FLASH radiotherapy enables dose escalation and improves survival in an orthotopic muscle-invasive bladder cancer mouse model

Abstract Objectives FLASH radiotherapy is an innovative technique that delivers radiation at ultra-high dose rates (UHDR), offering tumour control comparable to conventional (CONV) radiotherapy while significantly reducing normal tissue toxicity. Here we aim to determine the effects of FLASH compared to CONV radiotherapy in muscle-invasive bladder cancer (MIBC) models. Methods Using an in-house 6 MeV linear accelerator able to deliver electron beam at UHDR or CONV dose rate, we employed clonogenic survival assays, RNA sequencing (RNA-seq), and in vivo tumour growth analyses using MBT2 cells and C3H MIBC models. Both subcutaneous and orthotopic tumour models were used to assess tumour response, survival and treatment-related toxicity as demonstrated by weight loss. Results Clonogenic analysis demonstrated comparable cancer cell survival between FLASH and CONV irradiation in vitro. RNA-seq analysis of in vitro irradiated cells revealed similar gene expression at 5 Gy but significant transcriptional divergence at 10 Gy. Intestinal organoids exhibited preserved growth after FLASH compared with CONV irradiation, consistent with a normal tissue sparing effect. In subcutaneous models, FLASH and CONV radiotherapy exhibited similar tumour responses. However, in the orthotopic model, FLASH radiotherapy enabled dose escalation, significantly extending survival at 15 Gy (p = 0.02) and 17.5 Gy (p = 0.004). Dose rate (100 vs 106 Gy/s) did not significantly affect survival. The benefit of single-fraction FLASH was not retained with fractionated (3 × 7.3 Gy) delivery. Conclusion FLASH radiotherapy demonstrates significant potential for treating MIBC, offering enhanced survival through effective dose escalation. These findings support continued investigation into optimal FLASH parameters and its clinical application. Advances in knowledge