Dual Vascular Endothelial Growth Factor Receptor and Fibroblast Growth Factor Receptor Inhibition Elicits Antitumor Immunity and Enhances Programmed Cell Death-1 Checkpoint Blockade in Hepatocellular Carcinoma

Abstract

Background and aims: Combining anti-angiogenic therapy with immune checkpoint blockade with anti-programmed cell death-1 (PD-1) antibodies is a promising treatment for hepatocellular carcinoma (HCC). Tyrosine kinase inhibitors are well-known anti-angiogenic agents and offer potential for combination with anti-PD-1 antibodies. This study investigated the possible underlying immunomodulatory mechanisms of combined therapy.

Methods: HCC tissue samples for RNA-sequencing (RNA-seq) were obtained from patients with differential prognoses following anti-PD-1 treatment. Recombinant basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) were used to stimulate T cells following lenvatinib or sorafenib treatment, respectively. T cell function was analyzed by flow cytometry and lactate dehydrogenase assay. In vivo experiments were conducted in murine H22 and Hepa 1-6 competent models of HCC. Local immune infiltration in the tumor microenvironment (TME) was assessed using multicolor flow cytometry. Gene regulation was evaluated by RNA-seq. Microvascular density was measured by immunohistochemistry, and PD-1 ligand (PD-L1) induction was quantified by western blot.

Results: The baseline expression of VEGF and fibroblast growth factor (FGF) in patients with progressive disease was significantly higher than in patients achieving stable disease following anti-PD-1 treatment. VEGFA and bFGF significantly upregulated the expression of PD-1, cytotoxic T-lymphocyte-associated protein-4, and Tim-3 on T cells, while inhibiting the secretion of interferon gamma (IFNG) and granzyme B and suppressing T cell cytotoxicity. This immunosuppressive effect was reverted by lenvatinib but not sorafenib. Furthermore, dual lenvatinib/anti-PD-1 antibody therapy led to better antitumor effects than either sorafenib or fibroblast growth factor receptor (FGFR) inhibitor (BGJ398) in H22 murine models of HCC. Combined lenvatinib/anti-PD-1 treatment also led to long-term immune memory formation, while synergistically modulating the TME and enhancing the cytotoxic effect of T cells. Finally, lenvatinib inhibited PD-L1 expression on human umbilical vein endothelial cells, which improved the function of T cells.

Conclusions: Inhibition of vascular endothelial growth factor receptor and FGFR augmented the efficacy of anti-PD-1 antibodies. Combined lenvatinib/anti-PD-1 treatment appears to exert antitumor activity by synergistically modulating effector T cell function in the TME and by mutually regulating tumor vessel normalization.

Keywords: Immune checkpoint; Immunomodulatory; Lenvatinib; Liver cancer; Programmed cell death-1 antibodies; Sorafenib; Vascular endothelial growth factor A; bFGF.

Conflict of interest statement

The authors have no conflict of interest.

Copyright © 2020 by S. Karger AG, Basel.

Figures

Fig. 1

The expression of angiogenesis-related factors in patients with HCC before initiating anti-PD-1 antibody treatment. Baseline tumor tissue samples from HCC patients treated with PD-1 antibodies were analyzed using RNA-seq. According to treatment response assessed using imaging evaluation (mRECIST), the RNA expression was analyzed (3 patients with partial response [PD] vs. 2 with SD). a Treatment pattern diagram and disease progression for HCC patients. b Relative mRNA expression of angiogenesis-related factors. c FGF family. Tyrosine kinase receptors (d) and chemokines (e) in patients achieving a PD and SD as tested by RNA-seq. fThe secretion level of VEGFA in HCC cell lines tested by enzyme-linked immunosorbent assay and read by microplate reader at 450 nm. * p < 0.05, ** p < 0.01, **** p < 0.0001 (two-way ANOVA). PD-1, programmed cell death-1; VEGFA, vascular endothelial growth factor A; VEGFB, vascular endothelial growth factor B; VEGFC, vascular endothelial growth factor C; FGF, fibroblast growth factor; FGFR1, FGF receptor 1; IL, interleukin; PDGFD, platelet-derived growth factor D; PDGFC, platelet-derived growth factor C; MMP9, matrix metalloprotein; SD, stable disease; PD, progressive disease; TKR, tyrosine kinase receptors; KDR, kinase insert domain receptor; PDGFRA, platelet-derived growth factor receptor A; PDGFRB, platelet-derived growth factor receptor B; CXCL, chemokine (C-X-C motif) ligand.

