Abstract 3320: Single-cell RNA sequencing analysis and transgenic mouse models reveal differential effects of flash vs. standard proton radiotherapy on gastrointestinal tissues and tumors
Authors: Verginadis Ioannis I. , Martinez Clara Morral , Lim Tristan , Chowdhury Priyanka , Skoufos Giorgos , Kim Michele M. , Velalopoulou Anastasia , Goia Denisa , Luo Leo , Foley Caitlin J. , Reitman Zachary J. , Daniel Andrea R. , Diffenderfer Eric , Dong Lei , Metz James , Kirsch David G. , Minn Andy , Koumenis Constantinos 
Published: June 2022

Abstract
Recent studies suggest that ultrahigh-dose-rate, “FLASH,” radiation therapy (RT) decreases normal tissue damage while maintaining tumor response compared with conventional dose rate RT. Our group has designed and tested the first system to accurately deliver dosimetrically identical FLASH Proton RT (F-PRT; 60-110 Gy/sec) or Standard Proton RT (S-PRT; 0.5-1 Gy/sec) using double-scattered protons. Our purpose is to identify if F-PRT is superior to S-PRT in protecting normal tissues, while equipotent in controlling tumor growth. We previously demonstrated that compared to S-PRT, F-PRT significantly decreases overall mortality from late epithelial gastrointestinal (GI) fibrosis (p&lt;0.005) in mouse models following 14 (males) and 15 Gy (females) whole-abdomen and 18 Gy focal intestinal RT, whereas tumor growth inhibition is essentially indistinguishable between the two modalities. We also found that F-PRT-treated mice displayed a significantly higher percentage of regenerated crypts (EdU incorporation) in comparison to the S-PRT (p&lt;0.01). To better understand the differential responses of the normal intestine between the two treatment modalities, we performed single-cell RNA sequencing on 14 Gy F-PRT and S-PRT-treated intestines at 2, 10, and 20 days post-RT. Analysis revealed enrichment of stem/progenitor epithelial cell populations with increased proliferative signatures and expression of genes related to the interferon-alpha signature in epithelial and immune cells post F-PRT treatment compared to the S-PRT. Immunofluorescence staining of F-PRT and S-PRT treated intestinal tissues confirmed a) the higher proliferation rate of epithelial cells observed in F-PRT-treated mice, b) significantly higher number of infiltrated immune cells in F-PRT- compared to the S-PRT-treated mice and c) higher expression of interferon signaling in immune cells in F-PRT treated mice. Collectively, our preliminary findings suggest that F-PRT may enhance a regenerative or facultative stem cell program that is associated with greater and more persistent IFN Type I signaling. Moreover, using a model of epithelial-specific knockout of p53 (Villin-Cre;p53), we found that mice treated with 13 Gy S-PRT quickly succumb to acute radiation GI syndrome, while F-PRT treated mice had significantly improved survival (median survival: 8.5 days for S-PRT vs 164 days for F-PRT; p&lt;0.05). This suggests that F-PRT may preserve a higher proportion of progenitor epithelial cells with enhanced regeneration potential. Understanding the cellular and molecular basis for the effects of F-PRT provides a framework for clinical application of this novel modality with the potential to improve the therapeutic outcome and quality of life of cancer patients.
Citation Format: Ioannis I. Verginadis, Clara Morral Martinez, Tristan Lim, Priyanka Chowdhury, Giorgos Skoufos, Michele M. Kim, Anastasia Velalopoulou, Denisa Goia, Leo Luo, Caitlin J. Foley, Zachary J. Reitman, Andrea R. Daniel, Eric Diffenderfer, Lei Dong, James Metz, David G. Kirsch, Andy Minn, Constantinos Koumenis. Single-cell RNA sequencing analysis and transgenic mouse models reveal differential effects of flash vs. standard proton radiotherapy on gastrointestinal tissues and tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3320.