Acquired resistance to immune checkpoint inhibitors is associated with hypoxia and ECM remodeling in colorectal cancer

Acquired resistance to immune checkpoint inhibitors (ICIs) limits the durability of therapeutic responses across multiple cancer types, yet the underlying mechanisms remain poorly defined. Using the murine MC38 colorectal cancer model, we established an in vivo model recapitulating clinical response patterns, including complete regression, primary resistance, and acquired resistance. Tumors with acquired resistance progressed after initial benefit from combined anti-PD-1 and anti-CTLA-4 therapy and maintained resistance upon retransplantation into naive hosts, indicating a cancer cell-intrinsic driver of resistance. Whole-genome sequencing showed no mutations in antigen presentation or IFN-{gamma} signaling pathways previously described in single clinical cases of acquired resistance, and functional assays confirmed preserved antigenicity and IFN-{gamma} responsiveness. Transcriptomic and metabolic profiling of resistant cancer cells instead revealed metabolic reprogramming characterized by enhanced mitochondrial respiration and enrichment of hypoxia-related gene signatures, suggesting cancer cell-intrinsic adaptations that reshape the tumor microenvironment. Tumors with acquired resistance exhibited an increase in tumor-associated macrophages, and these macrophages displayed enriched transcriptional signatures of hypoxia, angiogenesis, and extracellular matrix (ECM) remodeling. Proteomic analysis of ECM-enriched tumor fragments showed accumulation of proteoglycans and enzymes such as lysyl oxidase, consistent with active matrix-remodeling. These changes coincided with altered T cell activation characterized by reduced cytotoxic gene expression and decreased CD44 expression on tumor-infiltrating T cells, suggesting impaired effector functions within the resistant microenvironment. This study identifies non-genetic mechanisms of acquired resistance to ICIs and highlights metabolic and ECM remodeling programs as promising therapeutic targets to prevent or reverse acquired resistance to ICIs.