PD-1 inhibitor improves radiosensitivity by tumor vessel normalization.

BACKGROUND: Host immunity status and hypoxia are the hallmarks of radiosensitivity. Induction of anti-PD-1 immunotherapy demonstrates promise in locally advanced tumor radiotherapy, but whether anti-PD-1 immunotherapy improves radiosensitivity is unclear. METHODS: In vivo experiments were performed in mouse models (4T1 and LLC) treated with anti-PD-1 antibodies or X-ray irradiation. Tumor tissues were subjected to bulk-RNA sequencing. The immune cell profile was characterised by flow cytometry. The hypoxia level was detected by immunofluorescence and Hypoxyprobe and measured using the hypoxia gene score. Vessel normalization was determined by the pericyte-endothelial cell ratio. Anti-CD4 and anti-CD8 monoclonal antibodies were used to deplete CD4(+) and CD8(+) immune cells, respectively, in mice. The differences among anti-PD-1 immunotherapy, anti-PD-1 immunotherapy without CD4(+) cells, and anti-PD-1 immunotherapy without CD8(+) cells were compared using transcriptome analysis. The spatial immunophenotype was investigated using multi-marker immunofluorescence and HALO analyses. RESULTS: Induction immunotherapy increased radiosensitivity and maximized anti-tumor response compared with concurrent administration of immunotherapy by mitigating hypoxia. Immune cell profile analysis showed that the number of CD4(+) IFNγ(+) T cells, but not CD8(+) IFNγ(+) T cells, increased significantly after the induction of anti-PD-1 therapy combined with radiotherapy compared with concurrent radioimmunotherapy. Using antibodies to deplete CD4(+) or CD8(+) T cells, we confirmed that CD4(+) T cells contribute to PD-1 inhibitor-induced vessel normalization and reduced hypoxia. Spatially, more CD4(+) T cells infiltrate the tumor invasive margin and are located around CD31(+) endothelial cells after anti-PD-1 immunotherapy. CONCLUSIONS: PD-1 inhibitors improve radiosensitivity through vasculature and immune reprogramming, and vessel normalization may be a biomarker for distinguishing patients who will benefit from radiotherapy after induction immunotherapy.