ScRNA-Seq Deciphers an Autocrine EFNA1-EPHA1 Loop That Reprograms the Microenvironment in Hepatocellular Carcinoma

Background: Hepatocytes demonstrate significant heterogeneity between normal liver tissue and hepatocellular carcinoma (HCC), with malignant hepatocytes playing a crucial role in remodeling the tumor microenvironment through specific ligand-receptor interactions. However, the mechanisms by which hepatocytes drive HCC progression at the single-cell level remain poorly understood. Methods: We analyzed single-cell RNA sequencing datasets (GSE174748 and GSE166635) from the GEO database using CellChat to decode intercellular communication networks, which specifically revealed enhanced EPHA signaling in HCC hepatocytes and identified EFNA1-EPHA1 as the most prominent ligand-receptor pair. This key finding was validated through immunofluorescence analysis in both clinical HCC tissues and HepG2/LO2 cell lines, and its clinical relevance was assessed using the TCGA-LIHC dataset via UALCAN. Results: Single-cell analysis revealed that HCC hepatocytes act as both senders and receivers of pro-tumorigenic signals, with upregulated expression of malignancy-related genes (AFP, ACSL4, and SERPINA1). CellChat inference demonstrated significantly strengthened outgoing interaction signals from hepatocytes in the HCC microenvironment. The EFNA1-EPHA1 axis was identified as a key mediator of hepatocyte-microenvironment crosstalk, showing marked activation in HCC tissues and high co-expression in HepG2 cells. TCGA analysis confirmed EFNA1 upregulation in HCC, correlating with advanced clinical stage, higher tumor grade, and metastatic events. Conclusion: Our study provides single-cell resolution evidence that malignant hepatocytes promote HCC progression through autocrine and paracrine signaling via the EFNA1-EPHA1 axis, reshaping the tumor microenvironment. These findings delineate a key autocrine-paracrine mechanism in HCC progression and nominate the EFNA1-EPHA1 axis as a promising candidate for therapeutic development.