Background: Tumor metastasis is the leading cause of high mortality in hepatocellular carcinoma (HCC). Moreover, the HCC microenvironment is characterised by high heterogeneity. Single-cell RNA sequencing (scRNA-seq) may aid in determining specific cell clusters that could regulate the immune microenvironment of HCC.

Methods: The scRNA-seq data of 10 HCC samples were collected from the Gene Expression Omnibus (GEO) database GSE124395. Subcluster analyses were conducted to identify malignant hepatocytes. Correlations between key gene expression and clinicopathological data were determined using public databases. Immune cell infiltration analysis was performed and verified by immunohistochemistry and immunofluorescent staining.

Results: Nine malignant hepatocyte clusters with different marker genes and biological functions were identified. C3_Hepatocyte-SERF2 and C6_Hepatocyte-IL13RA2 were mainly involved in the regulation of the immune microenvironment, which was also a significant pathway in regulating HCC metastasis. Key genes in malignant hepatocyte clusters that were associated with HCC metastasis were further screened by LASSO regression analysis. TPI1, a key gene in C6_Hepatocyte-IL13RA2, presented a significant role in regulating the HCC immune microenvironment in the Cancer Genome Atlas (TCGA) and Tumor Immune Estimation Resource (TIMER) databases. Moreover, immunohistochemistry analysis demonstrated that TPI1 expression was positively correlated with HCC metastasis and poor prognosis, while it was negatively correlated with CD8⁺ T cell infiltration. The negative correlation between TPI1 expression and CD8⁺ T cell infiltration was further confirmed by immunofluorescence staining.

Conclusion: In summary, a cluster of TPI1⁺ malignant hepatocytes was associated with the suppression of CD8⁺ T cell infiltration and HCC metastasis, providing new insights into novel biomarkers and immunotherapeutic targets for HCC.