Proton FLASH Exposure Preserves Gut Commensal Microbiomes and Spares Intestinal Stem Cells

Emerging evidence shows Proton FLASH radiotherapy can spare normal tissues while maintaining anti-tumor efficacy. However, its impact on intestinal stem cell (ISC) populations and the gut microbiome remains unclear. This is critical, as the gut microbiome influences ISC radiosensitivity. In a mouse model of radiation-induced gastrointestinal syndrome, FLASH-irradiated mice exhibited better survival and less crypt-villus damage compared to mice exposed to conventional proton irradiation. Using scRNA-sequencing, we demonstrated that proton FLASH exposure using pulsed pencil beam scanning spares two distinct ISC populations— Lgr5+ CBCs and a Clu+, Mif+, Fabp2+, Anxa2+ revival stem cell (revSC) population—by modulating oxidative stress and cell cycle progression. Analysis of alpha and beta diversity demonstrated that FLASH modulates gut microbiota composition without compromising overall species richness. Notably, FLASH-irradiated mice had higher abundances of Alistipes sp. and Akkermensia sp., both known for protective effects on ISCs and the intestinal mucosa. The critical role of microbiome in FLASH-mediated sparing effect against radiation toxicity was further confirmed by fecal microbiota transplantation, where FLASH-donor microbiota demonstrated reduced lethality in recipients exposed to proton irradiation with conventional dose rate. Our findings highlight the crucial role of the microbiome in the FLASH-mediated sparing of the mucosal epithelium.