Integrative transcriptome profiling elucidates molecular and immunovascular characteristics of macrotrabecular hepatocellular carcinoma

Background and Aims: Recent advances in single-cell RNA sequencing (scRNA-seq) technologies have enabled us to clarify gene regulatory networks and immune cell compositions. In this study, integration of large-scale bulk and single-cell datasets refined hepatocellular carcinoma (HCC) classification and shed light on the characteristics of its aggressive subtype. Approach and Results: Single-cell analysis of 228,564 live cells from six scRNA-seq datasets identified five major clusters of HCC cells with high mitotic activity (Cluster 1), activated Wnt/β-catenin signaling (Cluster 2), elevated glycolysis (Cluster 3) and lipogenesis (Clusters 4 and 5). Aggressive HCC subtype defined in bulk RNA-seq analysis of 691 tumor samples comprised a combination of Cluster 1 with Clusters 3, 4 or 5. Gene regulatory network analysis and gene set enrichment analysis highlighted the essential roles of p53 and MYC in aggressive HCC/Cluster 1, and cell composition analysis elucidated T cell depletion as an immune resistance mechanism. In a syngeneic mouse model, Trp53 knockout and MYC overexpression caused highly mitotic, tumorigenic and metastatic phenotypes, characterized by a macrotrabecular (MT) pattern, vascular encapsulation (VE) and T cell exclusion. Angiogenesis inhibition disrupted MT/VE formation, resulting in T cell recruitment, and its combination with immune checkpoint blockade achieved remission. Conclusions: Single-cell analysis has deepened our understanding of the molecular mechanism and tumor microenvironment in aggressive HCC. The combination of targeting tumor vasculature and blocking immune checkpoints represents a promising therapeutic strategy for this subtype.