Exploring the role of hydrogen peroxide in the immune microenvironment of hypoxic tumors

Background and purpose Hypoxia remains a critical driver of radioresistance. While previous radiosensitizers have been limited by systemic toxicities, combining hydrogen peroxide (H 2 O 2 ) with sodium hyaluronate (KORTUC) enables safe intratumoral administration. This study investigates how this reoxygenation modulates the tumor immune microenvironment. Materials and methods Release of damage-associated molecular patterns (DAMPs) was assessed in H 2 O 2 -treated hypoxic CT26 and DLD-1. In vivo, CT26-tumor-bearing mice received i.t H 2 O 2 with sodium hyaluronate and irradiation (IR). Immune infiltration was analyzed via flow cytometry and validated in vitro using human CD3 + T cells and murine bone marrow-derived macrophages (BMDMs) under hypoxia. Hypoxic regions and CD206 + macrophages were spatially quantified in histological sections using HALO software. Results H 2 O 2 induced dose-dependent DAMPs levels in hypoxic tumor cells. In vivo and in vitro analysis revealed that H 2 O 2 preserved T-cell infiltration, proliferation and IFN-γ H 2 O 2 significantly reduced the anti-inflammatory macrophages (Mφ2) phenotype (CD206, Arg1) and induced a functional shift towards pro-inflammatory (Mφ1) activity (iNOS), further validated by qPCR in hypoxic BMDMs. Spatial analysis confirmed sustained reoxygenation and a pronounced reduction in Mφ2 colocalization within remaining hypoxic niches. Conclusions Clinically approved H 2 O 2 -based radiosensitizer enhances radioresponse through improved reoxygenation while also favoring remodeling of the tumor microenvironment. H 2 O 2 promotes immunogenic cell death, preserves T cell function, and selectively reduces immunosuppressive Mφ2. Collectively, these findings provide a strong rationale for combining H 2 O 2 with IR to alleviate hypoxia-driven immunosuppression and improve clinical outcomes in solid tumors.