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Purpose: To investigate ultra-high-dose rate helium ion irradiation and its potential FLASH sparing effect with the endpoint acute brain injury in preclinical in vivo settings., Material and Methods: Raster-scanned helium ion beams were administered to explore and compare the impact of dose rate variations between standard dose rate (SDR at 0.2 Gy/s) and FLASH (at 141 Gy/s) radiotherapy (RT). Irradiation-induced brain injury was investigated in healthy C57BL/6 mice via DNA damage response kinetic studies using nuclear γH2AX as a surrogate for double-strand breaks (DSB). The integrity of the neurovascular and immune compartments was assessed via CD31+ microvascular density and microglia/macrophages activation. Iba1+ ramified and CD68+ phagocytic microglia/macrophages were quantified, together with the expression of inducible nitric oxide synthetase (iNOS)., Results: Helium FLASH RT significantly prevented acute brain tissue injury compared with SDR. This was demonstrated by reduced levels of DSB and structural preservation of the neurovascular endothelium after FLASH RT. Moreover, FLASH RT exhibited reduced activation of neuroinflammatory signals compared with SDR, as detected by quantification of CD68+ iNOS+ microglia/macrophages., Conclusion: To our knowledge, this is the first report on the FLASH-sparing neuroprotective effect of raster scanning helium ion radiotherapy in vivo.

Bovine milk and meat factors (BMMFs) are plasmid-like DNA molecules isolated from bovine milk and serum, as well as the peritumor of colorectal cancer (CRC) patients. BMMFs have been proposed as zoonotic infectious agents and drivers of indirect carcinogenesis of CRC, inducing chronic tissue inflammation, radical formation and increased levels of DNA damage. Data on expression of BMMFs in large clinical cohorts to test an association with co-markers and clinical parameters were not previously available and were therefore assessed in this study. Tissue sections with paired tumor-adjacent mucosa and tumor tissues of CRC patients [individual cohorts and tissue microarrays (TMAs) (n = 246)], low-/high-grade dysplasia (LGD/HGD) and mucosa of healthy donors were used for immunohistochemical quantification of the expression of BMMF replication protein (Rep) and CD68/CD163 (macrophages) by co-immunofluorescence microscopy and immunohistochemical scoring (TMA). Rep was expressed in the tumor-adjacent mucosa of 99% of CRC patients (TMA), was histologically associated with CD68 + /CD163 + macrophages and was increased in CRC patients when compared to healthy controls. Tumor tissues showed only low stromal Rep expression. Rep was expressed in LGD and less in HGD but was strongly expressed in LGD/HGD-adjacent tissues. Albeit not reaching statistical significance, incidence curves for CRC-specific death were increased for higher Rep expression (TMA), with high tumor-adjacent Rep expression being linked to the highest incidence of death. BMMF Rep expression might represent a marker and early risk factor for CRC. The correlation between Rep and CD68 expression supports a previous hypothesis that BMMF-specific inflammatory regulations, including macrophages, are involved in the pathogenesis of CRC., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Epithelial membrane protein 3 (EMP3) is an N-glycosylated tetraspanin with a putative trafficking function. It is highly expressed in isocitrate dehydrogenase-wild-type glioblastoma (IDH-wt GBM), and its high expression correlates with poor survival. However, the exact trafficking role of EMP3 and how it promotes oncogenic signaling in GBM remain unclear. Here, we show that EMP3 promotes EGFR/CDK2 signaling by regulating the trafficking and enhancing the stability of EGFR. BioID2-based proximity labeling revealed that EMP3 interacts with endocytic proteins involved in the vesicular transport of EGFR. EMP3 knockout (KO) enhances epidermal growth factor (EGF)-induced shuttling of EGFR into RAB7 + late endosomes, thereby promoting EGFR degradation. Increased EGFR degradation is rescued by the RAB7 negative regulator and novel EMP3 interactor TBC1D5. Phosphoproteomic and transcriptomic analyses further showed that EMP3 KO converges into the inhibition of the cyclin-dependent kinase CDK2 and the repression of EGFR-dependent and cell cycle transcriptional programs. Phenotypically, EMP3 KO cells exhibit reduced proliferation rates, blunted mitogenic response to EGF, and increased sensitivity to the pan-kinase inhibitor staurosporine and the EGFR inhibitor osimertinib. Furthermore, EGFR-dependent patient-derived glioblastoma stem cells display a transcriptomic signature consistent with reduced CDK2 activity, as well as increased susceptibility to CDK2 inhibition upon EMP3 knockdown. Lastly, using TCGA data, we showed that GBM tumors with high EMP3 expression have increased total and phosphorylated EGFR levels. Collectively, our findings demonstrate a novel EMP3-dependent mechanism by which EGFR/CDK2 activity is sustained in GBM. Consequently, EMP3's stabilizing effect provides an additional layer of tumor cell resistance against targeted kinase inhibition., (© 2023. The Author(s).)

