Abstract

Abstract Background Cholangiocarcinoma (CCA), a malignancy arising from biliary epithelial cells, features a tumor microenvironment (TME) characterized by metabolic dysregulation and immunosuppression. Although diverse metabolic aberrations have been observed, the dominant metabolic driver remains unclear. Therefore, the study aims to elucidate the molecular connections between metabolic reprogramming and immune evasion, and to provide the basis for developing TME-targeted therapies. Methods First, untargeted metabolomics was performed to identify key metabolite classes involved in CCA progression. We subsequently used next-generation sequencing combined with database analysis to identify the key genes involved in tumor fatty acid metabolism. Metabolomics, proteomics and metabolic phenotyping experiments were subsequently performed to investigate this metabolic process. Tumor malignancy was evaluated using a range of in vitro and in vivo models. Single-cell RNA sequencing, Olink proteomics, flow cytometry and multiplex immunohistochemistry were performed to analyze the changes in the TME. Finally, we used humanized NOG mice to evaluate the efficacy of the combination therapy. Results Integrated multi-omics analysis revealed that long-chain unsaturated fatty acids (LCUFAs) were enriched in CCA tissues and closely associated with tumor progression. Ubiquitin-specific peptidase 1 (USP1) was upregulated in CCA tissues, driving dynamic lipid metabolic reprogramming and was associated with poor prognosis. Mechanistically, USP1 orchestrated LCUFAs accumulation through coordinating the enhancement of lipophagy and lipogenesis, establishing a systems-level metabolic regulatory network. Tumor-secreted LCUFAs were preferentially internalized by tumor-associated macrophages (TAMs) through FABP5, thereby suppressing cytotoxic T cells activity.