SPINT2 Is Associated with Dual-Phenotype Hepatocellular Carcinoma and Aggressive Tumor Features

Background: Hepatocellular carcinoma (HCC) heterogeneity, particularly the presence of tumor cells co-expressing hepatocytic and cholangiocytic markers, contributes to therapeutic resistance and poor prognosis, yet its molecular correlates remain incompletely understood. This study aimed to characterize dual-phenotype HCC (DPHCC) and investigate the association and functional relevance of SPINT2 in tumor progression. Methods: We integrated multiplex immunofluorescence, CyTOF mass cytometry, single-cell RNA sequencing, and functional assays across clinical cohorts and cell lines. Results: Multiplex staining identified DPHCC in 22.89% of patients, correlating significantly with younger age, elevated AFP, and inferior recurrence-free and overall survival compared to non-DPHCC cases. Parallel scRNA-seq and CyTOF analyses showed that DPHCC-associated cells were enriched for stemness- and EMT-related features, with SPINT2 emerging as a top candidate gene in the transcriptomic analysis. Immunohistochemistry showed higher SPINT2 expression in DPHCC tissues, and high SPINT2 expression was associated with shorter overall survival in univariate Kaplan–Meier analysis and frequently co-localized with CK19. Functionally, SPINT2 modulation produced context-dependent changes in invasion, proliferation-related CCK-8 readouts, wound closure, sphere formation, and xenograft tumor growth in MHCC-97H and MHCC-LM3 cells. Collectively, these findings characterize DPHCC as a clinically relevant dual-lineage phenotypic state associated with aggressive tumor features and identify SPINT2 as a candidate molecule associated with this phenotype. Conclusions: SPINT2 is associated with the DPHCC-related state and aggressive tumor-cell phenotypes, but its mechanistic and clinical significance requires further validation.