Integrative Spatial Transcriptomics and Experimental Validation Reveal UBC-Mediated EMT Associated with Immune Evasion in Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) exhibits pronounced spatial heterogeneity that limits therapeutic efficacy. The contribution of epithelial-mesenchymal transition (EMT) to regional tumor progression and immune evasion remains incompletely understood. Methods: We integrated bulk transcriptomic datasets (TCGA, GSE14520), 34 single-cell RNA-seq samples, and 15 spatial transcriptomic datasets to delineate EMT activity across distinct HCC regions. Immune infiltration profiling, pathway enrichment, and multi-model machine learning were used to identify candidate EMT regulators. Functional validation was performed in Hep3B cells through wound healing, Transwell migration/invasion, and immunofluorescence assays. Results: EMT activity was significantly elevated at tumor margins (fold change = 2.7, p < 0.001) and was associated with poorer overall survival (HR = 2.15, 95% CI: 1.41-3.27, p < 0.001). Regions with high EMT signatures showed reduced CD8⁺ T-cell infiltration and increased immunosuppressive cells, including MDSCs, M2 macrophages, and Tregs, along with elevated expression of immune checkpoints (PDCD1, CTLA4, LAG3). Among candidate regulators, UBC was consistently ranked as a top EMT-associated gene across all models. Functional assays confirmed that UBC overexpression enhanced migration, invasion, and vimentin expression, whereas UBC knockdown reversed these effects. Conclusion: Through integrative spatial multi-omics and experimental validation, we identify UBC as a key mediator of EMT and immune suppression at HCC margins. These findings provide mechanistic insight into spatial heterogeneity and suggest that targeting UBC could have translational potential for overcoming immune evasion in HCC.