modalities may provide incremental information related to plaque biology, they are expected to provide synergistic effects in detecting high risk patients and lesions. The aim of this article is to review existing literature on intravascular molecular imaging approaches, and to provide clinically oriented proposals of their application. In addition, we assembled an overview of future developments in this field geared towards detection of patients at risk for cardiovascular events.

2022 Review in Advanced Drug Delivery Reviews Adv. Drug Deliv. Rev. 189:114506 (2022)

Liu, N.# ; Mishra, K.# ; Stiel, A.-C. ; Gujrati, V. ; Ntziachristos, V.

The sound of drug delivery: Optoacoustic imaging in pharmacology.

Optoacoustic (photoacoustic) imaging offers unique opportunities for visualizing biological function in vivo by achieving high-resolution images of optical contrast much deeper than any other optical technique. The method detects ultrasound waves that are generated inside tissue by thermo-elastic expansion, i.e., the conversion of light absorption by tissue structures to ultrasound when the tissue is illuminated by the light of varying intensity. Listening instead of looking to light offers the major advantage of image formation with a resolution that obeys ultrasonic diffraction and not photon diffusion laws. While the technique has been widely used to explore contrast from endogenous photo-absorbing molecules, such as hemoglobin or melanin, the use of exogenous agents can extend applications to a larger range of biological and possible clinical applications, such as image-guided surgery, disease monitoring, and the evaluation of drug delivery, biodistribution, and kinetics. This review summarizes recent developments in optoacoustic agents, and highlights new functions visualized and potent pharmacology applications enabled with the use of external contrast agents.

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.

Lecture Notes in Computer Science In:. 2022. 383-391 (Lect. Notes Comput. Sc. ; 12963 LNCS)

Mächler, L. ; Ezhov, I. ; Kofler, F. ; Shit, S. ; Paetzold, J.C. ; Loehr, T. ; Zimmer, C. ; Wiestler, B. ; Menze, B.H.

FedCostWAvg: A new averaging for better federated learning.

We propose a simple new aggregation strategy for federated learning that won the MICCAI Federated Tumor Segmentation Challenge 2021 (FETS), the first ever challenge on Federated Learning in the Machine Learning community. Our method addresses the problem of how to aggregate multiple models that were trained on different data sets. Conceptually, we propose a new way to choose the weights when averaging the different models, thereby extending the current state of the art (FedAvg). Empirical validation demonstrates that our approach reaches a notable improvement in segmentation performance compared to FedAvg.

2022 Scientific Article in Nature Communications Nat. Commun. 13:4448 (2022)

Stylogiannis, A. ; Prade, L. ; Glasl, S. ; Mustafa, Q. ; Zakian Dominguez, C.M. ; Ntziachristos, V.

Frequency wavelength multiplexed optoacoustic tomography.

Optoacoustics (OA) is overwhelmingly implemented in the Time Domain (TD) to achieve high signal-to-noise ratios by maximizing the excitation light energy transient. Implementations in the Frequency Domain (FD) have been proposed, but suffer from low signal-to-noise ratios and have not offered competitive advantages over time domain methods to reach high dissemination. It is therefore commonly believed that TD is the optimal way to perform optoacoustics. Here we introduce an optoacoustic concept based on pulse train illumination and frequency domain multiplexing and theoretically demonstrate the superior merits of the approach compared to the time domain. Then, using recent advances in laser diode illumination, we launch Frequency Wavelength Multiplexing Optoacoustic Tomography (FWMOT), at multiple wavelengths, and experimentally showcase how FWMOT optimizes the signal-to-noise ratios of spectral measurements over time-domain methods in phantoms and in vivo. We further find that FWMOT offers the fastest multi-spectral operation ever demonstrated in optoacoustics.

2022 Review in Nature Protocols Nat. Protoc. 17, 2188-2215 (2022)

Mai, H.# ; Rong, Z.# ; Zhao, S.# ; Cai, R. ; Steinke, H. ; Bechmann, I. ; Ertürk, A.

Scalable tissue labeling and clearing of intact human organs.

Advances in tissue labeling and clearing methods include improvement of tissue transparency, better preservation of fluorescence signal, compatibility with immunostaining and large sample volumes. However, as existing methods share the common limitation that they can only be applied to human tissue slices, rendering intact human organs transparent remains a challenge. Here, we describe experimental details of the small-micelle-mediated human organ efficient clearing and labeling (SHANEL) pipeline, which can be applied for cellular mapping of intact human organs. We have successfully cleared multiple human organs, including kidney, pancreas, heart, lung, spleen and brain, as well as hard tissue like skull. We also describe an advanced volumetric imaging system using a commercial light-sheet fluorescence microscope that can accommodate most human organs and a pipeline for whole-organ imaging and visualization. The complete experimental process of labeling and clearing whole human organs takes months and the analysis process takes several weeks, depending on the organ types and sizes.

2022 Scientific Article in Cell Chemical Biology Cell Chem. Bio. 29, 1434-1445.e7 (2022)

Emslander, Q. ; Vogele, K. ; Braun, P. ; Stender, J. ; Willy, C. ; Joppich, M. ; Hammerl, J.A. ; Abele, M. ; Meng, C. ; Pichlmair, A. ; Ludwig, C. ; Bugert, J.J. ; Simmel, F.C. ; Westmeyer, G.G.

Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria.

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.

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 Nanophotonics Nanophotonics 11, 4637–4647 (2022)

Liu, N. ; O'Connor, P. ; Gujrati, V.&deg ; Anzenhofer,P. ; Klemm, U. ; Kleigrewe, K. ; Sattler, M. ; Plettenburg, O. ; Ntziachristos, V.&deg

Multifunctional croconaine nanoparticles for efficient optoacoustic imaging of deep tumors and photothermal therapy.

The proper design of near-infrared light-absorbing agents enables efficient optoacoustic imaging-guided phototherapy. In particular, several croconaine-based organic agents with excellent optical properties have been recently reported for this purpose. However, most of them absorb light below 800 nm, limiting deep-tissue imaging applications. To this end, we utilized a recently described novel croconaine derivative (CR880) to develop CR880-based nanoparticles (CR880-NPs) for effective in vivo delivery, deep tissue optoacoustic imaging and photothermal therapy applications. Radicals and strong π-πstacking in CR880 result in an 880 nm absorption peak with no blue-shift upon condensing to the solid phase. DSPE-PEG2000-formulated CR880-NPs exhibited high optoacoustic generation efficiency and photostability, and could be visualized in the tumors of three different mouse tumor models (breast, brain, and colon tumor) with high image contrast. The high photothermal conversion efficiency of CR880-NPs (∼58%) subsequently enabled efficient in vivo tumor elimination using a low energy laser, while remaining biocompatible and well-tolerated. This work introduces a promising novel agent for cancer theranostics of challenging deep-seated tumors.

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 Journal of Biophotonics J. Biophotonics 15:e202200032 (2022)

Englert, L. ; Riobo, L. ; Schönmann, C. ; Ntziachristos, V.&deg ; Aguirre Bueno, J.&deg

Enabling the autofocus approach for parameter optimization in planar measurement geometry clinical optoacoustic imaging.

In optoacoustic (photoacoustic) tomography, several parameters related to tissue and detector features are needed for image formation, but they may not be known a priori. An autofocus (AF) algorithm is generally used to estimate these parameters. However, the algorithm works iteratively and is therefore impractical for clinical imaging with planar geometry systems due to the long reconstruction times. We have developed a fast autofocus (FAF) algorithm for 3D optoacoustic systems with planar geometry. Such an algorithm exploits the symmetries of the planar geometry and a virtual source concept to reduce the dimensionality of the parameter estimation problem. The dimensionality reduction makes FAF much simpler computationally than the conventional AF algorithm. We show that the FAF algorithm required about 5 sec to provide accurate estimates of the speed of sound in simulated data and experimental data obtained using an imaging system that is poised to enter the clinic. The applicability of FAF for estimating other image formation parameters is discussed. We expect the FAF algorithm to contribute decisively to the clinical use of optoacoustic tomography systems with planar geometry.

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 Photoacoustics Photoacoustics 26:100362 (2022)

Vonk, J.# ; Kukacka, J.# ; Steinkamp, P.J. ; de Wit, J.G. ; Voskuil, F.J. ; Hooghiemstra, W.T.R. ; Bader, M. ; Jüstel, D. ; Ntziachristos, V. ; van Dam, G.M. ; Witjes, M.J.H.

Multispectral optoacoustic tomography for in vivo detection of lymph node metastases in oral cancer patients using an EGFR-targeted contrast agent and intrinsic tissue contrast: A proof-of-concept study.

Oral cancer patients undergo diagnostic surgeries to detect occult lymph node metastases missed by preoperative structural imaging techniques. Reducing these invasive procedures that are associated with considerable morbidity, requires better preoperative detection. Multispectral optoacoustic tomography (MSOT) is a rapidly evolving imaging technique that may improve preoperative detection of (early-stage) lymph node metastases, enabling the identification of molecular changes that often precede structural changes in tumorigenesis. Here, we characterize the optoacoustic properties of cetuximab-800CW, a tumor-specific fluorescent tracer showing several photophysical properties that benefit optoacoustic signal generation. In this first clinical proof-of-concept study, we explore its use as optoacoustic to differentiate between malignant and benign lymph nodes. We characterize the appearance of malignant lymph nodes and show differences in the distribution of intrinsic chromophores compared to benign lymph nodes. In addition, we suggest several approaches to improve the efficiency of follow-up studies.

2022 Review in Medical Sciences Med. Sci. 10:23 (2022)

Mirastschijski, U. ; Jiang, D. ; Rinkevich, Y.

Genital wound repair and scarring.

Skin wound repair has been the central focus of clinicians and scientists for almost a century. Insights into acute and chronic wound healing as well as scarring have influenced and ameliorated wound treatment. Our knowledge of normal skin notwithstanding, little is known of acute and chronic wound repair of genital skin. In contrast to extra-genital skin, hypertrophic scarring is uncommon in genital tissue. Chronic wound healing disorders of the genitals are mostly confined to mucosal tissue diseases. This article will provide insights into the differences between extra-genital and genital skin with regard to anatomy, physiology and aberrant wound repair. In light of fundamental differences between genital and normal skin, it is recommended that reconstructive and esthetic surgery should exclusively be performed by specialists with profound expertise in genital wound repair.

2022 Scientific Article in Cerebral Cortex Cereb. Cortex, DOI: 10.1093/cercor/bhac121 (2022)

Merino-Serrais, P. ; Plaza-Alonso, S. ; Hellal, F. ; Valero-Freitag, S. ; Kastanauskaite, A. ; Muñoz, A. ; Plesnila, N. ; DeFelipe, J.

Microanatomical study of pyramidal neurons in the contralesional somatosensory cortex after experimental ischemic stroke.

At present, many studies support the notion that after stroke, remote regions connected to the infarcted area are also affected and may contribute to functional outcome. In the present study, we have analyzed possible microanatomical alterations in pyramidal neurons from the contralesional hemisphere after induced stroke. We performed intracellular injections of Lucifer yellow in pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere in an ischemic stroke mouse model. A detailed 3-dimensional analysis of the neuronal complexity and morphological alterations of dendritic spines was then performed. Our results demonstrate that pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere show selective changes in their dendritic arbors, namely, less dendritic complexity of the apical dendritic arbor-but no changes in the basal dendritic arbor. In addition, we found differences in spine morphology in both apical and basal dendrites comparing the contralesional hemisphere with the lesional hemisphere. Our results show that pyramidal neurons of remote areas connected to the infarct zone exhibit a series of selective changes in neuronal complexity and morphological distribution of dendritic spines, supporting the hypothesis that remote regions connected to the peri-infarcted area are also affected after stroke.

2022 Scientific Article in Acta Neuropathologica Communications Acta Neuropathol. Commun. 10:51 (2022)

Ramos-Vega, M.&deg ; Kjellman, P. ; Todorov, M.I. ; Kylkilahti, T.M. ; Bäckström, B.T. ; Ertürk, A. ; Madsen, C.D. ; Lundgaard, I.&deg

Mapping of neuroinflammation-induced hypoxia in the spinal cord using optoacoustic imaging.

Recent studies suggest that metabolic changes and oxygen deficiency in the central nervous system play an important role in the pathophysiology of multiple sclerosis (MS). In our present study, we investigated the changes in oxygenation and analyzed the vascular perfusion of the spinal cord in a rodent model of MS. We performed multispectral optoacoustic tomography of the lumbar spinal cord before and after an oxygen enhancement challenge in mice with experimental autoimmune encephalomyelitis (EAE), a model for MS. In addition, mice were transcardially perfused with lectin to label the vasculature and their spinal columns were optically cleared, followed by light sheet fluorescence microscopy. To analyze the angioarchitecture of the intact spine, we used VesSAP, a novel deep learning-based framework. In EAE mice, the spinal cord had lower oxygen saturation and hemoglobin concentration compared to healthy mice, indicating compromised perfusion of the spinal cord. Oxygen administration reversed hypoxia in the spinal cord of EAE mice, although the ventral region remained hypoxic. Additionally, despite the increased vascular density, we report a reduction in length and complexity of the perfused vascular network in EAE. Taken together, these findings highlight a new aspect of neuroinflammatory pathology, revealing a significant degree of hypoxia in EAE in vivo that is accompanied by changes in spinal vascular perfusion. The study also introduces optoacoustic imaging as a tractable technique with the potential to further decipher the role of hypoxia in EAE and to monitor it in MS patients.

2022 Scientific Article in Oecologia Australis Oecologia Australis 26, 326-338 (2022)

Enrich-Prast, A. ; Figueiredo, V. ; Machado-Silva, F. ; Peixoto, R.B. ; Amora-Nogueira, L. ; Cugler, G. ; Dos Santos, M.C.B. ; de Sá Felizardo, J.P. ; Valle das Neves, J. ; Barreto, D.P. ; Valladares, L. ; Rodrigues, L. ; Santoro, A.L. ; Pinho, L.Q. ; Signori, C.N. ; Pollery, R. ; Silva, E. ; Marotta, H.

Inorganic nitrogen stimulates methane oxidation in coastal lagoon sediments.

Methane (CH4) oxidation is a critical process to reduce CH4 emissions from aquatic environments to the atmosphere. Considering the continuous increase in nitrogen in rivers, lakes, and lagoons from human sources, we re-evaluated the still controversial potential effect of inorganic nitrogen on CH4 oxidation. Here, we approached three shallow coastal lagoons that represent great environmental heterogeneity and used slurry sediments as a model system. The addition of ammonium chloride (NH4 Cl) and potassium nitrate (KNO3) significantly stimulated CH4 oxidation in the sediments of all studied lagoons, indicating the potential limitation of nitrogen for the growth of CH4 oxidizing bacteria. Our findings contrast to some previous reports, where ammonium and nitrate inhibited CH4 oxidation in sediments. Indeed, our experiment was performed in a more realistic range in relation to natural concentrations of inorganic nitrogen in aquatic systems (0.5 to 1 mM) and was opposed to extreme concentrations previously used (2 to 50 mM). Our results point to the need to further assess the connection between nitrogen inputs and CH4 budgets in aquatic sediments, considering the potential fuel for CH4 oxidation that may affect the global greenhouse gas balance.

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 Photoacoustics Photoacoustics 26:100354 (2022)

Visscher, M.# ; Pleitez, M.A.#&deg ; Van Gaalen, K. ; Nieuwenhuizen-Bakker, I.M. ; Ntziachristos, V. ; Van Soest, G.&deg

Label-free analytic histology of carotid atherosclerosis by mid-infrared optoacoustic microscopy.

Background and aims: Analysis of atherosclerotic plaque composition is a vital tool for unraveling the pathological metabolic processes that contribute to plaque growth. Methods: We visualize the constitution of human carotid plaques by mid-infrared optoacoustic microscopy (MiROM), a method for label-free analytic histology that requires minimal tissue preparation, rapidly yielding large field-of-view en-face images with a resolution of a few micrometers. We imaged endarterectomy specimens (n = 3, 12 sections total) at specific vibrational modes, targeting carbohydrates, lipids and proteins. Additionally, we recorded spectra at selected tissue locations. We identified correlations in the variability in this high-dimensional data set using non-negative matrix factorization (NMF). Results: We visualized high-risk plaque features with molecular assignment. Consistent NMF components relate to different dominant tissue constituents, dominated by lipids, proteins, and cholesterol and carbohydrates respectively. Conclusions: These results introduce MiROM as an innovative, stain-free, analytic histology technology for the biochemical characterization of complex human vascular pathology.

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.

In: (2022 IEEE/CVF Winter Conference on Applications of Computer Vision (WACV), 4-8 January 2022, Waikoloa). 2022. 3230-3240

Horvath, I.# ; Paetzold, J.C.# ; Schoppe, O. ; Al-Maskari, R. ; Ezhov, I. ; Shit, S. ; Li, H. ; Ertürk, A. ; Menze, B.

METGAN: Generative tumour inpainting and modality synthesis in light sheet microscopy.

Novel multimodal imaging methods are capable of generating extensive, super high resolution datasets for preclinical research. Yet, a massive lack of annotations prevents the broad use of deep learning to analyze such data. In this paper, we introduce a novel generative method which leverages real anatomical information to generate realistic image-label pairs of tumours. We construct a dualpathway generator, for the anatomical image and label, trained in a cycle-consistent setup, constrained by an independent, pretrained segmentor. Our method performs two concurrent tasks: domain adaptation and semantic synthesis, which, to our knowledge, has not been done before. The generated images yield significant quantitative improvement compared to existing methods that specialize in either of these tasks. To validate the quality of synthesis, we train segmentation networks on a dataset augmented with the synthetic data, substantially improving the segmentation over the baseline.

2022 Scientific Article in Photoacoustics Photoacoustics 26, 100343 (2022)

Kukacka, J. ; Metz, S. ; Dehner, C. ; Muckenhuber, A. ; Paul-Yuan, K. ; Karlas, A. ; Fallenberg, E.M. ; Rummeny, E. ; Jüstel, D. ; Ntziachristos, V.

Image processing improvements afford second-generation handheld optoacoustic imaging of breast cancer patients.

Background: Since the initial breast transillumination almost a century ago, breast cancer imaging using light has been considered in different implementations aiming to improve diagnostics, minimize the number of available biopsies, or monitor treatment. However, due to strong photon scattering, conventional optical imaging yields low resolution images, challenging quantification and interpretation. Optoacoustic imaging addresses the scattering limitation and yields high-resolution visualization of optical contrast, offering great potential value for breast cancer imaging. Nevertheless, the image quality of experimental systems remains limited due to a number of factors, including signal attenuation with depth and partial view angle and motion effects, particularly in multi-wavelength measurements. Methods: We developed data analytics methods to improve the accuracy of handheld optoacoustic breast cancer imaging, yielding second-generation optoacoustic imaging performance operating in tandem with ultrasonography. Results: We produced the most advanced images yet with handheld optoacoustic examinations of the human breast and breast cancer, in terms of resolution and contrast. Using these advances, we examined optoacoustic markers of malignancy, including vasculature abnormalities, hypoxia, and inflammation, on images obtained from breast cancer patients. Conclusions: We achieved a new level of quality for optoacoustic images from a handheld examination of the human breast, advancing the diagnostic and theranostic potential of the hybrid optoacoustic-ultrasound (OPUS) examination over routine ultrasonography.

2022 Scientific Article in International Journal of Molecular Sciences Int. J. Mol. Sci. 23:2169 (2022)

Pazzaglia, S.&deg ; Tanno, B. ; De Stefano, I. ; Giardullo, P. ; Leonardi, S. ; Merla, C. ; Babini, G. ; Tuncay Cagatay, S. ; Mayah, A. ; Kadhim, M. ; Lyng, F.M. ; von Toerne, C. ; Zhan, Z.N. ; Subedi, P. ; Tapio, S. ; Saran, A. ; Mancuso, M.&deg

Micro-RNA and proteomic profiles of plasma-derived exosomes from irradiated mice reveal molecular changes preventing apoptosis in neonatal cerebellum.

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.

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 IEEE Robotics and Automation Letters IEEE Robot. Autom. Lett. 7, 6638-6645 (2022)

Bi, Y.&deg ; Jiang, Z.&deg ; Gao, Y. ; Wendler, T. ; Karlas, A. ; Navab, N.

VesNet-RL: Simulation-based reinforcement learning for real-world US probe navigation.

Ultrasound (US) is one of the most common medical imaging modalities since it is radiation-free, low-cost, and real-time. In freehand US examinations, sonographers often navigate a US probe to visualize standard examination planes with rich diagnostic information. However, reproducibility and stability of the resulting images often suffer from intra- and inter-operator variation. Reinforcement learning (RL), as an interaction-based learning method, has demonstrated its effectiveness in visual navigating tasks; however, RL is limited in terms of generalization. To address this challenge, we propose a simulation-based RL framework for real-world navigation of US probes towards the standard longitudinal views of vessels. A UNet is used to provide binary masks from US images; thereby, the RL agent trained on simulated binary vessel images can be applied in real scenarios without further training. To accurately characterize actual states, a multi-modality state representation structure is introduced to facilitate the understanding of environments. Moreover, considering the characteristics of vessels, a novel standard view recognition approach based on the minimum bounding rectangle is proposed to terminate the searching process. To evaluate the effectiveness of the proposed method, the trained policy is validated virtually on 3D volumes of a volunteer’s in-vivo carotid artery, and physically on custom-designed gel phantoms using robotic US. The results demonstrate that proposed approach can effectively and accurately navigate the probe towards the longitudinal view of vessels.

2022 Scientific Article in Cancers Cancers 14:2462 (2022)

Zhao, X. ; Gabriels, R.Y. ; Hooghiemstra, W.T.R. ; Koller, M. ; Meersma, G.J. ; Buist-Homan, M. ; Visser, L. ; Robinson, D.J. ; Tenditnaya, A. ; Gorpas, D. ; Ntziachristos, V. ; Karrenbeld, A. ; Kats-Ugurlu, G. ; Fehrmann, R.S.N. ; Nagengast, W.B.

Validation of novel molecular imaging targets identified by functional genomic mRNA profiling to detect dysplasia in Barrett's Esophagus.

Barrett's esophagus (BE) is the precursor of esophageal adenocarcinoma (EAC). Dysplastic BE (DBE) has a higher progression risk to EAC compared to non-dysplastic BE (NDBE). However, the miss rates for the endoscopic detection of DBE remain high. Fluorescence molecular endoscopy (FME) can detect DBE and mucosal EAC by highlighting the tumor-specific expression of proteins. This study aimed to identify target proteins suitable for FME. Publicly available RNA expression profiles of EAC and NDBE were corrected by functional genomic mRNA (FGmRNA) profiling. Following a class comparison between FGmRNA profiles of EAC and NDBE, predicted, significantly upregulated genes in EAC were prioritized by a literature search. Protein expression of prioritized genes was validated by immunohistochemistry (IHC) on DBE and NDBE tissues. Near-infrared fluorescent tracers targeting the proteins were developed and evaluated ex vivo on fresh human specimens. In total, 1976 overexpressed genes were identified in EAC (n = 64) compared to NDBE (n = 66) at RNA level. Prioritization and IHC validation revealed SPARC, SULF1, PKCι, and DDR1 (all p < 0.0001) as the most attractive imaging protein targets for DBE detection. Newly developed tracers SULF1-800CW and SPARC-800CW both showed higher fluorescence intensity in DBE tissue compared to paired non-dysplastic tissue. This study identified SPARC, SULF1, PKCι, and DDR1 as promising targets for FME to differentiate DBE from NDBE tissue, for which SULF1-800CW and SPARC-800CW were successfully ex vivo evaluated. Clinical studies should further validate these findings.

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 Nanomaterials Nanomaterials 12:1657 (2022)

Gabashvili, A.N. ; Efremova, M.V. ; Vodopyanov, S.S. ; Chmelyuk, N.S. ; Oda, V.V. ; Sarkisova, V.A. ; Leonova, M.K. ; Semkina, A.S. ; Ivanova, A.V. ; Abakumov, M.A.

New approach to non-invasive tumor model monitoring via self-assemble iron containing protein nanocompartments.

According to the World Health Organization, breast cancer is the most common oncological disease worldwide. There are multiple animal models for different types of breast carcinoma, allowing the research of tumor growth, metastasis, and angiogenesis. When studying these processes, it is crucial to visualize cancer cells for a prolonged time via a non-invasive method, for example, magnetic resonance imaging (MRI). In this study, we establish a new genetically encoded material based on Quasibacillus thermotolerans (Q.thermotolerans, Qt) encapsulin, stably expressed in mouse 4T1 breast carcinoma cells. The label consists of a protein shell containing an enzyme called ferroxidase. When adding Fe2+, a ferroxidase oxidizes Fe2+ to Fe3+, followed by iron oxide nanoparticles formation. Additionally, genes encoding mZip14 metal transporter, enhancing the iron transport, were inserted into the cells via lentiviral transduction. The expression of transgenic sequences does not affect cell viability, and the presence of magnetic nanoparticles formed inside encapsulins results in an increase in T2 relaxivity.

Methods in Molecular Biology In: Atherosclerosis. 2022. 747-763 (Methods Mol. Biol. ; 2419)

Sun, T. ; Li, Y. ; Förstera, B. ; Stanic Aguilera, K.N. ; Lu, S. ; Steffens, S. ; Yin, C. ; Ertürk, A. ; Megens, R.T.A. ; Weber, C. ; Habenicht, A.J. ; Mohanta, S.K.

Tissue clearing approaches in atherosclerosis.

Recent advances in cardiovascular research have led to a more comprehensive understanding of molecular mechanisms of atherosclerosis. It has become apparent that the disease involves three layers of the arterial wall: the intima, the media, and a connective tissue coat termed the adventitia. It is also now appreciated that arteries are surrounded by adipose and neuronal tissues. In addition, adjacent to and within the adventitia, arteries are embedded in a loose connective tissue containing blood vessels (vasa vasora) and lymph vessels, artery-draining lymph nodes and components of the peripheral nervous system, including periarterial nerves and ganglia. During atherogenesis, each of these tissues undergoes marked structural and cellular alterations. We propose that a better understanding of these cell-cell and cell-tissue interactions may considerably advance our understanding of cardiovascular disease pathogenesis. Methods to acquire subcellular optical access to the intact tissues surrounding healthy and diseased arteries are urgently needed to achieve these aims. Tissue clearing is a landmark next-generation, three-dimensional (3D) microscopy technique that allows to image large-scale hitherto inaccessible intact deep tissue compartments. It allows for detailed reconstructions of arteries by a combination of labelling, clearing, advanced microscopies and other imaging and data-analysis tools. Here, we describe two distinct tissue clearing protocols; solvent-based modified three-dimensional imaging of solvent-cleared organs (3DISCO) clearing and another using aqueous-based 2,2'-thiodiethanol (TDE) clearing, both of which complement each other.

2022 Scientific Article in Cells Cells 11:775 (2022)

Essien, E.I. ; Hofer, T.P. ; Atkinson, M.J. ; Anastasov, N.

Combining HDAC and MEK inhibitors with radiation against glioblastoma-derived spheres.

Glioblastoma stem-like cells (GSLCs) in glioblastoma limit effective treatment and promote therapeutic resistance and tumor recurrence. Using a combined radiation and drugscreening platform, we tested the combination of a histone deacetylase inhibitor (HDACi) and MAPK/ERK kinase inhibitor (MEKi) with radiation to predict the efficacy against GSLCs. To mimic a stem-like phenotype, glioblastoma-derived spheres were used and treated with a combination of HDACi (MS-275) and MEKi (TAK-733 or trametinib) with 4 Gy irradiation. The sphere-forming ability after the combined radiochemotherapy was investigated using a sphere formation assay, while the expression levels of the GSLC markers (CD44, Nestin and SOX2) after treatment were analyzed using Western blotting and flow cytometry. The combined radiochemotherapy treatment inhibited the sphere formation in both glioblastoma-derived spheres, decreased the expression of the GSLC markers in a cell-line dependent manner and increased the dead cell population. Finally, we showed that the combined treatment with radiation was more effective at reducing the GSLC markers compared to the standard treatment of temozolomide and radiation. These results suggest that combining HDAC and MEK inhibition with radiation may offer a new strategy to improve the treatment of glioblastoma.

2022 Scientific Article in Journal of Biomedical Optics J. Biomed. Opt. 27:074704 (2022)

Sterkenburg, A.J.# ; Hooghiemstra, W.T.R.# ; Schmidt, I. ; Ntziachristos, V. ; Nagengast, W.B.&deg ; Gorpas, D.&deg

Standardization and implementation of fluorescence molecular endoscopy in the clinic.

SIGNIFICANCE: Near-infrared fluorescence molecular endoscopy (NIR-FME) is an innovative technique allowing for in vivo visualization of molecular processes in hollow organs. Despite its potential for clinical translation, NIR-FME still faces challenges, for example, the lack of consensus in performing quality control and standardization of procedures and systems. This may hamper the clinical approval of the technology by authorities and its acceptance by endoscopists. Until now, several clinical trials using NIR-FME have been performed. However, most of these trials had different study designs, making comparison difficult. AIM: We describe the need for standardization in NIR-FME, provide a pathway for setting up a standardized clinical study, and describe future perspectives for NIR-FME. Body: Standardization is challenging due to many parameters. Invariable parameters refer to the hardware specifications. Variable parameters refer to movement or tissue optical properties. Phantoms can be of aid when defining the influence of these variables or when standardizing a procedure. CONCLUSION: There is a need for standardization in NIR-FME and hurdles still need to be overcome before a widespread clinical implementation of NIR-FME can be realized. When these hurdles are overcome, clinical outcomes can be compared and systems can be benchmarked, enabling clinical implementation.

2022 Scientific Article in Photoacoustics Photoacoustics 25:100333 (2022)

Ali, Z. ; Zakian Dominguez, C.M. ; Li, Q. ; Gloriod, J. ; Crozat, S. ; Bouvet, F. ; Pierre, G. ; Sarantos, V. ; Di Pietro, M. ; Flisikowski, K. ; Andersen, P.M. ; Drexler, W. ; Ntziachristos, V.

360º optoacoustic capsule endoscopy at 50 Hz for esophageal imaging.

Gastrointestinal (GI) endoscopy is a common medical diagnostic procedure used for esophageal cancer detection. Current emerging capsule optoacoustic endoscopes, however, suffer from low pulse repetition rates and slow scanning units limit attainable imaging frame rates. Consequently, motion artifacts result in inaccurate spatial mapping and misinterpretation of data. To overcome these limitations, we report a 360º, 50 Hz frame rate, distal scanning capsule optoacoustic endoscope. The translational capability of the instrument for human GI tract imaging was characterized with an Archimedean spiral phantom consisting of twelve 100 µm sutures, a stainless steel mesh with a pitch of 3 mm and an ex vivo pig esophagus sample. We estimated an imaging penetration depth of ~0.84 mm in vivo by immersing the mesh phantom in intralipid solution to simulate light scattering in human esophageal tissue and validated our findings ex vivo using pig esophagus. This proof-of-concept study demonstrates the translational potential of the proposed video-rate endoscope for human GI tract imaging.

