Xinglong Zhang, Huarong Fu, Jun Lu
Background: Microvascular invasion (MVI) is a critical determinant of early recurrence and poor prognosis in hepatocellular carcinoma (HCC). The cellular and molecular mechanisms driving MVI, particularly the role of endothelial heterogeneity, remain incompletely understood. Methods: We integrated single-cell and spatial transcriptomics from multiple HCC cohorts to identify MVI-associated endothelial subpopulations. GeneNMF and hdWGCNA were applied to define stable gene modules, while CellChat and NicheNet analyzed intercellular signaling. Prognostic relevance was assessed using machine learning-based risk models across TCGA-LIHC and external datasets. Functional assays including EdU proliferation, tube formation, and Western blot were conducted to validate the role of IGF2BP3 in tumor-associated endothelial cells. Results: We identified a distinct MVI-associated endothelial subpopulation (MVI Endo) enriched in pro-angiogenic, EMT-like, and TGF-β-responsive pathways, predominantly localized near tumor vasculature. MVI Endo engaged in robust VEGF, ANGPT2, and TGF-β signaling with tumor and stromal cells. Machine learning-derived prognostic models highlighted IGF2BP3 as a central regulator, and functional assays demonstrated that IGF2BP3 enhances endothelial proliferation and tube formation via upregulation of VEGF-A and ANGPT2, supporting its involvement in the vascular phenotype associated with MVI. Conclusion: This study characterizes the endothelial landscape associated with MVI, implicates IGF2BP3 as a critical regulator of vascular reprogramming, and provides a foundation for precision vascular-targeted therapies in HCC.