Background: IDH mutant gliomas are grouped into astrocytomas or oligodendrogliomas depending on the codeletion of chromosome arms 1p and 19q. Although the genomic alterations of IDH mutant gliomas have been well described, transcriptional changes unique to either tumor type have not been fully understood. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas., Methods: We used several cell lines, including patient-derived oligodendroglioma tumorspheres, to knock down or overexpress TRIM67. We coupled high-throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS), and coimmunoprecipitation (co-IP)-MS with functional assays including immunofluorescence (IF) staining, co-IP, and western blotting (WB) to assess the in vitro phenotype associated with TRIM67. Patient-derived oligodendroglioma tumorspheres were orthotopically implanted in mice to determine the effect of TRIM67 on tumor growth and survival., Results: TRIM67 overexpression alters the abundance of cytoskeletal proteins and induces membrane bleb formation. TRIM67-associated blebbing was reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. NOGO-A/Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression, pointing to the underlying mechanism for TRIM67-induced blebbing. Phenotypically, TRIM67 expression resulted in higher cell motility and reduced cell adherence. In orthotopic implantation models of patient-derived oligodendrogliomas, TRIM67 accelerated tumor growth, reduced overall survival, and led to increased vimentin expression at the tumor margin., Conclusions: Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Glioblastoma (GBM) is an aggressive tumor that frequently exhibits gain of chromosome 7, loss of chromosome 10, and aberrantly activated receptor tyrosine kinase signaling pathways. Previously, we identified Mesenchyme Homeobox 2 (MEOX2), a gene located on chromosome 7, as an upregulated transcription factor in GBM. Overexpressed transcription factors can be involved in driving GBM. Here, we aimed to address the role of MEOX2 in GBM., Methods: Patient-derived GBM tumorspheres were used to constitutively knockdown or overexpress MEOX2 and subjected to in vitro assays including western blot to assess ERK phosphorylation. Cerebral organoid models were used to investigate the role of MEOX2 in growth initiation. Intracranial mouse implantation models were used to assess the tumorigenic potential of MEOX2. RNA-sequencing, ACT-seq, and CUT&Tag were used to identify MEOX2 target genes., Results: MEOX2 enhanced ERK signaling through a feed-forward mechanism. We identified Ser155 as a putative ERK-dependent phosphorylation site upstream of the homeobox-domain of MEOX2. S155A substitution had a major effect on MEOX2 protein levels and altered its subnuclear localization. MEOX2 overexpression cooperated with p53 and PTEN loss in cerebral organoid models of human malignant gliomas to induce cell proliferation. Using high-throughput genomics, we identified putative transcriptional target genes of MEOX2 in patient-derived GBM tumorsphere models and a fresh frozen GBM tumor., Conclusions: We identified MEOX2 as an oncogenic transcription regulator in GBM. MEOX2 increases proliferation in cerebral organoid models of GBM and feeds into ERK signaling that represents a core signaling pathway in GBM., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: To investigate brain tissue response to ultra-high dose rate (uHDR, FLASH) and standard dose rate (SDR) proton irradiations in the Bragg peak region., Methods and Materials: Active scanning uHDR delivery was established for proton beams for investigation of dose rate effects between clinical SDR and uHDR at ∼10 Gy in the Bragg peak region (dose-averaged linear energy transfer [LET D ] ranging from 4.5 to 10.2 keV µm -1 ). Radiation- induced injury of neuronal tissue was assessed by studying the DNA double strand break repair kinetics surrogated by nuclear γH2AX staining (radiation induced foci [RIF]), microvascular density and structural integrity (MVD, CD31+ endothelium), and inflammatory microenvironmental response (CD68+ microglia/macrophages and high mobility group box protein 1[HMGB]) in healthy C57BL/6 mouse brains., Results: Averaged dose rates achieved were 0.17 Gy/s (SDR) and 120 Gy/s (uHDR). The fraction of RIF-positive cells increased after SDR ∼10-fold, whereas a significantly lower fraction of RIF-positive cells was found after uHDR versus SDR (∼2 fold, P < .0001). Moreover, uHDR substantially preserved the microvascular architecture and reduced microglia/macrophage regulated associated inflammation as compared with SDR., Conclusions: The feasibility of uHDR raster scanning proton irradiation is demonstrated to elicit FLASH sparing neuroprotective effects compared to SDR in a preclinical in vivo model., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The survival rate among children with relapsed tumors remains poor, due to tumor heterogeneity, lack of directly actionable tumor drivers and multidrug resistance. Novel personalized medicine approaches tailored to each tumor are urgently needed to improve cancer treatment. Current pediatric precision oncology platforms, such as the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study, reveal that molecular profiling of tumor tissue identifies targets associated with clinical benefit in a subgroup of patients only and should be complemented with functional drug testing. In such an approach, patient-derived tumor cells are exposed to a library of approved oncological drugs in a physiological setting, e.g., in the form of animal avatars injected with patient tumor cells. We used molecularly fully characterized tumor samples from the INFORM study to compare drug screen results of individual patient-derived cell models in functional assays: (i) patient-derived spheroid cultures within a few days after tumor dissociation; (ii) tumor cells reisolated from the corresponding mouse PDX; (iii) corresponding long-term organoid-like cultures and (iv) drug evaluation with the corresponding zebrafish PDX (zPDX) model. Each model had its advantage and complemented the others for drug hit and drug combination selection. Our results provide evidence that in vivo zPDX drug screening is a promising add-on to current functional drug screening in precision medicine platforms.

