FLASH X-ray spares intestinal crypts from pyroptosis initiated by cGAS-STING activation upon radioimmunotherapy

Abstract

DNA-damaging treatments such as radiotherapy (RT) have become promising to improve the efficacy of immune checkpoint inhibitors by enhancing tumor immunogenicity. However, accompanying treatment-related detrimental events in normal tissues have posed a major obstacle to radioimmunotherapy and present new challenges to the dose delivery mode of clinical RT. In the present study, ultrahigh dose rate FLASH X-ray irradiation was applied to counteract the intestinal toxicity in the radioimmunotherapy. In the context of programmed cell death ligand-1 (PD-L1) blockade, FLASH X-ray minimized mouse enteritis by alleviating CD8 + T cell-mediated deleterious immune response compared with conventional dose rate (CONV) irradiation. Mechanistically, FLASH irradiation was less efficient than CONV X-ray in eliciting cytoplasmic double-stranded DNA (dsDNA) and in activating cyclic GMP-AMP synthase (cGAS) in the intestinal crypts, resulting in the suppression of the cascade feedback consisting of CD8 + T cell chemotaxis and gasdermin E-mediated intestinal pyroptosis in the case of PD-L1 blocking. Meanwhile, FLASH X-ray was as competent as CONV RT in boosting the antitumor immune response initiated by cGAS activation and achieved equal tumor control in metastasis burdens when combined with anti–PD-L1 administration. Together, the present study revealed an encouraging protective effect of FLASH X-ray upon the normal tissue without compromising the systemic antitumor response when combined with immunological checkpoint inhibitors, providing the rationale for testing this combination as a clinical application in radioimmunotherapy.

FLASH Irradiation Regulates IFN-β induction by mtDNA via Cytochrome c Leakage

“…3a, b), since mitochondrial DNA accounts for only 0.25% of the whole human cellular DNA 38 . In the prior investigations conducted by Shi et al 13 involving FLASH X-rays irradiation (120 Gy/s), a significant reduction in cytosolic dsDNA of intestinal organoids was observed through PicoGreen fluorescence quantification, which is a much stronger indicator than what we observed. Nevertheless, the normal human breast cell line MCF-10A used for our study is immortalized rather than the primary cells isolated from animal tissues, such that genes involved in cell cycle regulation, apoptosis, or DNA repair may exhibit elevated expression levels, and increase the intrinsic genome instability.…”

“…1e are obtained by using the single-hit, multi-target model 31 SF=1-(1exp(-kD)) n , the fitting parameters (k, n) for MCF-10A, MDA-MB-231, and 4T1-luc cell lines are (0.46, 2.94), (0.63, 2.43), and (0.49, 1.91), respectively. The cGAS-STING activation post electron irradiation was investigated to find out the potential dose rate impact, as reported by Shi et al in their FLASH X-ray experiments 13 . The fluorescence intensity of the cytosolic dsDNA extract was normalized to that of the corresponding whole-cell extract under irradiation with different dose rates.…”

“…Several hypotheses for the FLASH effect have been proposed from both simulation and experimental work, such as the radiolytic oxygen depletion 9,10 , circulating immune cells protection 12 , and relatively intact DNA integrity post FLASH irradiation 13 . A recent in vitro study 14 discovered that FLASH proton irradiation at 100 Gy/s maintained the morphology and function of mitochondria within normal human lung fibroblasts compared to the case post 0.33 Gy/s irradiation, which could be associated with the radiation induced dephosphorylation of the p-Drp1 protein and mitochondrial fission.…”

Absence of Tissue-Sparing Effects in Partial Proton FLASH Irradiation in Murine Intestine

“…Ruan et al found improvements in crypt survival and fewer changes to the microbiota after FLASH irradiation with a 6 MeV electron linear accelerator [ 34 ]. Ultra-high dose rate photon irradiation reportedly reduced intestinal pyroptosis and attenuated abdominal toxicity [ 35 ]. Less mortality was also observed following proton FLASH exposure when the mouse was placed either in the plateau region before the Bragg peak or within the spread-out Bragg peak (SOBP) [ 13 , 36 ].…”

The dose-related plateau effect of surviving fraction in normal tissue during the ultra-high-dose-rate radiotherapy

“…Although FLASH has been intensively researched, the mechanism is still subject to debate, including oxygen depletion (Jansen et al 2021), radical recombination (Labarbe et al 2020), change in endogenous reactive oxygen species (ROS) (Spitz et al 2019, Sultana et al 2022, immune response (Wilson et al 2020), radiation induced genomic instability (Shi et al 2022). The organisms damage from ionizing radiation primarily by shattering the DNA structure, which can be classified into direct damage and indirect damage.…”