2022 Review in Pharmaceutics Pharmaceutics 14:362 (2022)

Kyrkou, S.G. ; Vrettos, E.I. ; Gorpas, D. ; Crook, T. ; Syed, N. ; Tzakos, A.G.

Design principles governing the development of theranostic anticancer agents and their nanoformulations with photoacoustic properties.

The unmet need to develop novel approaches for cancer diagnosis and treatment has led to the evolution of theranostic agents, which usually include, in addition to the anticancer drug, an imaging agent based mostly on fluorescent agents. Over the past few years, a non-invasive pho-toacoustic imaging modality has been effectively integrated into theranostic agents. Herein, we shed light on the design principles governing the development of theranostic agents with photoa-coustic properties, which can be formulated into nanocarriers to enhance their potency. Specifi-cally, we provide an extensive analysis of their individual constituents including the imaging dyes, drugs, linkers, targeting moieties, and their formulation into nanocarriers. Along these lines, we present numerous relevant paradigms. Finally, we discuss the clinical relevance of the specific strategy, as also the limitations and future perspectives, and through this review, we envisage paving the way for the development of theranostic agents endowed with photoacoustic properties as effective anticancer medicines.

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 Journal of Biophotonics J. Biophotonics 15:e202100334 (2022)

Muhammad, M.H. ; Prakash, J. ; Liapis, E. ; Ntziachristos, V. ; Jüstel, D.

Weighted model-based optoacoustic reconstruction for partial-view geometries.

Acoustic heterogeneities in biological samples are known to cause artefacts in tomographic optoacoustic (photoacoustic) image reconstruction. A statistical weighted model-based reconstruction approach was previously introduced to mitigate such artefacts. However, this approach does not reliably provide high-quality reconstructions for partial-view imaging systems, which are common in preclinical and clinical optoacoustics. In this paper, the capability of the weighted model-based algorithm is extended to generate optoacoustic reconstructions with less distortions for partial-view geometry data. This is achieved by manipulating the weighting scheme based on the detector geometry. Using partial-view optoacoustic tomography data from a tissue-mimicking phantom containing a strong acoustic reflector, tumors grafted onto mice, and a mouse brain with intact skull, the proposed partial-view-corrected weighted model-based algorithm is shown to mitigate reflection artefacts in reconstructed images without distorting structures or boundaries, compared to both conventional model-based and the weighted model-based algorithms. It is also demonstrated that the partial-view-corrected weighted model-based algorithm has the additional advantage of suppressing streaking artefacts due to the partial-view geometry itself in the presence of a very strong optoacoustic chromophore. Due to its enhanced performance, the partial-view-corrected weighted model-based algorithm may prove useful for improving the quality of partial-view multispectral optoacoustic tomography, leading to enhanced visualization of functional parameters such as tissue oxygenation. This article is protected by copyright. All rights reserved.

2022 Scientific Article in Nature Communications Nat. Commun. 13:2659 (2022)

Empl, L. ; Chovsepian, A. ; Chahin, M. ; Kan, W.Y.V. ; Fourneau, J. ; Van Steenbergen, V. ; Weidinger, S. ; Marcantoni, M. ; Ghanem, A. ; Bradley, P. ; Conzelmann, K.K. ; Cai, R. ; Ghasemigharagoz, A. ; Ertürk, A. ; Wagner, I. ; Kreutzfeldt, M. ; Merkler, D. ; Liebscher, S. ; Bareyre, F.M.

Selective plasticity of callosal neurons in the adult contralesional cortex following murine traumatic brain injury.

Traumatic brain injury (TBI) results in deficits that are often followed by recovery. The contralesional cortex can contribute to this process but how distinct contralesional neurons and circuits respond to injury remains to be determined. To unravel adaptations in the contralesional cortex, we used chronic in vivo two-photon imaging. We observed a general decrease in spine density with concomitant changes in spine dynamics over time. With retrograde co-labeling techniques, we showed that callosal neurons are uniquely affected by and responsive to TBI. To elucidate circuit connectivity, we used monosynaptic rabies tracing, clearing techniques and histology. We demonstrate that contralesional callosal neurons adapt their input circuitry by strengthening ipsilateral connections from pre-connected areas. Finally, functional in vivo two-photon imaging demonstrates that the restoration of pre-synaptic circuitry parallels the restoration of callosal activity patterns. Taken together our study thus delineates how callosal neurons structurally and functionally adapt following a contralateral murine TBI.

2022 Scientific Article in Science Translational Medicine Sci. Transl. Med. 14:eabm8059 (2022)

Hindelang, B. ; Nau, T. ; Englert, L. ; Berezhnoi, A. ; Lauffer, F. ; Darsow, U. ; Biedermann, T. ; Eyerich, K. ; Aguirre Bueno, J.&deg ; Ntziachristos, V.&deg

Enabling precision monitoring of psoriasis treatment by optoacoustic mesoscopy.

Psoriasis is a widespread inflammatory skin disease affecting about 2% of the general population. Recently, treatments that specifically target key proinflammatory cytokines driving the disease have been developed to complement conventional therapies with unspecific antiproliferative or anti-inflammatory effects. Efficient monitoring of treatment efficacy in the context of precision medicine and the assessment of new therapeutics require accurate noninvasive readouts of disease progression. However, characterization of psoriasis treatment remains subjective based on visual and palpatory clinical assessment of features observed on the skin surface. We hypothesized that optoacoustic (photoacoustic) mesoscopy could offer label-free assessment of inflammation biomarkers, extracted from three-dimensional (3D) high-resolution images of the human skin, not attainable by other noninvasive methods. We developed a second-generation ultra-broadband optoacoustic mesoscopy system, featuring sub-10-μm resolution and advanced motion correction technology, and performed 80 longitudinal measurements of 20 psoriatic skin plaques in humans under conventional inpatient treatment or receiving biologics with concomitant topical corticosteroid treatment. Optoacoustic image analysis revealed inflammatory and morphological skin features that indicated treatment efficacy with sensitivity, accuracy, and precision that was not possible using clinical metrics. We identify 3D imaging biomarkers that reveal responses to treatment and offer the potential to facilitate disease and treatment characterization. Our findings suggest that optoacoustic mesoscopy may offer a method of choice for yielding both qualitative and quantitative evaluations of skin treatments that are inaccessible by other methods, potentially enabling optimized therapies and precision medicine in dermatology.

2022 Scientific Article in Nature Communications Nat. Commun. 13:2803 (2022)

He, H. ; Schönmann, C. ; Schwarz, M. ; Hindelang, B. ; Berezhnoi, A. ; Steimle-Grauer, S.A. ; Darsow, U. ; Aguirre Bueno, J. ; Ntziachristos, V.

Fast raster-scan optoacoustic mesoscopy enables assessment of human melanoma microvasculature in vivo.

Melanoma is associated with angiogenesis and vascular changes that may extend through the entire skin depth. Three-dimensional imaging of vascular characteristics in skin lesions could therefore allow diagnostic insights not available by conventional visual inspection. Raster-scan optoacoustic mesoscopy (RSOM) images microvasculature through the entire skin depth with resolutions of tens of micrometers; however, current RSOM implementations are too slow to overcome the strong breathing motions on the upper torso where melanoma lesions commonly occur. To enable high-resolution imaging of melanoma vasculature in humans, we accelerate RSOM scanning using an illumination scheme that is coaxial with a high-sensitivity ultrasound detector path, yielding 15 s single-breath-hold scans that minimize motion artifacts. We apply this Fast RSOM to image 10 melanomas and 10 benign nevi in vivo, showing marked differences between malignant and benign lesions, supporting the possibility to use biomarkers extracted from RSOM imaging of vasculature for lesion characterization to improve diagnostics.

2022 Scientific Article in Medical Image Analysis Med. Image Anal. 77:102364 (2022)

Ayhan, M.S.# ; Kuemmerle, L.# ; Kühlewein, L. ; Inhoffen, W. ; Aliyeva, G. ; Ziemssen, F. ; Berens, P.

Clinical validation of saliency maps for understanding deep neural networks in ophthalmology.

Deep neural networks (DNNs) have achieved physician-level accuracy on many imaging-based medical diagnostic tasks, for example classification of retinal images in ophthalmology. However, their decision mechanisms are often considered impenetrable leading to a lack of trust by clinicians and patients. To alleviate this issue, a range of explanation methods have been proposed to expose the inner workings of DNNs leading to their decisions. For imaging-based tasks, this is often achieved via saliency maps. The quality of these maps are typically evaluated via perturbation analysis without experts involved. To facilitate the adoption and success of such automated systems, however, it is crucial to validate saliency maps against clinicians. In this study, we used three different network architectures and developed ensembles of DNNs to detect diabetic retinopathy and neovascular age-related macular degeneration from retinal fundus images and optical coherence tomography scans, respectively. We used a variety of explanation methods and obtained a comprehensive set of saliency maps for explaining the ensemble-based diagnostic decisions. Then, we systematically validated saliency maps against clinicians through two main analyses — a direct comparison of saliency maps with the expert annotations of disease-specific pathologies and perturbation analyses using also expert annotations as saliency maps. We found the choice of DNN architecture and explanation method to significantly influence the quality of saliency maps. Guided Backprop showed consistently good performance across disease scenarios and DNN architectures, suggesting that it provides a suitable starting point for explaining the decisions of DNNs on retinal images.

2022 Scientific Article in Engineering in Life Sciences Eng. Life Sci. 22, 100-114 (2022)

Verbelen, B. ; Girardi, T. ; Sulima, S.O. ; Vereecke, S. ; Verstraete, P. ; Verbeeck, J. ; Royaert, J. ; Cinque, S. ; Montanaro, L. ; Penzo, M. ; Imbrechts, M. ; Geukens, N. ; Geuens, T. ; Dierckx, K. ; Pepe, D. ; Kampen, K. ; De Keersmaecker, K.

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells.

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10-R98S). Our data support that RPL10-R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock-in of RPL10-R98S was associated with a 1.7- to 2.5-fold increased production of four transiently expressed recombinant proteins and 1.7-fold for one out of two stably expressed proteins. In CHO-S cells, eGFP reached a 2-fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10-R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10-R98S phenotypes can maximize its exploitability for the mammalian protein production industry.

2022 Scientific Article in Drug Delivery Drug Deliv. 29, 214-228 (2022)

Yan, C. ; Chen, J. ; Wang, C. ; Yuan, M. ; Kang, Y. ; Wu, Z. ; Li, W. ; Zhang, G. ; Machens, H.G. ; Rinkevich, Y. ; Chen, Z.&deg ; Yang, X.&deg ; Xu, X.&deg

Milk exosomes-mediated miR-31-5p delivery accelerates diabetic wound healing through promoting angiogenesis.

The refractory diabetic wound has remained a worldwide challenge as one of the major health problems. The impaired angiogenesis phase during diabetic wound healing partly contributes to the pathological process. MicroRNA (miRNA) is an essential regulator of gene expression in crucial biological processes and is a promising nucleic acid drug in therapeutic fields of the diabetic wound. However, miRNA therapies have limitations due to lacking an effective delivery system. In the present study, we found a significant reduction of miR-31-5p expression in the full-thickness wounds of diabetic mice compared to normal mice. Further, miR-31-5p has been proven to promote the proliferation, migration, and angiogenesis of endothelial cells. Thus, we conceived the idea of exogenously supplementing miR-31-5p mimics to treat the diabetic wound. We used milk-derived exosomes as a novel system for miR-31-5p delivery and successfully encapsulated miR-31-5p mimics into milk exosomes through electroporation. Then, we proved that the miR-31-5p loaded in exosomes achieved higher cell uptake and was able to resist degradation. Moreover, our miRNA-exosomal formulation demonstrated dramatically improved endothelial cell functions in vitro, together with the promotion of angiogenesis and enhanced diabetic wound healing in vivo. Collectively, our data showed the feasibility of milk exosomes as a scalable, biocompatible, and cost-effective delivery system to enhance the bioavailability and efficacy of miRNAs.

2022 Scientific Article in EJNMMI Research EJNMMI Res. 12:2 (2022)

Marcazzan, S. ; Braz Carvalho, M.J. ; Konrad, M. ; Strangmann, J. ; Tenditnaya, A. ; Baumeister, T. ; Schmid, R.M. ; Wester, H.J. ; Ntziachristos, V. ; Gorpas, D. ; Wang, T.C. ; Schottelius, M. ; Quante, M.

CXCR4 peptide-based fluorescence endoscopy in a mouse model of Barrett's esophagus.

BACKGROUND: Near-infrared (NIR) fluorescence imaging has been emerging as a promising strategy to overcome the high number of early esophageal adenocarcinomas missed by white light endoscopy and random biopsy collection. We performed a preclinical assessment of fluorescence imaging and endoscopy using a novel CXCR4-targeted fluorescent peptide ligand in the L2-IL1B mouse model of Barrett's esophagus. METHODS: Six L2-IL1B mice with advanced stage of disease (12-16 months old) were injected with the CXCR4-targeted, Sulfo-Cy5-labeled peptide (MK007), and ex vivo wide-field imaging of the whole stomach was performed 4 h after injection. Before ex vivo imaging, fluorescence endoscopy was performed in three L2-IL1B mice (12-14 months old)  by a novel imaging system with two L2-IL1B mice used as negative controls. RESULTS: Ex vivo imaging and endoscopy in L2-IL1B mice showed that the CXCR4-targeted MK007 accumulated mostly in the dysplastic lesions with a mean target-to-background ratio > 2. The detection of the Sulfo-Cy5 signal in dysplastic lesions and its co-localization with CXCR4 stained cells  by confocal microscopy further confirmed the imaging results. CONCLUSIONS: This preliminary preclinical study shows that CXCR4-targeted fluorescence endoscopy using MK007 can detect dysplastic lesions in a mouse model of Barrett's esophagus. Further investigations are needed to assess its use in the clinical setting.

2022 Scientific Article in Journal of Nuclear Medicine J. Nucl. Med. 63, 640-645 (2022)

Heeman, W. ; Vonk, J. ; Ntziachristos, V. ; Pogue, B.W. ; Dierckx, R.A.J.O. ; Kruijff, S. ; van Dam, G.M.

A guideline for clinicians performing clinical studies with fluorescence imaging.

Fluorescence imaging is an emerging imaging technique that has shown many benefits for clinical care. Currently, the field is in rapid clinical translation, and an unprecedented number of clinical trials are performed. Clinicians are inundated with numerous opportunities and combinations of different imaging modalities. To streamline this process, a multidisciplinary approach is needed with drug discovery, software and systems engineering, and translational medicine. Here, we discuss the main constituents of a uniform fluorescence imaging protocol to match the clinical need and ensure consistent study designs and reliable data collection in clinical trials. In an era in which the potential of fluorescence imaging has become evident, consistent conduct of studies, data analysis, and data interpretation is essential for implementation into the standard of care.

2022 Scientific Article in Nanomedicine Nanomed. 40:102511 (2022)

Khalin, I.&deg ; Severi, C. ; Heimburger, D. ; Wehn, A. ; Hellal, F. ; Reisch, A.&deg ; Klymchenko, A.S.&deg ; Plesnila, N.&deg

Dynamic tracing using ultra-bright labelling and multi-photon microscopy identifies endothelial uptake of poloxamer 188 coated poly(lactic-co-glycolic acid) nano-carriers in vivo.

The potential of poly(lactic-co-glycolic acid) (PLGA) to design nanoparticles (NPs) and target the central nervous system remains to be exploited. In the current study we designed fluorescent 70-nm PLGA NPs, loaded with bulky fluorophores, thereby making them significantly brighter than quantum dots in single-particle fluorescence measurements. The high brightness of NPs enabled their visualization by intravital real-time 2-photon microscopy. Subsequently, we found that PLGA NPs coated with pluronic F-68 circulated in the blood substantially longer than uncoated NPs and were taken up by cerebro-vascular endothelial cells. Additionally, confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1hour after systemic injection and were more readily taken up by endothelial cells in peripheral organs. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers.

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 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 Nature Methods Nat. Methods 19, 171–178 (2022)

Palla, G.# ; Spitzer, H.# ; Klein, M. ; Fischer, D.S. ; Schaar, A. ; Kuemmerle, L. ; Rybakov, S. ; Ibarra Del Rio, I.A. ; Holmberg, O. ; Virshup, I. ; Lotfollahi, M. ; Richter, S. ; Theis, F.J.

Squidpy: A scalable framework for spatial omics analysis.

Spatial omics data are advancing the study of tissue organization and cellular communication at an unprecedented scale. Flexible tools are required to store, integrate and visualize the large diversity of spatial omics data. Here, we present Squidpy, a Python framework that brings together tools from omics and image analysis to enable scalable description of spatial molecular data, such as transcriptome or multivariate proteins. Squidpy provides efficient infrastructure and numerous analysis methods that allow to efficiently store, manipulate and interactively visualize spatial omics data. Squidpy is extensible and can be interfaced with a variety of already existing libraries for the scalable analysis of spatial omics data.

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 Optica Optica 9, 32-41 (2022)

Li, J. ; Wang, C. ; Chen, T. ; Lu, T. ; Li, S. ; Sun, B.&deg ; Gao, F.&deg ; Ntziachristos, V.&deg

Deep learning-based quantitative optoacoustic tomography of deep tissues in the absence of labeled experimental data.

Deep learning (DL) shows promise for quantitating anatomical features and functional parameters of tissues in quantitative optoacoustic tomography (QOAT), but its application to deep tissue is hindered by a lack of ground truth data. We propose DL-based "QOAT-Net,"which functions without labeled experimental data: A dual-path convolutional network estimates absorption coefficients after training with data-label pairs generated via unsupervised "simulation-to-experiment"data translation. In simulations, phantoms, and ex vivo and in vivo tissues, QOAT-Net affords quantitative absorption images with high spatial resolution. This approach makes DL-based QOAT and other imaging applications feasible in the absence of ground truth data.

2022 Scientific Article in Photoacoustics Photoacoustics 25:100301 (2022)

Fuenzalida Werner, J.P. ; Mishra, K. ; Stankevych, M. ; Klemm, U. ; Ntziachristos, V. ; Stiel, A.-C.

Alginate beads as a highly versatile test-sample for optoacoustic imaging.

Test-samples are necessary for the development of emerging imaging approaches such as optoacoustics (OA); these can be used to benchmark new labeling agents and instrumentation, or to characterize image analysis algorithms or the inversion required to form the three-dimensional reconstructions. Alginate beads (AlBes) loaded with labeled mammalian or bacterial cells provide a method of creating defined structures of controllable size and photophysical characteristics and are well-suited for both in vitro and in vivo use. Here we describe a simple and rapid method for efficient and reproducible production of AlBes with specific characteristics and show three example applications with multispectral OA tomography imaging. We show the advantage of AlBes for studying and eventually improving photo-switching OA imaging approaches. As highly defined, homogeneous, quasi point-like signal sources, AlBes might hold similar advantages for studying other agents, light-fluence models, or the impact of detection geometries on correct image formation in the near future.

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: Molecular Imaging (Second Edition). 2021. 143-152

Gorpas, D. ; Ntziachristos, V. ; Tian, J.

Principles and practice of intraoperative fluorescence imaging.

Fluorescence-guided surgery (FGS) is an optical-based, real-time, intraoperative imaging technique, which can effectively expand the visual range of surgeons and assist clinical decision-making. Due to the radioactivity and the difficulty of long-term, continuous, real-time in vivo imaging, traditional imaging methods for preoperative diagnosis and postoperative evaluation are inappropriate for the continuous, real-time, in vivo imaging that is necessary for surgical guidance. Thus, visual and tactile sensing combined with the experience of the physician are still the key factors affecting the surgical outcome. Therefore, there is an urgent need for an intraoperative imaging technique to distinguish lesions from normal tissues, important vessels, and organs. FGS has the advantages of high sensitivity, contrast, and specificity, without exposing the patient to ionizing radiation. It also has the capability to identify the boundaries and reflect the position of superficial lesions during a surgical operation, thus providing the technical means for the early detection and precise resection of small lesions. The validation of FGS technology’s performance through both imaging equipment development and standardization and clinical trials is expected to reduce iatrogenic trauma and improve the postoperative survival rates and quality of life. This chapter focuses on excitation fluorescence imaging and its clinical applications. Following a description of the principles underlying this technology, the three main fluorescent agents currently in clinical use are presented. Some representative clinical FGS systems, either for open or endoscopic surgery, are described, and the still unmet need for standardization of FGS systems and methods is discussed.

In: (European Conference on Biomedical Optics, 20–24 June 2021, Munich Germany). 2021. DOI: 10.1117/12.2614318 ( ; 11923)

Dehner, C. ; Olefir, I. ; Basak, K. ; Jüstel, D. ; Ntziachristos, V.

Deep-learning-based electrical noise removal for localized spectral optoacoustic contrast in deep tissue.

Image contrast in multispectral optoacoustic tomography can be reduced by electrical noise. We present a deep learning method to remove electrical noise from optoacoustic signals and thereby significantly enhance morphological and spectral contrast.

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.

In: (European Conferences on Biomedical Optics 2021, 20–24 June 2021, Munich, Germany). 2021.

Subochev, P.&deg ; Deán-Ben, X.L. ; Chen, Z. ; Orlova, A. ; Razansky, D.&deg

PVDF spherical matrix array for high resolution cerebral optoacoustic micro-angiography of rodents.

We developed high-density spherical matrix array based on polyvinylidene difluoride films. Ultrawide bandwidth (0.3-38 MHz) and sub-millimeter sized elements enabled non-invasive cerebrovascular imaging of adult mouse with ~60 µm resolution.

In: (European Conference on Biomedical Optics 2021, 20–24 June 2021, Munich, Germany). 2021.

Jüstel, D.&deg ; Basak, K.&deg ; Bader, M.&deg ; Dehner, C.&deg ; Ntziachristos, V.&deg

Impulse response correction enables high-resolution clinical hand-held optoacoustics.

The total impulse response of a clinical optoacoustic system is characterized by combining experimentally acquired signals with a numerical model of the spatial impulse response, resulting in high-resolution images in clinical applications.

In: (European Conference on Biomedical Optics 2021, 20–24 June 2021, Munich, Germany). 2021.

Pieters, C. ; Westerveld, W.J. ; Mahmud-Ul-Hasan, M. ; Severi, S. ; Shnaiderman, R. ; Ntziachristos, V. ; Billen, M. ; Kjellman, J. ; Jansen, R. ; Rochus, V. ; Rottenberg, X.

Sensitive optomechanical ultrasound sensor in a silicon photonic chip towards single-shot photoacoustic imaging with an ultrasound sensor matrix.

We propose a new opto-mechanical ultrasound sensor (OMUS) enabled by an innovative silicon photonics waveguide. We present experimental results up to 30 MHz, a 10-sensor array proof-of-concept and our latest findings.

2021 Scientific Article in Frontiers in Cardiovascular Medicine Front. Cardiovasc. Med. 8:755968 (2021)

Alkhodari, M.&deg ; Jelinek, H.F. ; Karlas, A. ; Soulaidopoulos, S. ; Arsenos, P. ; Doundoulakis, I. ; Gatzoulis, K.A. ; Tsioufis, K. ; Hadjileontiadis, L.J. ; Khandoker, A.H.&deg

Deep learning predicts heart failure with preserved, mid-range, and reduced left ventricular ejection fraction from patient clinical profiles.

Background: Left ventricular ejection fraction (LVEF) is the gold standard for evaluating heart failure (HF) in coronary artery disease (CAD) patients. It is an essential metric in categorizing HF patients as preserved (HFpEF), mid-range (HFmEF), and reduced (HFrEF) ejection fraction but differs, depending on whether the ASE/EACVI or ESC guidelines are used to classify HF. Objectives: We sought to investigate the effectiveness of using deep learning as an automated tool to predict LVEF from patient clinical profiles using regression and classification trained models. We further investigate the effect of utilizing other LVEF-based thresholds to examine the discrimination ability of deep learning between HF categories grouped with narrower ranges. Methods: Data from 303 CAD patients were obtained from American and Greek patient databases and categorized based on the American Society of Echocardiography and the European Association of Cardiovascular Imaging (ASE/EACVI) guidelines into HFpEF (EF > 55%), HFmEF (50% ≤ EF ≤ 55%), and HFrEF (EF < 50%). Clinical profiles included 13 demographical and clinical markers grouped as cardiovascular risk factors, medication, and history. The most significant and important markers were determined using linear regression fitting and Chi-squared test combined with a novel dimensionality reduction algorithm based on arc radial visualization (ArcViz). Two deep learning-based models were then developed and trained using convolutional neural networks (CNN) to estimate LVEF levels from the clinical information and for classification into one of three LVEF-based HF categories. Results: A total of seven clinical markers were found important for discriminating between the three HF categories. Using statistical analysis, diabetes, diuretics medication, and prior myocardial infarction were found statistically significant (p < 0.001). Furthermore, age, body mass index (BMI), anti-arrhythmics medication, and previous ventricular tachycardia were found important after projections on the ArcViz convex hull with an average nearest centroid (NC) accuracy of 94%. The regression model estimated LVEF levels successfully with an overall accuracy of 90%, average root mean square error (RMSE) of 4.13, and correlation coefficient of 0.85. A significant improvement was then obtained with the classification model, which predicted HF categories with an accuracy ≥93%, sensitivity ≥89%, 1-specificity <5%, and average area under the receiver operating characteristics curve (AUROC) of 0.98. Conclusions: Our study suggests the potential of implementing deep learning-based models clinically to ensure faster, yet accurate, automatic prediction of HF based on the ASE/EACVI LVEF guidelines with only clinical profiles and corresponding information as input to the models. Invasive, expensive, and time-consuming clinical testing could thus be avoided, enabling reduced stress in patients and simpler triage for further intervention.

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 Physics in Medicine and Biology Phys. Med. Biol. 66:245020 (2021)

Wieser, H.P.#&deg ; Huang, Y.# ; Schauer, J.# ; Lascaud, J. ; Wuerl, M. ; Lehrack, S. ; Radonic, D. ; Vidal, M. ; Herault, J. ; Chmyrov, A. ; Ntziachristos, V. ; Assmann, W. ; Parodi, K.&deg ; Dollinger, G.

Experimental demonstration of accurate Bragg peak localization with ionoacoustic tandem phase detection (iTPD).

Accurate knowledge of the exact stopping location of ions inside the patient would allow full exploitation of their ballistic properties for patient treatment. The localized energy deposition of a pulsed particle beam induces a rapid temperature increase of the irradiated volume and leads to the emission of ionoacoustic (IA) waves. Detecting the time-of-flight (ToF) of the IA wave allows inferring information on the Bragg peak location and can henceforth be used for in-vivo range verification. A challenge for IA is the poor signal-to-noise ratio at clinically relevant doses and viable machines. We present a frequency-based measurement technique, labeled as ionoacoustic tandem phase detection (iTPD) utilizing lock-in amplifiers. The phase shift of the IA signal to a reference signal is measured to derive the ToF. Experimental IA measurements with a 3.5 MHz lead zirconate titanate (PZT) transducer and lock-in amplifiers were performed in water using 22 MeV proton bursts. A digital iTPD was performed in-silico at clinical dose levels on experimental data obtained from a clinical facility and secondly, on simulations emulating a heterogeneous geometry. For the experimental setup using 22 MeV protons, a localization accuracy and precision obtained through iTPD deviates from a time-based reference analysis by less than 15 mu m. Several methodological aspects were investigated experimentally in systematic manner. Lastly, iTPD was evaluated in-silico for clinical beam energies indicating that iTPD is in reach of sub-mm accuracy for fractionated doses < 5 Gy. iTPD can be used to accurately measure the ToF of IA signals online via its phase shift in frequency domain. An application of iTPD to the clinical scenario using a single pulsed beam is feasible but requires further development to reach <1 Gy detection capabilities.

2021 Scientific Article in Scientific Reports Sci. Rep. 11:24430 (2021)

Yun, M. ; You, S.H. ; Nguyen, V.H. ; Prakash, J. ; Glasl, S. ; Gujrati, V. ; Choy, H.E. ; Stiel, A.-C. ; Min, J.J.&deg ; Ntziachristos, V.&deg

Reporter gene-based optoacoustic imaging of E. coli targeted colon cancer in vivo.

Bacteria-mediated cancer-targeted therapy is a novel experimental strategy for the treatment of cancers. Bacteria can be engineered to overcome a major challenge of existing therapeutics by differentiating between malignant and healthy tissue. A prerequisite for further development and study of engineered bacteria is a suitable imaging concept which allows bacterial visualization in tissue and monitoring bacterial targeting and proliferation. Optoacoustics (OA) is an evolving technology allowing whole-tumor imaging and thereby direct observation of bacterial colonization in tumor regions. However, bacterial detection using OA is currently hampered by the lack of endogenous contrast or suitable transgene fluorescent labels. Here, we demonstrate improved visualization of cancer-targeting bacteria using OA imaging and E. coli engineered to express tyrosinase, which uses L-tyrosine as the substrate to produce the strong optoacoustic probe melanin in the tumor microenvironment. Tumors of animals injected with tyrosinase-expressing E. coli showed strong melanin signals, allowing to resolve bacterial growth in the tumor over time using multispectral OA tomography (MSOT). MSOT imaging of melanin accumulation in tumors was confirmed by melanin and E. coli staining. Our results demonstrate that using tyrosinase-expressing E. coli enables non-invasive, longitudinal monitoring of bacterial targeting and proliferation in cancer using MSOT.

2021 Scientific Article in Cell Reports Cell Rep. 37:110161 (2021)

Grimm, C. ; Frässle, S. ; Steger, C. ; von Ziegler, L. ; Sturman, O. ; Shemesh, N. ; Peleg-Raibstein, D. ; Burdakov, D. ; Bohacek, J. ; Stephan, K.E. ; Razansky, D. ; Wenderoth, N. ; Zerbi, V.

Optogenetic activation of striatal D1R and D2R cells differentially engages downstream connected areas beyond the basal ganglia.

The basal ganglia (BG) are a group of subcortical nuclei responsible for motor and executive function. Central to BG function are striatal cells expressing D1 (D1R) and D2 (D2R) dopamine receptors. D1R and D2R cells are considered functional antagonists that facilitate voluntary movements and inhibit competing motor patterns, respectively. However, whether they maintain a uniform function across the striatum and what influence they exert outside the BG is unclear. Here, we address these questions by combining optogenetic activation of D1R and D2R cells in the mouse ventrolateral caudoputamen with fMRI. Striatal D1R/D2R stimulation evokes distinct activity within the BG-thalamocortical network and differentially engages cerebellar and prefrontal regions. Computational modeling of effective connectivity confirms that changes in D1R/D2R output drive functional relationships between these regions. Our results suggest a complex functional organization of striatal D1R/D2R cells and hint toward an interconnected fronto-BG-cerebellar network modulated by striatal D1R and D2R cells.

2021 Scientific Article in Biomedicines Biomedicines 9:1696 (2021)

Karlas, A.# ; Nunes, A.# ; Driessen, W.&deg ; Liapis, E. ; Reber, J.&deg

Multi-aspect optoacoustic imaging of breast tumors under chemotherapy with exogenous and endogenous contrasts: Focus on apoptosis and hypoxia.

