ESTRO 2023 - Abstract Book

S330

Sunday 14 May 2023

ESTRO 2023

Materials and Methods C57BL/6 mice were inoculated in the flank with UPPL 1591 mouse bladder tumour cells when starting low (0.2% cellulose) and high fibre (5% psyllium, 5% psyllium plus 10% resistant starch/10% inulin) diets (N=30) and tumours allowed to grow to 100 mm3. In another cohort, the tumours were irradiated with 6 Gy ionising radiation (IR) when they reached 100 mm3 (N=60) and allowed to grow up to 700 mm3. Gut microbiota, metabolomics, and immunity analyses were performed by using 16S rRNA sequencing, ion chromatography-mass spectrometry and IHC. For normal tissue studies, mice received 10- 14 Gy (N=84, acute) or 5x5Gy (N=40, late) IR to their lower abdomen, and intestines were studied by crypt assay at 3.75 days. Results Psyllium plus inulin significantly decreased tumour (P=0.007) and delayed tumour growth following IR (P<0.001) compared to 0.2% cellulose. It raised Bifidobacterium relative abundance up to 40% (P<0.001) and intratumoural CD8+ cell numbers (P=0.0129 compared to psyllium plus RS). Post-IR, tumour control positively correlated to Lachnospiraceae family abundance (R2=0.534, P=0.016) and caecal butyrate levels (P=0.041). In responders to psyllium plus inulin, increased cytokine and receptor and T cell functional pathways and decreased exhausted CD8+ cells in tumours were found. Psyllium plus RS significantly radiosensitised the tumours compared to 0.2% cellulose and psyllium (P=0.004 and <0.001) and this positively correlated to Bacteroides genus abundance (R2=0.573, P=0.011). Although local immunity was suppressed, psyllium plus RS increased the caecal isoferulic acid level (R2=0.472, P=0.028), associated with a favourable response. Gut microbes also involved in tumour control by psyllium plus resistant starch included Peptostreptococcaceae family, Parasutterella and Faecalibaculum genera, and Muribulaceae family. Furthermore, the two diets mitigated the radiation injury caused by 14 Gy in intestinal crypt assays (P=0.08 and 0.011). All psyllium-containing diets increased caecal short- chain fatty acids (P<0.001), and prevented the delayed weight gain seen with 0.2% cellulose after IR. Conclusion Since psyllium plus either RS or inulin improved the radiation therapeutic ratio, prebiotics may be useful in pelvic cancer patients receiving radiotherapy. OC-0429 Interplay of replication stress and immune signaling drives radioresistance in BRCA1 mutated cells S. Classen 1 , E. Rahlf 1 , J. Jungwirth 2 , S. Gehre 3 , M. Rückert 3 , H. Pospiech 2,4 , K. Rothkamm 1 , U.S. Gaipl 3 , C. Petersen 5 , K. Borgmann 1 1 University Medical Center Hamburg-Eppendorf, Laboratory for Radiobiology and Experimental Radiooncology, Hamburg, Germany; 2 Leibniz Institute on Aging - Fritz Lipmann Institute, Project Group Biochemistry, Jena, Germany; 3 Universitätsklinikum Erlangen, Translational Radiobiology, Department of Radiation Oncology, Erlangen, Germany; 4 University of Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu, Finland; 5 University Medical Center Hamburg- Eppendorf, Department of Radiotherapy and Radiation Oncology, Hamburg, Germany Purpose or Objective Expanding the number of patients that could benefit from immunotherapy is a major aim of many ongoing studies. Lately it has become evident, that an increase in replication stress can lead to accumulation of cytosolic DNA, followed by activation of the intracellular immune response via the cGAS/STING pathway. Subsequently the inflammatory signaling is activated. Enhancing this activation by combined treatment with irradiation, chemotherapeutics or DNA damage response and immune checkpoint inhibitors might lead to new therapeutic options. This project therefore aims to maximize the anti- tumor immune effects by increasing replication stress in BRCA1 mutated breast cancer cell lines using novel combinations of radio-chemotherapy. Materials and Methods Isogenic MCF7 and MDA-MB231 clones with mutations in BRCA1 were generated using CRISPR/Cas9. Illumina next-generation sequencing was used for analysis of the mutations. HR-capacities were determined by plasmid-reconstruction assay and cell cycle distributions were analyzed by propidium iodide staining. For determination of the radio- and chemoresistance the colony formation assay was used. BRCA1, RAD51, RPA and yH2AX foci formation, micronuclei formation and IRF3 translocation were analyzed by (immuno)fluorescence microscopy. To analyze DNA replication, the DNA fiber assay was conducted. Cytosolic DNA was measured using the PicoGreen Assay. PD-L1 expression on the cell surface was analyzed by multicolor flow cytometry. Results To our surprise, mutations in non-functional domains of BRCA1 resulted in resistance, as well as sensitivity against different DNA damaging agents and irradiation (IR) in the generated clones, despite a similar significant reduction of the homologous recombination (HR)-capacity (p ≤ 0.001). One BRCA1 mutated clone showed indeed significantly increased survival after treatment with mitomycin C and the PARP1 inhibitor talazoparib, which both cause damages mainly repaired by HR, compared to the parental cell line. We revealed that this resistance is likely associated with (I) more efficient DNA repair, shown by high RAD51 foci formation (p ≤ 0.05) (II) avoidance of DNA replication stress, indicated by efficient replication fork restart after IR (p ≤ 0.001) and low rates of stalled replication forks after hydroxyurea treatment (p ≤ 0.01) (III) differences in the activation of immune signaling in response to DNA damage, shown by cytosolic DNA after IR, micronuclei formation and IRF3 translocation. Further, a change in PD-L1 surface expression was observed. Conclusion Our results indicate that radiochemoresistance may be linked to DNA repair, replication stress and immune signaling in the analyzed cell lines. Targeting at least two of these pathways at the same time may offer a new therapeutic approach to treat tumors that have been shown to be therapy resistant before. OC-0430 Class I histone deacetylase inhibitor Entinostat sensitises prostate cancer cells to radiation B. McCullough 1 , J. Manley 1 , S. McDade 2 , M. LaBonte Wilson 1 1 Queen's University Belfast, Patrick G Johnson Centre for Cancer Research, Belfast, United Kingdom; 2 Queen's University Belfast, Patrick G Johnson Centre for Cancer Research, Belfast , United Kingdom