Background and Aims: Ambiguous understanding of tumors and the tumor microenvironments (TMEs) hinders accurate diagnosis and available treatment for multifocal hepatocellular carcinoma (HCC) covering intrahepatic metastasis (IM) and multicentric occurrence (MO). Here, we characterized the diverse TMEs of IM and MO identified by whole-exome sequencing (WES) at single-cell resolution. Approach and Results: We performed parallel WES and single-cell RNA sequencing (scRNA-seq) on twenty-three samples from seven patients to profile their TMEs when major results were validated by immunohistochemistry in the additional cohort. Integrative analysis of WES and scRNA-seq found that malignant cells in IM showed higher intra-tumor heterogeneity, stemness and more activated metabolism than those in MO. Tumors from IM shared similar TMEs while distinct TMEs were noticed in those from MO. Furthermore, CD20+ B cells, plasma cells and conventional type II dendritic cells (cDC2s) were decreased in IM relative to MO while T cells in IM exhibited a more terminally exhausted capacity with a higher proportion of proliferative/exhausted T cells than that in MO. Both CD20 and CD1C correlated with better prognosis in multifocal HCC. Additionally, MMP9+ tumor-associated macrophages were enriched across IM and MO which formed cellular niches with regulatory T cells (Tregs) and proliferative/exhausted T cells. Conclusions: Our findings deeply decipher the heterogeneous TMEs between IM and MO, which provide a comprehensive landscape of multifocal HCC. Export

Histone lactylation acts as a master regulator in tumor development, but its role in a noncoding RNA (ncRNA) network remains unclear. This study aims to reveal the interaction between H3K18la and the lncRNA-miRNA-mRNA regulatory network in hepatocellular carcinoma (HCC). Transcriptome sequencing and ChIP sequencing were performed in HCC and adjacent normal tissues. Cut&Run and qPCR were used to validate the H3K18la enrichment on LINC02732 and CD44 promoter. Dual luciferase reporter assay, qPCR and Western blotting were used to verify the LINC02732-miR-1291-ASF1B axis. Co-Immunoprecipitation was performed to validate ASF1B recruiting p300. CCK8 and mouse subcutaneous tumor formation were performed to demonstrate this axis promoting HCC. H3K18la enrichment on LINC02732 promoter elevates its expression in both HCC samples and cell lines, therefore enhancing ASF1B expression via sponging miR-1291. Moreover, ASF1B, a histone chaperone, promotes H3K18la by recruiting lactyltransferase p300, forming an ASF1B-H3K18la positive feedback loop. The axis upregulates CD44 expression and promotes HCC in vitro and in vivo. These findings demonstrated the influence of H3K18la on the LINC02732-miR-1291-ASF1B axis and the novel role of ASF1B in histone lactylation by recruiting p300, which together promoted HCC proliferation.