Breast cancer is a complex tumor type involving many biological processes. Most chemotherapeutic agents exert their antitumoral effects by rapid induction of apoptosis. Another main feature of breast cancer is hypoxia, which may drive malignant progression and confer resistance to various forms of therapy. Thus, multi-aspect imaging of both tumor apoptosis and oxygenation in vivo would be of enormous value for the effective evaluation of therapy response. Herein, we demonstrate the capability of a hybrid imaging modality known as multispectral optoacoustic tomography (MSOT) to provide high-resolution, simultaneous imaging of tumor apoptosis and oxygenation, based on both the exogenous contrast of an apoptosis-targeting dye and the endogenous contrast of hemoglobin. MSOT imaging was applied on mice bearing orthotopic 4T1 breast tumors before and following treatment with doxorubicin. Apoptosis was monitored over time by imaging the distribution of xPLORE-APOFL750©, a highly sensitive poly-caspase binding apoptotic probe, within the tumors. Oxygenation was monitored by tracking the distribution of oxy- and deoxygenated hemoglobin within the same tumor areas. Doxorubicin treatment induced an increase in apoptosis-depending optoacoustic signal of xPLORE-APOFL750© at 24 h after treatment. Furthermore, our results showed spatial correspondence between xPLORE-APO750© and deoxygenated hemoglobin. In vivo apoptotic status of the tumor tissue was independently verified by ex vivo fluorescence analysis. Overall, our results provide a rationale for the use of MSOT as an effective tool for simultaneously investigating various aspects of tumor pathophysiology and potential effects of therapeutic regimes based on both endogenous and exogenous molecular contrasts.

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 European Journal of Nuclear Medicine and Molecular Imaging Eur. J. Nucl. Med. Mol. Imaging, DOI: 10.1007/s00259-021-05582-y (2021)

Fang, H.Y.# ; Stangl, S.# ; Marcazzan, S.# ; Carvalho, M.J.B. ; Baumeister, T. ; Anand, A. ; Strangmann, J. ; Huspenina, J.S. ; Wang, T.C. ; Schmid, R.M. ; Feith, M. ; Friess, H. ; Ntziachristos, V. ; Multhoff, G. ; Gorpas, D.&deg ; Quante, M.&deg

Targeted Hsp70 fluorescence molecular endoscopy detects dysplasia in Barrett's esophagus.

PURPOSE: The incidence of esophageal adenocarcinoma (EAC) has been increasing for decades without significant improvements in treatment. Barrett's esophagus (BE) is best established risk factor for EAC, but current surveillance with random biopsies cannot predict progression to cancer in most BE patients due to the low sensitivity and specificity of high-definition white light endoscopy. METHODS: Here, we evaluated the membrane-bound highly specific Hsp70-specific contrast agent Tumor-Penetrating Peptide (Hsp70-TPP) in guided fluorescence molecular endoscopy biopsy. RESULTS: Hsp70 was significantly overexpressed as determined by IHC in dysplasia and EAC compared with non-dysplastic BE in patient samples (n = 12) and in high-grade dysplastic lesions in a transgenic (L2-IL1b) mouse model of BE. In time-lapse microscopy, Hsp70-TPP was rapidly taken up and internalized  by human BE dysplastic patient-derived organoids. Flexible fluorescence endoscopy of the BE mouse model allowed a specific detection of Hsp70-TPP-Cy5.5 that corresponded closely with the degree of dysplasia but not BE. Ex vivo application of Hsp70-TPP-Cy5.5 to freshly resected whole human EAC specimens revealed a high (> 4) tumor-to-background ratio and a specific detection of previously undetected tumor infiltrations. CONCLUSION: In summary, these findings suggest that Hsp70-targeted imaging using fluorescently labeled TPP peptide may improve tumor surveillance in BE patients.

2021 Scientific Article in Biochemistry Biochemistry 86, 1434-1445 (2021)

Balint, V. ; Stanisavljevic Ninkovic, D. ; Anastasov, N. ; Lazic, S. ; Kovacevic-Grujicic, N. ; Stevanovic, M. ; Lazic, A.

Inhibition of miR-21 promotes cellular senescence in NT2-derived astrocytes.

Abstract: Astrocytes are the main homeostatic cells in the central nervous system (CNS) that provide mechanical, metabolic, and trophic support to neurons. Disruption of their physiological role or acquisition of senescence-associated phenotype can contribute to the CNS dysfunction and pathology. However, molecular mechanisms underlying the complex physiology of astrocytes are explored insufficiently. Recent studies have shown that miRNAs are involved in the regulation of astrocyte function through different mechanisms. Although miR-21 has been reported as an astrocytic miRNA with an important role in astrogliosis, no link between this miRNA and cellular senescence of astrocytes has been identified. To address the role of miR-21 in astrocytes, with special focus on cellular senescence, we used NT2/A (astrocytes derived from NT2/D1 cells). Downregulation of miR-21 expression in both immature and mature NT2/A by the antisense technology induced the arrest of cell growth and premature cellular senescence, as indicated by senescence hallmarks such as increased expression of cell cycle inhibitors p21 and p53 and augmented senescence-associated β-galactosidase activity. Additionally, in silico analysis predicted many of the genes, previously shown to be upregulated in astrocytes with the irradiation-induced senescence, as miR-21 targets. Taken together, our results point to miR-21 as a potential regulator of astrocyte senescence. To the best of our knowledge, these are the first data showing the link between miR-21 and cellular senescence of astrocytes. Since senescent astrocytes are associated with different CNS pathologies, development of novel therapeutic strategies based on miRNA manipulation could prevent senescence and may improve the physiological outcome.

2021 Scientific Article in ACS Photonics ACS Photonics 8, 3346–3356 (2021)

Kaydanov, N.&deg ; Perevoschikov, S. ; German, S.V. ; Romanov, S.A. ; Ermatov, T. ; Kozyrev, A.A. ; Cvjetinovic, J. ; MacHnev, A. ; Noskov, R.E. ; Kosolobov, S.S. ; Skibina, J.S. ; Nasibulin, A.G. ; Zakian Dominguez, C.M. ; Lagoudakis, P.G. ; Gorin, D.A.&deg

Optoacoustic effect in a hybrid multilayered membrane deposited on a hollow-core microstructured optical waveguide.

Modern imaging technologies, including optoacoustic endoscopy, are based on the optoacoustic effect. Much promise is offered by the all-optical fiber-based approach, because fiber has a miniature cross section, is highly sensitive, and can be used in a variety of imaging and therapeutic techniques. We developed a probe based on a hollow-core microstructured optical waveguide (HC-MOW) with a hybrid nanostructured membrane. The membrane consisted of a free-standing single-walled carbon nanotube film and a Bragg reflector, which can be used as a source and a detector of ultrasound. Membrane vibrations were excited with an IR laser pulse and were read out by recording the intensity of the reflected visible CW laser light. We explained the nature of the intensity modulation of the reflected light and supported our explanation with numerical simulations of the membrane's vibration eigenfrequencies and thermal distribution. The membrane vibrations were also observed with raster-scanning optoacoustic mesoscopy. The transmittance of the HC-MOW between 400 nm and 6.5 μm and that of the hybrid nanostructured membrane in the NIR range enable potential optoacoustic sensing in the IR fingerprint region of biomolecules. This permits the optoacoustic probe to be used for medical endoscopic purposes.

2021 Scientific Article in Analytical Chemistry Anal. Chem. 93, 15323-15330 (2021)

Yuan, T. ; Pleitez, M.A.&deg ; Gasparin, F. ; Ntziachristos, V.&deg

Wide-field mid-infrared hyperspectral imaging by snapshot phase contrast measurement of optothermal excitation.

Vibrational microscopy methods based on Raman scattering or infrared absorption provide a label-free approach for chemical-contrast imaging, but employ point-by-point scanning and impose a compromise between the imaging speed and field-of-view (FOV). Optothermal microscopy has been proposed as a promising imaging modality to avoid this compromise, although at restrictively small FOVs capable of imaging only few cells. Here, we present wide-field optothermal mid-infrared microscopy (WOMiM) for wide-field chemical-contrast imaging based on snapshot pump-probe detection of optothermal signal, using a custom-made condenser-free phase contrast microscopy to capture the phase change of samples after mid-infrared irradiation. We achieved chemical contrast for FOVs up to 180 μm in diameter, yielding 10-fold larger imaging areas than the state-of-the-art, at imaging speeds of 1 ms/frame. The maximum possible imaging speed of WOMiM was determined by the relaxation time of optothermal heat, measured to be 32.8 μs in water, corresponding to a frame rate of μ30 kHz. This proof-of-concept demonstrates that vibrational imaging can be achieved at an unprecedented imaging speed and large FOV with the potential to significantly facilitate label-free imaging of cellular dynamics.

Optics InfoBase Conference Papers In: (2021 European Conferences on Biomedical Optics, ECBO 2021, 20-24 June 2021, Virtual, Online). 2021.:ES1C.1 (Optics InfoBase Conference Papers)

Dehner, C. ; Olefir, I. ; Basak, K. ; Jüstel, D. ; Ntziachristos, V.

Deep learning based electrical noise removal for localized spectral optoacoustic contrast in deep tissue.

Image contrast in multispectral optoacoustic tomography can be reduced by electrical noise. We present a deep learning method to remove electrical noise from optoacoustic signals and thereby significantly enhance morphological and spectral contrast.

2021 Scientific Article in International Journal of Molecular Sciences Int. J. Mol. Sci. 22:12275 (2021)

Gabashvili, A.N. ; Vodopyanov, S.S. ; Chmelyuk, N.S. ; Sarkisova, V.A. ; Fedotov, K.A. ; Efremova, M.V. ; Abakumov, M.A.

Encapsulin based self‐assembling iron‐containing protein nanoparticles for stem cells mri visualization.

Over the past decade, cell therapy has found many applications in the treatment of different diseases. Some of the cells already used in clinical practice include stem cells and CAR‐T cells. Compared with traditional drugs, living cells are much more complicated systems that must be strictly controlled to avoid undesirable migration, differentiation, or proliferation. One of the approaches used to prevent such side effects involves monitoring cell distribution in the human body by any noninvasive technique, such as magnetic resonance imaging (MRI). Long‐term tracking of stem cells with artificial magnetic labels, such as magnetic nanoparticles, is quite problematic because such labels can affect the metabolic process and cell viability. Additionally, the concentration of exogenous labels will decrease during cell division, leading to a corresponding decrease in signal intensity. In the current work, we present a new type of genetically encoded label based on encapsulin from Myxococcus xanthus bacteria, stably expressed in human mesenchymal stem cells (MSCs) and coexpressed with ferroxidase as a cargo protein for nanoparticles’ synthesis inside encapsulin shells. mZip14 protein was expressed for the enhancement of iron transport into the cell. Together, these three proteins led to the synthesis of iron‐containing nanoparticles in mesenchymal stem cells—without affecting cell viability—and increased contrast properties of MSCs in MRI.

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 Nature Biotechnology Nat. Biotechnol., DOI: 10.1038/s41587-021-01100-5 (2021)

Mishra, K.# ; Fuenzalida Werner, J.P.# ; Pennacchietti, F. [extern]# ; Janowski, R.# ; Chmyrov, A. ; Huang, Y. ; Zakian Dominguez, C.M. ; Klemm, U. ; Testa, I. [extern] ; Niessing, D. ; Ntziachristos, V. ; Stiel, A.-C.

Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging.

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.Calcium and other analytes can be imaged at super-resolution and in vivo with photo-switchable sensors.

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 Review in Hautarzt, Der Hautarzt 72, 1025-1038 (2021)

Nau, T. ; Schneider, S.A. ; Aguirre Bueno, J. ; Ntziachristos, V. ; Biedermann, T. ; Darsow, U.

Optoakustische Bildgebung – innovative Bildgebungsverfahren auf dem Vormarsch.

Die optoakustische Bildgebung (OAB) hat sich in den letzten Jahren stetigweiterentwickelt. Mittels teils gepulsten Lichts in verschiedenen Wellenlängen werdenunterschiedliche Farbträger (Chromophore) zur Bildung von Schallwellen angeregt.Diese werden von den neu entwickelten Systemen detektiert und mittels verschiedenerAlgorithmen in dreidimensionale Bilder umgewandelt. Die Technik zeichnet sich durchein gutes Verhältnis von Kontrast und Eindringtiefe aus und kann aufgrund ihrerSkalierbarkeit makro-, meso- und mikroskopische Bilder erzeugen. Die optoakustischeMakroskopie bestrahlt das zu untersuchende Areal breit mit Laserlicht. Hierdurchkönnen Abbildungen mit hoher Eindringtiefe erzeugt werden, jedoch lediglich miteiner mittleren Auflösung. Klinisch interessante Anwendungsfelder sind z.B. dieErgebnisse von ex-vivo untersuchten Sentinellymphknoten mittels makroskopischerOptoakustik. Aufgrund der Fähigkeit der OAB Melanin darzustellen zeigte sicheine bisherigen bildgebenden Methoden, jedoch nicht der Histologie überlegeneDetektionsrate für Metastasen. Die Fähigkeit, mit einer guten Auflösung dermale undepidermale Strukturen, besonders Gefäße, darzustellen, macht sich die optoakustischeMesoskopie bei der Untersuchung entzündlicher Hauterkrankungen zunutze undkönnte künftig zur Überprüfung des Therapieerfolges, z.B. durch Biologika bei Psoriasisvulgaris oder dem atopischen Ekzem, beitragen. Die bisher v. a. auf präklinische In-vivo-Forschung beschränkte optoakustische Mikroskopie könnte künftig zur Detektion nochfeinerer Gefäßstrukturen und deren Veränderungen verwendet werden. Die klinischenMöglichkeiten der OAB scheinen bisher sehr großen Nutzen bieten zu können und sindein aktuell stark untersuchtes Forschungsfeld.

Lecture Notes in Computer Science In: (12th International Workshop on Machine Learning in Medical Imaging, MLMI 2021, 27 September 2021, Virtual, Online). 2021. 596-605 (Lect. Notes Comput. Sc. ; 12966 LNCS)

Dima, A. ; Paetzold, J.C. ; Jungmann, F. ; Lemke, T. ; Raffler, P. ; Kaissis, G. ; Rueckert, D. ; Braren, R.

Segmentation of peripancreatic arteries in multispectral computed tomography imaging.

Pancreatic ductal adenocarcinoma is an aggressive form of cancer with a poor prognosis, where the operability and hence chance of survival is strongly affected by the tumor infiltration of the arteries. In an effort to enable an automated analysis of the relationship between the local arteries and the tumor, we propose a method for segmenting the peripancreatic arteries in multispectral CT images in the arterial phase. A clinical dataset was collected, and we designed a fast semi-manual annotation procedure, which requires around 20 min of annotation time per case. Next, we trained a U-Net based model to perform binary segmentation of the peripancreatic arteries, where we obtained a near perfect segmentation with a Dice score of 95.05 % in our best performing model. Furthermore, we designed a clinical evaluation procedure for our models; performed by two radiologists, yielding a complete segmentation of 85.31 % of the clinically relevant arteries, thereby confirming the clinical relevance of our method.

2021 Review in Nature biomedical engineering Nat. Bio. Eng. 6, 503-514 (2021)

Voskuil, F.J.# ; Vonk, J.# ; van der Vegt, B. ; Kruijff, S. ; Ntziachristos, V. ; van der Zaag, P.J. ; Witjes, M.J.H. ; van Dam, G.M.

Intraoperative imaging in pathology-assisted surgery.

The pathological assessment of surgical specimens during surgery can reduce the incidence of positive resection margins, which otherwise can result in additional surgeries or aggressive therapeutic regimens. To improve patient outcomes, intraoperative spectroscopic, fluorescence-based, structural, optoacoustic and radiological imaging techniques are being tested on freshly excised tissue. The specific clinical setting and tumour type largely determine whether endogenous or exogenous contrast is to be detected and whether the tumour specificity of the detected biomarker, image resolution, image-acquisition times or penetration depth are to be prioritized. In this Perspective, we describe current clinical standards for intraoperative tissue analysis and discuss how intraoperative imaging is being implemented. We also discuss potential implementations of intraoperative pathology-assisted surgery for clinical decision-making.

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 Scientific Reports Sci. Rep. 11:18370 (2021)

Afshari, P. ; Zakian Dominguez, C.M. ; Bachmann, J. ; Ntziachristos, V.

Speckle reduction in ultrasound endoscopy using refraction based elevational angular compounding.

Endoscopic ultrasonography (EUS) is a safe, real-time diagnostic and therapeutic tool. Speckle noise, inherent to ultrasonography, degrades the diagnostic precision of EUS. Elevational angular compounding (EAC) can provide real-time speckle noise reduction; however, EAC has never been applied to EUS because current implementations require costly and bulky arrays and are incompatible with the tight spatial constraints of hollow organs. Here we develop a radial implementation of a refraction-based elevational angular compounding technique (REACT) for EUS and demonstrate for the first time spatial compounding in a radial endoscopy. The proposed implementation was investigated in cylindrical phantoms and demonstrated superior suppression of ultrasound speckle noise and up to a two-fold improvement in signal- and contrast- ratios, compared to standard image processing techniques and averaging. The effect of elevational angular deflection on image fidelity was further investigated in a phantom with lymph node-like structures to determine the optimum elevational angular width for high speckle reduction efficiency while maintaining image fidelity. This study introduces REACT as a potential compact and low-cost solution to impart current radial echo-endoscopes with spatial compounding, which could enable accurate identification and precise sizing of lymph nodes in staging of gastrointestinal tract cancers.

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 Communications Biology Comm. Biol. 4:1040 (2021)

Seeger, M. ; Dehner, C. ; Jüstel, D. ; Ntziachristos, V.

Label-free concurrent 5-modal microscopy (Co5M) resolves unknown spatio-temporal processes in wound healing.

The non-invasive investigation of multiple biological processes remains a methodological challenge as it requires capturing different contrast mechanisms, usually not available with any single modality. Intravital microscopy has played a key role in dynamically studying biological morphology and function, but it is generally limited to resolving a small number of contrasts, typically generated by the use of transgenic labels, disturbing the biological system. We introduce concurrent 5-modal microscopy (Co5M), illustrating a new concept for label-free in vivo observations by simultaneously capturing optoacoustic, two-photon excitation fluorescence, second and third harmonic generation, and brightfield contrast. We apply Co5M to non-invasively visualize multiple wound healing biomarkers and quantitatively monitor a number of processes and features, including longitudinal changes in wound shape, microvascular and collagen density, vessel size and fractality, and the plasticity of sebaceous glands. Analysis of these parameters offers unique insights into the interplay of wound closure, vasodilation, angiogenesis, skin contracture, and epithelial reformation in space and time, inaccessible by other methods. Co5M challenges the conventional concept of biological observation by yielding multiple simultaneous parameters of pathophysiological processes in a label-free mode.

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 Carcinogenesis Carcinogenesis 42, 1068-1078 (2021)

Baumeister, T. ; Ingermann, J. ; Marcazzan, S. ; Fang, H. ; Oellinger, R. ; Rad, R. ; Engleitner, T. ; Kleigrewe, K. ; Anand, A. ; Strangmann, J. ; Schmid, R.M. ; Wang, T.C. ; Quante, M.

Anti-inflammatory chemoprevention attenuates the phenotype in a mouse model of esophageal adenocarcinoma.

Barrett's esophagus (BE) is the main known precursor condition of esophageal adenocarcinoma (EAC). BE is defined by the presence of metaplasia above the normal squamous columnar junction and has mainly been attributed to gastroesophageal reflux disease and chronic reflux esophagitis. Thus, the rising incidence of EAC in the Western world is probably mediated by chronic esophageal inflammation, secondary to gastroesophageal reflux disease in combination with environmental risk factors such as a Western diet and obesity. However, (at present) risk prediction tools and endoscopic surveillance have shown limited effectiveness. Chemoprevention as an adjunctive approach remains an attractive option to reduce the incidence of neoplastic disease. Here, we investigate the feasibility of chemopreventive approaches in BE and EAC via inhibition of inflammatory signaling in a transgenic mouse model of BE and EAC (L2-IL1B mice), with accelerated tumor formation on a high-fat diet (HFD). L2-IL1B mice were treated with the IL-1 receptor antagonist Anakinra and the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin or Sulindac. Interleukin-1b antagonism reduced tumor progression in L2-IL1B mice with or without a HFD, whereas both NSAIDs were effective chemoprevention agents in the accelerated HFD-fed L2-IL1B mouse model. Sulindac treatment also resulted in a marked change in the immune profile of L2-IL1B mice. In summary, anti-inflammatory treatment of HFD-treated L2-IL1B mice acted protectively on disease progression. These results from a mouse model of BE support results from clinical trials that suggest that anti-inflammatory medication may be effective in the chemoprevention of EAC.

2021 Scientific Article in Theranostics Theranostics 11, 7813-7828 (2021)

Liapis, E. ; Karlas, A. ; Klemm, U. ; Ntziachristos, V.

Chemotherapeutic effects on breast tumor hemodynamics revealed by eigenspectra multispectral optoacoustic tomography (eMSOT).

Non-invasive monitoring of hemodynamic tumor responses to chemotherapy could provide unique insights into the development of therapeutic resistance and inform therapeutic decision-making in the clinic. Methods: Here, we examined the longitudinal and dynamic effects of the common chemotherapeutic drug Taxotere on breast tumor (KPL-4) blood volume and oxygen saturation using eigenspectra multispectral optoacoustic tomography (eMSOT) imaging over a period of 41 days. Tumor vascular function was assessed by dynamic oxygen-enhanced eMSOT (OE-eMSOT). The obtained in vivo optoacoustic data were thoroughly validated by ex vivo cryoimaging and immunohistochemical staining against markers of vascularity and hypoxia. Results: We provide the first preclinical evidence that prolonged treatment with Taxotere causes a significant drop in mean whole tumor oxygenation. Furthermore, application of OE-eMSOT showed a diminished vascular response in Taxotere-treated tumors and revealed the presence of static blood pools, indicating increased vascular permeability. Conclusion: Our work has important translational implications and supports the feasibility of eMSOT imaging for non-invasive assessment of tumor microenvironmental responses to chemotherapy.

2021 Scientific Article in Medical Image Analysis Med. Image Anal. 73:102166 (2021)

Sekuboyina, A. ; Husseini, M.E. ; Bayat, A. ; Löffler, M. ; Liebl, H. ; Li, H. ; Tetteh, G. ; Kukacka, J. ; Payer, C. ; Štern, D. ; Urschler, M. ; Chen, M. ; Cheng, D.S. ; Lessmann, N. ; Hu, Y. ; Wang, T. ; Yang, D. ; Xu, D. ; Ambellan, F. ; Amiranashvili, T. ; Ehlke, M. ; Lamecker, H. ; Lehnert, S. ; Lirio, M. ; Olaguer, N.P.d. ; Ramm, H. ; Sahu, M. ; Tack, A. ; Zachow, S. ; Jiang, T. ; Ma, X. ; Angerman, C. ; Wang, X. ; Brown, K. ; Kirszenberg, A. ; Puybareau, É. ; Chen, D. ; Bai, Y. ; Rapazzo, B.H. ; Yeah, T. ; Zhang, A. ; Xu, S. ; Hou, F. ; He, Z. ; Zeng, C. ; Xiangshang, Z. ; Liming, X. ; Netherton, T.J. ; Mumme, R.P. ; Court, L.E. ; Huang, Z. ; He, C. ; Wang, L.W. ; Ling, S.H. ; Huỳnh, L.D. ; Boutry, N. ; Jakubicek, R. ; Chmelik, J. ; Mulay, S. ; Sivaprakasam, M. ; Paetzold, J.C. ; Shit, S. ; Ezhov, I. ; Wiestler, B. ; Glocker, B. ; Valentinitsch, A. ; Rempfler, M. ; Menze, B.H. ; Kirschke, J.S.

VERSE: A Vertebrae labelling and segmentation benchmark for multi-detector CT images.

Vertebral labelling and segmentation are two fundamental tasks in an automated spine processing pipeline. Reliable and accurate processing of spine images is expected to benefit clinical decision support systems for diagnosis, surgery planning, and population-based analysis of spine and bone health. However, designing automated algorithms for spine processing is challenging predominantly due to considerable variations in anatomy and acquisition protocols and due to a severe shortage of publicly available data. Addressing these limitations, the Large Scale Vertebrae Segmentation Challenge (VERSE) was organised in conjunction with the International Conference on Medical Image Computing and Computer Assisted Intervention (MICCAI) in 2019 and 2020, with a call for algorithms tackling the labelling and segmentation of vertebrae. Two datasets containing a total of 374 multi-detector CT scans from 355 patients were prepared and 4505 vertebrae have individually been annotated at voxel level by a human-machine hybrid algorithm (, A total of 25 algorithms were benchmarked on these datasets. In this work, we present the results of this evaluation and further investigate the performance variation at the vertebra level, scan level, and different fields of view. We also evaluate the generalisability of the approaches to an implicit domain shift in data by evaluating the top-performing algorithms of one challenge iteration on data from the other iteration. The principal takeaway from VERSE: the performance of an algorithm in labelling and segmenting a spine scan hinges on its ability to correctly identify vertebrae in cases of rare anatomical variations. The VERSE content and code can be accessed at:

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 Nature Cell Biology Nat. Cell Biol. 23, 652-663 (2021)

Truong, D.J.J. ; Phlairaharn, T. ; Eßwein, B. ; Gruber, C. ; Tümen, D. ; Baligács, E. ; Armbrust, N. ; Vaccaro, F.L. ; Lederer, E.-M. ; Beck, E.M. ; Geilenkeuser, J. ; Göppert, S. ; Krumwiede, L. ; Grätz, C. ; Raffl, G. ; Schwarz, D. ; Zirngibl, M. ; Živanić, M. ; Beyer, M. ; Körner, J.D. ; Santl, T. ; Evsyukov, V. ; Strauß, T. ; Schwarz, S.C. ; Höglinger, G.U. ; Heutink, P. ; Doll, S. ; Conrad, M. ; Giesert, F. ; Wurst, W. ; Westmeyer, G.G.

Non-invasive and high-throughput interrogation of exon-specific isoform expression.

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

2021 Review in Methods in Enzymology Methods Enzymol. 657, 365-383 (2021)

Stankevych, M. ; Mishra, K. ; Ntziachristos, V. ; Stiel, A.-C.

A practical guide to photoswitching optoacoustics tomography.

Photochromic proteins and photoswitching optoacoustics (OA) are a promising combination, that allows OA imaging of even small numbers of cells in whole live animals and thus can facilitate a more wide-spread use of OA in life-science and preclinical research. The concept relies on exploiting the modulation achieved by the photoswitching to discriminate the agents' signal from the non-modulating background. Here we share our analysis approaches that can be readily used on data generated with commercial OA tomography imaging instrumentation allowing—depending on the used photoswitching agent and sample—routine visualizations of as little as several hundreds of transgene labeled cells per imaging volume in the live animal.

2021 Scientific Article in Photoacoustics Photoacoustics 23:100283 (2021)

Karlas, A.# ; Kallmayer, M.# ; Bariotakis, M. ; Fasoula, N.-A. ; Liapis, E. ; Hyafil, F. ; Pelisek, J. ; Wildgruber, M. ; Eckstein, H.H. ; Ntziachristos, V.

Multispectral optoacoustic tomography of lipid and hemoglobin contrast in human carotid atherosclerosis.

Several imaging techniques aim at identifying features of carotid plaque instability but come with limitations, such as the use of contrast agents, long examination times and poor portability. Multispectral optoacoustic tomography (MSOT) employs light and sound to resolve lipid and hemoglobin content, both features associated with plaque instability, in a label-free, fast and highly portable way. Herein, 5 patients with carotid atherosclerosis, 5 healthy volunteers and 2 excised plaques, were scanned with handheld MSOT. Spectral unmixing allowed visualization of lipid and hemoglobin content within three ROIs: whole arterial cross-section, plaque and arterial lumen. Calculation of the fat-blood-ratio (FBR) value within the ROIs enabled the differentiation between patients and healthy volunteers (P = 0.001) and between plaque and lumen in patients (P = 0.04). Our results introduce MSOT as a tool for molecular imaging of human carotid atherosclerosis and open new possibilities for research and clinical assessment of carotid plaques.

2021 Scientific Article in Proceedings of SPIE Proc. SPIE 11629:116292D (2021)

Degtyaruk, O. ; Mc Larney, B. ; Deán-Ben, X.L. ; Shoham, S. ; Razansky, D.

Optoacoustic visualization of GCaMP6f labeled deep brain activity in a murine intracardiac perfusion model.

The inability to directly visualize large-scale neural dynamics across the entire mammalian brain in the millisecond temporal resolution regime is among the main limitations of existing neuroimaging methods. Recent advances in optoacoustic imaging systems have led to the establishment of this technology as an alternative method for real-time deep-tissue observations. Particularly, functional optoacoustic neurotomography (FONT) has recently been suggested for three-dimensional imaging of both direct calcium activity and cerebral hemodynamic parameters in rodents. However, the lack of suitable calcium indicators featuring optical absorption peaks within the so-called near-infrared window has hampered the applicability of FONT for imaging neuronal activity deep within the mammalian brain. To surmount this challenge, we developed and validated an intracardially perfused murine brain model labelled with genetically encoded calcium indicator GCaMP6f that closely simulates in vivo conditions. Penetration of light through skull and skin is greatly facilitated after blood is substituted by artificial cerebrospinal fluid (ACSF). The new preparation enabled here the observation of stimulus-evoked calcium dynamics within the mouse brain at penetration depths and spatio-temporal resolution scales not attainable with other neuroimaging techniques.

2021 Scientific Article in Advanced Optical Materials Adv. Opt. Mater. 9:2100256 (2021)

Shnaiderman, R.&deg ; Mustafa, Q. ; Ülgen, O. ; Wissmeyer, G. ; Estrada, H. ; Razansky, D. ; Chmyrov, A. ; Ntziachristos, V.&deg

Silicon-photonics point sensor for high-resolution optoacoustic imaging.

The recent development of ultrasound sensing using the silicon-photonics platform has enabled super-resolution optoacoustic imaging not possible by piezoelectric technology or polymeric optical microresonators. The silicon waveguide etalon detector (SWED) design exploits the sub-micrometer light confinement in the cross-section of a silicon strip waveguide to achieve a sensor aperture which is 13-fold to 30-fold smaller than the cutoff wavelength of the sensor. While its performance in near-field scanning optoacoustic imaging has been previously studied, the operational characteristics of this sensor as it relates to conventional optoacoustic imaging applications are not known. Here, for the first time, the application of the SWED in optoacoustic mesoscopy is investigated, the interaction of the sensor with ultrasound in the far-field is characterized, the acoustic point spread function up to a depth of 10 mm is measured, and 3D vasculature-mimicking phantoms are imaged. The measured point spread function of the sensor shows that surface acoustic waves can degrade the lateral resolution. Nevertheless, superior resolution is demonstrated over any state-of-the-art ultrasound sensor, over the whole range of imaging depths that are of interest to optoacoustic mesoscopy. Silicon photonics is proposed as a powerful and promising new platform for ultrasonics and optoacoustics.

