TM4SF1 Overexpression in Tumor-associated Endothelial Cells Promotes Microvascular Invasion in Hepatocellular Carcinoma

Junwu Guo Liangrui Chen Binghua Dai Chengjun Sui ^* Zhitao Dong Keji Chen ^* Kecai Duan ^* Kunpeng Fang ^* Aijun Li ^* Kui Wang ^* Li Geng ^*

• Eastern Hepatobiliary Surgery Hospital, Shanghai, China

Background: Microvascular invasion (MVI) is linked to poor prognosis, early recurrence and postsurgical 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 TEC in microvascular invasion in HCC.Methods: Single-cell RNA sequencing (scRNA-seq) data from two patients with MVI and three 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. TEC subgroup developmental trajectory was analyzed using MVI. Six 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 TEC subgroups had significant heterogeneity between datasets from MVI and non-MVI patients. MVI samples had TEC 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.TECs with elevated TM4SF1 expression facilitate MVI during HCC via an effect on the EMT, suggesting the potential of TM4SF1 as a therapeutic target.