Large-scale single-cell analysis and in silico perturbation reveal dynamic evolution of HCC: from initiation to therapeutic targeting.

The extensive intratumoral and microenvironmental heterogeneity of hepatocellular carcinoma (HCC) remains a major therapeutic barrier. Integrating single-cell transcriptomics samples spanning normal liver, primary tumors, portal vein tumor thrombus (PVTT), and metastatic lymph nodes (MLN) with spatial profiling, we systematically dissected cellular ecosystems driving HCC progression. Malignant hepatocytes segregated into four transcriptional meta-programs with divergent clinical trajectories: Diff-Metabolic, Prolif-Stress, MYC-Biosynth-Immune, and EMT-Inflammatory states. Diff-Metabolic cells retained liver-specific functions with favorable prognosis, whereas the other three programs correlated with disease advancement; notably, all four states exhibited differential therapeutic vulnerabilities, including sorafenib resistance. Within the tumor microenvironment, immunosuppressive Macro-SPP1 and Macro-TREM2 populations expanded during tumor progression. Spatial mapping revealed organized stromal territories where Endo-ESM1 endothelial cells and Fib-POSTN/Fib-CD36 fibroblasts establish TGFβ-enriched niches spatially correlating with Prolif-Stress and EMT-Inflammatory tumor cells, linking stromal architecture to malignant phenotypes. Endothelial-fibroblast crosstalk intensified through extracellular matrix and angiogenic signaling during progression. Geneformer-based virtual knockout screening identified HSP90B1 as a convergent dependency, validated by its cancer cell essentiality, HCC overexpression, abundance in treatment-resistant tumors, and association with adverse survival. This integrated atlas establishes a framework for targeting tumor-intrinsic states and microenvironmental dependencies in HCC.