ESTRO 2020 Abstract Book

S121 ESTRO 2020

the tumor growth and enhanced radiosensitivity in a PANC- 1-xenograft model. The expression of nuclear BCL10, BCL3, and NF-κB (p65) were also downregulated in the shBCL10-transfected PANC-1 xenograft tissues. Conclusion Our findings indicated that nuclear BCL10 plays an important role in controlling tumor growth and radiosensitivity of PDAC cells by activating AKT/mTOR, DNA-repair, and NF-κB-related signaling pathways. PH-0236 CD44v3-10 affects G2-arrest and radiosensitivity of S-phase cells via homologous recombination M. Al Bazaz 1 , S. Lerch 1 , R. Hannen 1 , K. Lauber 2 , E. Dikomey 1,3 , R. Engenhart-Cabillic 1 , U. Schötz 1 1 Philipps-University Marburg- University Hospital Gießen and Marburg, Department of Radiotherapy and Radiooncology, Marburg, Germany ; 2 University Hospital- LMU Munich, Department of Radiation Oncology, Munich, Germany ; 3 University Medical Center Hamburg Eppendorf, Laboratory for Radiobiology & Experimental Radiooncology-, Hamburg, Germany Purpose or Objective Locally advanced head and neck squamous cell carcinomas (HNSCC) exhibit frequent therapeutic failure, despite intense radiotherapeutic treatment regimens. Expression of tumor stem cell marker CD44 was shown to be negatively associated with survival of HNSCC-patients receiving radiochemotherapy. The most prominent isoform of CD44 in keratinocytes is CD44v3-10, a hyaluronic acid receptor with coreceptor function in activating MAPK- signaling. Functionally, we wanted to understand the role of the receptor in response towards irradiation and clarify its potential to serve as marker for innovative treatment options, specifically carbon ion irradiation (12C). Initial preclinical in vitro studies in our lab focus on examination of proliferation, cell cycle regulation and DNA repair. Material and Methods In vitro the HNSCC cell lines UPCI:SCC-040 (high CD44v3- 10 expression) and UPCI:SCC-131 (low CD44v3-10 expression) were used. CD44v3-10 was overexpressed in UPCI:SCC-131 and transcripts were knocked down in UPCI:SCC-040 via a specific inhibitory siRNA. Clonogenic survival after irradiation (photon and 12C) was measured via colony formation assay. Cell cycle was measured in ethanol-fixed cells stained with propidium iodide using a flow cytometer. To synchronize cells in G1- and late S- phase, a double thymidine block was performed. DNA repair was examined by immunofluorescent visualization of double-strand breaks (γH2AX/53BP1 foci) after irradiation and Olaparib treatment. Results The presence of CD44v3-10 transcripts stimulates proliferation. For photon irradiation, overexpression of CD44v3-10 led to a decreased, while knock-down led to an increased cellular sensitivity. Using synchronized cells, effect of knock-down on radiosensitivity was found to be pronounced for cells in late S-phase, while no effect was seen for G1-phase cells. This finding suggests that CD44v3- 10 is involved in DNA damage response by homologous recombination (HR). In line with this, knockdown of CD44v3-10 was found to enlarge the radiation-induced G2/M-arrest and for these cells radiosensitivity was enhanced when treated by Olaparib. This decrease in clonogenic survival could be attributed to an enhanced number of residual double strand breaks. No difference in radiosensitivity was measured for CD44v3-10 depleted cells when exposed to 12C irradiation. Conclusion An overexpression of the tumor stem cell marker CD44v3- 10 plays a relevant role in radiotherapeutic treatment options. The data suggests for the first time, that CD44v3- 10 affects radiation-associated cell cycle regulation, repopulation and DNA repair, because of its relevance for

HR. 12C irradiation sensitizes all cells, independently from variations in CD44v3-10 status to a similar level. Further validation experiments are on the way. Acknowledgements: This project is funded by MIT- Forschung, Philipps-University Marburg PH-0237 Radiotherapy-induced mesenchymal gene signatures attenuated by VEGF-A blockade in mouse glioblastoma A.E. Nieto 1 , K. Unger 2,3 , D. Fleischmann 1 , D. Piehlmaier 2 , 1 University Hospital- LMU Munich- Germany, Department of Radiation Oncology, Munich, Germany ; 2 Helmholtz Zentrum München, Research Unit of Radiation Cytogenetics, Munich, Germany ; 3 German Research Center for Environmental Health GmbH, Clinical Cooperation Group 'Personalized Radiotherapy in Head and Neck Cancer' Helmholtz Center Munich, Neuherberg, Germany ; 4 University Medical Center Regensburg, Department of Neurosurgery, Regensburg, Germany ; 5 German Cancer Consortium DKTK, Partner Site Munich, Munich, Germany Purpose or Objective Modern glioblastoma (GBM) research has progressed to the consensus that the mesenchymal subtype is associated with worse prognosis, treatment resistance, and therapeutic failure. Mesenchymal gene expression is composed of GBM tumor cell-intrinsic and stromal contributions. GBM growth outpaces its demands for adequate blood supply by producing tortuous feeder arteries and draining veins via abnormal arterio- and venogenesis. VEGF-A is intricately involved in these processes, namely by stabilizing and maturing new blood vessels and acting as a potent chemokine for certain immune cells, particularly of the myeloid lineage. The VEGF-A-targeting antibody Bevacizumab improves patients' quality of life, reduces the need for steroid treatment, and prolongs progression-free survival when combined with radio(chemo)therapy. Additionally, Bevacizumab was very recently reported to reduce normal brain toxicity during reirradiation of GBM. Here, we elucidate the molecular mechanisms of the above mentioned effects of VEGF-A blockade in an orthotopic mouse GBM model. Material and Methods Mouse GL261 GBM cells were implanted orthotopically into C57BL/6 mice. Established tumors were subjected to fractionated radiation (RTX) over 2 weeks with 5x 2 Gy per week with or without concomitant anti-VEGF-A treatment. Tumor growth was monitored via contrast enhanced conebeam CT scans, and mice were sacrificed upon reaching pre-defined humane endpoints. Explanted tumor and brain tissues were processed for microarray-based gene expression analyses. Immunogenomic and gene set enrichment analyses (GSEA) were performed using R and Cytoscape. Reference data and gene sets were accessed from public resources. Results GL261 cells represent a model system of mesenchymal GBM as extracted from GSEA of intracranial transplants and public data sets. Mesenchymal gene expression is attributable – in part – to the tumor cell-intrinsic transcriptome as observed in vitro. In vivo, myeloid immune cell signatures, including microglial, monocytic, and neutrophilic expression patterns contribute to "mesenchymality". RTX enforces enrichment of mesenchymal and derichment of proneural and classical gene expression patterns, paralleled by enrichment of myeloid immune cell gene signatures. Anti-VEGF-A treatment reduces mesenchymal gene expression and attenuates radiation-induced "mesenchymalization". In normal brain tissue, RTX induces expression of necrosis- V. Albrecht 1 , J. Maas 1 , M. Proescholdt 4 , H. Zitzelsberger 2,3 , C. Belka 1,3,5 , K. Lauber 1,3,5