Exploratory single-nucleus transcriptomic analysis of endothelial heterogeneity and ferroptosis-related gene patterns in a human hepatocellular carcinoma sample

Abstract

Background Endothelial heterogeneity may contribute to vascular remodeling in hepatocellular carcinoma (HCC), while ferroptosis-related iron handling intersects with hypoxic, inflammatory, and metabolic stress. We therefore characterized endothelial transcriptional states in a publicly available HCC single-nucleus RNA-sequencing dataset. Methods We re-analyzed GEO GSE212046/GSE212047 and analyzed library GSM6508441. After quality control, 67,481 cells were retained, including 754 endothelial cells for re-clustering. Endothelial states were evaluated using canonical markers, differential expression, a literature-derived microvascular invasion (MVI)-associated gene-set score, hypoxia/angiogenesis scores, transcription-factor expression, and exploratory PAGA connectivity. Supportive hypoxia-response qRT-PCR assays were performed in HepG2 and Huh7 cells. Results Two endothelial states were identified: EC-A (492 cells) and vEC (262 cells). EC-A showed higher expression of ESM1, PLVAP, ANGPT2, RAMP3, and IGFBP3 and a higher MVI-associated gene-set score, while vEC showed a higher hypoxia score. VWF, CD36, and HSPG2 were among the leading EC-A differential genes, and FTL was broadly expressed. Hypoxia increased PLVAP, ESM1, FTL, and CLEC4G expression in HepG2/Huh7 cells, but these tumor-cell assays were not interpreted as direct endothelial validation. Conclusion This single-library reanalysis reveals substantial endothelial heterogeneity and linked vascular, iron-handling, hypoxic, inflammatory, and metabolic transcriptional features in HCC. The findings are exploratory and require patient-level replication and direct endothelial validation.