Genomic and the tumor microenvironment heterogeneity in multifocal hepatocellular carcinoma

Abstract

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.