Single-Cell Transcriptomics Dissects the Molecular Mechanism by Which Macrophage Polarization Drives Hepatocellular...

Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy whose progression is tightly linked to the immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages (TAMs), central regulatory components of the TME, foster HCC metastasis by mediating immune evasion and epithelial-mesenchymal transition (EMT). The underlying molecular mechanisms remain to be elucidated. Clarifying how TAM polarization intersects with EMT will provide a rational basis for targeted HCC therapies.

We integrated scRNA-seq and copy-number variation profiling to delineate TAM subpopulations. CellChat was employed to construct cell-cell communication networks and screen for pivotal TAM-EMT signals. Expression and prognostic relevance of key molecules were validated in the TCGA-LIHC cohort. qRT-PCR, WB, Transwell, CCK-8, and flow cytometry were used for functional characterization.

scRNA-seq resolved eight major cell types, including T/NK cells, epithelial cells, macrophages, monocytes, endothelial cells, fibroblasts, B cells, and dendritic cells. Four TAM subpopulations were identified, among which M2-like macrophages dominated both primary and metastatic HCC lesions. Cell-cell communication analysis revealed that M2-like macrophages engaged epithelial cells via the SPP1-(ITGA5 + ITGB1) signaling. According to clinical data, the activity of this signaling correlated with poor prognosis in HCC patients. Functional assays confirmed that knocking down ITGA5 reversed M2 macrophage polarization and suppressed HCC cell proliferation, apoptosis resistance, migration, and invasion.

ITGA5 is a master regulator of the pro-tumorigenic functions of TAMs. The SPP1-(ITGA5 + ITGB1) signaling represents a novel immunotherapeutic target in HCC. Targeting TAM polarization may reprogram the immunosuppressive microenvironment and improve patient outcomes.