Humanized disulfide‐stabilized diabody against fibroblast growth factor‐2 inhibits PD‐L1 expression and epithelial‐mesenchymal transition in hepatoma cells through STAT3

Abstract

AbstractFibroblast growth factor 2 (FGF2) plays an important role in tumor angiogenesis. Humanized disulfide‐stable double‐chain antibody against fibroblast growth factor‐2 (anti‐FGF2 ds‐Diabody) is a small molecule antibody with good tissue permeability and low immunogenicity, which has potential in tumor‐targeted therapy. This study intended to investigate the effect of anti‐FGF2 ds‐Diabody on the migration and expression of programmed death‐ligand1 (PD‐L1) in hepatocellular carcinoma (HCC) cells. The anti‐FGF2 ds‐Diabody was expressed under methanol induction and purified with Ni2+‐affinity chromatography. Anti‐FGF2 ds‐Diabody significantly inhibited cell viability and proliferation in SK‐Hep1 and HepG2 cells as confirmed by CCK‐8 assays and colony formation assays. Western blot assays indicated that the proliferation of SK‐Hep1 and HepG2 cells was inhibited by anti‐FGF2 ds‐Diabody through inhibiting the phosphorylation activation of AKT and MAPK. The results of transwell and western blot assays showed that the migration and invasion of SK‐Hep1 and HepG2 cells were suppressed by anti‐FGF2 ds‐Diabody by affecting the epithelial‐mesenchymal transition (EMT) process. Meanwhile, anti‐FGF2 ds‐Diabody inhibited the expression of PD‐L1, and STAT3 participated in this process. Analysis of RT‐PCR and Western blot suggested that fibroblast growth factor receptor 4 inhibitor 1 (FGFR4‐IN‐1) suppressed the expression of PD‐L1, while STAT3 overexpression reversed this inhibitory effect. In addition, overexpression of STAT3 promoted migration and invasion and restored the suppressive effect of anti‐FGF2 ds‐Diabody on EMT. In conclusion, anti‐FGF2 ds‐Diabody could inhibit the expression of PD‐L1 and EMT of hepatoma cells through FGF2/FGFR4/STAT3 axis. These results suggested that anti‐FGF2 ds‐Diabody has potential clinical application in inhibiting metastasis and immune escape of hepatocellular carcinoma.