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 Review in Methods in Enzymology Methods Enzymol. 657, 349-364 (2021)

Gujrati, V. ; Ntziachristos, V.

Bioengineered bacterial vesicles for optoacoustics-guided phototherapy.

Genetically engineered bacterial outer membrane vesicles (OMVs) offer promising applications for gene therapy, immunotherapy, and vaccine delivery. Importantly, OMVs are biocompatible, biodegradable, and easy to engineer and produce on a large scale. In this chapter, we discuss the development and application of bioengineered OMVs for optoacoustics-guided phototherapy applications (theranostics). We provide detailed protocols for OMVs preparation, characterization, and in vitro and in vivo validation. The engineered OMVs carry the biopolymer melanin, which generates a strong optoacoustic (OA) signal and intense heat upon absorption of near-infrared (NIR) light, enabling optoacoustics-guided cancer diagnosis and photothermal therapy in vivo.

2021 Review in Frontiers in Neuroscience Front. Neurosci. 15:655247 (2021)

Bodea, S.V.&deg ; Westmeyer, G.G.&deg

Photoacoustic neuroimaging - Perspectives on a maturing imaging technique and its applications in neuroscience.

A prominent goal of neuroscience is to improve our understanding of how brain structure and activity interact to produce perception, emotion, behavior, and cognition. The brain's network activity is inherently organized in distinct spatiotemporal patterns that span scales from nanometer-sized synapses to meter-long nerve fibers and millisecond intervals between electrical signals to decades of memory storage. There is currently no single imaging method that alone can provide all the relevant information, but intelligent combinations of complementary techniques can be effective. Here, we thus present the latest advances in biomedical and biological engineering on photoacoustic neuroimaging in the context of complementary imaging techniques. A particular focus is placed on recent advances in whole-brain photoacoustic imaging in rodent models and its influential role in bridging the gap between fluorescence microscopy and more non-invasive techniques such as magnetic resonance imaging (MRI). We consider current strategies to address persistent challenges, particularly in developing molecular contrast agents, and conclude with an overview of potential future directions for photoacoustic neuroimaging to provide deeper insights into healthy and pathological brain processes.

2021 Scientific Article in Journal of Biophotonics J. Biophotonics 14:e202100048 (2021)

Kellnberger, S.# ; Wissmeyer, G.# ; Albaghdadi, M. ; Piao, Z. ; Li, W. ; Mauskapf, A. ; Rauschendorfer, P. ; Tearney, G.J. ; Ntziachristos, V. ; Jaffer, F.A.

Intravascular molecular-structural imaging with a miniaturized integrated near-infrared fluorescence and ultrasound catheter.

Coronary artery disease (CAD) remains a leading cause of mortality and warrants new imaging approaches to better guide clinical care. We report on a miniaturized, hybrid intravascular catheter and imaging system for comprehensive coronary artery imaging in vivo. Our catheter exhibits a total diameter of 1.0 mm (3.0 French), equivalent to standalone clinical intravascular ultrasound (IVUS) catheters but enables simultaneous near-infrared fluorescence (NIRF) and IVUS molecular-structural imaging. We demonstrate NIRF-IVUS imaging in vitro in coronary stents using NIR fluorophores, and compare NIRF signal strengths for prism and ball lens sensor designs in both low and high scattering media. Next, in vivo intravascular imaging in pig coronary arteries demonstrates simultaneous, co-registered molecular-structural imaging of experimental CAD inflammation on IVUS and distance-corrected NIRF images. The obtained results suggest substantial potential for the NIRF-IVUS catheter to advance standalone IVUS, and enable comprehensive phenotyping of vascular disease to better assess and treat patients with CAD.

2021 Scientific Article in Proceedings of SPIE Proc. SPIE 11642:116422K (2021)

Özsoy, Ç. ; Periyasamy, V. ; Reiss, M. ; Deán-Ben, X.L. ; Razansky, D.

Concurrent in vivo tumor ablation and real-time optoacoustic monitoring with a pulsed 1064 nm laser source.

Laser ablation (LA) is gaining acceptance for the treatment of tumors as a viable alternative to surgical resection. In parallel, optoacoustic tomography (OAT) has enabled defining new regimes for diagnosis and characterization of malignant neoplastic lesions with high sensitivity and specificity. Even though pulsed nanosecond lasers are commonly used for both imaging and therapeutic purposes, real-time thermal treatment monitoring with a single laser source has not been previously attempted. Herein, we demonstrate the feasibility of combined OAT and LA by percutaneous irradiation of subcutaneous tumors with a 100 mJ short-pulsed (∼5 ns) laser operating at 1064 nm and 100 Hz pulse repetition frequency. The OAT images rendered with a spherical ultrasound transducer array enabled real-time monitoring of the LA lesion progression, which is essential for determining the optimal treatment end-point. Local changes in the optoacoustic signal intensity associated with the induced temperature changes as well as structural alterations in the tumor vasculature could clearly be observed. The optoacoustic volumetric projections further correlated with crosssections extracted from the excised tumors. This newly enabled capability anticipates new theranostic approaches in cancer research and treatment with potential applicability in a clinical setting.

2021 Scientific Article in Proceedings of SPIE Proc. SPIE 11642:116420A (2021)

Kalva, S.K. ; Ron, A. ; Periyasamy, V. ; Reiss, M. ; Deán-Ben, X.L. ; Razansky, D.

Whole-body visualization of nanoagent kinetics in mice with flash scanning volumetric optoacoustic tomography.

Visualizing whole-body dynamics across entire living organisms is crucial for understanding complex biology, disease progression as well as evaluating efficacy of new drugs and therapies. Existing small animal functional and molecular imaging modalities either suffer from low spatial and temporal resolution, limited penetration depth or poor contrast. In this work, we present flash scanning volumetric optoacoustic tomography (fSVOT) imaging system that enables the acquisition speeds required for visualizing fast kinetics and biodistribution of optical contrast agents across whole mice. fSVOT can render images of intricate vascular and organ anatomy with rich contrast by capitalizing on the large angular coverage of a spherical matrix array transducer rapidly scanned around the mouse. Volumetric (three-dimensional) images with 200 μm resolution can be acquired within 45 seconds, which corresponds to an imaging speed gain of an order of magnitude with respect to existing state-of-the-art modalities offering comparable resolution performance. We demonstrate volumetric tracking and quantification of gold nanorod kinetics and their differential uptake across the spleen, liver and kidneys. Overall, fSVOT offers unprecedented capabilities for multi-scale imaging of pharmacokinetics and bio-distribution of agents with high contrast, resolution and image acquisition speed.

2021 Scientific Article in Proceedings of SPIE Proc. SPIE 11642:116422L (2021)

Lafci, B. ; Mercep, E. ; Herraiz, J.L. ; Deán-Ben, X.L. ; Razansky, D.

Transmission-reflection optoacoustic ultrasound (TROPUS) imaging of mammary tumors.

Ultrasound (US) and optoacoustic (OA) imaging provide complementary information for quantitative analysis of the tumor microenvironment. Herein, we demonstrate the unique capabilities of transmission-reflection optoacoustic ultrasound (TROPUS) for characterizing breast cancer in tumor-bearing mice. For this, 4 different mice featuring orthotopic tumor of different sizes were scanned with a full-ring ultrasound transducer array to simultaneously render pulse-echo US images, speed of sound (SoS) maps and OA images. The tumor size, vascular density and its elastic parameters were further quantified in the images. Our results pave the way toward clinical translation of the hybrid TROPUS imaging for tumor detection and characterization.

2021 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 40, 3349-3357 (2021)

Mustafa, Q. ; Omar, M. ; Prade, L. ; Mohajerani, P. ; Stylogiannis, A. ; Ntziachristos, V. ; Zakian Dominguez, C.M.

In vivo three-dimensional Raster Scan Optoacoustic Mesoscopy using Frequency Domain Inversion.

Optoacoustic signals are typically reconstructed into images using inversion algorithms applied in the time-domain. However, time-domain reconstructions can be computationally intensive and therefore slow when large amounts of raw data are collected from an optoacoustic scan. Here we consider a fast weighted ω-κ (FWOK) algorithm operating in the frequency domain to accelerate the inversion in raster-scan optoacoustic mesoscopy (RSOM), while seamlessly incorporating impulse response correction with minimum computational burden. We investigate the FWOK performance with RSOM measurements from phantoms and mice in vivo and obtained 360-fold speed improvement over inversions based on the back-projection algorithm in the time-domain. This previously unexplored inversion of in vivo optoacoustic data with impulse response correction in frequency domain reconstructions points to a promising strategy of accelerating optoacoustic imaging computations, toward video-rate tomography.

2021 Scientific Article in Biological Trace Element Research Biol. Trace Elem. Res. 200, 1688-1698 (2021)

Aouey, B. ; Boukholda, K. ; Gargouri, B. ; Bhatia, H.S. ; Attaai, A. ; Kebieche, M. ; Bouchard, M. ; Fetoui, H.

Silica nanoparticles induce hepatotoxicity by triggering oxidative damage, apoptosis, and bax-Bcl2 signaling pathway.

The increase in the usage of silica nanoparticles (SiNPs) in the industrial and medical fields has raised concerns about their possible adverse effects on human health. The present study aimed to investigate the potential adverse effects of SiNPs at daily doses of 25 and 100 mg/kg body weight intraperitoneally (i.p.) for 28 consecutive days on markers of liver damage in adult male rats. Results revealed that SiNPs induced a marked increase in serum markers of liver damage, including lactate dehydrogenase (LDH), alanine aminotransferase (ALAT), and aspartate aminotransferase (ASAT). SiNPs also induced an elevation of reactive oxygen species (ROS) production in liver, along with an increase in oxidative stress markers (NO, MDA, PCO, and H2O2), and a decrease in antioxidant enzyme activities (CAT, SOD, and GPx). Quantitative real-time PCR showed that SiNPs also induced upregulation of pro-apoptotic gene expression (including Bax, p53, Caspase-9/3) and downregulation of anti-apoptotic factors Bcl-2. Moreover, histopathological analysis revealed that SiNPs induced hepatocyte alterations, which was accompanied by sinusoidal dilatation, Kupffer cell hyperplasia, and the presence of inflammatory cells in the liver. Taken together, these data showed that SiNPs trigger hepatic damage through ROS-activated caspase signaling pathway, which plays a fundamental role in SiNP-induced apoptosis in the liver.

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 Science Advances Sci. Adv. 7:eabd1505 (2021)

Huang, Y. ; Omar, M. ; Tian, W. ; López-Schier, H. ; Westmeyer, G.G. ; Chmyrov, A. ; Sergiadis, G. ; Ntziachristos, V.

Noninvasive visualization of electrical conductivity in tissues at the micrometer scale.

Despite its importance in regulating cellular or tissue function, electrical conductivity can only be visualized in tissue indirectly as voltage potentials using fluorescent techniques, or directly with radio waves. These either requires invasive procedures like genetic modification or suffers from limited resolution. Here, we introduce radio-frequency thermoacoustic mesoscopy (RThAM) for the noninvasive imaging of conductivity by exploiting the direct absorption of near-field ultrashort radio-frequency pulses to stimulate the emission of broadband ultrasound waves. Detection of ultrasound rather than radio waves enables micrometer-scale resolutions, over several millimeters of tissue depth. We confirm an imaging resolution of <30 μm in phantoms and demonstrate microscopic imaging of conductivity correlating to physical structures in 1- and 512-cell zebrafish embryos, as well as larvae. These results support RThAM as a promising method for high-resolution, label-free assessment of conductivity in tissues.

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 Nature Reviews - Endocrinology Nat. Rev. Endocrinol., DOI: 10.1038/s41574-021-00515-z (2021)

Karlas, A. ; Pleitez, M.A. ; Aguirre Bueno, J. ; Ntziachristos, V.

Author Correction: Optoacoustic imaging in endocrinology and metabolism (Nature Reviews Endocrinology, (2021), 17, 6, (323-335), 10.1038/s41574-021-00482-5).

In the original version of this article, in Fig. 1c, the icons for oxygenated haemoglobin and deoxygenated haemoglobin were incorrectly shown outside of the artery and vein. This error has now been corrected in the HTML and PDF versions of the article.

2021 Scientific Article in Cell Metabolism Cell Metab. 33, 1155-1170.e10 (2021)

Gruber, T. ; Pan, C. ; Contreras, R. ; Wiedemann, T. ; Morgan, D.A. ; Skowronski, A.A. ; Lefort, S. ; De Bernardis Murat, C. ; Le Thuc, O. ; Legutko, B. ; Ruiz Ojeda, F.J. ; Fuente-Fernández, M. ; García-Villalón, A.L. ; González-Hedström, D. ; Huber, M. ; Szigeti-Buck, K. ; Müller, T.D. ; Ussar, S. ; Pfluger, P.T. ; Woods, S.C. ; Ertürk, A. ; LeDuc, C.A. ; Rahmouni, K. ; Granado, M. ; Horvath, T.L. ; Tschöp, M.H. ; García-Cáceres, C.

Obesity-associated hyperleptinemia alters the gliovascular interface of the hypothalamus to promote hypertension.

Pathologies of the micro- and macrovascular systems are a hallmark of the metabolic syndrome, which can lead to chronically elevated blood pressure. However, the underlying pathomechanisms involved still need to be clarified. Here, we report that an obesity-associated increase in serum leptin triggers the select expansion of the micro-angioarchitecture in pre-autonomic brain centers that regulate hemodynamic homeostasis. By using a series of cell- and region-specific loss- and gain-of-function models, we show that this pathophysiological process depends on hypothalamic astroglial hypoxia-inducible factor 1α-vascular endothelial growth factor (HIF1α-VEGF) signaling downstream of leptin signaling. Importantly, several distinct models of HIF1α-VEGF pathway disruption in astrocytes are protected not only from obesity-induced hypothalamic angiopathy but also from sympathetic hyperactivity or arterial hypertension. These results suggest that hyperleptinemia promotes obesity-induced hypertension via a HIF1α-VEGF signaling cascade in hypothalamic astrocytes while establishing a novel mechanistic link that connects hypothalamic micro-angioarchitecture with control over systemic blood pressure.

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 Review in Nature Reviews - Endocrinology Nat. Rev. Endocrinol. 17, 323-335 (2021)

Karlas, A. ; Pleitez, M.A. ; Aguirre Bueno, J. ; Ntziachristos, V.

Optoacoustic imaging in endocrinology and metabolism.

Imaging is an essential tool in research, diagnostics and the management of endocrine disorders. Ultrasonography, nuclear medicine techniques, MRI, CT and optical methods are already used for applications in endocrinology. Optoacoustic imaging, also termed photoacoustic imaging, is emerging as a method for visualizing endocrine physiology and disease at different scales of detail: microscopic, mesoscopic and macroscopic. Optoacoustic contrast arises from endogenous light absorbers, such as oxygenated and deoxygenated haemoglobin, lipids and water, or exogenous contrast agents, and reveals tissue vasculature, perfusion, oxygenation, metabolic activity and inflammation. The development of high-performance optoacoustic scanners for use in humans has given rise to a variety of clinical investigations, which complement the use of the technology in preclinical research. Here, we review key progress with optoacoustic imaging technology as it relates to applications in endocrinology; for example, to visualize thyroid morphology and function, and the microvasculature in diabetes mellitus or adipose tissue metabolism, with particular focus on multispectral optoacoustic tomography and raster-scan optoacoustic mesoscopy. We explain the merits of optoacoustic microscopy and focus on mid-infrared optoacoustic microscopy, which enables label-free imaging of metabolites in cells and tissues. We showcase current optoacoustic applications within endocrinology and discuss the potential of these technologies to advance research and clinical practice.

Lecture Notes in Computer Science In: International workshop on Cerebral Aneurysm Detection. 2021. 51-57 (Lect. Notes Comput. Sc. ; 12643 LNCS)

Shit, S. ; Ezhov, I. ; Paetzold, J.C. ; Menze, B.

A ν -Net: Automatic detection and segmentation of aneurysm.

We propose an automatic solution for the CADA 2020 challenge to detect aneurysm from Digital Subtraction Angiography (DSA) images. Our method relies on 3D U-net as the backbone and heavy data augmentation with a carefully chosen loss function. We were able to generalize well using our solution (despite training on a small dataset) that is demonstrated through accurate detection and segmentation on the test data.

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 Pharmaceutics Pharmaceutics 13:397 (2021)

Efremova, M.V.&deg ; Bodea, S.V. ; Sigmund, F. ; Semkina, A.S. ; Westmeyer, G.G. ; Abakumov, M.A.&deg

Genetically encoded self-assembling iron oxide nanoparticles as a possible platform for cancer-cell tracking.

The study of growth and possible metastasis in animal models of tumors would benefit from reliable cell labels for noninvasive whole-organism imaging techniques such as magnetic resonance imaging. Genetically encoded cell-tracking reporters have the advantage that they are contrast-selective for viable cells with intact protein expression machinery. Besides, these reporters do not suffer from dilution during cell division. Encapsulins, which are bacterial protein nanocompartments, can serve as genetically controlled labels for multimodal detection of cells. Such nanocompartments can host various guest molecules inside their lumen. These include, for example, fluorescent proteins or enzymes with ferroxidase activity leading to biomineralization of iron oxide inside the encapsulin nanoshell. The aim of this work was to implement heterologous expression of encapsulin systems from Quasibacillus thermotolerans using the fluorescent reporter protein mScarlet-I and ferroxidase IMEF in the human hepatocellular carcinoma cell line HepG2. The successful expression of self-assembled encapsulin nanocompartments with functional cargo proteins was confirmed by fluorescence microscopy and transmission electron microscopy. Also, coexpression of encapsulin nanoshells, ferroxidase cargo, and iron transporter led to an increase in T2-weighted contrast in magnetic resonance imaging of HepG2 cells. The results demonstrate that the encapsulin cargo system from Q. thermotolerans may be suitable for multimodal imaging of cancer cells and could contribute to further in vitro and in vivo studies.

2021 Scientific Article in Journal of Biophotonics J. Biophotonics 14:e202000501 (2021)

Ülgen, O. ; Shnaiderman, R. ; Zakian Dominguez, C.M. ; Ntziachristos, V.

Interferometric optical fiber sensor for optoacoustic endomicroscopy.

Optical fiber sensors can offer robust and miniaturized detection of wideband ultrasound, yielding high sensitivity and immunity to electromagnetic interference. However, the lack of cost-effective manufacturing methods prevents the disseminated use of these sensors in biomedical applications. In this study, we developed and optimized a simple method to create optical cavities with high-quality mirrors for acoustic sensing based on micro-manipulation of UV-curable optical adhesives and electroless chemical silver deposition. This approach enables the manufacturing of ultrasound sensors based on Fabry-Pérot Interferometers (FPI) on optical fiber tips with minimal production costs. Characterization and high-resolution optoacoustic imaging experiments show that the manufacturing process yielded a fiber sensor with a small NEP (11 mPa/ Hz ) over a broad detection bandwidth (25 MHz), generally outperforming conventional piezoelectric based transducers. We discuss how the new manufacturing process leads to a high-performance acoustic detector that, due to low cost, can be used as a disposable sensor.

2021 Review in Molecular Systems Biology Mol. Syst. Biol. 17:e9807 (2021)

Molbay, M. ; Kolabas, Z.I. ; Todorov, M.I. ; Ohn, T.-L. ; Ertürk, A.

A guidebook for DISCO tissue clearing.

Histological analysis of biological tissues by mechanical sectioning is significantly time-consuming and error-prone due to loss of important information during sample slicing. In the recent years, the development of tissue clearing methods overcame several of these limitations and allowed exploring intact biological specimens by rendering tissues transparent and subsequently imaging them by laser scanning fluorescence microscopy. In this review, we provide a guide for scientists who would like to perform a clearing protocol from scratch without any prior knowledge, with an emphasis on DISCO clearing protocols, which have been widely used not only due to their robustness, but also owing to their relatively straightforward application. We discuss diverse tissue-clearing options and propose solutions for several possible pitfalls. Moreover, after surveying more than 30 researchers that employ tissue clearing techniques in their laboratories, we compiled the most frequently encountered issues and propose solutions. Overall, this review offers an informative and detailed guide through the growing literature of tissue clearing and can help with finding the easiest way for hands-on implementation.

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 Editorial in Nature Biotechnology Nat. Biotechnol. 39, 281-286 (2021)

Annabi, N. ; Baker, M. ; Boettiger, A. ; Chakraborty, D. ; Chen, Y. ; Corbett, K.S. ; Correia, B. ; Dahlman, J. ; de Oliveira, T. ; Ertürk, A. ; Yanik, M.F. ; Henaff, E. ; Huch, M. ; Iliev, I.D. ; Jacobs, T. ; Junca, H. ; Keung, A. ; Kolodkin-Gal, I. ; Krishnaswamy, S. ; Lancaster, M. ; Macosko, E. ; Martínez-Núñez, M.A. ; Miura, K. ; Molloy, J. ; Cruz, A.O. ; Platt, R.J. ; Posey, A.D. ; Shao, H. ; Simunovic, M. ; Slavov, N. ; Takebe, T. ; Vandenberghe, L.H. ; Varshney, R.K. ; Wang, J.

Voices of biotech research.

2021 Scientific Article in Advanced functional materials Adv. Func. Mat. 31:2101527 (2021)

Di Giosia, M. ; Solda, A. ; Seeger, M. ; Cantelli, A. ; Arnesano, F. ; Nardella, M.I. ; Mangini, V. ; Valle, F. ; Montalti, M. ; Zerbetto, F. ; Rapino, S. ; Calvaresi, M.&deg ; Ntziachristos, V.&deg

A bio-conjugated fullerene as a subcellular-targeted and multifaceted phototheranostic agent.

Fullerenes are candidates for theranostic applications because of their high photodynamic activity and intrinsic multimodal imaging contrast. However, fullerenes suffer from low solubility in aqueous media, poor biocompatibility, cell toxicity, and a tendency to aggregate. C70@lysozyme is introduced herein as a novel bioconjugate that is harmless to a cellular environment, yet is also photoactive and has excellent optical and optoacoustic contrast for tracking cellular uptake and intracellular localization. The formation, water-solubility, photoactivity, and unperturbed structure of C70@lysozyme are confirmed using UV-visible and 2D 1H, 15N NMR spectroscopy. The excellent imaging contrast of C70@lysozyme in optoacoustic and third harmonic generation microscopy is exploited to monitor its uptake in HeLa cells and lysosomal trafficking. Last, the photoactivity of C70@lysozyme and its ability to initiate cell death by means of singlet oxygen (1O2) production upon exposure to low levels of white light irradiation is demonstrated. This study introduces C70@lysozyme and other fullerene-protein conjugates as potential candidates for theranostic applications.

2021 Scientific Article in Nature Photonics Nat. Photonics 15, 341–345 (2021)

Westerveld, W.J. ; Mahmud-Ul-Hasan, M. ; Shnaiderman, R. ; Ntziachristos, V. ; Rottenberg, X.&deg ; Severi, S.&deg ; Rochus, V.

Sensitive, small, broadband and scalable optomechanical ultrasound sensor in silicon photonics.

Ultrasonography1 and photoacoustic2,3 (optoacoustic) tomography have recently seen great advances in hardware and algorithms. However, current high-end systems still use a matrix of piezoelectric sensor elements, and new applications require sensors with high sensitivity, broadband detection, small size and scalability to a fine-pitch matrix. This work demonstrates an ultrasound sensor in silicon photonic technology with extreme sensitivity owing to an innovative optomechanical waveguide. This waveguide has a tiny 15 nm air gap between two movable parts, which we fabricated using new CMOS-compatible processing. The 20 μm small sensor has a noise equivalent pressure below 1.3 mPa Hz−1/2 in the measured range of 3–30 MHz, dominated by acoustomechanical noise. This is two orders of magnitude better than for piezoelectric elements of an identical size4. The demonstrated sensor matrix with on-chip photonic multiplexing5–7 offers the prospect of miniaturized catheters that have sensor matrices interrogated using just a few optical fibres, unlike piezoelectric sensors that typically use an electrical connection for each element.

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 Nanomaterials Nanomaterials 11:1113 (2021)

Mukha, I.&deg ; Chepurna, O. ; Vityuk, N. ; Khodko, A. ; Storozhuk, L. ; Dzhagan, V. ; Zahn, D.R.T. ; Ntziachristos, V. ; Chmyrov, A. ; Ohulchanskyy, T.Y.&deg

Multifunctional magneto-plasmonic fe3o4/au nanocomposites: Approaching magnetophoretically-enhanced photothermal therapy.

Magneto-plasmonic nanocomposites can possess properties inherent to both individual components (iron oxide and gold nanoparticles) and are reported to demonstrate high potential in targeted drug delivery and therapy. Herein, we report on Fe O /Au magneto-plasmonic nanocomposites (MPNC) synthesized with the use of amino acid tryptophan via chemical and photochemical reduction of Au ions in the presence of nanosized magnetite. The magnetic field (MF) induced aggregation was accompanied by an increase in the absorption in the near-infrared (NIR) spectral region, which was demonstrated to provide an enhanced photothermal (PT) effect under NIR laser irradiation (at 808 nm). A possibility for therapeutic application of the MPNC was illustrated using cancer cells in vitro. Cultured HeLa cells were treated by MPNC in the presence of MF and without it, following laser irradiation and imaging using confocal laser scanning microscopy. After scanning laser irradiation of the MPNC/MF treated cells, a formation and rise of photothermally-induced microbubbles on the cell surfaces was observed, leading to a damage of the cell membrane and cell destruction. We conclude that the synthesized magneto-plasmonic Fe O /Au nanosystems exhibit magnetic field-induced reversible aggregation accompanied by an increase in NIR absorption, allowing for an opportunity to magnetophoretically control and locally enhance a NIR light-induced thermal effect, which holds high promise for the application in photothermal therapy. 3 4 3 4

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 Laser & Photonics Reviews Laser Photon. Rev. 15:2000484 (2021)

Ron, A. ; Kalva, S.K. ; Periyasamy, V. ; Deán-Ben, X.L. ; Razansky, D.

Flash scanning volumetric optoacoustic tomography for high resolution whole-body tracking of nanoagent kinetics and biodistribution.

Tracking of biodynamics across entire living organisms is essential for understanding complex biology and disease progression. The presently available small-animal functional and molecular imaging modalities remain constrained by factors including long image acquisition times, low spatial resolution, limited penetration or poor contrast. Here flash scanning volumetric optoacoustic tomography (fSVOT), a new approach for high-speed imaging of fast kinetics and biodistribution of optical contrast agents in whole mice that simultaneously provides reference images of vascular and organ anatomy with unrivaled fidelity and contrast, is presented. The imaging protocol employs continuous overfly scanning of a spherical matrix array transducer, accomplishing a 200 µm resolution 3D scan of the whole mouse body within 45 s without relying on signal averaging. This corresponds to an imaging speed gain of more than an order of magnitude compared with existing state-of-the-art implementations of comparable resolution performance. Volumetric tracking and quantification of gold nanoagent and near infrared (NIR)-II dye kinetics and their differential uptake in various organs are demonstrated. fSVOT thus offers unprecedented capabilities for multiscale imaging of pharmacokinetics and biodistribution with high contrast, resolution, and speed.

2021 Scientific Article in Sensors Sensors 21:1379 (2021)

Stylogiannis, A.# ; Kousias, N.# ; Kontses, A. ; Ntziachristos, L. ; Ntziachristos, V.

A low-cost optoacoustic sensor for environmental monitoring.

Attention to Black Carbon (BC) has been rising due to its effects on human health as well its contribution to climate change. Measurements of BC are challenging, as currently used devices are either expensive or impractical for continuous monitoring. Here, we propose an optoacoustic sensor to address this problem. The sensor utilizes a novel ellipsoidal design for refocusing the optoacoustic signal with minimal acoustic energy losses. To reduce the cost of the system, without sacrificing accuracy, an overdriven laser diode and a Quartz Tuning Fork are used as the light source and the sound detector, respectively. The prototype was able to detect BC particles and to accurately monitor changes in concentration in real time and with very good agreement with a reference instrument. The response of the sensor was linearly dependent on the BC particles concentration with a normalized noise equivalent absorption coefficient (NNEA) for soot equal to 7.39 × 10−9 W cm−1 Hz−1/2. Finally, the prototype was able to perform NO2 measurements, demonstrating its ability to accurately monitor both particulate and gaseous pollutants. The proposed sensor has the potential to offer a significant economic impact for BC environmental measurements and source appointment technologies.

2021 Scientific Article in International Journal of Molecular Sciences Int. J. Mol. Sci. 22:2175 (2021)

Wu, A.Z.&deg ; Ohn, T.-L. ; Shei, R.J. ; Wu, H.F. ; Chen, Y.C. ; Lee, H.C. ; Dai, D.F. ; Wu, S.N.&deg

Permissive modulation of sphingosine-1-phosphate-enhanced intracellular calcium on BKCa channel of chromaffin cells.

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa ) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca) ) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai ) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.

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. 47:101184 (2021)

Fasoula, N.-A. ; Karlas, A. ; Kallmayer, M. ; Milik, A.B. ; Pelisek, J. ; Eckstein, H.H. ; Klingenspor, M. ; Ntziachristos, V.

Multicompartmental non-invasive sensing of postprandial lipemia in humans with multispectral optoacoustic tomography.

OBJECTIVE: Postprandial lipid profiling (PLP), a risk indicator ofcardiometabolic disease, is based on frequent blood sampling over several hours after a meal, an approach that is invasive and inconvenient. Non-invasive PLP may offer an alternative for disseminated human monitoring. Herein, we investigate the use of clinical Multispectral Optoacoustic Tomography (MSOT) for the non-invasive, label-free PLP via direct lipid-sensing in human vasculature and soft tissues. METHODS: Four (n = 4) subjects (3 females and 1 male, age: 28 ± 7 years) were enrolled in the current pilot study. We longitudinally measured the lipid signals in arteries, veins, skeletal muscles and adipose tissues of all participants at 30 min-intervalsfor 6 hours after the oral consumption of a high-fat meal. RESULTS: Optoacoustic lipid-signal analysis showed on average a 63.4% intra-arterial increase at ∼4 hours postprandially, a 83.9% intra-venous increase at ∼3 hours, a 120.8% intra-muscular increase at ∼3 hours and a 32.8% subcutaneous fat increase at ∼4 hours. CONCLUSION: MSOT provides the potential to study lipid metabolism that could lead to novel diagnostics and prevention strategies by label-free, non-invasive detection of tissue biomarkers implicated in cardiometabolic diseases.

2021 Scientific Article in Nature Nature 592, 768–772 (2021)

Herud-Sikimić, O.# ; Stiel, A.-C.# ; Kolb, M.# ; Shanmugaratnam, S. ; Berendzen, K.W. ; Feldhaus, C. ; Höcker, B.&deg ; Jürgens, G.&deg

A biosensor for the direct visualization of auxin.

