Publications by authors named "Axel Walch"

242 Publications

Mass Spectrometry Imaging Identifies Metabolic Patterns Associated with Malignant Potential in Pheochromocytoma and Paraganglioma.

Eur J Endocrinol 2021 May 1. Epub 2021 May 1.

F Beuschlein, Klinik für Endokrinologie, Diabetologie und Klinische Ernährung, UniversitatsSpital Zurich, Zurich, 8091, Switzerland.

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.
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http://dx.doi.org/10.1530/EJE-20-1407DOI Listing
May 2021

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

Photoacoustics 2021 Jun 23;22:100263. Epub 2021 Mar 23.

Chair of Biological Imaging, Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich 81675, Germany.

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.
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http://dx.doi.org/10.1016/j.pacs.2021.100263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080078PMC
June 2021

Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond.

Front Oncol 2021 17;11:612354. Epub 2021 Mar 17.

Department of Radiation Oncology, University Hospital, LMU Munich, Munich, Germany.

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 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 . , 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 results the 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.
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http://dx.doi.org/10.3389/fonc.2021.612354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011354PMC
March 2021

HER2 Expression, Test Deviations, and Their Impact on Survival in Metastatic Gastric Cancer: Results From the Prospective Multicenter VARIANZ Study.

J Clin Oncol 2021 May 25;39(13):1468-1478. Epub 2021 Mar 25.

University Cancer Center Leipzig (UCCL), Leipzig University Medical Center, Leipzig, Germany.

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, 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 10.9 months, n = 65; hazard ratio, 0.42; 95% CI, 8.2 to 14.4; < .001). In the centrally confirmed cohort, significantly more tumor cells stained HER2+ than in the unconfirmed cohort, and the amplification ratio was significantly higher. A minimum of 40% HER2+ tumor cells and a 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 expression and amplification levels may improve selection of patients for HER2-directed treatment.
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http://dx.doi.org/10.1200/JCO.20.02761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099392PMC
May 2021

Facile Synthesis of a Croconaine-Based Nanoformulation for Optoacoustic Imaging and Photothermal Therapy.

Adv Healthc Mater 2021 May 18;10(9):e2002115. Epub 2021 Mar 18.

Chair of Biological Imaging, Center for Translational Cancer Research (TranslaTUM), Technical University of Munich, Munich, 81675, Germany.

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 α β 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.
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http://dx.doi.org/10.1002/adhm.202002115DOI Listing
May 2021

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

PLoS One 2021 16;16(3):e0248594. Epub 2021 Mar 16.

Research Unit Analytical Pathology, Helmholtz Zentrum München, Neuherberg, Germany.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248594PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963095PMC
March 2021

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

J Hepatol 2021 Feb 20. Epub 2021 Feb 20.

State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China; University of Chinese Academy of Sciences, Beijing 100049, China; Cancer Center, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai 200031, China; School of Life Science, Hangzhou Institute for Advance Study, University of Chinese Academy of Sciences, Hangzhou 310024, China. Electronic address:

Background & Aims: HBV remains a global threat to human health. It remains incompletely understood how HBV self-restricts in the host during most adult infections. Thus, we performed multi-omics analyses to systematically interrogate HBV-host interactions 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 the HBV genome; 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, a novel short isoform of HBX. Having confirmed their existence, we functionally characterized them as potent suppressors of HBV gene expression and genome replication. Mechanistically, HpZ/P' was found to repress HBV gene expression partially by interacting with, and sequestering SUPV3L1. Activation of the host immune system seemed to reduce the abundance of HBV mutants deficient in HpZ/P' or with disruptions in EnhI-SL. Finally, SRSF2, a host RNA spliceosome protein that is downregulated by HBV, was found to promote the splicing of viral pre-genomic RNA and HpZ/P' biogenesis.

Conclusion: This study has identified multiple self-restricting HBV-host interactions. In particular, SRSF2-HpZ/P' appeared to constitute another negative feedback mechanism in the HBV life cycle. Targeting host splicing machinery might thus represent a strategy to intervene in HBV-host interactions.

