CXCL9-driven immune rewiring and tumor-intrinsic ATR signaling inhibition restore radiotherapy response in lung cancer

Abstract Background Non-small cell lung cancer (NSCLC) accounts for ~ 85% of lung cancers and is associated with poor outcomes in advanced stages. Radiotherapy (RT) is central to NSCLC treatment, yet locoregional relapse remains frequent. Increasing evidence indicates that, beyond tumor-intrinsic adaptations, the tumor microenvironment (TME) and immune contexture critically influence response to irradiation. However, most radioresistance models rely on human cell lines unsuitable for immunocompetent systems, limiting insight into immune-tumor interactions. Methods We generated radioresistant (RR) isogenic variants of murine lung adenocarcinoma cell lines and investigated RT resistance using transcriptomics, 3D co-culture systems, and syngeneic mouse models. Results RR clones showed stable adaptations to DNA damage, including enhanced clonogenic survival, reduced immunogenic cell death, and constitutive activation of DNA damage response pathways, particularly ATR signaling. High ATR levels in NSCLC patients were significantly associated with immunosuppression. Transcriptomics revealed altered DNA repair and downregulation of type-I interferon and innate immune pathways. In vivo, RR tumors exhibited an immunosuppressive TME with increased T regulatory cells, M2-like macrophages, and myeloid-derived suppressor cells, alongside reduced cytotoxic T cell recruitment. In 3D co-cultures, immunosuppressive cells protected cancer cells from RT-induced cytotoxicity. Notably, RR tumors regained RT sensitivity in immunodeficient mice, highlighting the role of immune suppression in radioresistance. Therapeutically, activation of innate immunity using TLR3-TLR7/8 agonists (poly(I:C)+R848) restored antitumor responses and improved RT efficacy, partly via CXCL9/10-mediated immune recruitment. ATR inhibition with ceralasertib further sensitized RR tumors to RT. Conclusion These findings identify cancer cell-intrinsic ATR signaling and immune suppression as key drivers of radioresistance and support combined therapeutic strategies to prevent locoregional relapse in NSCLC after radiotherapy.