ESTRO 2023 - Abstract Book

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ESTRO 2023

Purpose or Objective Bladder cancer is the 9th most common cancer worldwide and associated with significant morbidity and mortality. The toxic effects of chemo-radiotherapy can be poorly tolerated, highlighting the need for novel systemic treatments to be used in conjunction with radiotherapy. Certain gut bacteria can produce short chain fatty acids (SCFAs), including acetate, butyrate, and propionate, that are cytotoxic to tumour cells and protective to normal tissues. Bacteroides acidifaciens , an acetate producer, has shown a promising correlation with tumour cell radiosensitisation in vivo , but further investigations into combinations of cross-feeding species and assessment of their metabolites are required. This study aims to assess the (1) cytotoxic and (2) radio-sensitising effects of extracts harvested from acetate producer Bacteroides uniformis ( BU ) and butyrate producer Faecalibacterium prausnitzii ( FP ) cultured alone and in combination ( BU+FP ), and supplemented with dietary fibres, on mouse bladder tumour (UPPL1591) cells, and (3) to quantify the resulting SCFAs. Materials and Methods (1) Cell viability: UPPL1591 cells were treated for 48 hr with extracts from BU , FP , and BU+FP, supplemented with inulin, psyllium, or glucose (n=3). Effect of the bacterial extracts on cell viability was measured using MTT assay. (2) Colony formation: UPPL1591 cells were seeded at 100 – 3200 cells/flask for 24 hr and treated for 24 hr with extract from BU+FP supplemented with inulin at IC10 or IC50 concentrations or cell medium only. Cells were irradiated (0 – 8Gy), colony formation was assessed after 7 days, and cell survival plotted (n=3). (3) Bacterial extracts were analysed using gas chromatography. Data were analysed using one-way analysis of variance. Results (1) Cell viability: extracts from co-culture of BU+FP significantly (p<0.001) reduced cell viability compared to BU or FP alone. Both inulin and psyllium supplementation significantly (p<0.001) reduced viability compared to glucose, displaying the cytotoxic potential of co-culturing the bacteria with fibre supplementation. (2) Colony formation: the IC50 concentration significantly reduced cell survival at 2 Gy (p<0.001), and 4 Gy (p<0.001), demonstrating that extracts can radio-sensitise tumour cells. (3) SCFA analysis: butyrate levels were significantly higher (p<0.001) in co-culture of BU+FP with glucose and inulin extracts compared to control, highlighting the co-dependence of BU+FP in butyrate production. Propionate concentrations were significantly (p<0.001) higher in BU+FP extracts supplemented with inulin and psyllium compared to glucose, which may indicate how fibre supplementation mediates its greater cytotoxic effect. Conclusion We have demonstrated the potential of combining selected gut bacteria fed on specific dietary fibres as an alternative to chemotherapy, as both cytotoxic and radio-sensitising to tumour cells, likely mediated by SCFA production. If they result in no worse normal tissue toxicity, such an approach could be useful clinically. C. Showalter 1 , T. Cui 1 , E. Sebastian 1 , P. Rajasekera 1 , J. McElroy 2 , A. Becker 1 , J. Fleming 1 , H. Manring 1 , S.J. Haque 1 , A. Chakravarti 1 1 The Ohio State University, Radiation Oncology, Columbus, USA; 2 The Ohio State University, Biomedical Informatics, Columbus, USA Purpose or Objective Glioblastoma (GBM; WHO grade IV) is the most common high-grade primary malignant tumor with a dismal five-year survival rate of only 5%. Patients with wild-type isocitrate dehydrogenase 1 and 2 (IDHwt) GBM have the worst survival outcomes and little progress has been made since 2005 to improve upon the current standard treatment for GBM consisting of maximal safe surgical tumor resection followed by radiation and concomitant and adjuvant temozolomide (TMZ). Therefore, there is an urgent need to develop novel treatment strategies and identify actionable targets in GBM. In our study we identified proteasome subunit alpha type-7 (PSMA7), a non-catalytic subunit of the 20S proteasome core particle involved in the regulated protein degradation via the ubiquitin-proteasome pathway and with proteasome-independent functions via interactions with HIF1 α , c-Abl, and PAC4 among other oncogenic proteins, to be upregulated in IDHwt GBM. PSMA7 is frequently upregulated in other cancer types and PSMA7 silencing reduces cell proliferation in cervical, colorectal, and gastric cancer. Despite the association between PSMA7 and tumor development in multiple tumor models, the role(s) that upregulated PSMA7 expression has in GBM biology and treatment response is largely unknown. In this study we show that silencing PSMA7 sensitizes GBM cells to ionizing radiation (IR)/TMZ-induced cell death and inhibits double-strand DNA damage repair following IR treatment. Materials and Methods PSMA7 was identified using publicly available mRNA profiling data of two cohorts of patients with primary glioma from the CGGA database. PSMA7 was functionally validated in vitro using RNAi-mediated knockdown (KD) approaches and IDHwt GBM cell lines. Stable cell lines with shRNA-mediated PSMA7 KD were generated. Protein expression was quantified by Western blot analysis. Cell viability was assessed by MTS or ATP assays. A biological irradiator was used to treat cells with IR in vitro to evaluate response to radiation. Results Primary glioma patients with IDHwt tumors had higher PSMA7 mRNA levels compared to those from patients with IDH mutant (IDHmut) tumors. Furthermore, PSMA7 mRNA levels increased as the tumor grade increased in IDHwt primary gliomas. Importantly, patients with primary IDHwt glioma and high PSMA7 mRNA levels (median split) had worse overall survival than patients with low PSMA7 mRNA levels. We found that RNAi-mediated KD of PSMA7 in IDHwt GBM cells increased IR-induced cell death in vitro. Additionally, IDHwt GBM cells with PSMA7 KD had prolonged expression of γ -H2AX after treatment with IR. Also, IDHwt GBM cells with PSMA7 KD demonstrated lower cell viability when treated with TMZ in vitro. Collectively, RNAi-mediated silencing of PSMA7 expression enhanced the sensitivity of GBM cells to IR and TMZ. PO-2218 RNAi-mediated silencing of PSMA7 sensitizes GBM cells to ionizing radiation and temozolomide

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