Lay Summary: There remain many unknowns about the natural history of HBV infection in adults. Herein, we identified new HBV-host mechanisms which could be responsible for self-restricting infections. Targeting these mechanisms could be a promising strategy for the treatment of HBV infections.
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http://dx.doi.org/10.1016/j.jhep.2021.02.009DOI Listing
February 2021

Transcriptomic landscape of radiation-induced murine thyroid proliferative lesions.

Endocr Relat Cancer 2021 Mar;28(3):213-224

Helmholtz Zentrum München - German Research Center for Environmental Health, Research Unit Radiation Cytogenetics, Neuherberg, Germany.

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.
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http://dx.doi.org/10.1530/ERC-21-0019DOI Listing
March 2021

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

Mol Oncol 2021 Apr 29;15(4):1040-1053. Epub 2020 Dec 29.

Department of Otorhinolaryngology, Head and Neck Surgery, Ludwig-Maximilians-University Munich, Germany.

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.
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http://dx.doi.org/10.1002/1878-0261.12886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024715PMC
April 2021

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

PLoS One 2020 9;15(12):e0243462. Epub 2020 Dec 9.

Research Unit Analytical Pathology, Helmholtz Zentrum München, Neuherberg, Germany.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243462PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725368PMC
January 2021

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

Life Sci Alliance 2021 01 30;4(1). Epub 2020 Nov 30.

Research Group Adipocytes and Metabolism, Institute for Diabetes and Obesity, Helmholtz Zentrum München, Neuherberg, Germany

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 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.
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http://dx.doi.org/10.26508/lsa.202000924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723269PMC
January 2021

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

Eur Radiol 2020 Nov 19. Epub 2020 Nov 19.

Physics Department, Technical University of Munich, Garching, Germany.

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.
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http://dx.doi.org/10.1007/s00330-020-07459-4DOI Listing
November 2020

Effect of Dietary Sodium Modulation on Pig Adrenal Steroidogenesis and Transcriptome Profiles.

Hypertension 2020 12 19;76(6):1769-1777. Epub 2020 Oct 19.

From the Medizinische Klinik und Poliklinik IV, Klinikum der Universität München (T.V., I.-S.K., M.R., T.A.W.), Ludwig-Maximilians-Universität München, Munich, Germany.

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 (=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 (<0.001) and an almost 3-fold upregulation of (<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.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.120.15998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011841PMC
December 2020

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

Anal Chim Acta 2020 Oct 13;1134:125-135. Epub 2020 Aug 13.

Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany and University of Tübingen, Tübingen, Germany. Electronic address:

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 [LPE]) 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.
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http://dx.doi.org/10.1016/j.aca.2020.08.005DOI Listing
October 2020

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

Sci Rep 2020 09 2;10(1):14461. Epub 2020 Sep 2.

Research Unit Analytical Pathology, Helmholtz Zentrum München, 8576, Neuherberg, Germany.

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.
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http://dx.doi.org/10.1038/s41598-020-71465-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468256PMC
September 2020

A multi-test strategy for adrenal tumours.

Lancet Diabetes Endocrinol 2020 09 23;8(9):733-734. Epub 2020 Jul 23.

Research Unit Analytical Pathology, Helmholtz Zentrum München-German Research Centre for Environmental Health, Neuherberg, Munich 85764, Germany. Electronic address:

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http://dx.doi.org/10.1016/S2213-8587(20)30224-2DOI Listing
September 2020

Morphometric Cell Classification for Single-Cell MALDI-Mass Spectrometry Imaging.

Angew Chem Int Ed Engl 2020 09 17;59(40):17447-17450. Epub 2020 Aug 17.

Maastricht MultiModal Molecular Imaging Institute (M4I), University of Maastricht, Universiteitssingel 50, 6200 MD, Maastricht, The Netherlands.

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.
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http://dx.doi.org/10.1002/anie.202007315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540554PMC
September 2020

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

Am J Cancer Res 2020 1;10(6):1785-1792. Epub 2020 Jun 1.

Clinical Research Unit, Department of Obstetrics and Gynecology, Technical University of Munich (TUM) Germany.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339269PMC
June 2020

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

Nat Commun 2020 06 17;11(1):3068. Epub 2020 Jun 17.

Helmholtz Zentrum München, Institute of Lung Biology and Disease, Regenerative Biology and Medicine, Member of the German Center for Lung Research (DZL), Munich, Germany.

