FLASH radiotherapy improves survival in mouse glioblastoma and spares circulating antigen reactive CD8 + T-cells

Introduction: Glioblastoma is a lethal brain tumor with poor response to current therapies, which include surgery, chemotherapy, and conventional radiation therapy (CONV-RT). Although, CONV-RT (0.01Gy/second) to brain tumors stimulate tumor antigen release, it also recruits immunosuppressive myeloid-derived suppressor cells and is associated with neurotoxicity. Ultrahigh-dose-rate or FLASH-RT, which delivers CONV-RT doses over a significantly shorter period (more than 40Gy/second) maintains tumor control, reduces normal tissue injury and is less immunosuppressive compared to CONV-RT across multiple cancer types. In our study, we compared the effects of FLASH-RT to CONV RT in syngeneic mouse GL261 glioblastoma-bearing mice. We hypothesized that FLASH-RT would be equally or more effective than CONV-RT for tumor control and result in less immunosuppression within the tumor and systemically. Methods: We stereotactically implanted 2 × 10 5 mouse GL261 cells into the right forebrain of C57BL/6 mice. Five days after tumor initiation, tumor bearing mice were treated with Sham-RT (control), CONV-RT (mean dose rate > 0.373 Gy/s), or FLASH-RT (mean dose rate >3.6x10 6 Gy/s). Brain tumor tissue and peripheral blood were collected on days 5 and 12 after treatment. To assess for changes in the tumor microenvironment after CONV-RT or FLASH-RT, we performed 10x Xenium spatial transcriptomics analysis (stRNA-seq; brain tumor, n = 6 mice per group), Lunaphore COMET multiplexed immunofluorescence assay (brain tumor, n = 6 mice per group), and flow cytometry (peripheral mononuclear cells, n = 6 mice per group). We also evaluated survival outcomes following treatment (n = 8 mice per group). Results: FLASH-RT significantly improved overall survival rate of GL261 bearing mice compared to CONV-RT ( P <0.05 ) and Sham-RT ( P < 0.001 ). FLASH-RT treatment markedly increased intratumoral CD8+ T-cell infiltration compared with CONV-RT ( P <0.01 ) and Sham-RT ( P < 0.01 ). We also found that compared to FLASH-RT, CONV-RT caused a significant decrease in circulating PD-1 + CD8 + T-cells ( P < 0.01 ), a potent antigen reactive cytotoxic T-cell population previously identified in human patients with glioblastoma. Conclusion: FLASH-RT is associated with better tumor control in mouse GL261 glioblastoma, increased intratumoral CD8+ T-cell infiltration, and preserves circulating antigen reactive PD1 + CD8 + T-cells. These results indicate that FLASH-RT may synergize better with immune checkpoint inhibitors to re-invigorate anti-tumor T-cell responses against glioblastoma.