Dual FGFR and VEGFR inhibition synergistically restrain hexokinase 2-dependent lymphangiogenesis and immune escape in intrahepatic cholangiocarcinoma

Abstract Background Intrahepatic cholangiocarcinoma (iCCA) patients often lose the chance of radical resection due to early lymphatic metastasis. And most of the therapy in cholangiocarcinoma has been limited and ineffective. Herein, we examined the role of FGF and VEGF pathways in regulating lymphangiogenesis and PD-L1 expression. We also validated the possibility to achieve a powerful anti-lymphangiogenesis effect and improve the immunocompetence with combination therapy in iCCA. Methods The expression of FGFR1 and VEGFR3 in primary lymphatic endothelial cells (LECs) were detected by Flow cytometry. The lymphngiogenic function of FGF and VEGF were evaluated in LECs and iCCA xenograft mice model. The relationship between VEGF and hexokinase2 (HK2) was validated in LECs by western blot, immunofluorescence and luciferase reporter assays. The therapeutic efficacy of infigratinib in combination with SAR131675 were assessed in LECs and xenograft models. Microarray analysis was used to evaluate the pathological relevance between FGFR1 and VEGFR3 with HK2 in human lymphatic vessels. PD-L1 expression in LECs affected by combined treatment were analysis by flow cytometry and western blot. Results FGF promoted lymphangiogenesis through c-MYC-dependent modulation of HK2. In addition, VEGFC also upregulated HK2 expression. Mechanically, VEGFC phosphorylated PI3K/Akt/mTOR axis to upregulate HIF-1α expression at the translational level, HIF-1α then bind to the HK2 promoter region for transcriptional activation. More importantly, dual FGFR and VEGFR inhibition with infigratinib and SAR131675 almost completely inhibited lymphangiogenesis in LECs and iCCA xenograft mice model. Histologically, high HK2 expression in lymphatic vessels were significantly associated with poor iCCA prognosis and the expression of HK2, FGFR1 and VEGFR3 in lymphatic vessels were related to lymph node metastasis in intrahepatic cholangiocarcinoma. Meanwhile, FGFR1 and VEGFR3 were significantly correlated with HK2 in lymphatic vessels. Furthermore, the combination of infigratinib and SAR131675 significantly reduced PD-L1 expression in LECs through inhibiting lactic acid production. Conclusions Dual FGFR and VEGFR inhibition restrain lymphangiogenesis through suppression c-MYC-dependent and HIF-1α-mediated HK2 expression respectively. Decreased HK2 down-regulated glycolytic activity and further attenuated PD-L1 expression. Our findings suggest that dual FGFR and VEGFR blocking is an effective novel combination strategy to inhibit lymphangiogenesis and improve the immunocompetence in iCCA.