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.
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http://dx.doi.org/10.1038/s41467-020-16893-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299976PMC
June 2020

The Intratumoral Heterogeneity Reflects the Intertumoral Subtypes of Glioblastoma Multiforme: A Regional Immunohistochemistry Analysis.

Front Oncol 2020 24;10:494. Epub 2020 Apr 24.

Division of Neuropathology, Technische Universität München, München, Germany.

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.
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http://dx.doi.org/10.3389/fonc.2020.00494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193089PMC
April 2020

In situ Metabolite Mass Spectrometry Imaging: New Insights into the Adrenal Gland.

Horm Metab Res 2020 Jun 29;52(6):435-447. Epub 2020 Apr 29.

Research Unit Analytical Pathology, German Research Center for Environmental Health, Helmholtz Zentrum München, München, Germany.

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.
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http://dx.doi.org/10.1055/a-1129-6947DOI Listing
June 2020

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

Mol Metab 2020 06 14;36:100953. Epub 2020 Feb 14.

Research Unit Analytical Pathology, Helmholtz Zentrum München - German Research Center for Environmental Health, Ingolstädter Landstraße 1, Neuherberg, 85764, Germany. Electronic address:

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, non-objective, 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.
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http://dx.doi.org/10.1016/j.molmet.2020.01.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149754PMC
June 2020

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

Cell Death Dis 2020 03 17;11(3):192. Epub 2020 Mar 17.

Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Würzburg, Würzburg, Germany.

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 EC values of 5.7 × 10, 8.1 × 10 and 2.1 × 10 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.
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http://dx.doi.org/10.1038/s41419-020-2385-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078189PMC
March 2020

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

J Cachexia Sarcopenia Muscle 2020 02 13;11(1):226-240. Epub 2019 Nov 13.

Research Unit Analytical Pathology, Helmholtz Zentrum München, Oberschleißheim, Germany.

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.
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http://dx.doi.org/10.1002/jcsm.12498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015243PMC
February 2020

Mass Spectrometry Imaging Establishes 2 Distinct Metabolic Phenotypes of Aldosterone-Producing Cell Clusters in Primary Aldosteronism.

Hypertension 2020 03 20;75(3):634-644. Epub 2020 Jan 20.

Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, LMU München, Germany (L.S.M., M.R., T.A.W.).

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.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.14041DOI Listing
March 2020

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

Sci Rep 2020 01 9;10(1):79. Epub 2020 Jan 9.

Department of Diagnostic and Interventional Radiology, School of Medicine & Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.

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 (7 T) 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.
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http://dx.doi.org/10.1038/s41598-019-57075-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952459PMC
January 2020

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".

Radiat Oncol 2020 Jan 6;15(1). Epub 2020 Jan 6.

Clinical Cooperation Group 'Personalized Radiotherapy in Head and Neck Cancer', Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany.

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.
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http://dx.doi.org/10.1186/s13014-019-1452-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945615PMC
January 2020

Dusp8 affects hippocampal size and behavior in mice and humans.

Sci Rep 2019 12 20;9(1):19483. Epub 2019 Dec 20.

Research Unit Neurobiology of Diabetes, Helmholtz Zentrum München, 85764, Neuherberg, Germany.

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 Dusp8 WT 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.
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http://dx.doi.org/10.1038/s41598-019-55527-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925303PMC
December 2019

Patch repair of deep wounds by mobilized fascia.

Nature 2019 12 27;576(7786):287-292. Epub 2019 Nov 27.

Group Regenerative Biology and Medicine, Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany.

Mammals form scars to quickly seal wounds and ensure survival by an incompletely understood mechanism. 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.
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http://dx.doi.org/10.1038/s41586-019-1794-yDOI Listing
December 2019

Multimodal Precision Imaging of Pulmonary Nanoparticle Delivery in Mice: Dynamics of Application, Spatial Distribution, and Dosimetry.

Small 2019 12 22;15(49):e1904112. Epub 2019 Oct 22.

Comprehensive Pneumology Center (CPC-M), Member of the German Center for Lung Research (DZL), Munich, 81377, Germany.

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.
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http://dx.doi.org/10.1002/smll.201904112DOI Listing
December 2019