Spatially fractionated radiotherapy with PD-L1 blockade achieves superior antitumor efficacy and safety in a preclinical model of peritoneal metastasis

Abstract

Abstract Peritoneal metastasis from gastrointestinal cancers has poor outcomes, and whole-abdominal radiotherapy (RT) is limited by toxicity to radiosensitive organs. Spatially fractionated radiotherapy (SFRT) delivers heterogeneous doses, potentially sparing normal tissue while stimulating antitumor immunity. This study aimed to evaluate the efficacy, safety, and immune modulation of SFRT combined with PD-L1 blockade in a syngeneic peritoneal metastasis model. Mouse-specific grid collimators (2–4 mm) were fabricated and tested. CT26-luc cells were injected intraperitoneally, followed by conventional RT (CONV) or 3 mm SFRT at 8–16 Gy, with or without anti–PD-L1. Tumor burden was assessed by bioluminescence, survival and weight monitored, and necropsy performed to evaluate toxicity. Immune profiling of ascites and blood was performed by flow cytometry, and functional outcomes by open-field testing. The 3 mm grid achieved a peak-to-valley dose ratio of ~ 11 and was used for therapy. At 8 Gy, SFRT provided tumor control comparable to CONV RT with less weight loss. At 10 Gy, CONV RT caused early mortality, whereas SFRT maintained 100% survival. SFRT plus anti–PD-L1 induced near-complete tumor regression, reduced ascites, and preserved intestinal length. Immune analysis showed increased CD4⁺/CD8⁺ T cells and M1 macrophages with SFRT; combination therapy further expanded T cells and yielded the lowest neutrophil-to-lymphocyte ratio. Functional testing demonstrated improved locomotion with SFRT plus anti–PD-L1. SFRT combined with PD-L1 blockade achieves potent tumor control, reduces toxicity, and enhances physical performance, supporting its potential as a safer immuno-radiotherapy strategy for peritoneal metastasis.