One of the most important regulatory small molecules in plants is indole-3-acetic acid, also known as auxin. Its dynamic redistribution has an essential role in almost every aspect of plant life, ranging from cell shape and division to organogenesis and responses to light and gravity1,2. So far, it has not been possible to directly determine the spatial and temporal distribution of auxin at a cellular resolution. Instead it is inferred from the visualization of irreversible processes that involve the endogenous auxin-response machinery3-7; however, such a system cannot detect transient changes. Here we report a genetically encoded biosensor for the quantitative in vivo visualization of auxin distribution. The sensor is based on the Escherichia coli tryptophan repressor8, the binding pocket of which is engineered to be specific to auxin. Coupling of the auxin-binding moiety with selected fluorescent proteins enables the use of a fluorescence resonance energy transfer signal as a readout. Unlike previous systems, this sensor enables direct monitoring of the rapid uptake and clearance of auxin by individual cells and within cell compartments in planta. By responding to the graded spatial distribution along the root axis and its perturbation by transport inhibitors-as well as the rapid and reversible redistribution of endogenous auxin in response to changes in gravity vectors-our sensor enables real-time monitoring of auxin concentrations at a (sub)cellular resolution and their spatial and temporal changes during the lifespan of a plant.

2021 Review in Nature materials Nat. Mater. 20, 585–592 (2021)

Farhadi, A. ; Sigmund, F. ; Westmeyer, G.G.&deg ; Shapiro, M.G.&deg

Genetically encodable materials for non-invasive biological imaging.

Many questions in basic biology and medicine require the ability to visualize the function of specific cells and molecules inside living organisms. In this context, technologies such as ultrasound, optoacoustics and magnetic resonance provide non-invasive imaging access to deep-tissue regions, as used in many laboratories and clinics to visualize anatomy and physiology. In addition, recent work has enabled these technologies to image the location and function of specific cells and molecules inside the body by coupling the physics of sound waves, nuclear spins and light absorption to unique protein-based materials. These materials, which include air-filled gas vesicles, capsid-like nanocompartments, pigment-producing enzymes and transmembrane transporters, enable new forms of biomolecular and cellular contrast. The ability of these protein-based contrast agents to be genetically encoded and produced by cells creates opportunities for unprecedented in vivo studies of cellular function, while their amenability to genetic engineering enables atomic-level design of their physical, chemical and biological properties.

2021 Scientific Article in Scientific Reports Sci. Rep. 11:2181 (2021)

Seeger, M. ; Stiel, A.-C.&deg ; Ntziachristos, V.&deg

In vitro optoacoustic flow cytometry with light scattering referencing.

Morphological and functional optoacoustic imaging is enhanced by dedicated transgene reporters, in analogy to fluorescence methods. The development of optoacoustic reporters using protein engineering and directed evolution would be accelerated by high-throughput in-flow screening for intracellular, genetically encoded, optoacoustic contrast. However, accurate characterization of such contrast is impeded because the optoacoustic signals depend on the cell’s size and position in the flow chamber. We report herein an optoacoustic flow cytometer (OA-FCM) capable of precise measurement of intracellular optoacoustic signals of genetically-encoded chromoproteins in flow. The novel system records light-scattering as a reference for the detected optoacoustic signals in order to account for cell size and position, as well as excitation light flux in the focal volume, which we use to reference the detected optoacoustic signals to enhance the system’s precision. The OA-FCM was calibrated using micrometer-sized particles to showcase the ability to assess in-flow objects in the size range of single-cells. We demonstrate the capabilities of our OA-FCM to identify sub-populations in a mixture of two E. coli stocks expressing different reporter-proteins with a precision of over 90%. High-throughput screening of optoacoustic labels could pave the way for identifying genetically encoded optoacoustic reporters by transferring working concepts of the fluorescence field such as directed evolution and activated cell sorting.

2021 Scientific Article in Scientific Reports Sci. Rep. 11:1654 (2021)

Ali, Z. ; Zakian Dominguez, C.M. ; Ntziachristos, V.

Ultra-broadband axicon transducer for optoacoustic endoscopy.

Image performance in optoacoustic endoscopy depends markedly on the design of the transducer employed. Ideally, high-resolution performance is required over an expanded depth of focus. Current optoacoustic focused transducers achieve lateral resolutions in the range of tens of microns in the mesoscopic regime, but their depth of focus is limited to hundreds of microns by the nature of their spherical geometry. We designed an ultra-broadband axicon detector with a 2 mm central aperture and investigated whether the imaging characteristics exceeded those of a spherical detector of similar size. We show a previously undocumented ability to achieve a broadband elongated pencil-beam optoacoustic sensitivity with an axicon detection geometry, providing approximately 40 μm-lateral resolution maintained over a depth of focus of 950 μm-3.8 times that of the reference spherical detector. This performance could potentially lead to optoacoustic endoscopes that can visualize optical absorption deeper and with higher resolution than any other optical endoscope today.

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 Optics Letters Opt. Lett. 46, 1-4 (2021)

Basak, K. ; Bader, M. ; Dehner, C. ; Jüstel, D. ; Ntziachristos, V.

Individual transducer impulse response characterization method to improve image quality of array-based handheld optoacoustic tomography.

The physical properties of each transducer element play a vital role in the quality of images generated in optoacoustic (photoacoustic) tomography using transducer arrays. Thorough experimental characterization of such systems is often laborious and impractical. A shortcoming of the existing impulse response correction methods, however, is the assumption that all transducers in the array are identical and therefore share one electrical impulse response (EIR). In practice, the EIRs of the transducer elements in the array vary, and the effect of this element-to-element variability on image quality has not been investigated so far, to the best of our knowledge. We hereby propose a robust EIR derivation for individual transducer elements in an array using sparse measurements of the total impulse response (TIR) and by solving the linear system for temporal convolution. Thereafter, we combine a simulated spatial impulse response with the derived individual EIRs to obtain a full characterization of the TIR, which we call individual synthetic TIR. Correcting for individual transducer responses, we demonstrate significant improvement in isotropic resolution, which further enhances the clinical potential of array-based. handheld transducers.

2021 Scientific Article in Cancers Cancers 13:126 (2021)

Mohr, H. ; Ballke, S. ; Bechmann, N. ; Gulde, S. ; Malekzadeh Najafabadi, J. ; Peitzsch, M. ; Ntziachristos, V. ; Steiger, K. ; Wiedemann, T. ; Pellegata, N.S.

Mutation of the cell cycle regulator p27kip1 drives pseudohypoxic pheochromocytoma development.

BACKGROUND: Pseudohypoxic tumors activate pro-oncogenic pathways typically associated with severe hypoxia even when sufficient oxygen is present, leading to highly aggressive tumors. Prime examples are pseudohypoxic pheochromocytomas and paragangliomas (p-PPGLs), neuroendendocrine tumors currently lacking effective therapy. Previous attempts to generate mouse models for p-PPGLs all failed. Here, we describe that the rat MENX line, carrying a Cdkn1b (p27) frameshift-mutation, spontaneously develops pseudohypoxic pheochromocytoma (p-PCC). METHODS: We compared rat p-PCCs with their cognate human tumors at different levels: histology, immunohistochemistry, catecholamine profiling, electron microscopy, transcriptome and metabolome. The vessel architecture and angiogenic potential of pheochromocytomas (PCCs) was analyzed by light-sheet fluorescence microscopy ex vivo and multi-spectral optoacoustic tomography (MSOT) in vivo. RESULTS: The analysis of tissues at various stages, from hyperplasia to advanced grades, allowed us to correlate tumor characteristics with progression. Pathological changes affecting the mitochrondrial ultrastructure where present already in hyperplasias. Rat PCCs secreted high levels of norepinephrine and dopamine. Transcriptomic and metabolomic analysis revealed changes in oxidative phosphorylation that aggravated over time, leading to an accumulation of the oncometabolite 2-hydroxyglutarate, and to hypermethylation, evident by the loss of the epigenetic mark 5-hmC. While rat PCC xenografts showed high oxygenation, induced by massive neoangiogenesis, rat primary PCC transcriptomes possessed a pseudohypoxic signature of high Hif2a, Vegfa, and low Pnmt expression, thereby clustering with human p-PPGL. CONCLUSION: Endogenous rat PCCs recapitulate key phenotypic features of human p-PPGLs. Thus, MENX rats emerge as the best available animal model of these aggressive tumors. Our study provides evidence of a link between cell cycle dysregulation and pseudohypoxia.

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 Theranostics Theranostics 11, 1864-1876 (2021)

Varasteh, Z.&deg ; De Rose, F. ; Mohanta, S. ; Li, Y. ; Zhang, X. ; Miritsch, B. ; Scafetta, G. ; Yin, C. ; Sager, H.B. ; Glasl, S. ; Gorpas, D. ; Habenicht, A.J.R. ; Ntziachristos, V. ; Weber, W.A. ; Bartolazzi, A.&deg ; Schwaiger, M. ; D'Alessandria, C.&deg

Imaging atherosclerotic plaques by targeting Galectin-3 and activated macrophages using (89Zr)-DFO- Galectin3-F(ab')2 mAb.

Rationale: The high expression of Galectin-3 (Gal3) in macrophages of atherosclerotic plaques suggests its participation in atherosclerosis pathogenesis, and raises the possibility to use it as a target to image disease severity in vivo. Here, we explored the feasibility of tracking atherosclerosis by targeting Gal3 expression in plaques of apolipoprotein E knockout (ApoE-KO) mice via PET imaging. Methods: Targeting of Gal3 in M0-, M1- and M2 (M2a/M2c)-polarized macrophages was assessed in vitro using a Gal3-F(ab’)2 mAb labeled with AlexaFluor®488 and 89Zr- desferrioxamine-thioureyl-phenylisothiocyanate (DFO). To visualize plaques in vivo, ApoE-KO mice were injected i.v. with 89Zr-DFO-Gal3-F(ab’)2 mAb and imaged via PET/CT 48 h post injection. Whole length aortas harvested from euthanized mice were processed for Sudan-IV staining, autoradiography, and immunostaining for Gal3, CD68 and α-SMA expression. To confirm accumulation of the tracer in plaques, ApoE-KO mice were injected i.v. with Cy5.5-Gal3-F(ab')2 mAb, euthanized 48 h post injection, followed by cryosections of the body and acquisition of fluorescent images. To explore the clinical potential of this imaging modality, immunostaining for Gal3, CD68 and α-SMA expression were carried out in human plaques. Single cell RNA sequencing (scRNA-Seq) analyses were performed to measure LGALS3 (i.e. a synonym for Gal3) gene expression in each macrophage of several subtypes present in murine or human plaques. Results: Preferential binding to M2 macrophages was observed with both AlexaFluor®488-Gal3-F(ab’)2 and 89Zr-DFO-Gal3-F(ab’)2 mAbs. Focal and specific 89Zr-DFO-Gal3-F(ab’)2 mAb uptake was detected in plaques of ApoE-KO mice by PET/CT. Autoradiography and immunohistochemical analyses of aortas confirmed the expression of Gal3 within plaques mainly in macrophages. Moreover, a specific fluorescent signal was visualized within the lesions of vascular structures burdened by plaques in mice. Gal3 expression in human plaques showed similar Gal3 expression patterns when compared to their murine counterparts. Conclusions: Our data reveal that 89Zr-DFO-Gal3-F(ab’)2 mAb PET/CT is a potentially novel tool to image atherosclerotic plaques at different stages of development, allowing knowledge-based tailored individual intervention in clinically significant disease.

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 Annals of Translational Medicine Ann. Transl. Med. 9:36 (2021)

Karlas, A.# ; Masthoff, M.# ; Kallmayer, M. ; Helfen, A. ; Bariotakis, M. ; Fasoula, N.-A. ; Schäfers, M. ; Seidensticker, M. ; Eckstein, H.H. ; Ntziachristos, V. ; Wildgruber, M.

Multispectral optoacoustic tomography of peripheral arterial disease based on muscle hemoglobin gradients-a pilot clinical study.

Background: Current imaging assessment of peripheral artery disease (PAD) relies on anatomical cross-sectional visualizations of the affected arteries. Multispectral optoacoustic tomography (MSOT) is a novel molecular imaging technique that provides direct and label-free visualizations of soft tissue perfusion and oxygenation. Methods: MSOT was prospectively assessed in a pilot trial in healthy volunteers (group n1=4, mean age 31, 50% male and group n3=4, mean age 37.3, 75% male) and patients with intermittent claudication (group n2=4, mean age 72, 75% male, PAD stage IIb). We conducted cuff-induced ischemia (group n1) and resting state measurements (groups n2 and n3) over the calf region. Spatially resolved mapping of oxygenated (HbO2), deoxygenated (Hb) and total (THb) hemoglobin, as well as oxygen saturation (SO2), were measured via hand-held hybrid MSOT-Ultrasound based purely on hemoglobin contrast. Results: Calf measurements in healthy volunteers revealed distinct dynamics for HbO2, Hb, THb and SO2 under cuff-induced ischemia. HbO2, THb and SO2 levels were significantly impaired in PAD patients compared to healthy volunteers (P<0.05 for all parameters). Revascularization led to significant improvements in HbO2 of the affected limb. Conclusions: Clinical MSOT allows for non-invasive, label-free and real-time imaging of muscle oxygenation in health and disease with implications for diagnostics and therapy assessment in PAD.

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 Proceedings of the National Academy of Sciences of the United States of America Proc. Natl. Acad. Sci. U.S.A. 118:e2008072118 (2021)

Yim, J.J.# ; Harmsen, S.# ; Flisikowski, K. ; Flisikowska, T. ; Namkoong, H. ; Garland, M. ; van den Berg, N.S. ; Vilches-Moure, J.G. ; Schnieke, A. ; Saur, D. ; Glasl, S. ; Gorpas, D. ; Habtezion, A. ; Ntziachristos, V. ; Contag, C.H. ; Gambhir, S.S. ; Bogyo, M.&deg ; Rogalla, S.&deg

A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions.

Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in highrisk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 μm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.

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 Journal of Biophotonics J. Biophotonics 14, e202000325 (2021)

Lu, T. ; Chen, T. ; Gao, F. ; Sun, B. ; Ntziachristos, V. ; Li, J.

LV-GAN: A deep learning approach for limited-view optoacoustic imaging based on hybrid datasets.

The optoacoustic imaging (OAI) methods are rapidly evolving for resolving optical contrast in medical imaging applications. In practice, measurement strategies are commonly implemented under limited-view conditions due to oversized image objectives or system design limitations. Data acquired by limited-view detection may impart artifacts and distortions in reconstructed optoacoustic (OA) images. We propose a hybrid data-driven deep learning approach based on generative adversarial network (GAN), termed as LV-GAN, to efficiently recover high quality images from limited-view OA images. Trained on both simulation and experiment data, LV-GAN is found capable of achieving high recovery accuracy even under limited detection angles less than 60 degrees. The feasibility of LV-GAN for artifact removal in biological applications was validated by ex vivo experiments based on two different OAI systems, suggesting high potential of a ubiquitous use of LV-GAN to optimize image quality or system design for different scanners and application scenarios.

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 Advanced Photonics Advanced Photonics 2:036003 (2020)

Gianani, I. ; Suprano, A. ; Giordani, T. ; Spagnolo, N. ; Sciarrino, F. ; Gorpas, D. ; Ntziachristos, V. ; Pinker, K. ; Biton, N. ; Kupferman, J. ; Arnon, S.

Transmission of vector vortex beams in dispersive media.

Scattering phenomena affect light propagation through any kind of medium from free space to biological tissues. Finding appropriate strategies to increase the robustness to scattering is the common requirement in developing both communication protocols and imaging systems. Recently, structured light has attracted attention due to its seeming scattering resistance in terms of transmissivity and spatial behavior. Moreover, correlation between optical polarization and orbital angular momentum (OAM), which characterizes the so-called vector vortex beams (VVBs) states, seems to allow for the preservation of the polarization pattern. We extend the analysis by investigating both the spatial features and the polarization structure of vectorial optical vortexes propagating in scattering media with different concentrations. Among the observed features, we find a sudden swift decrease in contrast ratio for Gaussian, OAM, and VVB modes for concentrations of the adopted scattering media exceeding 0.09%. Our analysis provides a more general and complete study on the propagation of structured light in dispersive and scattering media.

2020 Nature biomedical engineering Nat. Bio. Eng. 4:1120 (2020)

Gottschalk, S. ; Degtyaruk, O. ; Mc Larney, B. ; Rebling, J. ; Hutter, M.A. ; Shoham, S.&deg ; Razansky, D.&deg

Publisher Correction: Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain (Nature Biomedical Engineering, (2019), 3, 5, (392-401), 10.1038/s41551-019-0372-9).

The Author(s), under exclusive licence to Springer Nature Limited. In the HTML version of the Article originally published, Shy Shoham was mistakenly not denoted as a corresponding author; this has now been corrected. The PDF version was unaffected.

2020 Scientific Article in Cancers Cancers 12:3760 (2020)

Anastasov, N. ; Hirmer, E. ; Klenner, M. ; Ott, J. ; Falkenberg, N. ; Bao, X. ; Mutschelknaus, L. ; Mörtl, S. ; Combs, S.E. ; Atkinson, M.J. ; Schmid, T.E.

Mek1 inhibitor combined with irradiation reduces migration of breast cancer cells including mir-221 and zeb1 emt marker expression.

The miR-221 expression is dependent on the oncogenic RAS-RAF-MEK pathway activation and influences epithelial-to-mesenchymal transition (EMT). The Cancer Genome Atlas (TCGA) database analysis showed high gene significance for ZEB1 with EMT module analysis and miR-221 overexpression within the triple-negative breast cancer (TNBC) and HER2+ subgroups when compared to luminal A/B subgroups. EMT marker expression analysis after MEK1 (TAK-733) inhibitor treatment and irradiation was combined with miR-221 and ZEB1 expression analysis. The interaction of miR-221 overexpression with irradiation and its influence on migration, proliferation, colony formation and subsequent EMT target activation were investigated. The results revealed that MEK1 inhibitor treatment combined with irradiation could decrease the migratory potential of breast cancer cells including reduction of miR-221 and corresponding downstream ZEB1 (EMT) marker expression. The clonogenic survival assays revealed that miR-221 overexpressing SKBR3 cells were more radioresistant when compared to the control. Remarkably, the effect of miR-221 overexpression on migration in highly proliferative and highly HER2-positive SKBR3 cells remained constant even upon 8 Gy irradiation. Further, in naturally miR-221-overexpressing MDA-MB-231 cells, the proliferation and migration significantly decrease after miR-221 knockdown. This leads to the assumption that radiation alone is not reducing migration capacity of miR-221-overexpressing cells and that additional factors play an important role in this context. The miR-221/ZEB1 activity is efficiently targeted upon MEK1 inhibitor (TAK-733) treatment and when combined with irradiation treatment, significant reduction in migration of breast cancer cells was shown.

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 Physical Chemistry, Chemical Physics Phys. Chem. Chem. Phys. 22, 26728-26741 (2020)

Shams, S.F. ; Ghazanfari, M.R. ; Pettinger, S. ; Tavabi, A.H. ; Siemensmeyer, K. ; Smekhova, A. ; Dunin-Borkowski, R.E. ; Westmeyer, G.G. ; Schmitz-Antoniak, C.

Structural perspective on revealing heat dissipation behavior of CoFe2O4-Pd nanohybrids: Great promise for magnetic fluid hyperthermia.

Loss mechanisms in fluid heating of cobalt ferrite (CFO) nanoparticles and CFO-Pd heterodimer colloidal suspensions are investigated as a function of particle size, fluid concentration and magnetic field amplitude. The specific absorption rate (SAR) is found to vary with increasing particle size due to a change in dominant heating mechanism from susceptibility to hysteresis and frictional loss. The maximum SAR is obtained for particle diameters of 11-15 nm as a result of synergistic contributions of susceptibility loss, including Néel and Brownian relaxation and especially hysteresis loss, thereby validating the applicability of linear response theory to superparamagnetic CFO nanoparticles. Our results show that the ferrofluid concentration and magnetic field amplitude alter interparticle interactions and associated heating efficiency. The SAR of the CFO nanoparticles could be maximized by adjusting the synthesis parameters. Despite the paramagnetic properties of individual palladium nanoparticles, CFO-Pd heterodimer suspensions were observed to have surprisingly improved magnetization as well as SAR values, when compared with CFO ferrofluids. This difference is attributed to interfacial interactions between the magnetic moments of paramagnetic Pd and superparamagnetic/ferrimagnetic CFO. SAR values measured from CFO-Pd heterodimer suspensions were found to be 47-52 W gFerrite-1, which is up to a factor of two higher than the SAR values of commercially available ferrofluids, demonstrating their potential as efficient heat mediators. Our results provide insight into the utilization of CFO-Pd heterodimer suspensions as potential nanoplatforms for diagnostic and therapeutic biomedical applications, e.g., in cancer hyperthermia, cryopreserved tissue warming, thermoablative therapy, drug delivery and bioimaging.

2020 Scientific Article in Optics Letters Opt. Lett. 45, 6579-6582 (2020)

Stylogiannis, A. ; Riobo, L. ; Prade, L. ; Glasl, S. ; Klein, S. ; Lucidi, G. ; Fuchs, M. ; Saur, D. ; Ntziachristos, V.

Low-cost single-point optoacoustic sensor for spectroscopic measurement of local vascular oxygenation.

Optical sensors developed for the assessment of oxygen in tissue microvasculature, such as those based on near-infrared spectroscopy, are limited in application by light scattering. Optoacoustic methods are insensitive to light scattering, and therefore, they can provide higher specificity and accuracy when quantifying local vascular oxygenation. However, currently, to the best of our knowledge, there is no low-cost, single point, optoacoustic sensor for the dedicated measurement of oxygen saturation in tissue microvasculature. This work introduces a spectroscopic optoacoustic sensor (SPOAS) for the non-invasive measurement of local vascular oxygenation in real time. SPOAS employs continuous wave laser diodes and measures at a single point, which makes it low-cost and portable. The SPOAS performance was benchmarked using blood phantoms, and it showed excellent linear correlation (R2 = 0.98) with a blood gas analyzer. Subsequent measurements of local vascular oxygenation in living mice during an oxygen stress test correlated well with simultaneous readings from a reference instrument.

2020 Scientific Article in Cancer Research Cancer Res. 80, 5291-5304 (2020)

Liapis, E. ; Klemm, U. ; Karlas, A. ; Reber, J. ; Ntziachristos, V.

Resolution of spatial and temporal heterogeneity in bevacizumab-treated breast tumors by eigenspectra multispectral optoacoustic tomography.

Understanding temporal and spatial hemodynamic heterogeneity as a function of tumor growth or therapy affects the development of novel therapeutic strategies. In this study, we employed eigenspectra multispectral optoacoustic tomography (eMSOT) as a next-generation optoacousticmethod to impart high accuracy in resolving tumor hemodynamics during bevacizumab therapy in two types of breast cancer xenografts (KPL-4 and MDA-MB-468). Patterns of tumor total hemoglobin concentration (THb) and oxygen saturation (sO(2)) were imaged in control and bevacizumab-treated tumors over the course of 58 days (KPL-4) and 16 days (MDA-MB-468), and the evolution of functional vasculature "normalization" was resolved macroscopically. Aninitial sharp drop in tumor sO(2) andTHb content shortly after the initiation of bevacizumab treatment was followed by a recovery in oxygenation levels. Rim-core subregion analysis revealed steep spatial oxygenation gradients in growing tumors that were reduced after bevacizumab treatment. Critically, eMSOT imaging findings were validated directly by histopathologic assessment of hypoxia (pimonidazole) and vascularity (CD31). These data demonstrate how eMSOT brings new abilities for accurate observation of entire tumor responses to challenges at spatial and temporal dimensions not available by other techniques today.Significance: Accurate assessment of hypoxia and vascularization over space and time is critical for understanding tumor development and the role of spatial heterogeneity in tumor aggressiveness, metastasis, and response to treatment.

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 IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 39, 3643-3654 (2020)

Olefir, I.# ; Tzoumas, S.# ; Restivo, C. ; Mohajerani, P. ; Xing, L. ; Ntziachristos, V.

Deep learning-based spectral unmixing for optoacoustic imaging of tissue oxygen saturation.

Label free imaging of oxygenation distribution in tissues is highly desired in numerous biomedical applications, but is still elusive, in particular in sub-epidermal measurements. Eigenspectra multispectral optoacoustic tomography (eMSOT) and its Bayesian-based implementation have been introduced to offer accurate label-free blood oxygen saturation (sO(2)) maps in tissues. The method uses the eigenspectra model of light fluence in tissue to account for the spectral changes due to the wavelength dependent attenuation of light with tissue depth. eMSOT relies on the solution of an inverse problem bounded by a number of ad hoc hand-engineered constraints. Despite the quantitative advantage offered by eMSOT, both the non-convex nature of the optimization problem and the possible sub-optimality of the constraints may lead to reduced accuracy. We present herein a neural network architecture that is able to learn how to solve the inverse problem of eMSOT by directly regressing from a set of input spectra to the desired fluence values. The architecture is composed of a combination of recurrent and convolutional layers and uses both spectral and spatial features for inference. We train an ensemble of such networks using solely simulated data and demonstrate how this approach can improve the accuracy of sO(2) computation over the original eMSOT, not only in simulations but also in experimental datasets obtained from blood phantoms and small animals (mice) in vivo. The use of a deep-learning approach in optoacoustic sO(2) imaging is confirmed herein for the first time on ground truth sO(2) values experimentally obtained in vivo and ex vivo.

2020 Scientific Article in Optics Express Opt. Express. 28, 35427-35437 (2020)

Suprano, A. ; Giordani, T. ; Gianani, I. ; Spagnolo, N. ; Pinker, K. ; Kupferman, J. ; Arnon, S. ; Klemm, U. ; Gorpas, D. ; Ntziachristos, V. ; Sciarrino, F.

Propagation of structured light through tissue-mimicking phantoms.

Optical interrogation of tissues is broadly considered in biomedical applications. Nevertheless, light scattering by tissue limits the resolution and accuracy achieved when investigating sub-surface tissue features. Light carrying optical angular momentum or complex polarization profiles, offers different propagation characteristics through scattering media compared to light with unstructured beam profiles. Here we discuss the behaviour of structured light scattered by tissue-mimicking phantoms. We study the spatial and the polarization profile of the scattered modes as a function of a range of optical parameters of the phantoms, with varying scattering and absorption coefficients and of different lengths. These results show the non-trivial trade-off between the advantages of structured light profiles and mode broadening, stimulating further investigations in this direction.

2020 Scientific Article in Photoacoustics Photoacoustics 20:100203 (2020)

Chlis, N.-K.# ; Karlas, A.# ; Fasoula, N.-A. ; Kallmayer, M. ; Eckstein, H.H. ; Theis, F.J. ; Ntziachristos, V. ; Marr, C.

A sparse deep learning approach for automatic segmentation of human vasculature in multispectral optoacoustic tomography.

Multispectral Optoacoustic Tomography (MSOT) resolves oxy- (HbO2) and deoxy-hemoglobin (Hb) to perform vascular imaging. MSOT suffers from gradual signal attenuation with depth due to light-tissue interactions: an effect that hinders the precise manual segmentation of vessels. Furthermore, vascular assessment requires functional tests, which last several minutes and result in recording thousands of images. Here, we introduce a deep learning approach with a sparse-UNET (S-UNET) for automatic vascular segmentation in MSOT images to avoid the rigorous and time-consuming manual segmentation. We evaluated the S-UNET on a test-set of 33 images, achieving a median DICE score of 0.88. Apart from high segmentation performance, our method based its decision on two wavelengths with physical meaning for the task-at-hand: 850 nm (peak absorption of oxy-hemoglobin) and 810 nm (isosbestic point of oxy-and deoxy-hemoglobin). Thus, our approach achieves precise data-driven vascular segmentation for automated vascular assessment and may boost MSOT further towards its clinical translation.

2020 Scientific Article in Nature chemistry Nat. Chem. 12, 1123-1130 (2020)

Cosco, E. ; Spearman, A.L. ; Ramakrishnan, S. ; Lingg, J.G.P. ; Saccomano, M. ; Pengshung, M. ; Arus, B.A. ; Wong, K.C.Y. ; Glasl, S. ; Ntziachristos, V. ; Warmer, M. ; McLaughlin, R.R. ; Bruns, O.T.&deg ; Sletten, E.M.&deg

Shortwave infrared polymethine fluorophores matched to excitation lasers enable non-invasive, multicolour in vivo imaging in real time.

High-resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to substantial scattering and autofluorescence in tissue at visible (350-700 nm) and near-infrared (700-1,000 nm) wavelengths. Here, we enable real-time, non-invasive multicolour imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1,000-2,000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established relationships between structure and photophysical properties for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable near-infrared/SWIR excitation and single-channel detection, facilitating video-rate multicolour SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1,064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-colour imaging with high temporal and spatial resolutions.

2020 Scientific Article in Scientific Reports Sci. Rep. 10:18173 (2020)

Afshari, P. ; Zakian Dominguez, C.M. ; Ntziachristos, V.

Improving ultrasound images with elevational angular compounding based on acoustic refraction.

Ultrasound imaging is affected by coherent noise or speckle, which reduces contrast and overall image quality and degrades the diagnostic precision of the collected images. Elevational angular compounding (EAC) is an attractive means of addressing this limitation, since it reduces speckle noise while operating in real-time. However, current EAC implementations rely on mechanically rotating a one-dimensional (1D) transducer array or electronically beam steering of two-dimensional (2D) arrays to provide different elevational imaging angles, which increases the size and cost of the systems. Here we present a novel EAC implementation based on a 1D array, which does not necessitate mechanically rotating the transducer. The proposed refraction-based elevational angular compounding technique (REACT) instead utilizes a translating cylindrical acoustic lens that steers the ultrasound beam along the elevational direction. Applying REACT to investigate phantoms and excised tissue samples demonstrated superior suppression of ultrasound speckle noise compared to previous EAC methods, with up to a two-fold improvement in signal- and contrast-to-noise ratios. The effects of elevational angular width on speckle reduction was further investigated to determine the appropriate conditions for applying EAC. This study introduces acoustic refractive elements as potential low cost solutions to noise reduction, which could be integrated into current medical ultrasound devices.

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 Interface : journal of the Royal Society J. R. Soc. Interface 17:20200776 (2020)

Dutta, R.#&deg ; Mandal, S.#&deg ; Lin, H.-C. ; Raz, T. ; Kind, A. ; Schnieke, A. ; Razansky, D.

Brilliant cresyl blue enhanced optoacoustic imaging enables non-destructive imaging of mammalian ovarian follicles for artificial reproduction.

