Membrane and soluble VTCN1 (B7H4) converge on Src signaling to mediate gemcitabine resistance in intrahepatic cholangiocarcinoma.

RATIONALE: A combination of gemcitabine (GEM)-based chemotherapy and an immune checkpoint inhibitor is the standard treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA). However, 70% of patients develop progressive disease following GEM treatment, highlighting the urgent need for therapeutic strategies targeting GEM-resistant (GR) disease. METHODS: We established GR iCCA sublines and identified 9 upregulated genes by RNA-sequencing and cDNA microarray. Among the 9 genes, we demonstrated that VTCN1 expression improved GR. The enhanced effect of VTCN1 on GR was validated in a spontaneous rat iCCA model, a xenograft mouse model, an orthotopic mouse model, and iCCA specimens. A phospho-kinase array was used to identify the downstream effector of membrane and soluble VTCN1 (sVTCN1). RESULTS: VTCN1 expression was significantly elevated in GEM non-responders and independently predicted poor progression-free survival (HR = 2.038, P = 0.035). Plasma sVTCN1 levels correlated with tumor VTCN1 expression (r = 0.8752, P = 0.002) and tumor burden (r = 0.7696, P < 0.0001). Mechanistically, membrane-bound VTCN1 directly interacted with integrin β1 to enhance FAK phosphorylation, while sVTCN1 bound EGFR to create a positive feedback loop increasing EGFR phosphorylation. Both pathways converged on Src family kinase activation (Y419 phosphorylation) to drive GR. The VTCN1-Src axis was associated with upregulation of cytosolic 5'-nucleotidase 3 (NT5C3) expression to promote GEM resistance. VTCN1 depletion, Src depletion, or Src inhibition with dasatinib restored GEM sensitivity. Combining VTCN1 antibody or dasatinib with GEM significantly suppressed resistant tumor growth and enhanced CD8+ T cell infiltration in vivo. CONCLUSIONS: Membrane and soluble VTCN1 drive GR through dual pathways converging on Src activation. VTCN1 represents both a prognostic biomarker and therapeutic target, with Src inhibition providing a rational strategy to overcome chemotherapy resistance in iCCA.