Abstract
Background
Hepatocellular carcinoma (HCC) is characterized by aberrant angiogenesis and an immunosuppressive tumor microenvironment, both of which limit durable responses to PD-1 blockade. Strategies that concurrently target vascular dysfunction and immune inhibition may improve therapeutic efficacy.Methods
The interaction between S-propargyl-cysteine (SPRC) and cystathionine-gamma-lyase (CSE) was assessed by surface plasmon resonance. In human umbilical vein endothelial cells, NF-κB p65 and STAT3 phosphorylation, VEGFA expression, extracellular H2S production, CSE enzymatic activity, and tube formation were evaluated. In HepG2 and activated Jurkat-cell co-cultures, PD-L1 expression, T-cell phenotypes, and granzyme B secretion were analyzed. H3K27me3 enrichment at RELA and STAT3 was examined by ChIP-qPCR. Antitumor efficacy was assessed in an HCC mouse model treated with anti-PD-1 alone or in combination with SPRC. Tumor tissues were further analyzed for intratumoral H₂S levels, CSE enzymatic activity, NF-κB/STAT3 activation, VEGFA and PD-L1 expression, CD31-associated vascular density, immune-cell infiltration, metabolomic remodeling, and oxidative-stress status.Results
SPRC bound recombinant CSE with nanomolar affinity and enhanced CSE-dependent H2S generation and enzymatic activity. In endothelial cells, SPRC reduced NF-κB p65 and STAT3 phosphorylation, decreased VEGFA expression, and impaired tube formation, whereas these effects were attenuated by CSE knockdown. In co-culture, SPRC reduced PD-L1 expression on HepG2 cells and promoted a more cytotoxic T-cell phenotype, as indicated by increased CD8α, reduced FoxP3, and elevated granzyme B secretion. Rescue experiments showed that constitutively active STAT3 restored PD-L1 expression despite SPRC treatment, whereas p65 overexpression reversed SPRC-mediated suppression of VEGFA expression and endothelial tube formation. SPRC also increased H3K27me3 enrichment at the RELA and STAT3 promoters. In vivo, SPRC enhanced the antitumor efficacy of PD-1 blockade, increased intratumoral CD8⁺ T-cell infiltration, decreased Treg abundance, and reduced CD31-associated vascular density. Mechanistically, SPRC plus anti-PD-1 increased intratumoral H₂S levels and CSE enzymatic activity, suppressed NF-κB p65 and STAT3 phosphorylation, and decreased VEGFA and PD-L1 expression in tumor tissues. Metabolomic profiling further linked an amino-acid-centered metabolic module, particularly the arginine-ornithine-citrulline axis, to immune and vascular remodeling, accompanied by reduced MDA and increased SOD activity.Conclusions
SPRC activates a functional CSE/H2S-linked program that suppresses NF-κB/STAT3 signaling, inhibits angiogenesis, alleviates PD-L1-associated immune suppression, and enhances the efficacy of PD-1 blockade in HCC. These findings support SPRC as a promising adjunct strategy for microenvironment-targeted immunotherapy in HCC.