The invasiveness of late-stage cutaneous squamous cell carcinoma (cSCC) is associated with poor patients' prognosis and linked to strong upregulation of the glycoprotein Podoplanin (PDPN) in cancer cells. However, the function of PDPN in these processes in cSCC carcinogenesis has not been characterized in detail yet. Employing a CRISPR/Cas9-based loss-of-function approach on murine cSCC cells, we show that the loss of Pdpn results in decreased migration and invasion in vitro. Complementing these in vitro studies, labelled murine control and Pdpn knockout cells were injected orthotopically into the dermis of nude mice to recapitulate the formation of human cSCC displaying a well-differentiated morphology with a PDPN-positive reaction in fibroblasts in the tumor stroma. Smaller tumors were observed upon Pdpn loss, which is associated with reduced tumor cell infiltration into the stroma. Utilizing Pdpn mutants in functional experiments in vitro, we provide evidence that both the intra- and extracellular domains are essential for cancer cell invasion. These findings underline the critical role of PDPN in cSCC progression and highlight potential therapeutic strategies targeting PDPN-dependent cancer cell invasion, especially in late-stage cSCC patients., (© 2021 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Localized radiotherapy (RT) induces an immunogenic antitumor response that is in part counterbalanced by activation of immune evasive and tissue remodeling processes, e.g., via upregulation of programmed cell death-ligand 1 (PD-L1) and transforming growth factor β (TGF-β). We report that a bifunctional fusion protein that simultaneously inhibits TGF-β and PD-L1, bintrafusp alfa (BA), effectively synergizes with radiotherapy, leading to superior survival in multiple therapy-resistant murine tumor models with poor immune infiltration. The BA + RT (BART) combination increases tumor-infiltrating leukocytes, reprograms the tumor microenvironment, and attenuates RT-induced fibrosis, leading to reconstitution of tumor immunity and regression of spontaneous lung metastases. Consistently, the beneficial effects of BART are in part reversed by depletion of cytotoxic CD8 + T cells. Intriguingly, targeting of the TGF-β trap to PD-L1 + endothelium and the M2/lipofibroblast-like cell compartment by BA attenuated late-stage RT-induced lung fibrosis. Together, the results suggest that the BART combination has the potential to eradicate therapy-resistant tumors while sparing normal tissue, further supporting its clinical translation., Competing Interests: Declaration of interests Y.L., C.X., T.-L.Y., M.H.J., H.Y., B.M., G.Q., J.Q., G.L., M.G.D., F.J., and K.-M.L. are all employees of EMD Serono Research & Development Institute, Inc., Billerica, MA, USA, an affiliate of Merck KGaA, Darmstadt, Germany. K.-M.L. is the inventor on Patent Cooperation Treaty Publications WO2015/118115 and US 2015/0225483 held by applicant Merck Patent GmvH covering bintrafusp alfa, its methods of making, and its methods of use. J.D. has received research grants from Siemens Healthcare GmbH, Solution Akademia GmbH, ViewRay, The Clinical Research Institute GmbH, Accuray International Sàrl, RaySearch Laboratories AB, Vision RT, Merck Serono GmbH, Astellas Pharma GmbH, AstraZeneca GmbH, Ergomed, Quintiles GmbH, and Pharmaceutical Research Associates GmbH and declares grant support from EMD/Merck KGaA to his institution to conduct experiments directly related to the published study. A.A. has received research grants from Merck KGaA, FibroGen, and Bayer; has a consulting or advisory role with Roche, Merck KGaA, Merck Serono, FibroGen, BMS Brazil, Bayer Health, and BioMedX; and declares grant support from EMD/Merck KGaA to his institution to conduct experiments directly related to the published study. All other authors disclose no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell-cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.

The CD73 pathway is an important anti-inflammatory mechanism in various disease settings. Observations in mouse models suggested that CD73 might have a protective role in kidney damage; however, no direct evidence of its role in human kidney disease has been described to date. Here, we hypothesized that podocyte injury in human kidney diseases alters CD73 expression that may facilitate the diagnosis of podocytopathies. We assessed the expression of CD73 and one of its functionally important targets, the C-C chemokine receptor type 2 (CCR2), in podocytes from kidney biopsies of 39 patients with podocytopathy (including focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), membranous glomerulonephritis (MGN) and amyloidosis) and a control group. Podocyte CD73 expression in each of the disease groups was significantly increased in comparison to controls ( p < 0.001- p < 0.0001). Moreover, there was a marked negative correlation between CD73 and CCR2 expression, as confirmed by immunohistochemistry and immunofluorescence (Pearson r = -0.5068, p = 0.0031; Pearson r = -0.4705, p = 0.0313, respectively), thus suggesting a protective role of CD73 in kidney injury. Finally, we identify CD73 as a novel potential diagnostic marker of human podocytopathies, particularly of MCD that has been notorious for the lack of pathological features recognizable by light microscopy and immunohistochemistry.

Depending on context and tumor stage, deregulation of autophagy can either suppress tumorigenesis or promote chemoresistance and tumor survival. Histone deacetylases (HDACs) can modulate autophagy; however, the exact mechanisms are not fully understood. Here, we analyze the effects of the broad-spectrum HDAC inhibitors (HDACi) panobinostat and vorinostat on the transcriptional regulation of autophagy with respect to autophagy transcription factor activity (Transcription factor EB-TFEB, forkhead boxO-FOXO) and autophagic flux in neuroblastoma cells. In combination with the late-stage autophagic flux inhibitor bafilomycin A1, HDACis increase the number of autophagic vesicles, indicating an increase in autophagic flux. Both HDACi induce nuclear translocation of the transcription factors FOXO1 and FOXO3a, but not TFEB and promote the expression of pro-autophagic FOXO1/3a target genes. Moreover, FOXO1/3a knockdown experiments impaired HDACi treatment mediated expression of autophagy related genes. Combination of panobinostat with the lysosomal inhibitor chloroquine, which blocks autophagic flux, enhances neuroblastoma cell death in culture and hampers tumor growth in vivo in a neuroblastoma zebrafish xenograft model. In conclusion, our results indicate that pan-HDACi treatment induces autophagy in neuroblastoma at a transcriptional level. Combining HDACis with autophagy modulating drugs suppresses tumor growth of high-risk neuroblastoma cells. These experimental data provide novel insights for optimization of treatment strategies in neuroblastoma.

