WED-346 Single-cell profiling reveals altered natural killer cell composition and transcriptional programs in MASLD-related HCC

Background and aims: Liver cancer is a leading cause of cancer death, and its incidence is expected to rise over the next 20 years. Hepatocellular carcinoma (HCC), comprising 85–90% of primary liver cancers, has a poor prognosis. While viral-associated HCC is declining, metabolic dysfunction-associated steatotic liver disease (MASLD)-related HCC is increasing, and the role of NK cells in this context remains poorly understood. Method: Surgically resected non-MASLD-HCC (n=7) and MASLD HCC(n=6) and matched non-tumor liver tissues were collected from treatment-naïve patients. CD45+ cells were profiled by single-cell RNA sequencing using BD Rhapsody™, and 46,979 high-quality cells were analyzed for differential expression, pathway enrichment, and cell–cell communication. Results: Unsupervised clustering and marker gene expression analysis revealed 24 distinct immune cell populations, including three NK cell subsets: one circulating (cC4) and two tissue-resident (trC2, trC14). Total NK cells were reduced in tumors compared to liver-infiltrating cells, with the greatest decrease observed in trC2 cells, independent of HCC etiology. Tumor-infiltrating NK-cell subset transcriptional profiles differed between MASLD- and non–MASLD HCC. In MASLD-HCC, cC4 cells displayed altered immune regulatory pathways and upregulation of lipid-related metabolic pathways, suggesting adaptation to a lipid-rich microenvironment. In the same context, TrC2 and trC14 cells showed downregulation of pathways related to NK activation, cytotoxicity, neutrophil activation, and Tcell signaling, alongside enrichment of inflammatory and metabolic pathways. CellChat analysis revealed altered cell–cell interactions in MASLD-HCC, with cC4 showing the strongest reduction in incoming and outgoing signaling, while trC2 displayed high communication activity. TrC2 displayed increased interactions with macrophages, CD8+ Tcells, and proliferating cells, reduced interactions with MAIT and mastcells, and enhancedsignaling through FASLG, CADM, ICAM, and TRAIL pathways. Conclusion: All tumor NK cell subsets derived from MASLD-HCC upregulated lipid-related pathways, whereas only tissue-resident NK cells downregulated pathways associated with neutrophil activation and NK cell cytotoxicity. These findings indicate that all three NK cell subsets in MASLD-HCC exhibit metabolic and functional alterations, though the specific changes differ among the clusters, collectively contributing to a less cytotoxic phenotype and potentially impaired anti-tumor immunity.