Background Intrahepatic cholangiocarcinoma (ICC) is an aggressive and heterogeneous malignancy derived from the biliary tract in the liver, with poor prognosis and limited treatment options. In this study, we explore the role of METTL5 in promoting ICC progression by reshaping the tumor immune microenvironment and its therapeutic potential.

Methods Liver-specific Mettl5 knockout (cKO) mouse model and human ICC samples were employed to investigate the role of METTL5 in ICC progression. Functional and mechanistic studies were performed using single-cell transcriptomic (scRNA-seq) and T-cell receptor sequencing (scTCR-seq), flow cytometry and in vitro experiments. The efficacy of METTL5 knockdown using lipid nanoparticle-encapsulated siRNA and anti-PD-1 antibody was evaluated in vivo.

Results By establishing ICC tumors in Mettl5 wildtype and cKO mice, we show that Mettl5, as the 18S rRNA m⁶A methytransferase, promotes ICC progression via reshaping tumor immune microenvironment. scRNA-seq and scTCR-seq analysis unraveled intratumoral IFN-γ⁺CD8⁺ T cell infiltration and expansion as well as reduction of immunosuppressive Ms4a7⁺C1qa⁺ tumor-associated macrophages (TAMs) in Mettl5 cKO mice. Mechanistically, METTL5-mediated 18S rRNA m⁶A modification downregulated the mRNA translation of CXCL16 to exclude CD8⁺T cells, while upregulated CX3CL1 to recruit TAMs for ICC tumorigenicity. Concordantly, low METTL5 expression correlated with increased CD8⁺T cells and decreased TAMs in human ICCs, which correlated with better prognosis and responsiveness to immunotherapy. Combined METTL5 targeting using lipid nanoparticle encapsulated siRNA and PD-1 blockade provoked anti-tumor immunity to eradicate ICC tumors, highlighting the strong immuno-evasive ability of METTL5 as a promising therapeutic target.

Conclusions METTL5 regulates the translation of chemokine mRNA through ribosome 18S rRNA m⁶A modification, and targeting METTL5-mediated immunosuppression unleashes anti-tumor immunity and improves the efficacy of anti-PD-1 therapy.