Role of the SPP1–CD44–NETs Signaling Axis in EMT Regulation and Prognostic Model Construction of Hepatocellular Carcinoma Based on Multi-Cohort and Single-Cell Analysis

Background and Objective:Epithelial–mesenchymal transition (EMT) plays a crucial role in the initiation and progression of hepatocellular carcinoma (HCC), yet the prognostic value of its associated genes requires further investigation. This study aims to construct a prognostic model based on EMT-related genes and explore its association with the tumor immune microenvironment and mechanisms underlying HCC metastasis. Methods:EMT-related genes were identified from The Cancer Genome Atlas (TCGA) and GSE14520 datasets to construct a HCC prognostic model, which was validated using survival analysis, C-index, and hazard ratios (HRs). Functional enrichment analysis, GSEA, and immune cell infiltration assessment were performed to explore molecular mechanisms between high- and low-risk groups. Single-cell analysis using GSE189903 was conducted to further investigate the cellular distribution of key prognostic genes. In addition, immunohistochemistry (IHC) was performed on human HCC tissues and paired adjacent and non-tumor liver tissues to validate the expression pattern of SPP1. Results:The EMT-related prognostic model, consisting of MMP1, EZH2, SPP1, CCNA2, and MYCN, demonstrated robust predictive performance in both training and validation cohorts. High-risk tumors were enriched in proliferation, cell cycle, MYC targets, and EMT-related pathways, accompanied by immune microenvironment dysregulation and immune evasion. Single-cell analysis revealed that SPP1 was highly expressed in tumor-associated macrophages and closely associated with CD44-mediated signaling pathways. Immunohistochemical analysis further confirmed that SPP1 expression was significantly upregulated in HCC tissues compared with adjacent and normal liver tissues. We hypothesize that the SPP1–CD44 signaling axis may promote EMT and invasive potential in HCC by regulating neutrophil activation and neutrophil extracellular trap (NET) formation. Additionally, significant differences in tumor mutation burden (TMB) and driver gene mutation patterns were observed between risk groups. Conclusion:The EMT-related gene-based prognostic model effectively predicts survival risk in HCC patients and highlights a potential mechanism whereby SPP1 promotes EMT through macrophage–neutrophil interactions and NET formation. These findings provide novel molecular insights and potential therapeutic targets for HCC risk stratification and individualized immunotherapy.