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

ABSTRACT Emerging evidence shows that Proton FLASH radiotherapy can spare normal tissues while maintaining anti‐tumor efficacy. However, its impact on intestinal stem cells (ISCs) and the gut microbiome remains unclear. Gut microbiome influences ISC's radiosensitivity. In a mouse model of abdominal irradiation, Proton FLASH exposure exhibited improved survival and less crypt‐villus damage compared to Proton Conventional dose rate. Using scRNA‐sequencing, we demonstrated that Proton FLASH exposure using pulsed pencil beam scanning spares two distinct ISC populations, Lgr5+ Crypt‐based columnar cells (CBCs) and a Ly6a+ , Clu+, Areg+, Anxa2+ revival stem cell (revSC) population—by modulating oxidative stress and cell cycle progression. Analysis of α and β‐diversity demonstrated that Proton FLASH modulates gut microbiota composition without compromising overall species richness. Notably, Proton FLASH‐irradiated mice had higher abundances of Alistipes sp . and Akkermensia sp ., both known for protective effects on ISCs and the intestinal mucosa. The role of microbiome in Proton FLASH‐mediated sparing effect was further confirmed by fecal microbiota transplantation, where Proton FLASH‐donor microbiota demonstrated reduced lethality with protection of crypt villus morphology in recipient mice exposed to Proton Conventional dose rate. Our findings highlight the crucial role of the microbiome in the Proton FLASH‐mediated sparing of the mucosal epithelium.