In the field of reproductive biology, there is a strong need for a suitable tool capable of non-destructive evaluation of oocyte viability and function. We studied the application of brilliant cresyl blue (BCB) as an intra-vital exogenous contrast agent using multispectral optoacoustic tomography (MSOT) for visualization of porcine ovarian follicles. The technique provided excellent molecular sensitivity, enabling the selection of competent oocytes without disrupting the follicles. We further conducted in vitro embryo culture, molecular analysis (real-time and reverse transcriptase polymerase chain reaction) and DNA fragmentation analysis to comprehensively establish the safety of BCB-enhanced MSOT imaging in monitoring oocyte viability. Overall, the experimental results suggest that the method offers a significant advance in the use of contrast agents and molecular imaging for reproductive studies. Our technique improves the accurate prediction of ovarian reserve significantly and, once standardized for in vivo imaging, could provide an effective tool for clinical infertility management.

2020 Scientific Article in Nature Communications Nat. Commun. 11:5626 (2020)

Schoppe, O.&deg ; Pan, C. ; Coronel, J. ; Mai, H. ; Rong, Z. ; Todorov, M.I. ; Müskes, A. ; Navarro, F. ; Li, H. ; Ertürk, A.&deg ; Menze, B.H.&deg

Deep learning-enabled multi-organ segmentation in whole-body mouse scans.

Whole-body imaging of mice is a key source of information for research. Organ segmentation is a prerequisite for quantitative analysis but is a tedious and error-prone task if done manually. Here, we present a deep learning solution called AIMOS that automatically segments major organs (brain, lungs, heart, liver, kidneys, spleen, bladder, stomach, intestine) and the skeleton in less than a second, orders of magnitude faster than prior algorithms. AIMOS matches or exceeds the segmentation quality of state-of-the-art approaches and of human experts. We exemplify direct applicability for biomedical research for localizing cancer metastases. Furthermore, we show that expert annotations are subject to human error and bias. As a consequence, we show that at least two independently created annotations are needed to assess model performance. Importantly, AIMOS addresses the issue of human bias by identifying the regions where humans are most likely to disagree, and thereby localizes and quantifies this uncertainty for improved downstream analysis. In summary, AIMOS is a powerful open-source tool to increase scalability, reduce bias, and foster reproducibility in many areas of biomedical research.

2020 Scientific Article in Scientific Reports Sci. Rep. 10:16444 (2020)

Nitkunanantharajah, S. ; Haedicke, K. ; Moore, T.B. ; Manning, J.B. ; Dinsdale, G. ; Berks, M. ; Taylor, C. ; Dickinson, M.R. ; Jüstel, D. ; Ntziachristos, V. ; Herrick, A.L. ; Murray, A.K.

Three-dimensional optoacoustic imaging of nailfold capillaries in systemic sclerosis and its potential for disease differentiation using deep learning.

The autoimmune disease systemic sclerosis (SSc) causes microvascular changes that can be easily observed cutaneously at the finger nailfold. Optoacoustic imaging (OAI), a combination of optical and ultrasound imaging, specifically raster-scanning optoacoustic mesoscopy (RSOM), offers a noninvasive high-resolution 3D visualization of capillaries allowing for a better view of microvascular changes and an extraction of volumetric measures. In this study, nailfold capillaries of patients with SSc and healthy controls are imaged and compared with each other for the first time using OAI. The nailfolds of 23 patients with SSc and 19 controls were imaged using RSOM. The acquired images were qualitatively compared to images from state-of-the-art imaging tools for SSc, dermoscopy and high magnification capillaroscopy. The vascular volume in the nailfold capillaries were computed from the RSOM images. The vascular volumes differ significantly between both cohorts (0.216 +/- 0.085 mm(3) and 0.337 +/- 0.110 mm(3); p < 0.0005). In addition, an artificial neural network was trained to automatically differentiate nailfold images from both cohorts to further assess whether OAI is sensitive enough to visualize anatomical differences in the capillaries between the two cohorts. Using transfer learning, the model classifies images with an area under the ROC curve of 0.897, and a sensitivity of 0.783 and specificity of 0.895. In conclusion, this study demonstrates the capabilities of RSOM as an imaging tool for SSc and establishes it as a modality that facilitates more in-depth studies into the disease mechanisms and progression.

2020 Scientific Article in Neoplasia : An International Journal for Oncology Research Neoplasia 22, 770-777 (2020)

Lafci, B. ; Mercep, E. ; Herraiz, J.L. ; Deán-Ben, X.L. ; Razansky, D.

Noninvasive multiparametric charac-terization of mammary tumors with transmission-reflection optoacoustic ultrasound.

Development of imaging methods capable of furnishing tumor-specific morphological, functional, and molecular information is paramount for early diagnosis, staging, and treatment of breast cancer. Ultrasound (US) and optoacoustic (OA) imaging methods exhibit excellent traits for tumor imaging in terms of fast imaging speed, ease of use, excellent contrast, and lack of ionizing radiation. Here, we demonstrate simultaneous tomographic whole body imaging of optical absorption, US reflectivity, and speed of sound (SoS) in living mice. In vivo studies of 4T1 breast cancer xenografts models revealed synergistic and complementary value of the hybrid imaging approach for characterizing mammary tumors. While neovasculature surrounding the tumor areas were observed based on the vascular anatomy contrast provided by the OA data, the tumor boundaries could be discerned by segmenting hypoechoic structures in pulse-echo US images. Tumor delineation was further facilitated by enhancing the contrast and spatial resolution of the SoS maps with a full-wave inversion method. The malignant lesions could thus be distinguished from other hypoechoic regions based on the average SoS values. The reported findings corroborate the strong potential of the hybrid imaging approach for advancing cancer research in small animal models and fostering development of new clinical diagnostic approaches.

2020 Scientific Article in Nature Nature 585, 372-378 (2020)

Shnaiderman, R.&deg ; Wissmeyer, G. ; Ülgen, O. ; Mustafa, Q. ; Chmyrov, A. ; Ntziachristos, V.&deg

A submicrometre silicon-on-insulator resonator for ultrasound detection.

The widely available silicon-on-insulator technology is used to develop a miniaturized ultrasound detector, which is 200 times smaller than the wavelengths of sound that it can detect.Ultrasound detectors use high-frequency sound waves to image objects and measure distances, but the resolution of these readings is limited by the physical dimensions of the detecting element. Point-like broadband ultrasound detection can greatly increase the resolution of ultrasonography and optoacoustic (photoacoustic) imaging(1,2), but current ultrasound detectors, such as those used for medical imaging, cannot be miniaturized sufficiently. Piezoelectric transducers lose sensitivity quadratically with size reduction(3), and optical microring resonators(4)and Fabry-Perot etalons(5)cannot adequately confine light to dimensions smaller than about 50 micrometres. Micromachining methods have been used to generate arrays of capacitive(6)and piezoelectric(7)transducers, but with bandwidths of only a few megahertz and dimensions exceeding 70 micrometres. Here we use the widely available silicon-on-insulator technology to develop a miniaturized ultrasound detector, with a sensing area of only 220 nanometres by 500 nanometres. The silicon-on-insulator-based optical resonator design provides per-area sensitivity that is 1,000 times higher than that of microring resonators and 100,000,000 times better than that of piezoelectric detectors. Our design also enables an ultrawide detection bandwidth, reaching 230 megahertz at -6 decibels. In addition to making the detectors suitable for manufacture in very dense arrays, we show that the submicrometre sensing area enables super-resolution detection and imaging performance. We demonstrate imaging of features 50 times smaller than the wavelength of ultrasound detected. Our detector enables ultra-miniaturization of ultrasound readings, enabling ultrasound imaging at a resolution comparable to that achieved with optical microscopy, and potentially enabling the development of very dense ultrasound arrays on a silicon chip.

2020 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 39, 2931-2940 (2020)

Ding, L ; Razansky, D. ; Deán-Ben, X.L.

Model-based reconstruction of large three-dimensional optoacoustic datasets.

Iterative model-based algorithms are known to enable more accurate and quantitative optoacoustic (photoacoustic) tomographic reconstructions than standard back-projection methods. However, three-dimensional (3D) model-based inversion is often hampered by high computational complexity and memory overhead. Parallel implementations on a graphics processing unit (GPU) have been shown to efficiently reduce the memory requirements by on-the-fly calculation of the actions of the optoacoustic model matrix, but the high complexity still makes these approaches impractical for large 3D optoacoustic datasets. Herein, we show that the computational complexity of 3D model-based iterative inversion can be significantly reduced by splitting the model matrix into two parts: one maximally sparse matrix containing only one entry per voxel-transducer pair and a second matrix corresponding to cyclic convolution. We further suggest reconstructing the images by multiplying the transpose of the model matrix calculated in this manner with the acquired signals, which is equivalent to using a very large regularization parameter in the iterative inversion method. The performance of these two approaches is compared to that of standard back-projection and a recently introduced GPU-based model-based method using datasets from in vivo experiments. The reconstruction time was accelerated by approximately an order of magnitude with the new iterative method, while multiplication with the transpose of the matrix is shown to be as fast as standard back-projection.

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 IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 39, 3218-3230 (2020)

Chowdhury, S.P. ; Prakash, J. ; Karlas, A. ; Jüstel, D. ; Ntziachristos, V.

A synthetic total impulse response characterization method for correction of hand-held optoacoustic images.

The impulse response of optoacoustic (photoacoustic) tomographic imaging system depends on several system components, the characteristics of which can influence the quality of reconstructed images. The effect of these system components on reconstruction quality have not been considered in detail so far. Here we combine sparse measurements of the total impulse response (TIR) with a geometric acoustic model to obtain a full characterization of the TIR of a handheld optoacoustic tomography system with concave limited-view acquisition geometry. We then use this synthetic TIR to reconstruct data from phantoms and healthy human volunteers, demonstrating improvements in image resolution and fidelity. The higher accuracy of optoacoustic tomographic reconstruction with TIR correction further improves the diagnostic capability of handheld optoacoustic tomographic systems.

2020 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 39, 3250-3255 (2020)

Özbek, A. ; Dean-Ben, X.L. ; Razansky, D.

Compressed optoacoustic sensing of volumetric cardiac motion.

The recently developed optoacoustic tomography systems have attained volumetric frame rates exceeding 100 Hz, thus opening up new venues for studying previously invisible biological dynamics. Further gains in temporal resolution can potentially be achieved via partial data acquisition, though a priori knowledge on the acquired data is essential for rendering accurate reconstructions using compressed sensing approaches. In this work, we suggest a machine learning method based on principal component analysis for high-frame-rate volumetric cardiac imaging using only a few tomographic optoacoustic projections. The method is particularly effective for discerning periodic motion, as demonstrated herein by non-invasive imaging of a beating mouse heart. A training phase enables efficiently compressing the heart motion information, which is subsequently used as prior information for image reconstruction from sparse sampling at a higher frame rate. It is shown that image quality is preserved with a 64-fold reduction in the data flow. We demonstrate that, under certain conditions, the volumetric motion could effectively be captured by relying on time-resolved data from a single optoacoustic detector. Feasibility of capturing transient (non-periodic) events not registered in the training phase is further demonstrated by visualizing perfusion of a contrast agent in vivo. The suggested approach can be used to significantly boost the temporal resolution of optoacoustic imaging and facilitate development of more affordable and data efficient systems.

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 Lecture Notes in Computer Science Lect. Notes Comput. Sc. 12266 LNCS, 309-319 (2020)

Gerl, S. ; Paetzold, J.C. ; He, H. ; Ezhov, I. ; Shit, S. ; Kofler, F. ; Bayat, A.A. ; Tetteh, G. ; Ntziachristos, V. ; Menze, B.

A distance-based loss for smooth and continuous skin layer segmentation in optoacoustic images.

Raster-scan optoacoustic mesoscopy (RSOM) is a powerful, non-invasive optical imaging technique for functional, anatomical, and molecular skin and tissue analysis. However, both the manual and the automated analysis of such images are challenging, because the RSOM images have very low contrast, poor signal to noise ratio, and systematic overlaps between the absorption spectra of melanin and hemoglobin. Nonetheless, the segmentation of the epidermis layer is a crucial step for many downstream medical and diagnostic tasks, such as vessel segmentation or monitoring of cancer progression. We propose a novel, shape-specific loss function that overcomes discontinuous segmentations and achieves smooth segmentation surfaces while preserving the same volumetric Dice and IoU. Further, we validate our epidermis segmentation through the sensitivity of vessel segmentation. We found a 20% improvement in Dice for vessel segmentation tasks when the epidermis mask is provided as additional information to the vessel segmentation network.

2020 Scientific Article in British Journal of Dermatology - BJD Br. J. Dermatol., DOI: 10.1111/bjd.19463 (2020)

Hindelang, B. ; Aguirre Bueno, J. ; Berezhnoi, A. ; Biedermann, T. ; Darsow, U. ; Eberlein, B. ; Ntziachristos, V.

Quantification of skin sensitivity to ultraviolet radiation using ultra-wideband optoacoustic mesoscopy.

Phototesting is used to assess individual sensitivity to ultraviolet (UV) radiation in order to determine adequate UV dosage for phototherapy1 . In the standard procedure, small skin areas are exposed to increasing doses of UV radiation. The lowest UV dose that induces a delineated erythema at 24±2 h after UV exposure defines the minimal erythema dose (MED)2 . Visual assessment is the gold standard for MED determination; however, it is prone to observer variability3 . Optical methods have been considered to quantify the magnitude of erythema response. However, they are limited by light scattering therefore high-resolution is restricted to depths of <200 μm resulting in unreliable measurements4,5 .

2020 Scientific Article in Neoplasia : An International Journal for Oncology Research Neoplasia 22, 441-446 (2020)

Dean-Ben, X.L. ; Weidenfeld, I. ; Degtyaruk, O. ; Ntziachristos, V. ; Stiel, A.-C. ; Razansky, D.

Deep tissue volumetric optoacoustic tracking of individual circulating tumor cells in an in intracardially perfused mouse model.

Widespread metastasis is the major cause of death from melanoma and other types of cancer. At present, the dynamic aspects of the metastatic cascade remain enigmatic. The feasibility to track circulating melanoma cells deep within living intact organisms can greatly impact our knowledge on tumor metastasis, but existing imaging approaches lack the sensitivity, spatio-temporal resolution or penetration depth to capture flowing tumor cells over large fields of view within optically-opaque biological tissues. Vast progress with the development of optoacoustic tomography technologies has recently enabled two- and three-dimensional imaging at unprecedented frame rates in the order of hundreds of Hertz, effectively mapping up to a million image voxels within a single volumetric snapshot. Herein, we employ volumetric optoacoustic tomography for real-time visualization of passage and trapping of individual B16 melanoma cells in the whole mouse brain. Detection of individual circulating melanoma cells was facilitated by substituting blood with an artificial cerebrospinal fluid that removes the strong absorption background in the optoacoustic images. The approach can provide new opportunities for studying trafficking and accumulation of metastatic melanoma cells in different organs.

2020 Scientific Article in Photoacoustics Photoacoustics 20:100200 (2020)

Longo, A. ; Morscher, S. ; Malekzadeh Najafabadi, J. ; Jüstel, D. ; Zakian Dominguez, C.M. ; Ntziachristos, V.

Assessment of hessian-based Frangi vesselness filter in optoacoustic imaging.

The Hessian-based Frangi vesselness filter is commonly used to enhance vasculature in optoacoustic (photoacoustic) images, but its accuracy and limitations have never been rigorously assessed. Here we validate the ability of the filter to enhance vessel-like structures in phantoms, and we introduce an experimental approach that uses measurements before and after the administration of gold nanorods (AuNRs) to examine filter performance in vivo. We evaluate the influence of contrast, filter scales, angular tomographic coverage, out-of-plane signals and light fluence on image quality, and gain insight into the performance of the filter. We observe the generation of artifactual structures that can be misinterpreted as vessels and provide recommendations to ensure appropriate use of Frangi and other vesselness filters and avoid misinterpretation of post-processed optoacoustic images.

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 Analytical Chemistry Anal. Chem. 92, 10717-10724 (2020)

Fuenzalida Werner, J.P. ; Huang, Y. ; Mishra, K. ; Janowski, R. ; Vetschera, P. ; Heichler, C. ; Chmyrov, A. ; Neufert, C. ; Niessing, D. ; Ntziachristos, V. ; Stiel, A.-C.

Challenging a preconception: Optoacoustic spectrum differs from the absorption spectrum of proteins and dyes for molecular imaging.

Optoacoustic (photoacoustic) imaging has seen marked advances in detection and data analysis, but there is less progress in understanding the photophysics of common optoacoustic contrast agents. This gap blocks the development of novel agents and the accurate analysis and interpretation of multispectral optoacoustic images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. Herein, we employ MLS to analyze contrast agents (methylene blue, rhodamine 800, Alexa Fluor 750, IRDye 800CW, and indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720, and smURFP). We found that the optical absorption spectrum does not correlate with the optoacoustic spectrum for the majority of the analytes. We determined that for dyes, the transition underlying an aggregation state has more optoacoustic signal generation efficiency than the monomer transition. For proteins we found a favored optoacoustic relaxation that stems from the neutral or zwitterionic chromophores and unreported photoswitching behavior of tdTomato and HcRed. We then crystalized HcRed in its photoswitch optoacoustic state, confirming structurally the change in isomerization with respect to HcReds' fluorescence state. Finally, on the example of the widely used label tdTomato and the dye indocyanine green, we show the importance of correct photophysical (e.g., spectral and kinetic) information as a prerequisite for spectral-unmixing for in vivo imaging.

Vitamins and Hormones In:. 125 London Wall, London Ec2y 5as, England: Academic Press Ltd-elsevier Science Ltd, 2020. 1-21 (Vitam. Horm. ; 114)

Ovsepian, S.V. ; O´Leary, V.B. ; Vesselkin, N.P.

Evolutionary origins of chemical synapses.

Synaptic transmission is a fundamental neurobiological process by which neurons interact with each other and non-neuronal cells. It involves release of active substances from the presynaptic neuron onto receptive elements of postsynaptic cells, inducing waves of spreading electrochemical response. While much has been learned about the cellular and molecular mechanisms driving and governing transmitter release and sensing, the evolutionary origin of synaptic connections remains obscure. Herein, we review emerging evidence and concepts suggesting that key components of chemical synapse arose independently from neurons, in different functional and biological contexts, before the rise of multicellular living forms. We argue that throughout evolution, distinct synaptic constituents have been co-opted from ancestral forms for a new role in early metazoan, leading to the rise of chemical synapses and neurotransmission. Such a mosaic model of the origin of chemical synapses agrees with and supports the pluralistic hypothesis of evolutionary change.

Recent Results in Cancer Research In: Molecular Imaging in Oncology. 2020. 155-187 (Recent Results Cancer Res. ; 216)

Razansky, D. ; Ntziachristos, V.

Optical and optoacoustic imaging.

The present chapter summarizes progress with optical methods that go beyond human vision. The focus is on two particular technologies: fluorescence molecular imaging and optoacoustic (photoacoustic) imaging. The rationale for the selection of these two methods is that in contrast to optical microscopy techniques, both fluorescence and optoacoustic imaging can achieve large fields of view, i.e., spanning several centimeters in two or three dimensions. Such fields of views relate better to human vision and can visualize large parts of tissue, a necessary premise for clinical detection. Conversely, optical microscopy methods only scan millimeter-sized dimensions or smaller. With such operational capacity, optical microscopy methods need to be guided by another visualization technique in order to scan a very specific area in tissue and typically only provide superficial measurements, i.e., information from depths that are of the order of 0.05-1 mm. This practice has generally limited their clinical applicability to some niche applications, such as optical coherence tomography of the retina. On the other hand, fluorescence molecular imaging and optoacoustic imaging emerge as more global optical imaging methods with wide applications in surgery, endoscopy, and non-invasive clinical imaging, as summarized in the following. The current progress in this field is based on a volume of recent review and other literature that highlights key advances achieved in technology and biomedical applications. Context and figures from references from the authors of this chapter have been used here, as it reflects our general view of the current status of the field.

2020 Review in Biomolecules Biomolecules 10:966 (2020)

Gabashvili, A.N. ; Chmelyuk, N.S. ; Efremova, M.V. ; Malinovskaya, J.A. ; Semkina, A.S. ; Abakumov, M.A.

Encapsulins-bacterial protein nanocompartments: Structure, properties, and application.

Recently, a new class of prokaryotic compartments, collectively called encapsulins or protein nanocompartments, has been discovered. The shell proteins of these structures self-organize to form icosahedral compartments with a diameter of 25-42 nm, while one or more cargo proteins with various functions can be encapsulated in the nanocompartment. Non-native cargo proteins can be loaded into nanocompartments and the surface of the shells can be further functionalized, which allows for developing targeted drug delivery systems or using encapsulins as contrast agents for magnetic resonance imaging. Since the genes encoding encapsulins can be integrated into the cell genome, encapsulins are attractive for investigation in various scientific fields, including biomedicine and nanotechnology.

2020 Scientific Article in Frontiers in Neuroscience Front. Neurosci. 14:536 (2020)

Mc Larney, B.# ; Hutter, M.A.# ; Degtyaruk, O. ; Dean-Ben, X.L. ; Razansky, D.

Monitoring of stimulus evoked murine somatosensory cortex. hemodynamic activity with volumetric multi-. spectral optoacoustic. tomography.

Sensory stimulation is an attractive paradigm for studying brain activity using various optical-, ultrasound- and MRI-based functional neuroimaging methods. Optoacoustics has been recently suggested as a powerful new tool for scalable mapping of multiple hemodynamic parameters with rich contrast and previously unachievable spatio-temporal resolution. Yet, its utility for studying the processing of peripheral inputs at the whole brain level has so far not been quantified. We employed volumetric multi-spectral optoacoustic tomography (vMSOT) to non-invasively monitor the HbO, HbR, and HbT dynamics across the mouse somatosensory cortex evoked by electrical paw stimuli. We show that elevated contralateral activation is preserved in the HbO map (invisible to MRI) under isoflurane anesthesia. Brain activation is shown to be predominantly confined to the somatosensory cortex, with strongest activation in the hindpaw region of the contralateral sensorimotor cortex. Furthermore, vMSOT detected the presence of an initial dip in the contralateral hindpaw region in the delta HbO channel. Sensorimotor cortical activity was identified over all other regions in HbT and HbO but not in HbR. Pearson's correlation mapping enabled localizing the response to the sensorimotor cortex further highlighting the ability of vMSOT to bridge over imaging performance deficiencies of other functional neuroimaging modalities.

2020 Scientific Article in Science Advances Sci. Adv. 6:eaaz6293 (2020)

Mishra, K. ; Stankevych, M. ; Fuenzalida Werner, J.P. ; Grassmann, S. ; Gujrati, V. ; Huang, Y. ; Klemm, U. ; Buchholz, V.R. ; Ntziachristos, V. ; Stiel, A.-C.

Multiplexed whole-animal imaging with reversibly switchable optoacoustic proteins.

We introduce two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria, and tumors in the mouse body and brain. We developed machine learning-based software for commercial imaging systems for temporal unmixed OA imaging, enabling its routine use in life sciences.

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 Cell Cell 180, 796-812 (2020)

Zhao, S. ; Todorov, M.I. ; Cai, R. ; Ai-Maskari, R. ; Steinke, H. ; Kemter, E. ; Mai, H. ; Rong, Z. ; Warmer, M. ; Stanic Aguilera, K.N. ; Schoppe, O. ; Paetzold, J.C. ; Gesierich, B. ; Wong, M.N. ; Huber, T.B. ; Duering, M. ; Bruns, O.T. ; Menze, B. ; Lipfert, J. ; Puelles, V.G. ; Wolf, E. ; Bechmann, I. ; Ertürk, A.

Cellular and molecular probing of intact human organs.

Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.

2020 Scientific Article in Nature Communications Nat. Commun. 11:2910 (2020)

Seeger, M. ; Soliman, D. ; Aguirre Bueno, J. ; Diot, G. ; Wierzbowski, J. ; Ntziachristos, V.

Pushing the boundaries of optoacoustic microscopy by total impulse response characterization.

Optical microscopy improves in resolution and signal-to-noise ratio by correcting for the system's point spread function; a measure of how a point source is resolved, typically determined by imaging nanospheres. Optical-resolution optoacoustic (photoacoustic) microscopy could be similarly corrected, especially to account for the spatially-dependent signal distortions induced by the acoustic detection and the time-resolved and bi-polar nature of optoacoustic signals. Correction algorithms must therefore include the spatial dependence of signals' origins and profiles in time, i.e. the four-dimensional total impulse response (TIR). However, such corrections have been so far impeded by a lack of efficient TIR-characterization methods. We introduce high-quality TIR determination based on spatially-distributed optoacoustic point sources (SOAPs), produced by scanning an optical focus on an axially-translatable 250nm gold layer. Using a spatially-dependent TIR-correction improves the signal-to-noise ratio by >10dB and the axial resolution by similar to 30%. This accomplishment displays a new performance paradigm for optoacoustic microscopy.

2020 Scientific Article in Photoacoustics Photoacoustics 19:100193 (2020)

Lu, T. ; Wang, Y. ; Li, J. ; Prakash, J. ; Gao, F. ; Ntziachristos, V.

Full-frequency correction of spatial impulse response in back-projection scheme using space-variant filtering for optoacoustic mesoscopy.

The fidelity and quality of reconstructed images in optoacoustic mesoscopy (OPAM) can be significantly improved by considering the spatial impulse response (SIR) of the employed focused transducer into reconstruction. However, the traditional method fully taking the SIR into account can hardly meet the data-intensive requirements of high resolution OPAM because of excessive memory and time consumption. Herein, a modified back-projection method using a space-variant filter for full-frequency correction of the SIR is presented, and applied to the OPAM system with a sphere-focused transducer. The proposed method can readily manage the large datasets of the OPAM and effectively reduce the extra time consumption. The performance of the proposed method is showcased by simulations and experiments of phantoms and biological tissue. The results demonstrate that the modified back-projection method exhibits better image fidelity, resolution and contrast compared to the common and weighted back-projection methods that are not or not fully accounting for the SIR.

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 Journal of Biomedical Optics J. Biomed. Opt. 25, 1-15 (2020)

Ruiz, A.J. ; Wu, M. ; LaRochelle, E.P.M. ; Gorpas, D. ; Ntziachristos, V. ; Pfefer, T.J. ; Pogue, B.W.

Indocyanine green matching phantom for fluorescence-guided surgery imaging system characterization and performance assessment.

SIGNIFICANCE: Expanded use of fluorescence-guided surgery with devices approved for use with indocyanine green (ICG) has led to a range of commercial systems available. There is a compelling need to be able to independently characterize system performance and allow for cross-system comparisons. AIM: The goal of this work is to expand on previous proposed fluorescence imaging standard designs to develop a long-term stable phantom that spectrally matches ICG characteristics and utilizes 3D printing technology for incorporating tissue-equivalent materials. APPROACH: A batch of test targets was created to assess ICG concentration sensitivity in the 0.3- to 1000-nM range, tissue-equivalent depth sensitivity down to 6 mm, and spatial resolution with a USAF test chart. Comparisons were completed with a range of systems that have significantly different imaging capabilities and applications, including the Li-Cor® Odyssey, Li-Cor® Pearl, PerkinElmer® Solaris, and Stryker® Spy Elite. RESULTS: Imaging of the ICG-matching phantoms with all four commercially available systems showed the ability to benchmark system performance and allow for cross-system comparisons. The fluorescence tests were able to assess differences in the detectable concentrations of ICG with sensitivity differences >10× for preclinical and clinical systems. Furthermore, the tests successfully assessed system differences in the depth-signal decay rate, as well as resolution performance and image artifacts. The manufacturing variations, photostability, and mechanical design of the tests showed promise in providing long-term stable standards for fluorescence imaging. CONCLUSIONS: The presented ICG-matching phantom provides a major step toward standardizing performance characterization and cross-system comparisons for devices approved for use with ICG. The developed hybrid manufacturing platform can incorporate long-term stable fluorescing agents with 3D printed tissue-equivalent material. Further, long-term testing of the phantom and refinements to the manufacturing process are necessary for future implementation as a widely adopted fluorescence imaging standard.

2020 Review in Aktuelle Dermatologie Akt. Dermatol. 46, 171-178 (2020)

Hindelang, B.#&deg ; Schoenmann, C.#&deg ; Aguirre Bueno, J. ; Ntziachristos, V. ; Biedermann, T. ; Darsow, U.

Optoakustische Bildgebung – Licht rein, Schall raus?

Die optoakustische Bildgebung ist eine neuartige Bildgebungsmodalität, welche auf der Aufzeichnung und Verarbeitung von Ultraschallsignalen basiert, die im Gewebe durch Absorption von Laserlicht entstehen. Die Methode vereint guten, auf Lichtabsorption beruhenden Kontrast mit hoher Eindringtiefe und ermöglicht sowohl morphologische als auch molekulare und funktionelle Bildgebung. Sie kann mit oder ohne Kontrastmittel eingesetzt werden und ist frei von ionisierender Strahlung. Darüber hinaus ist die Technik skalierbar und kann somit zur makroskopischen, mesoskopischen und mikroskopischen Bildgebung verwendet werden. In den letzten Jahren wurden eine Reihe verschiedener Systeme zur optoakustischen Bildgebung entwickelt und in vielfältigen Bereichen der präklinischen und klinischen Forschung eingesetzt. In der Dermatologie zeigten sich vielversprechende Anwendungsgebiete der Optoakustik insbesondere in der Untersuchung von Melanomen, Wächterlymphknoten und nicht-melanozytärem Hautkrebs sowie in der Charakterisierung von entzündlichen Hauterkrankungen. In diesem Review sollen die technischen Grundlagen der optoakustischen Bildgebung erläutert sowie der derzeitige Stand der Forschung hinsichtlich Anwendungsbereiche makroskopischer, mesoskopischer und mikroskopischer optoakustischer Systeme beschrieben und diskutiert werden.

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 Proceedings of SPIE Proc. SPIE 11240 (2020)

Özsoy, Ç. ; Özbek, A. ; Dean-Ben, X.L. ; Razansky, D.

Ultrafast imaging of cardiac electromechanical wave propagation with volumetric optoacoustic tomography.

Understanding the mechanisms of cardiac disorders largely depends on availability of multi-dimensional and multiparametric imaging methods capable of quantitative assessment of cardiac morphology and function. The imaging modalities commonly employed in cardiac research, such as ultrasonography and magnetic resonance imaging, are lacking sufficient contrast and/or spatio-temporal resolution in 3D in order to reveal the multi-scale nature of rapid electromechanical activity in a beating heart. Our recently developed volumetric optoacoustic tomography (VOT) platform offers versatile observations of the heart function with rich optical contrast at otherwise unattainable temporal and spatial resolutions. Herein, we further advance the imaging performance by developing compressed acquisition scheme to boost the temporal resolution of VOT into the kilohertz range, thus enabling 3D mapping of electromechanical wave propagation in the heart. Experiments in isolated mouse hearts were performed by exciting the entire imaged tissue volume with nanosecond-duration laser pulses at 1 kHz repetition rate pulse operating at 532 nm and sparse tomographic signal sampling using a custom-made 512-element spherical matrix ultrasound array. By analyzing the strain maps obtained from the rapid VOT image sequence, it was possible to quantify the phase velocity of the electromechanical cardiac waves, in good agreement with previously reported values.

2020 Scientific Article in Proceedings of SPIE Proc. SPIE 11240 (2020)

Deán-Ben, X.L. ; Degtyaruk, O. ; Razansky, D.

