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

Chugh, R. M.; Setianegara, J.; Schueddig, E.; Lin, Y.; Bhanja, P.; Rehman, S.; Krepel, S.; Koestler, D.; Chen, R. C.; Cook, K. L.; Saha, S.

doi:10.1101/2025.09.28.679072

bioRxiv · 10.1101/2025.09.28.679072

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

Chugh, R. M.·Setianegara, J.·Schueddig, E.·Lin, Y.·Bhanja, P.·Rehman, S.·Krepel, S.·Koestler, D.·Chen, R. C.·Cook, K. L.·Saha, S.

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Abstract

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.

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Chugh, R. M., Setianegara, J., Schueddig, E., Lin, Y., Bhanja, P., Rehman, S., Krepel, S., Koestler, D., Chen, R. C., Cook, K. L., Saha, S.. 2025-09-30. Proton FLASH Exposure Preserves Gut Commensal Microbiomes and Spares Intestinal Stem Cells. https://doi.org/10.1101/2025.09.28.679072

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