Fig. 2

The effect of stimulation of VEGFA and bFGF on T cells derived from healthy donors and patients with HCC with or without PD-1 antibody treatment were analyzed for PD-1, CTLA-4, and Tim-3 expression and IFNG or GZMB secretion. a Representative images and histogram showing the expression of PD-1 on T cells stimulated by VEGFA (50 ng/mL), bFGF (50 ng/mL), or the combination of both. b Healthy donor-derived T cells were separated and RNA expression of bFGF (FGF2) was analyzed in remaining T cells and activated T cells by CD3 antibody (5 ng/mL) and CD28 antibody (2.5 ng/mL) and activated T cells treated with lenvatinib (100 nM), sorafenib (100 nM), and BGJ398 (100 nM). c Healthy donor-derived T cells were cultured with HepG2 supernatant and then stimulated by bFGF with gradient concentrations (0, 20, 50, 100 ng/mL). d Representative images showing CTLA-4 and Tim-3 expression on PD-1+/− T cells. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-way ANOVA). VEGF, vascular endothelial growth factor; FGF2, fibroblast growth factor 2; PD-1, programmed cell death-1; HCC, hepatocellular carcinoma; LEN, lenvatinib; SOR, Sorafenib; CD8, cluster of differentiation 8; CTLA4, cytotoxic lymphocyte antigen 4; FSC-H, forward scatter height.

Fig. 3

The effect of dual targeting of VEGFR and FGFR by lenvatinib on T cell function. HCC-patient-derived T cells were used to assess the effect of targeting VEGFR and FGFR on T cell function and cytotoxicity. a Representative images showing the expression of PD-1 on T cells stimulated by VEGFA (50 ng/mL) combined with bFGF (50 ng/mL), and then treated with lenvatinib (100 nM) and sorafenib (100 nM), respectively. b The effect of lenvatinib with different concentration gradients (0, 30, 60, 120, 240, 480, 960, 2,000, 3,000 nM) on T cell proliferation, tested by CCK8 and read by microplate reader at 450 nm. c HCC-patient-derived T cells were co-cultured with or without HepG2 cells and treated with or without lenvatinib (100 nM); T cells were cultured with HepG2 cells and treated with Blank, VEGFA (20 ng/mL), VEGFA (20 ng/mL) + lenvatinib (100 nM), and VEGFA (20 ng/mL) + sorafenib (100 nM). The LDH level was tested and read by microplate reader at 450 nm. Representative images showing the expression of IFNG (d) and GZMB (e) on T cells stimulated by VEGFA or bFGF, respectively, and the combination, and then treated with lenvatinib (100 nM), sorafenib (100 nM), and BGJ398 (FGFRi; 100 nM), respectively. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA). VEGF, vascular endothelial growth factor; LEN, lenvatinib; PD-1, programmed cell death-1; LDH, lactate dehydrogenase; IFNG, interferon gamma; GZMB, granzyme B; SSC-A, side scatter; PE-A, KO525, VF, and ECD-A are fluorescent dyes; HepG2 is a hepatoma cell line; BLANK is referred to the control group used in this experiment.

Fig. 4

The effect of lenvatinib, sorafenib, or BGJ398 combined with anti-PD-1 antibodies in vivo. In vivo antitumor experiments in murine models of HCC were performed to compare the anti-tumor effects of lenvatinib, sorafenib, and BGJ398 combined with PD-1 antibodies. a Tumor growth curves over 10 days for the control group (n = 5), lenvatinib combined with PD-1 antibodies group (10 mg/kg, 200 µg/3 days; n = 5), sorafenib combined with PD-1 antibodies group (50 mg/kg, 200 µg/3 days; n = 5) and BGJ398 combined with PD-1 antibodies group (10 mg/kg, 200 µg/3 days; n = 5) in Balb/C mice models subcutaneously implanted with H22 cells. b Mean weights for different treatment groups versus control in Balb/C mice models. c Tumor volume changes during treatment in the different groups and the final mean tumor volume at day 10 in the different treatment groups; individual tumor growth curves over the 10 days of treatment in the control group (d); lenvatinib combined with anti-PD-1 antibodies group (e); sorafenib combined with anti-PD-1 antibodies group (f); and BGJ398 combined with anti-PD-1 antibodies group (g). * p < 0.05, ** p < 0.01, **** p < 0.0001 (one-way/two-way ANOVA). LEN, lenvatinib; PD-1, programmed cell death-1.