Visualization of microparticle flow in the mouse brain in an intracardiac perfusion model.

Particles with sizes in the order of a few micrometers can significantly enhance the capabilities of optoacoustic imaging systems by improving visualization of arbitrarily oriented vascular structures and achieving resolution beyond the acoustic diffraction barrier. Particle tracking may also be used for mapping the blood flow in two and three dimensions. However, a trade-off exists between the particle absorption properties and size, whereas large sized microparticles also tend to arrest in the capillary network. We analyzed the flow of microparticles in an intracardiac perfusion mouse model in which blood is effectively substituted by artificial cerebrospinal fluid (ACSF). This enables mitigating the strong blood absorption background in the optoacoustic images thus facilitating the visualization of microparticles. A sequence of three-dimensional optoacoustic images of the mouse brain is then acquired at a high frame rate of 100 Hz after injection of the particles in the left heart ventricle. By visualizing the flow of particles of different sizes in microvascular structures it is possible to establish optimal trade-offs between the particle size, their optoacoustic signal and perfusion properties.

2020 Scientific Article in Nature biomedical engineering Nat. Bio. Eng. 4, 286-297 (2020)

Haedicke, K. ; Agemy, L. ; Omar, M. ; Berezhnoi, A. ; Roberts, S. ; Longo-Machado, C. ; Skubal, M. ; Nagar, K. ; Hsu, H.T. ; Kim, K. ; Reiner, T. ; Coleman, J. ; Ntziachristos, V. ; Scherz, A. ; Grimm, J.

High-resolution optoacoustic imaging of tissue responses to vascular-targeted therapies.

The monitoring of vascular-targeted therapies using magnetic resonance imaging, computed tomography or ultrasound is limited by their insufficient spatial resolution. Here, by taking advantage of the intrinsic optical properties of haemoglobin, we show that raster-scanning optoacoustic mesoscopy (RSOM) provides high-resolution images of the tumour vasculature and of the surrounding tissue, and that the detection of a wide range of ultrasound bandwidths enables the distinction of vessels of differing size, providing detailed insights into the vascular responses to vascular-targeted therapy. Using RSOM to examine the responses to vascular-targeted photodynamic therapy in mice with subcutaneous xenografts, we observed a substantial and immediate occlusion of the tumour vessels followed by haemorrhage within the tissue and the eventual collapse of the entire vasculature. Using dual-wavelength RSOM, which distinguishes oxyhaemoglobin from deoxyhaemoglobin, we observed an increase in oxygenation of the entire tumour volume immediately after the application of the therapy, and a second wave of oxygen reperfusion approximately 24 h thereafter. We also show that RSOM enables the quantification of differences in neoangiogenesis that predict treatment efficacy.

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 Scientific Article in Scientific Reports Sci. Rep. 10:4903 (2020)

Kimm, M.A.# ; Tzoumas, S.# ; Glasl, S. ; Omar, M. ; Symvoulidis, P. ; Olefir, I. ; Rummeny, E.J. ; Meier, R. ; Ntziachristos, V.

Longitudinal imaging of T cell-based immunotherapy with multi-spectral, multi-scale optoacoustic tomography.

Most imaging studies of immunotherapy have focused on tracking labeled T cell biodistribution in vivo for understanding trafficking and homing parameters and predicting therapeutic efficacy by the presence of transferred T cells at or in the tumour mass. Conversely, we investigate here a novel concept for longitudinally elucidating anatomical and pathophysiological changes of solid tumours after adoptive T cell transfer in a preclinical set up, using previously unexplored in-tandem macroscopic and mesoscopic optoacoustic (photoacoustic) imaging. We show non-invasive in vivo observations of vessel collapse during tumour rejection across entire tumours and observe for the first time longitudinal tumour rejection in a label-free manner based on optical absorption changes in the tumour mass due to cellular decline. We complement these observations with high resolution episcopic fluorescence imaging of T cell biodistribution using optimized T cell labeling based on two near-infrared dyes targeting the cell membrane and the cytoplasm. We discuss how optoacoustic macroscopy and mesoscopy offer unique contrast and immunotherapy insights, allowing label-free and longitudinal observations of tumour therapy. The results demonstrate optoacoustic imaging as an invaluable tool in understanding and optimizing T cell therapy.

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 Light: Science & Applications Light Sci. Appl. 9:57 (2020)

Li, J. ; Chekkoury, A. ; Prakash, J. ; Glasl, S. ; Vetschera, P. ; Koberstein-Schwarz, B. ; Olefir, I. ; Gujrati, V. ; Omar, M. ; Ntziachristos, V.

Spatial heterogeneity of oxygenation and haemodynamics in breast cancer resolved in vivo by conical multispectral optoacoustic mesoscopy.

Optoacoustic imaging: Revealing the details of tumour patterns A technique that can image the entire tumour volume with high resolution may help oncologists optimize specific treatments for breast cancer. Jiao Li (Tianjin University, China), Vasilis Ntziachristos (Technical University of Munich, Germany), and colleagues have designed a multispectral optoacoustic mesoscope (MSOM) that illuminates millimetre-sized tumours with laser light of various wavelengths, and detects the ultrasound waves generated by internal absorbers, such as haemoglobin, or external nanoparticle probes. By reconstructing the ultrasound signals over multiple frequencies, the team produced 3D images of features that included the vascular network of a tumour with micrometre-scale detail. Experiments with live mice demonstrated that specific tumours could be identified through differences in spatial patterns, such as altered oxygen levels between tumour cores and peripheries. The study highlights the power of MSOM as a tool for preclinical cancer studies.The characteristics of tumour development and metastasis relate not only to genomic heterogeneity but also to spatial heterogeneity, associated with variations in the intratumoural arrangement of cell populations, vascular morphology and oxygen and nutrient supply. While optical (photonic) microscopy is commonly employed to visualize the tumour microenvironment, it assesses only a few hundred cubic microns of tissue. Therefore, it is not suitable for investigating biological processes at the level of the entire tumour, which can be at least four orders of magnitude larger. In this study, we aimed to extend optical visualization and resolve spatial heterogeneity throughout the entire tumour volume. We developed an optoacoustic (photoacoustic) mesoscope adapted to solid tumour imaging and, in a pilot study, offer the first insights into cancer optical contrast heterogeneity in vivo at an unprecedented resolution of <50 mu m throughout the entire tumour mass. Using spectral methods, we resolve unknown patterns of oxygenation, vasculature and perfusion in three types of breast cancer and showcase different levels of structural and functional organization. To our knowledge, these results are the most detailed insights of optical signatures reported throughout entire tumours in vivo, and they position optoacoustic mesoscopy as a unique investigational tool linking microscopic and macroscopic observations.

2020 Scientific Article in BMC Biology BMC Biol. 18:42 (2020)

Grosch, M. ; Rusha, E. ; Ori, C. ; Truong, D.J.J. ; O'Neill, A.C. ; Pertek, A. ; Westmeyer, G.G. ; Drukker, M.

Nucleus size and DNA accessibility are linked to the regulation of paraspeckle formation in cellular differentiation.

Background Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo. Results As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages. We found that paraspeckles, which form by aggregation of the lncRNA NEAT1, are scaled by the size of the nucleus, and that small DNA-binding molecules promote the disintegration of paraspeckles and other lncRNA condensates. Furthermore, we found that paraspeckles regulate the differentiation of hPSCs. Conclusions Positive correlation between the size of the nucleus and the number of paraspeckles exist in numerous types of human cells. The tethering and structure of paraspeckles, as well as other lncRNAs, to the genome can be disrupted by small molecules that intercalate in DNA. The structure-function relationship of lncRNAs that regulates stem cell differentiation is likely to be determined by the dynamics of nucleus size and binding site accessibility.

2020 Scientific Article in Contact Dermatitis Contact Dermatitis 83, 206-214 (2020)

Hindelang, B.&deg ; Aguirre Bueno, J.&deg ; Berezhnoi, A. ; He, H. ; Eyerich, K. ; Ntziachristos, V. ; Biedermann, T. ; Darsow, U.

Optoacoustic mesoscopy shows potential to increase accuracy of allergy patch testing.

Background Differentiation between irritant and allergic skin reactions in epicutaneous patch testing is based largely on subjective clinical criteria, with the risk of high intraobserver and interobserver variability. Novel dermatological imaging using optoacoustic mesoscopy allows quantitative three-dimensional assessment of microvascular biomarkers.Objectives We investigated the potential of optoacoustic imaging to improve the precision of patch test evaluation.Methods Sixty-nine test reactions and 48 healthy skin sections in 52 patients with suspected type IV allergy were examined using raster-scan optoacoustic mesoscopy.Results We identified biomarkers from the optoacoustic images. Allergic reactions were associated with higher fragmentation of skin vasculature than irritant reactions (19.5 +/- 9.7 vs 14.3 +/- 3.7 fragments/100 pixels(2); P < .05), as well as lower ratio of low- to high-frequency acoustic signals (1.6 +/- 0.5 vs 2.0 +/- 0.6, P < .05). Allergic reactions graded "++" showed higher vessel fragmentation than reactions graded "+" (25.4 +/- 13.2 vs 17.1 +/- 6.5 fragments/100 pixels(2); P < .05). A linear model combining the biomarkers fragmentation and frequency ratio could differentiate allergic from irritant test reactions with an area under the receiving operator characteristic curve of 0.80 (95% confidence interval 0.64-0.91), reaching a sensitivity of 81% and specificity of 63%.Conclusions Optoacoustic mesoscopy shows potential to help in differentiating between allergic and irritant test reactions based on novel biomarkers that may reflect vasodilation, vessel tortuosity, and edema.

2020 Scientific Article in Optics Letters Opt. Lett. 45, 2006-2009 (2020)

Periyasamy, V. ; Özsoy, Ç. ; Reiss, M. ; Deán-Ben, X.L. ; Razansky, D.

In vivo optoacoustic monitoring of percutaneous laser ablation of tumors in a murine breast cancer model.

Laser ablation (LA) is a promising approach for minimally invasive cancer treatments. Its in vivo applicability is often impeded by the lack of efficient monitoring tools that can help to minimize collateral tissue damage and aid in determining the optimal treatment end-points. We have devised a new, to the best of our knowledge, hybrid LA approach combining simultaneous volumetric optoacoustic (OA) imaging to monitor the lesion progression accurately in real time and 3D. Time-lapse imaging of laser ablation of solid tumors was performed in a murine breast cancer model in vivo by irradiation of subcutaneous tumors with a 100 mJ short-pulsed (similar to 5 ns) laser operating at 1064 nm and 100 Hz pulse repetition frequency. Local changes in the OA signal intensity ascribed to structural alterations in the tumor vasculature were clearly observed, while the OA volumetric projections recorded in vivo appeared to correlate with cross sections of the excised tumors.

2020 Scientific Article in Optics Letters Opt. Lett. 45, 1695-1698 (2020)

Zhou, H. ; Cai, R. ; Quan, T. ; Liu, S. ; Li, S. ; Huang, Q. ; Ertürk, A. ; Zeng, S.

3D high resolution generative deep-learning network for fluorescence microscopy imaging.

Microscopic fluorescence imaging serves as a basic tool in many research areas including biology, medicine, and chemistry. With the help of optical clearing, large volume imaging of a mouse brain and even a whole body has been enabled. However, constrained by the physical principles of optical imaging, volume imaging has to balance imaging resolution and speed. Here, we develop a new, to the best of our knowledge, 3D deep learning network based on a dual generative adversarial network (dual-GAN) framework for recovering high-resolution (HR) volume images from high speed acquired low-resolution (LR) volume images. The proposed method does not require a precise image registration process and meanwhile guarantees the predicted HR volume image faithful to its corresponding LR volume image. The results demonstrated that our method can recover 20 x /1.0-NAvolume images from coarsely registered 5 x /0.16-NA volume images collected by light-sheet microscopy. This method. would provide great potential in applications which require high resolution volume imaging.

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 Photoacoustics Photoacoustics 19:100172 (2020)

Yang, H. ; Jüstel, D. ; Prakash, J. ; Karlas, A. ; Helfen, A. ; Masthoff, M. ; Wildgruber, M. ; Ntziachristos, V.

Soft ultrasound priors in optoacoustic reconstruction: Improving clinical vascular imaging.

Using the same ultrasound detector, hybrid optoacoustic-ultrasound (OPUS) imaging provides concurrent scans of tissue slices or volumes and visualizes complementary sound- and light-based contrast at similar resolutions. In addition to the benefit of hybrid contrast, spatial co-registration enables images from one modality to be employed as prior information for improving an aspect of the performance of the other modality. We consider herein a handheld OPUS system and utilize structural information from ultrasound images to guide regional Laplacian regularization-based reconstruction of optoacoustic images. Using phantoms and data from OPUS scans of human radial and carotid arteries, we show that ultrasound-driven optoacoustic inversion reduces limited-view artefacts and improves image contrast. In phantoms, prior-integrated reconstruction leads to a 50 % higher contrast-to-noise ratio (CNR) of the image than standard reconstruction, and a 17 % higher structural similarity (SSIM) index. In clinical data, prior-integrated reconstruction detects deep-seated radial arteries with higher CNR than the standard method at three different depths. In this way, the prior-integrated method offers unique insights into atherosclerotic carotid plaques in humans (with p < 0.01 between patients and healthy volunteers), potentially paving the way for new abilities in vascular imaging and more generally in optoacoustic imaging.

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 Sensors Sensors 20:766 (2020)

Yang, C. ; Jian, X. ; Zhu, X. ; Lv, J. ; Jiao, Y. ; Han, Z. ; Stylogiannis, A. ; Ntziachristos, V. ; Sergiadis, G. ; Cui, Y.

Sensitivity enhanced photoacoustic imaging using a high-frequency PZT transducer with an integrated front-end amplifier.

Photoacoustic (PA) imaging is a hybrid imaging technique that can provide both structural and functional information of biological tissues. Due to limited permissible laser energy deposited on tissues, highly sensitive PA imaging is required. Here, we developed a 20 MHz lead zirconium titanate (PZT) transducer (1.5 mm x 3 mm) with front-end amplifier circuits for local signal processing to achieve sensitivity enhanced PA imaging. The electrical and acoustic performance was characterized. Experiments on phantoms and chicken breast tissue were conducted to validate the imaging performance. The fabricated prototype shows a bandwidth of 63% and achieves a noise equivalent pressure (NEP) of 0.24 mPa/root Hz and a receiving sensitivity of 62.1 mu V/Pa at 20 MHz without degradation of the bandwidth. PA imaging of wire phantoms demonstrates that the prototype is capable of improving the detection sensitivity by 10 dB compared with the traditional transducer without integrated amplifier. In addition, in vitro experiments on chicken breast tissue show that structures could be imaged with enhanced contrast using the prototype and the imaging depth range was improved by 1 mm. These results demonstrate that the transducer with an integrated front-end amplifier enables highly sensitive PA imaging with improved penetration depth. The proposed method holds the potential for visualization of deep tissue structures and enhanced detection of weak physiological changes.

2020 Scientific Article in Science Translational Medicine Sci. Transl. Med. 12:eaaw3210 (2020)

Wang, X. ; Lou, N. ; Eberhardt, A. ; Yang, Y. ; Kusk, P. ; Xu, Q. ; Förstera, B. ; Peng, S. ; Shi, M. ; Ladrón-de-Guevara, A. ; Delle, C. ; Sigurdsson, B. ; Xavier, A.L.R. ; Ertürk, A. ; Libby, R.T. ; Chen, L. ; Thrane, A.S. ; Nedergaard, M.

An ocular glymphatic clearance system removes beta-amyloid from the rodent eye.

Despite high metabolic activity, the retina and optic nerve head lack traditional lymphatic drainage. We here identified an ocular glymphatic clearance route for fluid and wastes via the proximal optic nerve in rodents. beta-amyloid (A beta) was cleared from the retina and vitreous via a pathway dependent on glial water channel aquaporin-4 (AQP4) and driven by the ocular-cranial pressure difference. After traversing the lamina barrier, intra-axonal A beta was cleared via the perivenous space and subsequently drained to lymphatic vessels. Light-induced pupil constriction enhanced efflux, whereas atropine or raising intracranial pressure blocked efflux. In two distinct murine models of glaucoma, A. leaked from the eye via defects in the lamina barrier instead of directional axonal efflux. The results suggest that, in rodents, the removal of fluid and metabolites from the intraocular space occurs through a glymphatic pathway that might be impaired in glaucoma.

2020 Scientific Article in Photoacoustics Photoacoustics 19:100178 (2020)

Estrada, H.&deg ; Rebling, J. ; Hofmann, U. ; Razansky, D.&deg

Discerning calvarian microvascular networks by combined optoacoustic ultrasound microscopy.

Bone microvasculature plays a paramount role in bone marrow maintenance, development, and hematopoiesis. Studies of calvarian vascular patterns within living mammalian skull with the available intravital microscopy techniques are limited to small scale observations. We developed an optical-resolution optoacoustic microscopy method combined with ultrasound biomicroscopy in order to reveal and discern the intricate networks of calvarian and cerebral vasculature over large fields of view covering majority of the murine calvaria. The vasculature segmentation method is based on an angle-corrected homogeneous model of the rodent skull, generated using simultaneously acquired three-dimensional pulse-echo ultrasound images. The hybrid microscopy design along with the appropriate skull segmentation method enable high throughput studies of a living bone while facilitating correct anatomical interpretation of the vasculature images acquired with optical resolution optoacoustic microscopy.

2020 Scientific Article in Journal of Biophotonics J. Biophotonics 13:e201960169 (2020)

Karlas, A. ; Kallmayer, M. ; Fasoula, N.-A. ; Liapis, E. ; Bariotakis, M. ; Krönke, M. ; Anastasopoulou, M. ; Reber, J. ; Eckstein, H.H. ; Ntziachristos, V.

Multispectral optoacoustic tomography of muscle perfusion and oxygenation under arterial and venous occlusion: A human pilot study.

Perfusion and oxygenation are critical parameters of muscle metabolism in health and disease. They have been both the target of many studies, in particular using near-infrared spectroscopy (NIRS). However, difficulties with quantifying NIRS signals have limited a wide dissemination of the method to the clinics. Our aim was to investigate whether clinical multispectral optoacoustic tomography (MSOT) could enable the label-free imaging of muscle perfusion and oxygenation under clinically relevant challenges: the arterial and venous occlusion. We employed a hybrid clinical MSOT/ultrasound system equipped with a hand-held scanning probe to visualize hemodynamic and oxygenation changes in skeletal muscle under arterial and venous occlusions. Four (N = 4) healthy volunteers were scanned over the forearm for both 3-minute occlusion challenges. MSOT-recorded pathophysiologically expected results during tests of disturbed blood flow with high resolution and without the need for contrast agents. During arterial occlusion, MSOT-extracted Hb-values showed an increase, while HbO(2)- and total blood volume (TBV)-values remained roughly steady, followed by a discrete increase during the hyperemic period after cuff deflation. During venous occlusion, results showed a clear increase in intramuscular HbO(2), Hb and TBV within the segmented muscle area. MSOT was found to be capable of label-free non-invasive imaging of muscle hemodynamics and oxygenation under arterial and venous occlusion. We introduce herein MSOT as a novel modality for the assessment of vascular disorders characterized by disturbed blood flow, such as acute limb ischemia and venous thrombosis.

2020 Scientific Article in Photoacoustics Photoacoustics 17:100153 (2020)

Ovsepian, S.V.&deg ; Jiang, Y. ; Sardella, T.C.P. ; Malekzadeh Najafabadi, J. ; Burton, N.C. ; Yu, X. ; Ntziachristos, V.&deg

Visualizing cortical response to optogenetic stimulation and sensory inputs using multispectral handheld optoacoustic imaging.

To date, the vast majority of intra-vital neuroimaging systems applied in clinic and diagnostics is stationary with a rigid scanning element, requires specialized facilities and costly infrastructure. Here, we describe a simple yet radical approach for optoacoustic (photoacoustic) brain imaging in vivo using a light-weight handheld probe. It enables multispectral video-rate visualization of hemoglobin gradient changes in the cortex of adult rats induced by whisker and forelimb sensory inputs, as well as by optogenetic stimulation of intra-cortical connections. With superb penetration and molecular specificity, described here in method holds major promises for future applications in research, routine ambulatory neuroimaging, and diagnostics.

2020 Scientific Article in Nature Methods Nat. Methods 17, 442-449 (2020)

Todorov, M.I. ; Paetzold, J.C. ; Schoppe, O. ; Tetteh, G. ; Shit, S. ; Efremov, V. ; Todorov-Völgyi, K. ; Düring, M. ; Dichgans, M. ; Piraud, M. ; Menze, B.&deg ; Ertürk, A.&deg

Machine learning analysis of whole mouse brain vasculature.

Tissue clearing methods enable the imaging of biological specimens without sectioning. However, reliable and scalable analysis of large imaging datasets in three dimensions remains a challenge. Here we developed a deep learning-based framework to quantify and analyze brain vasculature, named Vessel Segmentation & Analysis Pipeline (VesSAP). Our pipeline uses a convolutional neural network (CNN) with a transfer learning approach for segmentation and achieves human-level accuracy. By using VesSAP, we analyzed the vascular features of whole C57BL/6J, CD1 and BALB/c mouse brains at the micrometer scale after registering them to the Allen mouse brain atlas. We report evidence of secondary intracranial collateral vascularization in CD1 mice and find reduced vascularization of the brainstem in comparison to the cerebrum. Thus, VesSAP enables unbiased and scalable quantifications of the angioarchitecture of cleared mouse brains and yields biological insights into the vascular function of the brain.VesSAP is a tissue clearing- and deep learning-based pipeline for comprehensively analyzing mouse vasculature, from large vessels to small capillaries.

2020 Scientific Article in Bone: Official Journal of the International Bone and Mineral Society Bone 133:115251 (2020)

Estrada, H. ; Rebling, J. ; Sivert, W. ; Hladik, D. ; Hofmann, U. ; Gottschalk, S. ; Tapio, S. ; Multhoff, G. ; Razansky, D.

Intravital optoacoustic and ultrasound bio-microscopy reveal radiation-inhibited skull angiogenesis.

Angiogenesis is critical in bone development and growth. Dense, large-scale, and multi-layered vascular networks formed by thin-walled sinusoidal vessels perfuse the plate bones and play an important role in bone repair. Yet, the intricate functional morphology of skull microvasculature remains poorly understood as it is difficult to visualize using existing intravital microscopy techniques. Here we introduced an intravital, fully-transcranial imaging approach based on hybrid optoacoustic and ultrasound bio-microscopy for large-scale observations and quantitative analysis of the vascular morphology, angiogenesis, vessel remodeling, and subsurface roughness in murine skulls. Our approach revealed radiation-inhibited angiogenesis in the skull bone. We also observed previously undocumented sinusoidal vascular networks spanning the entire skullcap, thus opening new vistas for studying the complex interactions between calvarial, pial, and cortical vascular systems.

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 Nature Reviews - Neuroscience Nat. Rev. Neurosci., DOI: 10.1038/s41583-020-0291-5 (2020)

Ueda, H.R. ; Ertürk, A. ; Gradinaru, V. ; Chédotal, A. ; Tomancak, P. ; Keller, P.J.

Publisher Correction: Tissue clearing and its applications in neuroscience (Nature Reviews Neuroscience, (2020), 21, 2, (61-79), 10.1038/s41583-019-0250-1).

In this Review Article, the accept date was inadvertently omitted. It should be 18 November 2019. This error has been corrected online. | Published online 09 March 2020.

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 Nature Reviews - Neuroscience Nat. Rev. Neurosci. 21, 61-79 (2020)

Ueda, H.R. ; Ertürk, A. ; Chung, K. ; Gradinaru, V. ; Chedotal, A. ; Tomancak, P. ; Keller, P.J.

Tissue clearing and its applications in neuroscience.

State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.Tissue-clearing methods are now allowing 3D imaging of intact tissues and some entire mammals. In this Review, Ueda and colleagues discuss the various tissue-clearing methods, related techniques and data analysis and management, as well as the application of these methods in neuroscience.

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 Biomedical Optics Express Biomed. Opt. Express 11, 1477-1488 (2020)

Subochev, P. ; Smolina, E. ; Sergeeva, E. ; Kirillin, M. ; Orlova, A. ; Kurakina, D. ; Emyanov, D. ; Razansky, D.

Toward whole-brain in vivo optoacoustic angiography of rodents: Modeling and experimental observations

Cerebrovascular imaging of rodents is one of the trending applications of optoacoustics aimed at studying brain activity and pathology. Imaging of deep brain structures is often hindered by sub-optimal arrangement of the light delivery and acoustic detection systems. In our work we revisit the physics behind opto-acoustic signal generation for theoretical evaluation of optimal laser wavelengths to perform cerebrovascular optoacoustic angiography of rodents beyond the penetration barriers imposed by light diffusion in highly scattering and absorbing brain tissues. A comprehensive model based on diffusion approximation was developed to simulate optoacoustic signal generation using optical and acoustic parameters closely mimicking a typical murine brain. The model revealed three characteristic wavelength ranges in the visible and near-infrared spectra optimally suited for imaging cerebral vasculature of different size and depth. The theoretical conclusions are confirmed by numerical simulations while in vivo imaging experiments further validated the ability to accurately resolve brain vasculature at depths ranging between 0.7 and 7 mm.

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 Proceedings of the National Academy of Sciences of the United States of America Proc. Natl. Acad. Sci. U.S.A. 117, 4007-4014 (2020)

Prakash, J.# ; Seyedebrahimi, M.M.# ; Ghazaryan, A.# ; Malekzadeh Najafabadi, J. ; Gujrati, V. ; Ntziachristos, V.

Short-wavelength optoacoustic spectroscopy based on water muting.

Infrared (IR) optoacoustic spectroscopy can separate a multitude of molecules based on their absorption spectra. However, the technique is limited when measuring target molecules in aqueous solution by strong water absorption at IR wavelengths, which reduces detection sensitivity. Based on the dependence of optoacoustic signal on the temperature of the probed medium, we introduce cooled IR optoacoustic spectroscopy (CIROAS) to mute water contributions in optoacoustic spectroscopy. We showcase that spectral measurements of proteins, lipids, and glucose in the short-wavelength IR region, performed at 4 degrees C, lead to marked sensitivity improvements over conventional optoacoustic or IR spectroscopy. We elaborate on the dependence of optoacoustic signals on water temperature and demonstrate polarity changes in the recorded signal at temperatures below 4 degrees C. We further elucidate the dependence of the optoacoustic signal and the muting temperature on sample concentration and demonstrate that changes in these dependences enable quantification of the solute concentration. We discuss how CIROAS may enhance abilities for molecular sensing in the IR.

2020 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 39, 1160-1169 (2020)

Knauer, N. ; Deán-Ben, X.L. ; Razansky, D.

Spatial compounding of volumetric data enables freehand optoacoustic angiography of large-scale vascular networks.

Optoacoustic tomography systems have attained unprecedented volumetric imaging speeds, thus enabling insights into rapid biological dynamics and marking a milestone in the clinical translation of this modality. Fast imaging performance often comes at the cost of limited field-of-view, which may hinder potential applications looking at larger tissue volumes. The imaged field-of-view can potentially be expanded via scanning and using additional hardware to track the position of the imaging probe. However, this approach turns impractical for high-resolution volumetric scans performed in a freehand mode along arbitrary trajectories. We have developed an accurate framework for spatial compounding of time-lapse optoacoustic data. The method exploits the frequency-domain properties of vascular networks in optoacoustic images and estimates the relative motion and orientation of the imaging probe. This allows rapidly combining sequential volumetric frames into large area scans without additional tracking hardware. The approach is universally applicable for compounding volumetric data acquired with calibrated scanning systems but also in a freehand mode with up to six degrees of freedom. Robust performance is demonstrated for whole-body mouse imaging with spiral volumetric optoacoustic tomography and for freehand visualization of vascular networks in humans using volumetric imaging probes. The newly introduced capability for angiographic observations at multiple spatial and temporal scales is expected to greatly facilitate the use of optoacoustic imaging technology in pre-clinical research and clinical diagnostics. The technique can equally benefit other biomedical imaging modalities, such as scanning fluorescence microscopy, optical coherence tomography or ultrasonography, thus optimizing their trade-offs between fast imaging performance and field-of-view.

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 Molecular Pharmaceutics Mol. Pharm. 17, 109-117 (2020)

Kimm, M.A. ; Haas, H. ; Stölting, M. ; Kuhlmann, M. ; Geyer, C. ; Glasl, S. ; Schäfers, M. ; Ntziachristos, V. ; Wildgruber, M. ; Höltke, C.

Targeting endothelin receptors in a murine model of myocardial infarction using a small molecular fluorescent probe.

The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyOmethyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.

2020 Scientific Article in Laser & Photonics Reviews Laser Photon. Rev. 14:1900070 (2020)

Chen, Z. ; Mc Larney, B. ; Rebling, J. ; Dean-Ben, X.L. ; Zhou, Q. ; Gottschalk, S. ; Razansky, D.

High-speed large-field Multifocal illumination fluorescence microscopy.

Scanning optical microscopy techniques are commonly restricted to a sub-millimeter field-of-view (FOV) or otherwise employ slow mechanical translation, limiting their applicability for imaging fast biological dynamics occurring over large areas. A rapid scanning large-field multifocal illumination (LMI) fluorescence microscopy technique is devised based on a beam-splitting grating and an acousto-optic deflector synchronized with a high-speed camera to attain real-time fluorescence microscopy over a centimeter-scale FOV. Owing to its large depth of focus, the approach allows noninvasive visualization of perfusion across the entire mouse cerebral cortex, not achievable with conventional wide-field fluorescence microscopy methods. The new concept can readily be incorporated into conventional wide-field microscopes to mitigate image blur due to tissue scattering and attain optimal trade-off between spatial resolution and FOV. It further establishes a bridge between conventional wide-field macroscopy and laser scanning confocal microscopy, thus it is anticipated to find broad applicability in functional neuroimaging, in vivo cell tracking, and other applications looking at large-scale fluorescent-based biodynamics.

2020 Scientific Article in Nature Biotechnology Nat. Biotechnol. 38, 293-296 (2020)

Pleitez, M.A.&deg ; Ali Khan, A. ; Solda, A. ; Chmyrov, A. ; Reber, J. ; Gasparin, F. ; Seeger, M. ; Schätz, B. ; Herzig, S. ; Scheideler, M. ; Ntziachristos, V.&deg

Label-free metabolic imaging by mid-infrared optoacoustic microscopy in living cells.

We develop mid-infrared optoacoustic microscopy (MiROM) for label-free, bond-selective, live-cell metabolic imaging, enabling spatiotemporal monitoring of carbohydrates, lipids and proteins in cells and tissues. Using acoustic detection of optical absorption, MiROM converts mid-infrared sensing into a positive-contrast imaging modality with negligible photodamage and high sensitivity. We use MiROM to observe changes in intrinsic carbohydrate distribution from a diffusive spatial pattern to tight co-localization with lipid droplets during adipogenesis.Mid-infrared optoacoustic microscopy enables label-free, bond-selective imaging in living cells

2020 Scientific Article in IEEE Transactions on Bio-Medical Electronics IEEE Trans. Bio. Med. Eng. 67, 185-192 (2020)

Gorpas, D. ; Koch, M. ; Anastasopoulou, M. ; Bozhko, D. ; Klemm, U. ; Nieberler, M. ; Ntziachristos, V.

