Tumor cell PD-L1 represses IFN signaling to limit anti-tumor immunity, despite an IFN-responsive immunophenotype predicting colorectal cancer response to immune checkpoint inhibitor therapy. Lipid nanoparticle (LNP)-encapsulated IFNα2-encoding nanoplasmid (LNP-mIFNα2) gene therapy suppresses tumor progression, however, the underlying mechanism and therapeutic trade-offs of the accompanying proinflammatory cytokine response triggered by nucleic acid delivery remain undefined. Here, we show that LNP-mIFNα2 selectively transfects tumor cells to restore local IFNα2 production in lung metastases, suppressing colon cancer lung metastasis in syngeneic and humanized mouse models and sensitizing tumors to ICI therapy. Efficacy required canonical IFNAR1 signaling, and was further enhanced by neutralizing co-induced IL6. Single-cell RNA sequencing revealed coordinated tumor microenvironment reprogramming with SPP1 + macrophages underwent apoptosis while incoming monocytes acquired an IFN-responsive identity, Tpex cells lost their quiescence program and expanded, and tumor cells lost their high-cycling phenotype while increasing antigen presentation and immune-cell-recruiting chemokines. Tumor cells also shifted away from a hypoxia/HIF-1α-driven cuproptosis-resistance program, with increased Fdx1 and copper-importer expression and decreased metallothionein Mt1, suggesting sensitization to coproptosis pathway. The LNP-mIFNα2-treated tumor microenvironment transcriptionally recapitulated T cell and myeloid signatures of pembrolizumab-responsive patients. Our findings establish LNP-delivered IFNα2 as a multi-compartment TME regulator that reprograms myeloid suppression, reinvigorates exhausted T cells, and restores tumor immunogenicity.