The survival rate among children with relapsed neuroblastomas continues to be poor, and thus new therapeutic approaches identified by reliable preclinical drug testing models are urgently needed. Zebrafish are a powerful vertebrate model in preclinical cancer research. Here, we describe a zebrafish neuroblastoma yolk sac model to evaluate efficacy and toxicity of histone deacetylase (HDAC) inhibitor treatments. Larvae were engrafted with fluorescently labeled, genetically diverse, established cell lines and short-term cultures of patient-derived primary cells. Engrafted tumors progressed locally and disseminated remotely in an intact environment. Combination treatments involving the standard chemotherapy doxorubicin and HDAC inhibitors substantially reduced tumor volume, induced tumor cell death, and inhibited tumor cell dissemination to the tail region. Hence, this model allows for fast, cost-efficient, and reliable in vivo evaluation of toxicity and response of the primary and metastatic tumor sites to drug combinations., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Sexual dimorphism arises from genetic differences between male and female cells, and from systemic hormonal differences 1-3 . How sex hormones affect non-reproductive organs is poorly understood, yet highly relevant to health given the sex-biased incidence of many diseases 4 . Here we report that steroid signalling in Drosophila from the ovaries to the gut promotes growth of the intestine specifically in mated females, and enhances their reproductive output. The active ovaries of the fly produce the steroid hormone ecdysone, which stimulates the division and expansion of intestinal stem cells in two distinct proliferative phases via the steroid receptors EcR and Usp and their downstream targets Broad, Eip75B and Hr3. Although ecdysone-dependent growth of the female gut augments fecundity, the more active and more numerous intestinal stem cells also increase female susceptibility to age-dependent gut dysplasia and tumorigenesis, thus potentially reducing lifespan. This work highlights the trade-offs in fitness traits that occur when inter-organ signalling alters stem-cell behaviour to optimize organ size.

