Comprehensive analysis of cellular heterogeneity in the tumor microenvironment reveals TUBG1 as an immunosuppressive regulator in hepatocellular carcinoma

Heterogeneity of the tumor microenvironment (TME) within and among patients with liver cancer has significant effects on their responses to therapies. Systematic and detailed characterization of the TME can guide precision therapy. To better understand this heterogeneity, we conducted unsupervised cell clustering by integrating two single-cell RNA sequencing datasets. Cell signaling activity was assessed in each cluster and analysis of cell communications was performed using Cellchat. Hepatocellular carcinoma (HCC) was classified into subtypes through consensus clustering based on immune cell infiltration scores. Tissue microarray (TMA) was used to explore the expression of tubulin, gamma 1 (TUBG1) in tumor versus normal tissues and examine its prognostic significance. Gene silencing was performed using small interfering RNA targeting TUBG1, followed by evaluation of major histocompatibility complex class I (MHC-I) antigen molecules. We profiled 70918 cells, identifying 13 major cell types with 27 sub-clusters. We observed that C17-FN1-TAMs cluster showed high expression levels of hypoxia, Transforming growth factor β (TGF-β), and vascular endothelial growth factor (VEGF) signaling. Two HCC subtypes were identified based on immune infiltration, with the immune-hot group exhibiting better prognosis. A favorable-related immune signature (FRIS) was constructed that positively correlated with survival, and TUBG1 expression showed an inverse correlation with FRIS. TUBG1 was overexpressed in tumors and linked to poorer outcomes and reduced immune infiltration. Moreover, in vitro studies demonstrated that suppressing TUBG1 activity resulted in elevated levels of MHC-I protein presentation on tumor cells. Our study expands existing knowledge on intratumoral heterogeneity in the TME of liver cancer and provides evidence of TUBG1 as a potential therapeutic target.