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.