Disseminated tumor cells (dTCs) can frequently be detected in the bone marrow (BM) of colorectal cancer (CRC) patients, raising the possibility that the BM serves as a reservoir for metastatic tumor cells. Identification of dTCs in BM aspirates harbors the potential of assessing therapeutic outcome and directing therapy intensity with limited risk and effort. Still, the functional and prognostic relevance of dTCs is not fully established. We have previously shown that CRC cell clones can be traced to the BM of mice carrying patient-derived xenografts. However, cellular interactions, proliferative state and tumorigenicity of dTCs remain largely unknown. Here, we applied a coculture system modeling the microvascular niche and used immunofluorescence imaging of the murine BM to show that primary CRC cells migrate toward endothelial tubes. dTCs in the BM were rare, but detectable in mice with xenografts from most patient samples (8/10) predominantly at perivascular sites. Comparable to primary tumors, a substantial fraction of proliferating dTCs was detected in the BM. However, most dTCs were found as isolated cells, indicating that dividing dTCs rather separate than aggregate to metastatic clones-a phenomenon frequently observed in the microvascular niche model. Clonal tracking identified subsets of self-renewing tumor-initiating cells in the BM that formed tumors out of BM transplants, including one subset that did not drive primary tumor growth. Our results indicate an important role of the perivascular BM niche for CRC cell dissemination and show that dTCs can be a potential source for tumor relapse and tumor heterogeneity., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Radiation-induced normal tissue toxicity often limits the curative treatment of cancer. Moreover, normal tissue relative biological effectiveness data for high-linear energy transfer particles are urgently needed. We propose a strategy based on transcriptome analysis of patient-derived human intestinal organoids (HIO) to determine molecular surrogates for radioresponse of gastrointestinal (GI) organs at risk in a personalized manner. HIO were generated from induced pluripotent stem cells (iPSC), which were derived from skin biopsies of three patients, including two patients with FANCA deficiency as a paradigm for enhanced radiosensitivity. For the two Fanconi anemia (FA) patients (HIO-104 and 106, previously published as FA-A#1 IND-iPS1 and FA-A#2 IND-iPS3), FANCA expression was reconstituted as a prerequisite for generation of HIO via lentiviral expression of a doxycycline inducible construct. For radiosensitivity analysis, FANCA deficient and FANCA rescued as well as wtHIO were sham treated or irradiated with 4Gy photon, proton or carbon ions at HIT, respectively. Immunofluorescence staining of HIO for 53BP1-foci was performed 1 h post IR and gene expression analyses was performed 12 and 48 h post IR. 53BP1-foci numbers and size correlated with the higher RBE of carbon ions. A FANCA dependent differential gene expression in response to radiation was found ( p < 0.01, ANOVA; n = 1071 12 h; n = 1100 48 h). Pathways associated with FA and DNA-damage repair i.e., transcriptional coupled nucleotide excision repair, homology-directed repair and translational synthesis were found to be differentially regulated in FANCA deficient HIO. Next, differential regulated genes were investigated as a function of radiation quality (RQ, p < 0.05, ANOVA; n = 742 12 h; n = 553 48 h). Interestingly, a gradual increase or decrease of gene expression was found to correlate with the three main qualities, from photon to proton and carbon irradiation. Clustering separated high-linear energy transfer irradiation with carbons from proton and photon irradiation. Genes associated with dual incision steps of TC-NER were differentially regulated in photon vs. proton and carbon irradiation. Consequently, SUMO3, ALC1, POLE4, PCBP4, MUTYH expression correlated with the higher RBE of carbon ions. An interaction between the two studied parameters FA and RQ was identified ( p < 0.01, 2-way ANOVA n = 476). A comparison of genes regulated as a function of FA, RQ and RBE suggest a role for p53 interacting genes BRD7, EWSR1, FBXO11, FBXW8, HMGB1, MAGED2, PCBP4, and RPS27 as modulators of FA in response to radiation. This proof of concept study demonstrates that patient tailored evaluation of GI response to radiation is feasible via generation of HIO and comparative transcriptome profiling. This methodology can now be further explored for a personalized assessment of GI radiosensitivity and RBE estimation.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S -/- ) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S -/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objective: The susceptibility to abdominal obesity and the metabolic syndrome is determined to a substantial extent during childhood and adolescence, when key adipose tissue characteristics are established. Although the general impact of postnatal nutrition is well known, it is not clear how specific dietary components drive adipose tissue growth and how this relates to the risk of metabolic dysfunction in adulthood., Methods: Adipose tissue growth including cell proliferation was analyzed in juvenile mice upon dietary manipulation with in vivo nucleotide labeling. The proliferative response of progenitors to specific fatty acids was assayed in primary cultures. Long-term metabolic consequences were assessed through transient dietary manipulation post-weaning with a second obesogenic challenge in adulthood., Results: Dietary lipids stimulated adipose tissue progenitor cell proliferation in juvenile mice independently of excess caloric intake and calorie-dependent adipocyte hypertrophy. Excess calories increased mitogenic IGF-1 levels systemically, whereas palmitoleic acid was able to enhance the sensitivity of progenitors to IGF-1, resulting in synergistic stimulation of proliferation. Early transient consumption of excess lipids promoted hyperplastic adipose tissue expansion in response to a second dietary challenge in adulthood and this correlated with abdominal obesity and hyperinsulinemia., Conclusions: Dietary lipids and calories differentially and synergistically drive adipose tissue proliferative growth and the programming of the metabolic syndrome in childhood., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cytoplasmic dynein-1 is a large minus-end-directed microtubule motor complex involved in membrane trafficking, organelle positioning, and microtubule organization. The roles of dynein light intermediate chains (DLICs; DLIC1 and DLIC2) within the complex are, however, still largely undefined. In this study, we investigated the possible roles of DLICs in epithelial homeostasis and colon cancer development. Mutant clonal analysis of Drosophila Dlic in the follicular epithelium of Drosophila ovary showed defects in nuclear positioning, epithelial integrity, and apical cell polarity. Consistently, knockdown of human DLIC1 and DLIC2 in colon carcinoma cells resulted in damaged epithelial organization, disturbed lumen formation, and impaired apical polarity establishment in three-dimensional cell culture. Depletion of DLIC1 and DLIC2 led to reduced proliferation, enhanced apoptosis rates, disrupted mitotic spindle assembly, and induction of G2/M arrest in cell cycle progression. Moreover, reduced levels of DLIC1 in contrast to DLIC2 impaired the migratory ability. On the other hand, immunohistochemical examination of human colorectal tissue samples and further colorectal cancer dataset analysis showed a significant upregulation for DLIC1 in tumors, whereas DLIC2 expression was unchanged. In addition, the overexpression of DLIC1 caused increased proliferation, decreased apoptosis and enhanced migration, whereas DLIC2 overexpression did not result in any significant changes. Together, these results indicate that DLIC1 and DLIC2 contribute to the establishment and maintenance of epithelial homeostasis. Furthermore, these findings present the first evidence that DLIC1 and DLIC2 have distinct roles in colon cancer development and that DLIC1 may contribute to proliferative overgrowth and migratory characteristics., (© 2019 Federation of European Biochemical Societies.)

Most antidiabetic drugs treat disease symptoms rather than adipose tissue dysfunction as a key pathogenic cause in the metabolic syndrome and type 2 diabetes. Pharmacological targeting of adipose tissue through the nuclear receptor PPARg, as exemplified by glitazone treatments, mediates efficacious insulin sensitization. However, a better understanding of the context-specific PPARg responses is required for the development of novel approaches with reduced side effects. Here, we identified the transcriptional cofactor Cited4 as a target and mediator of rosiglitazone in human and murine adipocyte progenitor cells, where it promoted specific sets of the rosiglitazone-dependent transcriptional program. In mice, Cited4 was required for the proper induction of thermogenic expression by Rosi specifically in subcutaneous fat. This phenotype had high penetrance in females only and was not evident in beta-adrenergically stimulated browning. Intriguingly, this specific defect was associated with reduced capacity for systemic thermogenesis and compromised insulin sensitization upon therapeutic rosiglitazone treatment in female but not male mice. Our findings on Cited4 function reveal novel unexpected aspects of the pharmacological targeting of PPARg., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

