Single-Cell and Bulk Transcriptomics Uncover the Cellular Ecosystem of Vascular Invasion in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver malignancy with a rising global incidence and limited therapeutic options. Vascular invasion (VI) is a hallmark of advanced disease, correlating with early recurrence and dismal prognosis, yet its tumor microenvironment (TME) drivers remain elusive. We analyzed single-cell RNA sequencing (scRNA-seq) data from 25 ICC samples to systematically characterize the cellular composition and molecular features related to VI. By integrating bulk RNA-seq data, spatial transcriptomics, and multiplex immunofluorescence, we identified a distinct subset of tumor-like cancer-associated fibroblasts (CAFs), termed tCAFs, enriched in VI-positive tumors. Functional enrichment analyses revealed that tCAFs were prominently associated with hypoxia and angiogenesis pathways, findings corroborated by the significant upregulation of tCAF markers (<i>MME</i> and <i>NT5E</i>) in ICC-derived CAFs under hypoxic conditions in vitro. Cell-cell communication analysis and spatial mapping uncovered that tCAFs might promote VI primarily through VEGF signaling interactions with endothelial cells. Integrative bioinformatics and RT-qPCR validation identified three key functional genes in tCAFs: <i>SLC2A1</i>, <i>PTGS2</i>, and <i>PLOD2</i>. In endothelial sprouting assays, pharmacological inhibition of <i>SLC2A1</i> exerted a pronounced suppressive effect. Consistently, sprouting assays using ICC-derived CAFs with <i>SLC2A1</i> knockdown confirmed that its downregulation significantly reduced endothelial sprouting capacity. Importantly, administration of the <i>SLC2A1</i> inhibitor BAY-876 effectively suppressed tumor progression and intrahepatic metastasis in the orthotopic ICC mouse model. Our findings define a VI-associated cellular ecosystem and molecular landscape in ICC, unveiling a novel hypoxia-tCAFs-endothelial cells axis. Furthermore, we identify <i>SLC2A1</i> as a clinically relevant therapeutic target, offering new insights into tumor VI.