TMEM176B orchestrates intratumoral hierarchy and immunosuppression in hepatocellular carcinoma: Insights from single-cell transcriptomics

Background Hepatocellular carcinoma (HCC) exhibits profound intratumoral heterogeneity and a complex immune microenvironment, posing significant challenges for effective treatment. Methods To dissect the cellular heterogeneity of HCC, we performed single-cell RNA sequencing (scRNA-seq) on paired tumor and adjacent non-tumor tissues from five patients. Non-negative matrix factorization (NMF) and WGCNA were employed to identify transcriptional metaprograms and co-expression modules. CellPhoneDB were utilized to analyze intercellular communication and the spatial organization of TMEM176B. Furthermore Transwell, wound healing, and colony formation assays, were conducted in LM3 and Huh-7 cell lines to validate the functional role of TMEM176B in vitro. Results Our analysis established a single-cell transcriptomic atlas of HCC, revealing a highly ordered hierarchical intratumoral heterogeneity. We identified TMEM176B as a pivotal molecule negatively correlated with cellular differentiation potential (CytoTRACE score) and positively associated with immune infiltration and checkpoint expression. Functional validation demonstrated that TMEM176B overexpression significantly impaired HCC cell invasion, migration, and proliferation. Clinically, low TMEM176B expression was significantly associated with worse overall survival in HCC patients. Conclusions Our study delineates the malignant hierarchy in HCC and identifies TMEM176B as a critical driver linking tumor dedifferentiation and immune remodeling. TMEM176B is associated with patient prognosis and warrants further evaluation as a potential prognostic biomarker in multivariate settings.and a potential therapeutic target for reversing immune dysfunction in HCC.