The transient activation of inflammatory networks is required for adipose tissue remodeling including the "browning" of white fat in response to stimuli such as β3-adrenergic receptor activation. In this process, white adipose tissue acquires thermogenic characteristics through the recruitment of so-called beige adipocytes. We investigated the downstream signaling pathways impinging on adipocyte progenitors that promote de novo formation of adipocytes. We showed that the Jak family of kinases controlled TGFβ signaling in the adipose tissue microenvironment through Stat3 and thereby adipogenic commitment, a function that was required for beige adipocyte differentiation of murine and human progenitors. Jak/Stat3 inhibited TGFβ signaling to the transcription factors Srf and Smad3 by repressing local Tgfb3 and Tgfb1 expression before the core transcriptional adipogenic cascade was activated. This pathway cross-talk was triggered in stromal cells by ATGL-dependent adipocyte lipolysis and a transient wave of IL-6 family cytokines at the onset of adipose tissue remodeling induced by β3-adrenergic receptor stimulation. Our results provide insight into the activation of adipocyte progenitors and are relevant for the therapeutic targeting of adipose tissue inflammatory pathways., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Invariant natural killer T (iNKT) cells represent a unique population of CD1d-restricted T lymphocytes expressing an invariant T cell receptor encoded by Vα14-Jα18 and Vα24-Jα18 gene segments in mice and humans, respectively. Recognition of CD1d-loaded endogenous lipid antigen(s) on CD4/CD8-double positive (DP) thymocytes is essential for the development of iNKT cells. The lipid repertoire of DP thymocytes and the identity of the decisive endogenous lipid ligands have not yet been fully elucidated. Glycosphingolipids (GSL) were implicated to serve as endogenous ligands. However, further in vivo investigations were hampered by early embryonal lethality of mice deficient for the key GSL-synthesizing enzyme glucosylceramide (GlcCer) synthase [GlcCer synthase (GCS), EC 2.4.1.80]. We have now analyzed the GSL composition of DP thymocytes and shown that GlcCer represented the sole neutral GSL and the acidic fraction was composed of gangliosides. Furthermore, we report on a mouse model that by combination of Vav-promoter-driven iCre and floxed GCS alleles ( Vav Cre GCS f/f ) enabled an efficient depletion of GCS-derived GSL very early in the T cell development, reaching a reduction by 99.6% in DP thymocytes. Although the general T cell population remained unaffected by this depletion, iNKT cells were reduced by approximately 50% in thymus, spleen, and liver and showed a reduced proliferation and an increased apoptosis rate. The Vβ-chains repertoire and development of iNKT cells remained unaltered. The GSL-depletion neither interfered with expression of CD1d, SLAM, and Ly108 molecules nor impeded the antigen presentation on DP thymocytes. These results indicate that GlcCer-derived GSL, in particular GlcCer, contribute to the homeostatic development of iNKT cells.

γδ T cell subsets can be characterized, in part, by their secretion of select proinflammatory cytokines. The molecular mechanisms driving the diverse fates of γδ T cells have not been elucidated. We have previously shown that the attachment of myristic acid to the N-terminal glycine of proteins, termed N-myristoylation, is essential for αβ T cell development and activation. Here, we explore the potential role of this lipid modification on the activation of γδ T cells. In the absence of N-myristoylation, the CD27 + γδ T cell subset was dominantly affected. The cells produced high levels of IFN-γ upon stimulation. In addition, they were more sensitive to inhibition of the CaN-Nfat pathway than were γδ T cells with myristoylated CaN. N-Myristoylation was found to modulate activity of phosphatase CaN, a regulator of Nfat. In summary, the CaN-Nfat pathway regulates development and function of IFN-γ-producing γδ T cells, and its balanced activity is strongly dependent on CaN N-myristoylation., (© Society for Leukocyte Biology.)

The growing number of particle therapy facilities worldwide landmarks a novel era of precision oncology. Implementation of robust biophysical readouts is urgently needed to assess the efficacy of different radiation qualities. This is the first report on biophysical evaluation of Monte Carlo simulated predictive models of prescribed dose for four particle qualities i.e., proton, helium-, carbon- or oxygen ions using raster-scanning technology and clinical therapy settings at HIT. A high level of agreement was found between the in silico simulations, the physical dosimetry and the clonogenic tumor cell survival. The cell fluorescence ion track hybrid detector (Cell-Fit-HD) technology was employed to detect particle traverse per cell nucleus. Across a panel of radiobiological surrogates studied such as late ROS accumulation and apoptosis (caspase 3/7 activation), the relative biological effectiveness (RBE) chiefly correlated with the radiation species-specific spatio-temporal pattern of DNA double strand break (DSB) formation and repair kinetic. The size and the number of residual nuclear γ-H2AX foci increased as a function of linear energy transfer (LET) and RBE, reminiscent of enhanced DNA-damage complexity and accumulation of non-repairable DSB. These data confirm the high relevance of complex DSB formation as a central determinant of cell fate and reliable biological surrogates for cell survival/ RBE. The multi-scale simulation, physical and radiobiological characterization of novel clinical quality beams presented here constitutes a first step towards development of high precision biologically individualized radiotherapy., Competing Interests: The authors declare that no competing interests exist.

Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.

Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease.

