FLASH X-rays preserve neutrophil chromatin to boost antitumor immunity

Abstract

Ultrahigh dose-rate FLASH radiotherapy spares normal tissue while preserving antitumor activity, positioning it as a promising technology in radiation oncology, but its immunomodulatory mechanisms remain elusive. Here, using a bilateral subcutaneous tumor model in mice, we show that 8-Gy FLASH X-rays, versus an equal dose of conventional dose-rate (CONV) X-rays, elicit a robust abscopal antitumor effect with anti-PD-1 therapy. Single-cell transcriptome and T cell receptor (TCR) profiling reveal that FLASH X-rays plus anti-PD-1 enhance antitumor immune infiltration and TCR clonal expansion in both irradiated and abscopal tumor lesions. Mechanistically, the ultrashort temporal continuity of FLASH dose delivery conserves chromatin compaction in neutrophils during exposure and inhibits neutrophil extracellular trap (NET) formation, thereby dismantling NET-mediated CD8+ T-cell exclusion within primary tumors. Conversely, CONV or split-dose FLASH X-rays induce marked chromatin modifications prior to dose completion, leading to chromatin hyper-relaxation which promotes NETosis and consequently abrogates systemic antitumor immunity. Notably, this detrimental effect can be reversed by pharmacologically suppressing PARylation-mediated chromatin modification and decondensation in neutrophils. These findings identify radiation-induced aberrant chromatin relaxation in neutrophils as a critical determinant of radioimmunotherapy outcomes, providing a rationale for redefining the FLASH effect and advancing its clinical translation.