Fig. 5

Antitumor and immunoregulatory effect of combination treatment with lenvatinib and anti-PD-1 antibodies on murine models of HCC. The effect of combination treatment with lenvatinib and anti-PD-1 antibodies was investigated in 2 HCC murine models and the immune TME was tested by flow cytometry. a Mean tumor weights for the lenvatinib group (10 mg/kg), PD-1 antibodies group (100 µg/3 days), and lenvatinib combined with PD-1 antibodies group (10 mg/kg, 100 µg/3 days) versus control group treated for 21 days in Balb/C mice models subcutaneously implanted with H22 cells (n = 7 per group). b Mean tumor volume for the different treatment groups versus control over 21 days of treatment in C57BL/6 mice models subcutaneously implanted with Hepa 1–6 cells (n = 5 per group); survival curve of mice over 24 days of treatment for Balb/C (c) and C57BL/6 (d) mice in the different treatment groups; tumor re-challenge for CR mice in each group after a 50-day washout without treatment in Balb/C mice and C57BL/6 mice versus normal control (e). Histogram showing infiltration of CD4, CD8, NK, and the proportion of PD-1+ NK cells, and infiltration of dendritic cells and the portion of MHC+/PD-L1+ dendritic cells of Balb/C (f) and C57BL/6 mice (g) measured by flow cytometry. h Representative flow cytometry image showing the change of CD8 T cells in lenvatinib group versus control group; infiltration of PD-1+ CD8, PD-1+ CD4, and T regulatory cell of Balb/C and C57BL/6 mice measured by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA). LEN, lenvatinib; PD-1, programmed cell death-1; NK, natural killer; MHC+, major histocompatibility complex-positive; PD-L1, PD-1 ligand; DC, dendritic cell; PE-Cy7, a fluorescent dye; CON, control group.

Fig. 6

The effect of combination lenvatinib/anti-PD-1 therapy on the TME. Profiler Mice XL cytokine analysis, RNA-seq, IHC, and TSA-IHC were used to evaluate the effect of lenvatinib combined with PD-1 antibodies therapy on the TME. a Profiler Mice XL cytokine analysis of peripheral blood plasma from tumor-bearing Balb/C mice in the different treatment groups. b Venn diagram analysis of gene expression profiles in tumors in the different treatment groups tested by RNA-seq. c Markers of activated T cells in the different treatment groups tested by RNA-seq. d Gene expression of Cluster 4 in the different treatment groups tested by RNA-seq. e Representative images and histogram of CD31 immunohistochemical staining in the different treatment groups in Balb/C mice and C57BL/6 mice. f Representative images of CD8 and PD-1 tyramide signal amplification immunohistochemical staining in tumor tissues sampled from tumor-bearing Balb/C mice in the different treatment groups and representative images for PD-1+ CD8 distribution in the combination group versus the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA). PD-1, programmed cell death-1; LEN, lenvatinib; ANGPT, angiopoietin 1; TNF-a, tumor necrosis factor-a; ACTB, beta actin, GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Si-NC, the negative control of small interference RNA.

Fig. 7

The effect of lenvatinib on PD-L1 expression on HUVEC. HUVEC were used to test the effect of lenvatinib on PD-L1 expression in vitro. a The effect of VEGFA (20 ng/mL) and lenvatinib with gradient concentration (4, 30, 300, 3,000 nM) on PD-L1 expression following stimulation by IFNG (10 ng/mL); the effect of HepG2 supernatant on PD-L1 expression in HUVEC. b The effect of downregulating VEGFR2 (sh21, sh22, sh23; si-01, si-02) in HUVEC on p-STAT1 and PD-L1 following stimulation by IFNG (10 ng/mL). c The effect of downregulating PD-L1 (si-01, si-02, si-03) in HUVEC on p-VEGFR2 following stimulation by VEGFA (50 ng/mL). d Diagram showing that dual targeting VEGFR and FGFR elicits antitumor immunity and enhances PD-1 checkpoint blockade in HCC. VEGF, vascular endothelial growth factor; LEN, lenvatinib; PD-1, programmed cell death-1; IFNG, interferon gamma; PD-L1, PD-1 ligand; HN and NH are both amines