Background: Telomere length is a putative biomarker of ageing, morbidity and mortality. Its application is hampered by lack of widely applicable reference ranges and uncertainty regarding the present limits of measurement reproducibility within and between laboratories., Methods: We instigated an international collaborative study of telomere length assessment: 10 different laboratories, employing 3 different techniques [Southern blotting, single telomere length analysis (STELA) and real-time quantitative PCR (qPCR)] performed two rounds of fully blinded measurements on 10 human DNA samples per round to enable unbiased assessment of intra- and inter-batch variation between laboratories and techniques., Results: Absolute results from different laboratories differed widely and could thus not be compared directly, but rankings of relative telomere lengths were highly correlated (correlation coefficients of 0.63-0.99). Intra-technique correlations were similar for Southern blotting and qPCR and were stronger than inter-technique ones. However, inter-laboratory coefficients of variation (CVs) averaged about 10% for Southern blotting and STELA and more than 20% for qPCR. This difference was compensated for by a higher dynamic range for the qPCR method as shown by equal variance after z-scoring. Technical variation per laboratory, measured as median of intra- and inter-batch CVs, ranged from 1.4% to 9.5%, with differences between laboratories only marginally significant (P = 0.06). Gel-based and PCR-based techniques were not different in accuracy., Conclusions: Intra- and inter-laboratory technical variation severely limits the usefulness of data pooling and excludes sharing of reference ranges between laboratories. We propose to establish a common set of physical telomere length standards to improve comparability of telomere length estimates between laboratories., (© The Author 2014. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Purpose: To investigate chromosomal instability and radiation response mechanisms in glioblastoma cells., Methods and Materials: We undertook a comparative analysis of two patient-derived glioblastoma cell lines. Their resistance to low and high linear energy transfer (LET) radiation was assessed using clonogenic survival assay and their intrinsic chromosome instability status using fluorescence in situ hybridization. DNA damage was analyzed by pulsed-field gel electrophoresis and by γ-H2AX foci quantification. Expression of DNA damage response proteins was assessed by immunoblot., Results: Increased radioresistance to X-rays as well as carbon ions was observed in glioblastoma cells exhibiting high levels of naturally occurring chromosomal instability and impaired Ataxia-telangiectasia mutated (ATM) signaling, as reflected by lack of phosphorylation of ATM, CHK2 and p53 after double-strand breaks induction., Conclusion: Our results indicate the existence of highly radioresistant glioblastoma cells, characterized by dysfunctional ATM signaling and high levels of intrinsic chromosomal instability.

Constitutive heterochromatin is crucial for the integrity of chromosomes and genomic stability. Here, we show that the chromatin remodelling complex NoRC, known to silence a fraction of rRNA genes, also establishes a repressive heterochromatic structure at centromeres and telomeres, preserving the structural integrity of these repetitive loci. Knockdown of NoRC leads to relaxation of centromeric and telomeric heterochromatin, abnormalities in mitotic spindle assembly, impaired chromosome segregation and enhanced chromosomal instability. The results demonstrate that NoRC safeguards genomic stability by coordinating enzymatic activities that establish features of repressive chromatin at centromeric and telomeric regions, and this heterochromatic structure is required for sustaining genomic integrity.

To assess why during in vitro aging of fibroblasts the maintenance of chromosomal stability is effective or occasionally fails, a detailed cytogenetic analysis was performed in normal human IMR-90 fetal lung fibroblasts. The onset of senescence was inferred from proliferation activity, expression pattern of cell cycle regulating proteins, activity of β-galactosidase, and morphological features. Over the period of proliferation, a moderate increase of non-transmissible structural chromosomal aberrations was observed. In addition, using fluorescence in situ hybridization (mFISH and mBAND) techniques, we detected clonally expanding translocations in up to 70% of the analyzed metaphases, all involving one homolog of chromosome 9 as an acceptor. Notably, chromosomes are randomly involved as donor-chromosomes of the translocated terminal acentric fragments. These fragments result from duplication because the donor chromosomes are apparently unchanged. Interstitial telomeric signals were detectable at fusion sites, most likely belonging to chromosome 9. Quantitative fluorescence in situ hybridization (QFISH) detecting telomere sequences, followed by mFISH technique revealed that already in young cells the respective telomeres of one chromosome 9 were particularly short. For the first time, we have observed dysfunctional telomeres of one specific chromosome in normal human cells that have been stabilized by duplicated terminal sequences., (Copyright © 2011 Wiley Periodicals, Inc.)

The regenerative potential diminishes with age and this has been ascribed to functional impairments of adult stem cells. Cells in culture undergo senescence after a certain number of cell divisions whereby the cells enlarge and finally stop proliferation. This observation of replicative senescence has been extrapolated to somatic stem cells in vivo and might reflect the aging process of the whole organism. In this study we have analyzed the effect of aging on gene expression profiles of human mesenchymal stromal cells (MSC) and human hematopoietic progenitor cells (HPC). MSC were isolated from bone marrow of donors between 21 and 92 years old. 67 genes were age-induced and 60 were age-repressed. HPC were isolated from cord blood or from mobilized peripheral blood of donors between 27 and 73 years and 432 genes were age-induced and 495 were age-repressed. The overlap of age-associated differential gene expression in HPC and MSC was moderate. However, it was striking that several age-related gene expression changes in both MSC and HPC were also differentially expressed upon replicative senescence of MSC in vitro. Especially genes involved in genomic integrity and regulation of transcription were age-repressed. Although telomerase activity and telomere length varied in HPC particularly from older donors, an age-dependent decline was not significant arguing against telomere exhaustion as being causal for the aging phenotype. These studies have demonstrated that aging causes gene expression changes in human MSC and HPC that vary between the two different cell types. Changes upon aging of MSC and HPC are related to those of replicative senescence of MSC in vitro and this indicates that our stem and progenitor cells undergo a similar process also in vivo.

