Single-cell transcriptomic profile unveils distinct immune ecosystems in alcohol- and nonalcohol-associated hepatocellular carcinoma.

Alcohol-associated hepatocellular carcinoma (A-HCC) shows more aggressive progression and has a poorer prognosis than nonalcohol-associated HCC (NA-HCC), but the underlying tumor microenvironment (TME) heterogeneity remains poorly characterized. Here, we performed single-cell RNA sequencing (scRNA-seq) on tumor and adjacent normal tissues from two A-HCC patients, constructing the first single-cell transcriptomic profile of human A-HCC. This dataset was integrated with public NA-HCC scRNA-seq data to compare cellular composition, functional states, and intercellular communication. Both HCC subtypes displayed immunosuppressive TME features, which were significantly more pronounced in A-HCC. Specifically, A-HCC tumors showed greater enrichment of regulatory T cells (Tregs) with enhanced suppressive function, anti-inflammatory macrophage polarization, and more severe CD8+ T and NK cell depletion and dysfunction with reduced metabolic activity. Malignant hepatocytes in A-HCC exhibited increased copy-number variation, epithelial-mesenchymal transition (EMT), stemness, proliferation, and drug resistance associated gene signature scores. Cell-cell communication analysis further suggested A-HCC-specific upregulation of immunosuppressive signaling pathways (LTA/LTB, TGF-β, LGALS9-HAVCR2) that reinforce this pro-tumorigenic ecosystem. A Treg-derived gene signature from A-HCC was associated with worse overall survival in independent TCGA cohorts. This first comprehensive single-cell comparison uncovers a coordinated immunosuppressive and malignant ecosystem in A-HCC that may be distinct from NA-HCC, providing a mechanistic framework to understand its aggressive clinical behavior and to identify potential etiology-targeted therapeutic strategies. Despite the limited sample size, this study provides the first single-cell resource for A-HCC and lays the foundation for future large-scale validation.