Abstract
The combination of immune checkpoint inhibitors (ICIs) with anti-angiogenic agents has demonstrated efficacy in the clinical treatment of advanced hepatocellular carcinoma (HCC). This study seeks to elucidate the underlying mechanisms that contribute to the enhanced therapeutic effects of apatinib when administered in conjunction with ICIs for the treatment of advanced HCC. The effects of apatinib on the viability, clonal formation, and apoptosis of HCC cells were evaluated through in vitro experiments. Meanwhile, in vivo experiments were conducted to substantiate these findings and further investigate the synergistic effects of apatinib with PD-1 inhibitors on the immune microenvironment, particularly by activating the signal transducer and activator of transcription 1 (STAT1)/natural killer (NK) cell axis. In vitro experiments demonstrated that apatinib significantly suppressed HCC cell viability, colony formation capacity, and induced apoptosis. In tumor-bearing mouse models, the combination of apatinib with PD-1 inhibitors showed superior tumor growth inhibition compared to monotherapy (combination group exhibited the smallest tumor volume and 100% survival rate vs. 0% in PBS group, p < 0.001). Western blot and immunohistochemical analyses revealed STAT1/NK axis activation through combination therapy (upregulated STAT1 expression with increased CD8(+)T cell and NK cell infiltration, p < 0.001). In the mechanism discussion, STAT1-overexpressing Hepa1-6 cells confirmed the antitumor effect of STAT1 in the combination therapy. Subsequently, we validated our findings using the STAT1 inhibitor fludarabine or the NK cell-depleting agent Asialo GM1. Furthermore, combination therapy remodeled the tumor microenvironment by reducing CA IX (hypoxia marker), CD31 (angiogenesis marker), and α-SMA (stromal activation marker) expression (p < 0.05). Apatinib enhances the efficacy and responsiveness of PD-1 inhibition via the STAT1/NK axis, while the combination therapy remodels the tumor microenvironment to potentiate anti-tumor effects.