Telomerase- and telomere length regulation in normal human tissues is still poorly understood. We show here that telomerase is expressed in the epidermis in situ independent of age but was repressed upon the passaging of keratinocytes in monolayer culture. However, when keratinocytes were grown in organotypic cultures (OTCs), telomerase was re-established, indicating that telomerase activity is not merely proliferation-associated but is regulated in a tissue context-dependent manner in human keratinocytes. While not inducible by growth factors, treatment with the histone deacetylation inhibitor FK228 restored telomerase activity in keratinocytes grown in monolayer cultures. Accordingly, CHIP analyses demonstrated an acetylated, active hTERT promoter in the epidermis in situ and in the epidermis of OTCs but a deacetylated, silenced hTERT promoter with subsequent propagation in monolayer culture suggesting that histone acetylation is part of the regulatory program to guarantee hTERT expression/telomerase activity in the epidermis. In agreement with the loss of telomerase activity, telomeres shortened during continuous propagation in monolayer culture by an average of approximately 70 base pairs (bp) per population doubling (pd). However, telomere erosion varied strongly between different keratinocyte strains and even between individual cells within the same culture, thereby arguing against a defined rate of telomere loss per replication cycle. In the epidermis in situ, as determined from early-passage keratinocytes and tissue sections from different age donors, we calculated a telomere loss of only approximately 25 bp per year. Since we determined the same rate for the non-regenerating melanocytes and dermal fibroblasts, our data suggest that in human epidermis telomerase is a protective mechanism against excessive telomere loss during the life-long regeneration.

Liver sinusoidal endothelial cells (LSECs) are highly active professional scavenger cells using clathrin-mediated endocytosis to clear the blood from macromolecular waste products. Using confocal microscopy, we observed a remarkable net-like distribution of clathrin heavy chain (CHC) in LSECs while all other cell types examined including various primary endothelial cells and cell lines showed the well-known punctuate staining pattern representing clathrin-coated vesicles (CCV). The net-like distribution of CHC in LSECs co-localized fully with microtubules, but not with actin. Upon 3D imaging, the net-like distribution of CHC resolved into numerous CCVs organized along the microtubules. The CCVs only partially co-localized with early endosome antigen 1 (EEA1) and adaptor protein 2 (AP-2). Endocytic vesicles containing ligand destined for degradation (FITC-AHGG) were organized along the clathrin/tubulin net-like structures, whereas transferrin-containing recycling vesicles co-localized to a much lower extent. Disruption of the microtubules by nocodazole treatment caused a collapse of the net-like organization of CCVs as well as a profound redistribution of EEA1, AP-2 and FITC-AHGG-containing vesicles, while transferrin internalization and recycling remained unaffected.

Telomeres are specialized DNA-protein structures at the ends of the linear chromosomes. In mammalian cells, they are composed of multifold hexameric TTAGGG repeats and a number of associated proteins. The double-stranded telomeric DNA ends in a 3' single stranded overhang of 150 to 300 base pair (bp) which is believed to be required for a higher order structure (reviewed in (Blackburn, 2001)). One important model is that the telomeres form loop structures, the T-loops, and by invasion of the 3' overhang into the duplex region of the double stranded part protect the DNA against degradation and hinder the cellular machinery to recognize the ends as broken DNA, thus providing chromosomal integrity (Griffith et al, 1999). If telomeres become critically short they loose their capping function, become sticky, and are prone to illegitimate chromosome end-to-end fusions. The resulting dicentric chromosomes are highly unusable and because of bridge-fusion-breakage cycles they give rise to chromosomal translocations, deletions, and amplifications. Thus, critically short telomeres are thought to be responsible for the onset of genomic instability. In addition, we provide evidence that in a length-independent manner telomeres can confer to genomic instability by forming telomericaggregates which through chromosomal dys-locations contribute to chromosomal aberrations.

Telomeres are specialized structures at the ends of the chromosomes that, with the help of proteins--such as the telomere repeat-binding factor TRF2 -, form protective caps which are essential for chromosomal integrity. Investigating the structure and three-dimensional (3D) distribution of the telomeres and TRF2 in the nucleus, we now show that the telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells, while they extend more widely during mitosis. TRF2 is present at the telomeres at all cell cycle phases. During mitosis additional TRF2 protein concentrates all around the chromosomes. This change in staining pattern correlates with a significant increase in TRF2 protein at the S/G2 transition as seen in Western blots of synchronized cells and is paralleled by a cell cycle-dependent regulation of TRF2 mRNA, arguing for a specific role of TRF2 during mitosis. The distinct territorial localization of telomeres is abrogated in a HaCaT variant that constitutively expresses c-Myc--a protein known to contribute to genomic instability. These cells are characterized by overlapping telomere territories, telomeric aggregates (TAs), that are accompanied by an overall irregular telomere distribution and a reduced level in TRF2 protein. These TAs which are readily detectable in interphase nuclei, are similarly present in mitotic cells, including cells in telophase. Thus, we propose that TAs, which subsequently also cluster their respective chromosomes, contribute to genomic instability by forcing an abnormal chromosome segregation during mitosis.