Multi-parametric standardization of fluorescence imaging systems based on a composite phantom.

Objective: Fluorescence molecular imaging (FMI) has emerged as a promising tool for surgical guidance in oncology, with one of the few remaining challenges being the ability to offer quality control and data referencing. This paper investigates the use of a novel composite phantom to correct and benchmark FMI systems. Methods: This paper extends on previous work by describing a phantom design that can provide a more complete assessment of FMI systems through quantification of dynamic range and determination of spatial illumination patterns for both reflectance and fluorescence imaging. Various performance metrics are combined into a robust and descriptive "system benchmarking score," enabling not only the comprehensive comparison of different systems, but also for the first time, correction of the acquired data. Results: We show that systems developed for targeted fluorescence imaging can achieve benchmarking scores of up to 70 & x0025;, while clinically available systems optimized for indocyanine green are limited to 50 & x0025;, mostly due to greater leakage of ambient and excitation illumination and lower resolution. The image uniformity can also be approximated and employed for image flat-fielding, an important milestone toward data referencing. In addition, we demonstrate composite phantom use in assessing the performance of a surgical microscope and of a raster-scan imaging system. Conclusion: Our results suggest that the new phantom has the potential to support high-fidelity FMI through benchmarking and image correction. Significance: Standardization of the FMI is a necessary process for establishing good imaging practices in clinical environments and for enabling high-fidelity imaging across patients and multi-center imaging studies.

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 Other: Opinion in Gut (eGut) Gut 69, 406-410 (2020)

Tjalma, J.J.J. ; Koller, M. ; Linssen, M.D. ; Hartmans, E. ; de Jongh, S. ; Jorritsma-Smit, A. ; Karrenbeld, A. ; de Vries, E.G. ; Kleibeuker, J.H. ; Pennings, J.P. ; Havenga, K. ; Hemmer, P.H. ; Hospers, G.A.P. ; Van Etten, B. ; Ntziachristos, V. ; van Dam, G.M. ; Robinson, D.J. ; Nagengast, W.B.

Quantitative fluorescence endoscopy: An innovative endoscopy approach to evaluate neoadjuvant treatment response in locally advanced rectal cancer.

2020 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 39, 458-467 (2020)

Nitkunanantharajah, S. ; Zahnd, G. ; Olivo, M. ; Navab, N. ; Mohajerani, P. ; Ntziachristos, V.

Skin surface detection in 3D optoacoustic mesoscopy based on dynamic programming,

Optoacoustic (photoacoustic) mesoscopy offers unique capabilities in skin imaging and resolves skin features associated with detection, diagnosis, and management of disease. A critical first step in the quantitative analysis of clinical optoacoustic images is to identify the skin surface in a rapid, reliable, and automated manner. Nevertheless, most common edge- and surface-detection algorithms cannot reliably detect the skin surface on 3D raster-scan optoacoustic mesoscopy (RSOM) images, due to discontinuities and diffuse interfaces in the image. We present herein a novel dynamic programming approach that extracts the skin boundary as a 2D surface in one single step, as opposed to consecutive extraction of several independent 1D contours. A domain-specific energy function is introduced, taking into account the properties of volumetric optoacoustic mesoscopy images. The accuracy of the proposed method is validated on scans of the volar forearm of 19 volunteers with different skin complexions, for which the skin surface has been traced manually to provide a reference. In addition, the robustness and the limitations of the method are demonstrated on data where the skin boundaries are low-contrast or ill-defined. The automatic skin surface detection method can improve the speed and accuracy in the analysis of quantitative features seen on the RSOM images and accelerate the clinical translation of the technique. Our method can likely be extended to identify other types of surfaces in the RSOM and other imaging modalities.

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.

In: Lecture Notes in Bioengineering. 2019. 103-111

Mohajerani, P. ; Ntziachristos, V.

Classification of normal versus malignant cells in B-ALL microscopic images based on a tiled convolution neural network approach.

In this paper we present a method based on the existing convolution neural network architecture of AlexNet for the purpose of classifying microscopic images of B-ALL white blood cancer cells. This classification problem is especially challenging due to lack of conspicuous morphological differences between normal and malignant cell nuclei. Therefore, we designed a machine learning pipeline that focused on the texture of the staining images. Briefly, our approach divides the cell image into several overlapping tiles and trains a modified version of AlexNet on the tiles. Only those tiles are retained which are fully contained within the cell image. Several such networks were trained in an ensemble fashion using different training–validation data splits. For a given test image, the tiles are generated and ran through all the trained networks. The outputs of all networks along with the nucleus area are then fed into a simple decision tree, which generates the final prediction. The proposed method was developed in the context of the ISBI 2019 C-NMC challenge. The final testing results demonstrated a classification-weighted F1 score of 0.8307 using 2586 test images. The results demonstrate the possibility of making relatively accurate predictions using only local texture features.

2019 Review in Photoacoustics Photoacoustics 16:100144 (2019)

Attia, A.B.E. ; Balasundaram, G. ; Moothanchery, M. ; Dinish, U.S. ; Bi, R. ; Ntziachristos, V. ; Olivo, M.

A review of clinical photoacoustic imaging: Current and future trends.

Photoacoustic imaging (or optoacoustic imaging) is an upcoming biomedical imaging modality availing the benefits of optical resolution and acoustic depth of penetration. With its capacity to offer structural, functional, molecular and kinetic information making use of either endogenous contrast agents like hemoglobin, lipid, melanin and water or a variety of exogenous contrast agents or both, PAI has demonstrated promising potential in a wide range of preclinical and clinical applications. This review provides an overview of the rapidly expanding clinical applications of photoacoustic imaging including breast imaging, dermatologic imaging, vascular imaging, carotid artery imaging, musculoskeletal imaging, gastrointestinal imaging and adipose tissue imaging and the future directives utilizing different configurations of photoacoustic imaging. Particular emphasis is placed on investigations performed on human or human specimens.

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 Cell Cell 179, 1661-1676 (2019)

Pan, C. ; Schoppe, O. ; Parra-Damas, A. ; Cai, R. ; Todorov, M.I. ; Gondi, G. ; von Neubeck, B. ; Böğürcü-Seidel, N. ; Seidel, S. ; Sleiman, K. ; Veltkamp, C. ; Förstera, B. ; Mai, H. ; Rong, Z. ; Trompak, O. ; Ghasemigharagoz, A. ; Reimer, M.A. ; Cuesta, A.M. ; Coronel, J. ; Jeremias, I. ; Saur, D. ; Acker-Palmer, A. ; Acker, T. ; Garvalov, B.K. ; Menze, B. ; Zeidler, R. ; Ertürk, A.

Deep learning reveals cancer metastasis and therapeutic antibody targeting in entire body.

Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the preclinical stage.

2019 Scientific Article in Nature Neuroscience Nat. Neurosci. 22, 317-327 (2019)

Cai, R. ; Pan, C. ; Ghasemigharagoz, A. ; Todorov, M.I. ; Förstera, B. ; Zhao, S. ; Bhatia, H.S. ; Parra-Damas, A. ; Mrowka, L. ; Theodorou, D. ; Rempfler, M. ; Xavier, A.L.R. ; Kress, B.T. ; Benakis, C. ; Steinke, H. ; Liebscher, S. ; Bechmann, I. ; Liesz, A. ; Menze, B. ; Kerschensteiner, M. ; Nedergaard, M. ; Ertürk, A.

Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.

Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues has remained a challenge. Here, we developed vDISCO, a pressure-driven, nanobody-based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

2019 Scientific Article in Nature Neuroscience Nat. Neurosci. 22, 191-204 (2019)

Parhizkar, S. ; Arzberger, T. ; Brendel, M. ; Kleinberger, G. ; Deussing, M. ; Focke, C. ; Nuscher, B. ; Xiong, M. ; Ghasemigharagoz, A. ; Katzmarski, N. ; Krasemann, S. ; Lichtenthaler, S.F. ; Müller, S.A. ; Colombo, A. ; Monasor, L.S. ; Tahirovic, S. ; Herms, J. ; Willem, M. ; Pettkus, N. ; Butovsky, O. ; Bartenstein, P. ; Edbauer, D. ; Rominger, A. ; Ertürk, A. ; Grathwohl, S.A. ; Neher, J.J. ; Holtzman, D.M. ; Meyer-Luehmann, M. ; Haass, C.

Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE.

Coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with late-onset Alzheimer's disease (AD). We demonstrate that amyloid plaque seeding is increased in the absence of functional Trem2. Increased seeding is accompanied by decreased microglial clustering around newly seeded plaques and reduced plaque-associated apolipoprotein E (ApoE). Reduced ApoE deposition in plaques is also observed in brains of AD patients carrying TREM2 coding variants. Proteomic analyses and microglia depletion experiments revealed microglia as one origin of plaque-associated ApoE. Longitudinal amyloid small animal positron emission tomography demonstrates accelerated amyloidogenesis in Trem2 loss-of-function mutants at early stages, which progressed at a lower rate with aging. These findings suggest that in the absence of functional Trem2, early amyloidogenesis is accelerated due to reduced phagocytic clearance of amyloid seeds despite reduced plaque-associated ApoE.

2019 Scientific Article in Scientific Reports Sci. Rep. 9:18123 (2019)

Anastasopoulou, M. ; Gorpas, D. ; Koch, M. ; Liapis, E. ; Glasl, S. ; Klemm, U. ; Karlas, A. ; Lasser, T. ; Ntziachristos, V.

Fluorescence imaging reversion using spatially variant deconvolution.

Fluorescence imaging opens new possibilities for intraoperative guidance and early cancer detection, in particular when using agents that target specific disease features. Nevertheless, photon scattering in tissue degrades image quality and leads to ambiguity in fluorescence image interpretation and challenges clinical translation. We introduce the concept of capturing the spatially-dependent impulse response of an image and investigate Spatially Adaptive Impulse Response Correction (SAIRC), a method that is proposed for improving the accuracy and sensitivity achieved. Unlike classical methods that presume a homogeneous spatial distribution of optical properties in tissue, SAIRC explicitly measures the optical heterogeneity in tissues. This information allows, for the first time, the application of spatially-dependent deconvolution to correct the fluorescence images captured in relation to their modification by photon scatter. Using experimental measurements from phantoms and animals, we investigate the improvement in resolution and quantification over non-corrected images. We discuss how the proposed method is essential for maximizing the performance of fluorescence molecular imaging in the clinic.

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 Review in Trends in Biotechnology Trends Biotechnol. 37, 1315-1326 (2019)

Ovsepian, S.V.&deg ; Olefir, I. ; Ntziachristos, V.&deg

Advances in optoacoustic neurotomography of animal models.

Unlike traditional optical methods, optoacoustic imaging is less sensitive to scattering of ballistic photons, so it is capable of high-resolution interrogation at a greater depth. By integrating video-rate visualization with multiplexing and sensing a range of endogenous and exogenous chromophores, optoacoustic imaging has matured into a versatile noninvasive investigation modality with rapidly expanding use in biomedical research. We review the principal features of the technology and discuss recent advances it has enabled in structural, functional, and molecular neuroimaging in small-animal models. In extending the boundaries of noninvasive observation beyond the reach of customary photonic methods, the latest developments in optoacoustics have substantially advanced neuroimaging inquiry, with promising implications for basic and translational studies.

2019 Scientific Article in Nano research Nano Res. 12, 3037-3043 (2019)

Liu, N. ; Shi, Y. ; Guo, J. ; Li, H. ; Wang, Q. ; Song, M. ; Shi, Z. ; He, L. ; Su, X. ; Xie, J. ; Sun, X.

Radioiodinated tyrosine based carbon dots with efficient renal clearance for single photon emission computed tomography of tumor.

Nanoparticles with effective tumor accumulation and efficient renal clearance have attracted significant interests for clinical applications. We prepared 2.5 nm tyrosine based carbon dots (TCDs) with phenolic hydroxyl groups on the surface for directly I-125 labeling. The I-125 labeled polyethylene glycol (PEG) functionalized TCDs (I-125-TCDPEGs) showed excellent radiochemical stability both in vitro and in vivo. Due to the enhanced permeability and retention effect, these I-125-TCDPEGs demonstrated a tumor accumulation around 4%-5% of the injected dose per gram (ID/g) for U87MG, 4T1, HepG2 and MCF7 tumor-bearing mice at 1 h post-injection. Meanwhile, the I-125-TCDPEGs also could be fast renally excreted, with less than 0.6% ID/g left in the liver and spleen within 24 h. These radioactive carbon dots not only can be used for cellular fluorescence imaging due to their intrinsic optical property, but are also effective single photon emission computed tomography (SPECT) imaging agents for tumor. Together with their excellent biocompatibility and stability, we anticipate these I-125-TCDPEGs of great potential for early tumor diagnosis in clinic. What's more, our TCDPEGs are also proved to be feasible carriers for other iodine isotopes such as 127I and 131I for different biomedical application.

2019 Scientific Article in Optics Express Opt. Express. 27, 31644-31666 (2019)

Stefanoiu, A. ; Page, J. ; Symvoulidis, P. ; Westmeyer, G.G. ; Lasser, T.

Artifact-free deconvolution in light field microscopy.

The sampling patterns of the light field microscope (LFM) are highly depth-dependent, which implies non-uniform recoverable lateral resolution across depth. Moreover, reconstructions using state-of-the-art approaches suffer from strong artifacts at axial ranges, where the LFM samples the light field at a coarse rate. In this work, we analyze the sampling patterns of the LFM, and introduce a flexible light field point spread function model (LFPSF) to cope with arbitrary LFM designs. We then propose a novel aliasing-aware deconvolution scheme to address the sampling artifacts. We demonstrate the high potential of the proposed method on real experimental data.

2019 Scientific Article in Advanced functional materials Adv. Func. Mat. 29:1904992 (2019)

Rogalla, S. ; Flisikowski, K. ; Gorpas, D. ; Mayer, A.T. ; Flisikowska, T. ; Mandella, M.J. ; Ma, X. ; Casey, K.M. ; Felt, S.A. ; Saur, D. ; Ntziachristos, V. ; Schnieke, A. ; Contag, C.H.&deg ; Gambhir, S.S.&deg ; Harmsen, S.&deg

Biodegradable fluorescent nanoparticles for endoscopic detection of colorectal carcinogenesis.

Early and comprehensive endoscopic detection of colonic dysplasia-the most clinically significant precursor lesion to colorectal adenocarcinoma-provides an opportunity for timely, minimally invasive intervention to prevent malignant transformation. Here, the development and evaluation of biodegradable near-infrared fluorescent silica nanoparticles (FSN) that have the potential to improve adenoma detection during fluorescence-assisted white-light colonoscopic surveillance in rodent and human-scale models of colorectal carcinogenesis is described. FSNs are biodegradable (t(1/2) of 2.7 weeks), well-tolerated, and enable detection and delineation of adenomas as small as 0.5 mm(2) with high tumor-to-background ratios. Furthermore, in the human scale, APC(1311/+) porcine model, the clinical feasibility and benefit of using FSN-guided detection of colorectal adenomas using video-rate fluorescence-assisted white-light endoscopy is demon-strated. Since nanoparticles of similar size (e.g., 100-150 nm) or composition (i.e., silica and silica/gold hybrid) have already been successfully translated to the clinic, and clinical fluorescent/white-light endoscopy systems are becoming more readily available, there is a viable path towards clinical translation of the proposed strategy for early colorectal cancer detection and prevention in high-risk patients.

2019 Scientific Article in Nature Communications Nat. Commun. 10:5056 (2019)

Weidenfeld, I. ; Zakian Dominguez, C.M. ; Duewell, P. ; Chmyrov, A. ; Klemm, U. ; Aguirre Bueno, J. ; Ntziachristos, V. ; Stiel, A.-C.

Homogentisic acid-derived pigment as a biocompatible label for optoacoustic imaging of macrophages.

Macrophages are one of the most functionally-diverse cell types with roles in innate immunity, homeostasis and disease making them attractive targets for diagnostics and therapy. Photo- or optoacoustics could provide non-invasive, deep tissue imaging with high resolution and allow to visualize the spatiotemporal distribution of macrophages in vivo. However, present macrophage labels focus on synthetic nanomaterials, frequently limiting their ability to combine both host cell viability and functionality with strong signal generation. Here, we present a homogentisic acid-derived pigment (HDP) for biocompatible intracellular labeling of macrophages with strong optoacoustic contrast efficient enough to resolve single cells against a strong blood background. We study pigment formation during macrophage differentiation and activation, and utilize this labeling method to track migration of pro-inflammatory macrophages in vivo with whole-body imaging. We expand the sparse palette of macrophage labels for in vivo optoacoustic imaging and facilitate research on macrophage functionality and behavior.

2019 Scientific Article in Frontiers in Neuroscience Front. Neurosci. 13:1092 (2019)

Stroh, A. ; Kressel, J. ; Coras, R. ; Dreyer, A.Y. ; Fröhlich, W. ; Förschler, A. ; Lobsien, D. ; Blümcke, I. ; Zoubaa, S. ; Schlegel, J. ; Zimmer, C. ; Boltze, J.

A safe and effective magnetic labeling protocol for MRI-based tracking of human adult neural stem cells.

Magnetic resonance imaging (MRI) provides a unique tool for in vivo visualization and tracking of stem cells in the brain. This is of particular importance when assessing safety of experimental cell treatments in the preclinical or clinical setup. Yet, specific imaging requires an efficient and non-perturbing cellular magnetic labeling which precludes adverse effects of the tag, e.g., the impact of iron-oxide-nanoparticles on the critical differentiation and integration processes of the respective stem cell population investigated. In this study we investigated the effects of very small superparamagnetic iron oxide particle (VSOP) labeling on viability, stemness, and neuronal differentiation potential of primary human adult neural stem cells (haNSCs). Cytoplasmic VSOP incorporation massively reduced the transverse relaxation time T2, an important parameter determining MR contrast. Cells retained cytoplasmic label for at least a month, indicating stable incorporation, a necessity for long-term imaging. Using a clinical 3T MRI, 1 x 10(3) haNSCs were visualized upon injection in a gel phantom, but detection limit was much lower (5 x 10(4) cells) in layer phantoms and using an imaging protocol feasible in a clinical scenario. Transcriptional analysis and fluorescence immunocytochemistry did not reveal a detrimental impact of VSOP labeling on important parameters of cellular physiology with cellular viability, stemness and neuronal differentiation potential remaining unaffected. This represents a pivotal prerequisite with respect to clinical application of this method.

2019 Scientific Article in Proceedings of SPIE Proc. SPIE 11076, DOI: 10.1117/12.2527214 (2019)

Psycharakis, S.E. ; Liapis, E. ; Zacharopoulos, A. ; Oraiopoulou, M.E. ; Aivalioti, C. ; Sakkalis, V. ; Papamatheakis, J. ; Ripoll, J. ; Zacharakis, G.

High resolution 3D imaging of primary and secondary tumor spheroids using multicolor multi-angle Light Sheet Fluorescence Microscopy (LSFM).

Breast cancer and Glioblastoma brain cancer are aggressive malignancies with poor prognosis. In this study primary Glioblastoma and secondary breast cancer spheroids are formed and treated with the well-known Temozolomide and Doxorubicin chemotherapeutics, respectively. A custom multi-angle Light Sheet Fluorescence Microscope is employed for high resolution imaging of both cancer cell spheroids. Such a technique is successful in realizing pre-clinical drug screening, while enables the discrimination among physiologic tumor parameters. LSFM technique, parameters and method followed are also presented.

Proceedings of SPIE In: (European Conferences on Biomedical Optics, Munich, Germany). 2019. (Proc. SPIE ; 11077)

Seyedebrahimi, M.M. ; Pleitez, M.A. ; Mohajerani, P. ; Ntziachristos, V.

Non-invasive In-vivo sensing of metabolites with a novel Optoacoustic Spectroscope in the SWIR.

In this work we developed a novel near-infrared two-path optoacoustic spectrometer (NiR-TAOS) that could sense OA intensity changes due to metabolite concentration changes in-vivo. The main aim of dividing the optical path in two is 1) perform real time correction of the laser emission profile of the laser source at different wavelengths and, 2) perform pulse to pulse correction to remove laser beam fluctuation and instability to increase signal to noise ratio. Signal to noise ratio improvement was significant not only at spectral peaks, but also at all other wavelengths. The system can be used for broad applications in biomedical measurements such as various metabolites in the SWIR.

2019 Scientific Article in Proceedings of SPIE Proc. SPIE 11079:1107913 (2019)

Gorpas, D. ; Anastasopoulou, M. ; Koch, M. ; Klemm, U. ; Nieberler, M. ; Ntziachristos, V.

Standardization phantom for intra-operative fluorescence molecular imaging.

Fluorescence-guided intervention is increasingly considered for real-time intra-operative oncological applications. Herein we propose a novel composite phantom for standardization and quality control, which could serve as a framework toward good clinical practices.

2019 Scientific Article in Proceedings of SPIE Proc. SPIE 11077 (2019)

Periyasamy, V. ; Özsoy, Ç. ; Reiss, M. ; Deán-Ben, X.L. ; Razansky, D.

Tumor ablation and volumetric optoacoustic monitoring with a short-pulsed laser source.

Laser ablation (LA) represents a minimally invasive intervention that is gaining acceptance for the treatment of different types of cancer, leading to important advantages such as less pain and shorter recovery time. Accurate monitoring of ablation progression is crucial to prevent damage of non-cancerous tissues and optimize the outcome of the intervention. To this end, imaging techniques such as ultrasound, computed tomography or magnetic resonance imaging have been used for monitoring LA. However, these techniques feature important drawbacks such as the need of contrast agents, poor spatio-temporal resolution or high cost. Optoacoustics (OA, photoacoustic) has recently been shown to provide unique properties to monitor thermal treatments. Herein, we demonstrate the feasibility of optoacoustic laser-ablation (OLA) monitoring in a murine breast tumor model using a single short-pulsed 1064 nm laser source. The effect of irradiation was volumetrically tracked with the OA images acquired with a 256-element spherical array. Structural damage of the tissue was clearly seen during the LA procedure.

2019 Scientific Article in Proceedings of SPIE Proc. SPIE 11077 (2019)

Seeger, M. ; Westmeyer, G.G. ; Ntziachristos, V.

In vivo hybrid microscopy of small model organisms.

We present the investigation of in vivo small model organisms, which are well established in biological and biomedical research, using a hybrid multiphoton and optoacoustic microscope (HyMPOM). The unique capabilities of HyMPOM for multimodal and potentially label-free signal acquisition, high resolution, as well as deep and fast imaging allow extraction of detailed information across large areas of living tissue on the microscale. Applying HyMPOM to living zebrafish-like fish larvae allowed exploration of the structural composition of the entire brain, including the brain vasculature and the neuronal network. Applying HyMPOM to the ears of living mice enabled accurate imaging of vasculature, connective tissue, keratinocytes, and sebaceous glands. The hybrid microscope proposed here constitutes a novel approach to explore small model organisms in vivo in great detail by revealing the spatial distribution and interplay of various tissue compartments on the microscale.

2019 Scientific Article in Physical Review Letters Phys. Rev. Lett. 123:174301 (2019)

Dean-Ben, X.L. ; Özbek, A. ; López-Schier, H. ; Razansky, D.

Acoustic scattering mediated single detector optoacoustic tomography.

Optoacoustic image formation is conventionally based upon ultrasound time-of-flight readings from multiple detection positions. Herein, we exploit acoustic scattering to physically encode the position of optical absorbers in the acquired signals, thus reducing the amount of data required to reconstruct an image from a single waveform. This concept is experimentally tested by including a random distribution of scatterers between the sample and an ultrasound detector array. Ultrasound transmission through a randomized scattering medium was calibrated by raster scanning a light-absorbing microparticle across a Cartesian grid. Image reconstruction from a single time-resolved signal was then enabled with a regularized model-based iterative algorithm relying on the calibration signals. The signal compression efficiency is facilitated by the relatively short acquisition time window needed to capture the entire scattered wave field. The demonstrated feasibility to form an image using a single recorded optoacoustic waveform paves a way to the development of faster and affordable optoacoustic imaging systems.

2019 Scientific Article in Plant and Soil Plant Soil 444, 519-534 (2019)

Möller, B. ; Chen, H. ; Schmidt, T. ; Zieschank, A. ; Patzak, R. ; Türke, M. ; Weigelt, A. ; Posch, S.

rhizoTrak: A flexible open source Fiji plugin for user-friendly manual annotation of time-series images from minirhizotrons.

Background and aims Minirhizotrons are commonly used to study root turnover which is essential for understanding ecosystem carbon and nutrient cycling. Yet, extracting data from minirhizotron images requires extensive annotation effort. Existing annotation tools often lack flexibility and provide only a subset of the required functionality. To facilitate efficient root annotation in minirhizotrons, we present the user-friendly open source tool rhizoTrak. Methods and results rhizoTrak builds on TrakEM2 and is publicly available as Fiji plugin. It uses treelines to represent branching structures in roots and assigns customizable status labels per root segment. rhizoTrak offers configuration options for visualization and various functions for root annotation mostly accessible via keyboard shortcuts. rhizoTrak allows time-series data import and particularly supports easy handling and annotation of time-series images. This is facilitated via explicit temporal links (connectors) between roots which are automatically generated when copying annotations from one image to the next. rhizoTrak includes automatic consistency checks and guided procedures for resolving inconsistencies. It facilitates easy data exchange with other software by supporting open data formats. Conclusions rhizoTrak covers the full range of functions required for user-friendly and efficient annotation of time-series images. Its flexibility and open source nature will foster efficient data acquisition procedures in root studies using minirhizotrons.

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 bioRxiv bioRxiv, accepted (2019)

Todorov, M.I. ; Paetzold, J.C. ; Schoppe, O. ; Tetteh, G. ; Efremov, V. ; Völgyi, K. ; Düring, M. ; Dichgans, M. ; Piraud, M. ; Menze, B. ; Ertürk, A.

Automated analysis of whole brainvasculature using machine learning.

Tissue clearing methods enable imaging of intactbiological specimens without sectioning. Howev-er, reliable and scalable analysis of such largeimaging data in 3D remains a challenge. Towardsthis goal, we developed a deep learning-basedframework to quantify and analyze the brain vas-culature, named Vessel Segmentation & AnalysisPipeline ( VesSAP). Our pipeline uses a fully con-volutional network with a transfer learning a p-proach for segmentation. We systematically ana-lyzed vascular features of the whole brains i n-cluding their length, bifurcation points and radiusat the micrometer scale by registering them to the Allen mouse brain atlas. We reported the firstevidence of secondary intracranial collateral vas-cularization in CD1-Elite mice and found reducedvascularization in the brainstem as compared to the cerebrum. VesSAP thus enables unbiasedand scalable quantifications for the angioarchi-tecture of the cleared intact mouse brain andyields new biological insights related to the vas-cular brain function.

2019 Scientific Article in bioRxiv bioRxiv, accepted (2019)

Zhao, S. ; Todorov, M.I. ; Cai, R. ; Steinke, H. ; Kemter, E. ; Wolf, E. ; Lipfert, J. ; Bechmann, I. ; Ertürk, A.

Cellular and molecular probing of intact transparent human organs.

Optical tissue transparency permits cellular and molecular investigation of complex tissues in 3D, a fundamental need in biomedical sciences. Adult human organs are particularly challenging for this approach, owing to the accumulation of dense and sturdy molecules in decades-aged human tissues. Here, we introduce SHANEL method utilizing a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to generate the first intact transparent adult human brain and kidney, and perform 3D histology using antibodies and dyes in centimeters depth. Thereby, we revealed structural details of sclera, iris and suspensory ligament in the human eye, and the vessels and glomeruli in the human kidney. We also applied SHANEL on transgenic pig organs to map complex structures of EGFP expressing beta cells in >10 cm size pancreas. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of intact large mammalian organs.

2019 Scientific Article in bioRxiv bioRxiv (2019)

Hang, Z. ; Shiwei, L. ; Qing, H. ; Shijie, L. ; Tingwei, Q. ; Cai, R. ; Ertürk, A. ; Shaoqun, Z.

A 3D high resolution generative deep-learning network for fluorescence microscopy image.

Deep learning technology enables us acquire high resolution image from low resolution image in biological imaging free from sophisticated optical hardware. However, current methods require a huge number of the precisely registered low-resolution (LR) and high-resolution (HR) volume image pairs. This requirement is challengeable for biological volume imaging. Here, we proposed 3D deep learning network based on dual generative adversarial network (dual-GAN) framework for recovering HR volume images from LR volume images. Our network avoids learning the direct mappings from the LR and HR volume image pairs, which need precisely image registration process. And the cycle consistent network makes the predicted HR volume image faithful to its corresponding LR volume image. The proposed method achieves the recovery of 20x/1.0 NA volume images from 5x/0.16 NA volume images collected by light-sheet microscopy. In essence our method is suitable for the other imaging modalities.

2019 Scientific Article in Biomedical Optics Express Biomed. Opt. Express 10, 5093-5102 (2019)

Chen, Z. ; Dean-Ben, X.L. ; Liu, N. ; Gujrati, V. ; Gottschalk, S. ; Ntziachristos, V. ; Razansky, D.

Concurrent fluorescence and volumetric optoacoustic tomography of nanoagent perfusion and bio-distribution in solid tumors.

Intravenously administered liposomes and other nano-sized particles are known to passively accumulate in solid tumors via the enhanced permeability and retention (EPR) effect, which is extensively explored toward the improvement of diagnosis and drug delivery in oncology. Agent extravasation into tumors is often hampered by the mononuclear phagocytic and renal systems, which sequester and/or eliminate most of the nanoparticles from the body. Dynamic imaging of the tumor microcirculation and bolus perfusion can thus facilitate optimization of the nanoparticle delivery. When it comes to non-invasive visualization of rapid biological dynamics in whole tumors, the currently available preclinical imaging modalities are commonly limited by shallow penetration, lack of suitable contrast or otherwise insufficient spatial or temporal resolution. Herein, we demonstrate the unique capabilities of a combined epi-fluorescence and optoacoustic tomography (FLOT) system for characterizing contrast agent dynamics in orthotopic breast tumors in mice. A liposomal indocyanine green (Lipo-ICG) preparation was administered intravenously with the time-lapse data continuously acquired during and after the injection procedure. In addition to the highly sensitive detection of the fluorescence agent by the epi-fluorescence modality, the volumetric multi-spectral optoacoustic tomography readings further enabled resolving deep-seated vascular structures with high spatial resolution and hence provided accurate readings of the dynamic bio-distribution of nanoparticles in the entire tumor in 3D. The synergetic combination of the two modalities can become a powerful tool in cancer research and potentially aid the diagnosis, staging and treatment guidance of certain types of cancer in the clinical setting.

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.