Tumor-intrinsic stratifin contributes to NECTIN2–TIGIT-mediated NK-cell suppression and therapeutic vulnerability in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with limited responsiveness to immunotherapy. Natural killer (NK) cells can eliminate tumor cells without prior antigen presentation, but their cytotoxic activity is frequently suppressed within the tumor microenvironment. The tumor-intrinsic mechanisms that restrain NK-cell activity and reshape tumor–NK cell communication in ICC remain unclear. Bulk RNA sequencing, single-cell RNA sequencing, proteomics, tissue microarrays, and external cohorts were integrated to characterize immune remodeling in ICC and prioritize candidate regulators. Functional studies were performed using ICC cell lines, NK-cell co-culture systems, and murine ICC models. NK-cell infiltration and activity were assessed by flow cytometry, immunofluorescence, immunohistochemistry, and functional assays. CellChat-based ligand–receptor inference was used to nominate candidate tumor–NK signaling axes, whereas co-culture, genetic rescue, and receptor-blockade assays were used to evaluate their functional relevance. Co-immunoprecipitation, ubiquitination assays, ChIP-qPCR, and dual-luciferase reporter assays were used to investigate SFN-associated signaling and NECTIN2/TIGIT-mediated tumor–NK cell communication. Stratifin (SFN) was prioritized as a candidate tumor-intrinsic regulator based on its high expression in ICC tumor cells, association with poor outcomes, and negative relationship with immune infiltration. SFN-high tumors showed reduced intratumoral NK-cell abundance and impaired NK-cell functional signatures. SFN knockdown increased NK-cell infiltration and effector activity in the mIC-23 orthotopic ICC model, whereas NK-cell depletion attenuated the antitumor effect of SFN knockdown. CellChat-based ligand–receptor inference and functional perturbation experiments supported the involvement of NECTIN2/TIGIT signaling in SFN-associated NK-cell suppression. Mechanistically, SFN interacted with PAK1 and was associated with reduced TRIM21-related K48-linked ubiquitination and increased PAK1 stability. SFN-associated PAK1–AKT/GSK-3β signaling contributed to MYC activation, and MYC participated in NECTIN2 transcriptional regulation. Chemotherapy combined with PD-1 and TIGIT blockade enhanced antitumor responses and NK cell activity in the mIC-23 orthotopic ICC model. Exploratory retrospective analyses suggested a potential association between elevated SFN and reduced immunotherapy responsiveness. These findings support a model in which tumor-intrinsic SFN signaling contributes to NECTIN2/TIGIT-mediated tumor–NK cell communication through a PAK1–MYC-associated signaling axis. This study provides mechanistic insight into NK-cell dysfunction in ICC and supports further evaluation of chemotherapy combined with PD-1/TIGIT blockade.