Hypoxic tumor exosomes suppress macrophage inflammation and ferroptosis via NDUFV2 to enhance bystander tumor radioresistance.

Hypoxia-induced radioresistance presents a major barrier to effective radiotherapy of non-small cell lung cancer (NSCLC), while the underlying role of macrophages remaining elusive. In this study, we found that, compared with normoxia-derived tumor exosomes, hypoxia-derived tumor exosomes more potently promoted M2 polarization of macrophages and TGF-β1/IL-10 secretion via NADH: ubiquinone oxidoreductase core subunit V2 (NDUFV2), as evidenced by the increased mitochondrial functions, including oxidative phosphorylation, ATP levels and mitochondrial membrane potentials, which collectively suppressed macrophage ferroptosis in an NDUFV2-dependent manner. When co-cultured with these exosome-educated macrophages, bystander normoxic NSCLC cells acquired radioresistance, which was attributed to the inhibition of ferroptosis. Notably, NDUFV2-knockdown macrophages abrogated this exosome-induced radioresistance. Mechanistically, IL-10 might be a pivotal signaling messenger rendering radioresistance of bystander normoxic cells. In vivo, hypoxic exosomes also conferred radioresistance to NSCLC xenografts in mice with intact macrophage populations but not in those with macrophage depletion. Immunohistochemical analysis revealed that irradiated xenografts treated with hypoxic exosomes exhibited significantly higher expression levels of GPX4, Ki67, HIF1α, CD163, and NDUFV2. Furthermore, GPX4 knockdown in xenografts reversed the radioresistance induced by hypoxic exosomes and was associated with reduced infiltration of M2-polarized macrophages, supporting the central role of ferroptosis in macrophage-regulated, hypoxia-induced radioresistance. Clinical tissue microarray analysis further confirmed that NDUFV2 was significantly upregulated in NSCLC tissues and correlated with advanced stages. Collectively, this study disclosed for the first time that hypoxic exosomes suppressed macrophage inflammatory responses and ferroptosis via NDUFV2, thereby enhancing radioresistance in bystander normoxic NSCLC cells.