An etiology-stratified single-cell atlas identifies FABP4 as a prognostic marker for MASLD-related HCC

The impact of HBV infection and metabolic dysfunction-associated steatotic liver disease (MASLD) on the tumor microenvironment in hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to compare the tumor microenvironment among HBV^-MASLD^-, HBV^-MASLD⁺, HBV⁺MASLD^-, HBV⁺MASLD⁺ HCC, and to identify etiology-specific prognostic biomarkers.

Single-cell RNA-sequencing was performed on paired tumor and adjacent tissues from 12 patients with HCC. Bulk RNA-sequencing data from the TCGA-LIHC and two immunotherapy-treated HCC cohorts were analyzed to assess FABP4's role in vascular normalization and prognosis. Validation included immunohistochemistry on 224 samples, multiplex immunohistochemistry, in vitro and in vivo experiments.

We compared the prognosis of patients with different etiologies of HCC and found that those with HBV⁺MASLD⁺ HCC exhibited the most favorable outcome following immunotherapy. Compared to MASLD^- HCC, MASLD⁺ HCC showed increased infiltration of CD8⁺ T cells and reduced macrophage abundance. In HBV⁺ HCC, tumor-reactive and tumor-specific T-cell scores, and M1-like macrophage signatures were significantly higher than in HBV^- cases. HBV⁺MASLD⁺ HCC also featured a higher proportion of precursor-exhausted CD8⁺ T cells. Additionally, FABP4 was predominantly expressed in tumor endothelial cells in MASLD-related HCC, regulated by PPARγ. FABP4 facilitated vascular normalization and enhanced CD8⁺ T-cell infiltration. Mechanistically, FABP4 overexpression upregulated multiple vascular-stabilizing genes and downregulated destabilizing genes, associated with Notch1 pathway activation. Inhibition of FABP4 using BMS309403 impaired anti-PD-1 efficacy and attenuated vascular normalization and cytotoxic CD8⁺ T-cell infiltration in MASLD-related HCC mouse models. Clinically, high FABP4 expression correlated with better prognosis.

These findings reveal etiology-heterogeneous immune landscapes in HCC and identify FABP4 as a potential prognostic biomarker to guide immunotherapy in MASLD-related HCC.

This study systematically analyzed the impact of HBV, metabolic dysfunction-associated steatotic liver disease (MASLD), and their coexistence on responses to immunotherapy and outcomes in hepatocellular carcinoma (HCC). By integrating single-cell sequencing of patient samples, it highlights the importance of etiological heterogeneity, particularly from the perspective of CD8⁺ T cells. The study identifies FABP4 as being highly expressed in MASLD-related HCC and implicates it in promoting vascular normalization and CD8⁺ T-cell infiltration. Most importantly, FABP4 emerges as a potential prognostic biomarker for HCC, especially in MASLD-related cases, offering a practical tool to identify patients who are more likely to benefit from immunotherapy.