ESTRO 2020 Abstract Book

S120 ESTRO 2020

model, and the consenquences of these microbial function changes using LC-MS. Conclusion The changes in structures and diversities of intestinal microorganisms in radiation enteritis were dependent on time-dose relationship, and they activated the change of metabolic function as well as the symbiotic environment of intestine-microorganism. However, the fluctuation of intestinal microorganisms was normalized as the radiation interference terminated. It indicates that the microbial ecology can be improved by controlling the dose constraints of the normal tissues in radiotherapy. PH-0234 The Adaptive Resistome in TNBC: Functional Targeting in Patient-derived Organoids and Cell Lines S. Classen 1 , E. Rahlf 1 , J. Görte 2 , A.C. Parplys 1 , U. Schumacher 3 , C. Petersen 4 , K. Rothkamm 1 , M. Toulany 5 , N. Cordes 2 , K. Borgmann 1 1 University Medical Center Hamburg-Eppendorf, Laboratory for Radiobiology and Experimental Radiooncology, Hamburg, Germany ; 2 OncoRay – National Center for Radiation Research in Oncology- Technische Universität Dresden, Molecular and Cellular Radiobiology, Dresden, Germany ; 3 University Medical Center Hamburg-Eppendorf, Department of Anatomy and Experimental Morphology, Hamburg, Germany ; 4 University Medical Center Hamburg-Eppendorf, Department of Radiotherapy and Radiation Oncology, Hamburg, Germany ; 5 University of Tuebingen, Division of Radiobiology & Molecular Environmental Research Department of Radiation Oncology, Tuebingen, Germany Purpose or Objective Despite a good initial response to radio- and chemotherapy, triple-negative breast cancer (TNBC) remains the subtype with the worst overall prognosis and survival. One major challenge in TNBC treatment is the acquired resistance against radio- and chemotherapy of recurrent tumors. Resensitizing the tumor cells by targeted inhibition of resistome-related factors seems to be a promising therapeutic approach. Therefore, this project aims to elicit druggable factors associated with DNA replication and repair pathways, which are known to play an important role in the adaptive resistome. Material and Methods Patient-derived organoids and isogenic MCF7 clones with different BRCA1 expression levels were grown in 3D cell culture. For validation of the 3D architecture, the organoids were stained with hematoxylin and eosin. As the BRCA1 level has a direct impact on homologous recombination (HR) and radioresistance, the viability of the clones after irradiation up to 12 Gy was monitored using an ATP-luminescence assay. Additionally, the DNA fiber assay was employed to investigate the replication stress level after irradiation with 6 Gy. The number of stem cells in the cultures was detected by analysis of the stem cell marker aldehyde dehydrogenase 1 (ALDH1) using the Aldefluor TM assay. Results The histological analysis revealed a successful growth of the patient-derived organoids and 3D organized spheroids of the isogenic MCF7 clones. As expected, the radiosensitivity was dependent on the BRCA1 expression level. Likewise, sensitization to irradiation correlated with an increase in the replication stress level represented by fork stalling after 6 Gy irradiation (R 2 = 0.96), whereas resistance to irradiation was accompanied by an increase up to 25 % in the number of ALDH1-positive stem cells. Dependent on the results of the viability analysis and replication stress level after irradiation the clones were classified as radio-resistant or sensitive. Since this classification could be successfully employed, we are currently conducting the PAMgene technology to characterize the serine/threonine kinase profiles of radio- resistant and –sensitive organoids to identify possible

druggable kinases. The radioresistant organoids will be treated with a combination of clinically relevant inhibitors against the identified kinases to determine the resensitizing effects. Conclusion Collectively, these results indicate that we can identify radio-resistant or -sensitive tumors by monitoring the survival and replication stress level after irradiation. This classification enables the targeted analysis of factors (e.g. kinases) in radioresistant cells, thus, providing possible new targets to resensitize the cells to radiotherapy. Clinically, this would offer a new approach for the treatment of radioresistant TNBCs. PH-0235 Downregulation of BCL10 attenuates tumor growth and enhances radiosensitivity in pancreatic cancer M. Wei 1 , S. Yang 1 , K. Yeh 1 , H. Lee 1 , Y. Tien 2 , A. Cheng 1 , S. Kuo 3 1 National Taiwan University Hospital, Department of Oncology, Taipei, Taiwan ; 2 National Taiwan University Hospital, Department of Surgery, Taipei, Taiwan ; 3 National Taiwan University Hospital, Division of Radiation Oncology- Department of Oncology, Taipei, Taiwan Purpose or Objective We previously reported that in breast cancer cell lines and diffuse large B-cell lymphoma cell lines, activation of TNF- α upregulates NF-κB signaling and nuclear translocation of BCL10. Because NF-κB activation has been shown to be involved in the pathogenesis and the radioresistance of pancreatic ductal adenocarcinoma (PDAC), we investigated whether inhibition of BCL10 can attenuate tumor growth and enhance radiosensitivity of PDAC cells. Material and Methods Three PDAC cell lines (mutant K-RAS lines: PANC-1 and AsPC-1; wild type K-RAS line: BxPC-3) were used in this study. We transfected three PDAC cell lines with BCL10 short hairpin RNA (shBCL10), and discovered that the majority of BCL10 translocated to cytoplasm from nuclei. Clonogenic assay was used to determine the radiosensitivity, whereas cell cycle was analyzed by flow cytometry. The expressions of BCL10-, cell cycle-, AKT/mTOR signaling pathway (p-mTOR, p-eIF4E, and p- rpS6), and NF-κB-related signaling molecules were assessed by immunoblotting. The DNA damage-related (γ- H2AX staining), and DNA double-strand break (DSB) repair- related (p-DNA-PKcs and p-ATM) molecules were also assessed. The differences of NF-κB activity between control cells and shBCL10 cells of PDAC were determined by NF-κB luciferase assay. The biological significances of BCL10 in determining tumor growth and radiotherapy effect of PDAC cell lines were further investigated in a PANC-1-xenograft model. Results We found that shBCL10 inhibited the cell viability and enhanced cytotoxicities of gemcitabine and oxaliplatin in three PDAC cell lines. Furthermore, shBCL10 differentially blocked cell cycle progression in PDAC cell lines; G1 phase arrest in wild-type K-RAS cell lines (downregulation of cyclin D1 expression in BxPC-3) and G2/M phase arrest in mutant K-RAS cell lines (downregulation of Cdc2 expression in PANC-1). ShBCL10 downregulated expressions of nuclear BCL10, BCL3, p-IκBα, and NF-κB (p65), and NF-κB activation. We observed that shBCL10 significantly inhibited cell viability and increased apoptosis (sub-G1 arrest) following 5 Gy irradiation in PANC-1 and BxPC-3 cells. When compared with normal PDAC cells treated with 5 Gy irradiation, the expression of AKT/mTOR signaling and DNA DSB repair-related molecules were downregulated, whereas cleaved caspase-3, cleaved PARP, and γ-H2AX expressions were upregulated in shBCL10-transfected PDAC cells treated with 5 Gy irradiation. Inhibition of BCL10 expression also reduced