TM4SF1 overexpression in tumor-associated endothelial cells promotes microvascular invasion in hepatocellular carcinoma

Background: Microvascular invasion (MVI) is linked to poor prognosis, early recurrence and post-surgical intrahepatic metastasis of hepatocellular carcinoma (HCC) but roles of tumor-associated endothelial cells (TECs) remain unclear. The aim of the current study was to investigate the role of TECs in microvascular invasion in HCC. Methods: Single-cell RNA sequencing (scRNA-seq) data from three patients with MVI and two patients with non-MVI HCC were used to identify TECs subpopulations via Seurat R package. Using bioinformatics analysis identified co-expression modules associated with MVI in TECs. Differential gene expression analysis, KME values and Gene Expression Profiling Interactive Analysis (GEPIA) survival were utilized to identify genes with significant involvement. TECs subgroup developmental trajectory was analyzed using monocle2. Five additional spatial transcriptomics (ST) datasets and four HCC postoperative pathological specimens were used to validate the differential expression of subgroups of TECs and hub genes between MVI and non-MVI groups. Results: Distinct TECs subgroups had significant heterogeneity between datasets from MVI and non-MVI patients. MVI samples had TECs subgroups with increased levels of the epithelial-mesenchymal transition (EMT), endothelial cell migration and angiogenesis. Opposing EMT development was found in MVI TECs relative to non-MVI TECs. TM4SF1 was highly expressed in TECs undergoing the EMT and is thought to be linked to MVI. Conclusion: TECs with elevated TM4SF1 expression facilitate MVI during HCC via an effect on the EMT, suggesting the potential of TM4SF1 as a therapeutic target.