Vascular Endothelial Growth Factor Receptor 2-Targeted Therapy Suppresses the Progression of Alpha-Fetoprotein-Positive Hepatocellular Carcinoma After Combination Therapy With Anti-Programmed Death-Ligand 1 and Anti-Vascular Endothelial Growth Factor-A Antibodies.

Background and Aims: Combination therapy with the anti-programmed death-ligand 1 (anti-PD-L1) antibody atezolizumab and the anti-vascular endothelial growth factor-A (anti-VEGF-A) antibody bevacizumab (Atezolizumab/Bevacizumab) has commonly been used as first-line treatment for advanced hepatocellular carcinoma (HCC). However, effective second-line treatment options remain under debate. The anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody ramucirumab has shown promise in unresectable HCC with high serum alpha-fetoprotein (AFP) levels, but its efficacy after Atezolizumab/Bevacizumab is unclear. This study investigated the effects of VEGFR2 inhibition on the tumor microenvironment and cancer stem cells (CSCs) in HCC after anti-PD-L1/anti-VEGF-A treatment. Methods: AFP-positive human and mouse HCC cell lines were used to evaluate the effects of the antimouse VEGFR2 antibody DC101. Syngeneic mouse models were employed to analyze the impact of DC101 as a secondary treatment following anti-PD-L1/anti-VEGF-A treatment. Various molecular biological analyses were conducted to assess tumor growth, gene expression, cellular localization, cell-cell interactions, and alterations in the tumor microenvironment. Results: DC101 significantly inhibited tumor growth and disrupted cell-cell interactions between AFP-positive HCC cells and VEGFR2-positive endothelial cells (ECs) in human HCC xenograft models. DC101 treatment following anti-PD-L1/anti-VEGF-A treatment showed notable antitumor effects in a syngeneic mouse model, with reduced expression of CSC and EC markers. Comprehensive gene expression analysis revealed that DC101 downregulates pathways associated with cancer stemness. Furthermore, single-cell analysis demonstrated that DC101 suppresses CSCs by disrupting their interaction with ECs and induces alterations in the tumor immune microenvironment. Conclusion: VEGFR2-targeted therapy not only suppressed tumor angiogenesis but also inhibited CSCs and enhanced antitumor immune activity, suggesting its potential utility as a second-line treatment following Atezolizumab/Bevacizumab.