Role of the HGF/c-MET pathway in resistance to immune checkpoint inhibitors in advanced non-small cell lung cancer

Abstract

Most of advanced non-small cell lung cancer (NSCLC) patients will experience tumor progression with immunotherapy (IO). Preliminary data suggested an association between high plasma HGF levels and poor response to IO in advanced NSCLC. Our study aimed to evaluate further the role of the HGF/MET pathway in resistance to IO in advanced NSCLC. We included retrospectively 82 consecutive NSCLC patients from two academic hospitals. Among them, 49 patients received ICIs alone or in combination with chemotherapy (CT), while 33 patients received chemotherapy alone as the control group. We analyzed plasma HGF levels by ELISA and expression of PD-L1, MET/phospho-MET, and CD8+ T-Cell infiltration on lung tumor tissue by immunohistochemistry. We investigated the contribution of HGF/MET to IO response by culturing peripheral blood mononuclear cells (PBMC) with or without pembrolizumab, with recombinant HGF, or cocultured with NSCLC patients-derived explants. Additionally, c-MET inhibitors were used to evaluate the contribution of MET activation in NSCLC-mediated immunosuppression. High HGF levels were associated with high progression rate with IO (p = 0.0092), but not with CT. ELISA analysis of supernatants collected from cultured NSCLC cells showed that HGF was produced by tumor cells. Furthermore, when activated PBMCs were cultured in the presence of recombinant HGF or on NSCLC monolayer, the proliferation of CD3+CD8+ lymphocytes was inhibited, even in the presence of pembrolizumab. The addition of HGF/MET inhibitors restored lymphocyte activation and induced IFNγ production. In conclusion, inhibiting the HGF/MET signaling pathway could be a promising approach to enhance the efficacy of immunotherapy.

Keywords: HGF; MET; immunotherapy; non-small cell lung cancer; resistance.

Fig. 1

Plasma HGF levels at baseline and first evaluation according to patient’s outcomes. Plasma samples were obtained from NSCLC patients at baseline and during the first evaluation. ELISA assays were utilized to quantify HGF levels. Subsequently, expression values were categorized based on drug response and disease progression, with A the group receiving ICIs alone and B the group receiving chemotherapy alone. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01

Fig. 2

Patient’s survival according to HGF expression levels. Kaplan–Meier analyses of patient survival (PFS and OS) were conducted based on baseline HGF levels (low or high) within each treatment group, including immunotherapy alone (A), immunotherapy associated with chemotherapy (B), and chemotherapy alone (C). The log-rank test was utilized to compare survival within each subgroup

Fig. 3

HGF production by NSCLC cells and inhibition of lymphocyte activation in the presence of pembrolizumab (“Pembro”) A expression levels of HGF in NSCLC cell cultures were assessed using an ELISA assay. B Peripheral blood mononuclear cells (PBMCs) were stained with CFSE and activated with phytohemagglutinin (PHA) alone or in combination with pembrolizumab. C PHA-activated cells were cultured either alone or on a primary NSCLC monolayer in the presence of pembrolizumab and/or crizotinib. After 96 h of incubation, CFSE-stained cells were collected and further stained with PE-Cy7-conjugated anti-CD3, BV421-conjugated anti-CD45, and APC-conjugated anti-CD8 antibodies. The cells were analyzed using flow cytometry (B). The presented data are representative of at least 6 patients

Fig. 4

MET inhibitors suppress the immunosuppressive capabilities of recombinant HGF. A, B Peripheral blood mononuclear cells (PBMCs) activated with phytohemagglutinin (PHA, 10 µg/ml) were cultured either alone (A) or in the presence of hepatocyte growth factor (HGF, 75 ng/mL) and/or MET inhibitors (crizotinib, SGX-523). The MTS proliferation assay was employed to quantify lymphocyte/PBMC proliferation at 72 h, while cell supernatants were collected at 24 h for interferon-gamma (IFNγ) quantification. C, D Activated PBMCs were stained with CFSE and treated for 96 h with HGF (75 ng/mL) alone or in combination with pembrolizumab (20 nm) and/or MET inhibitors (crizotinib, SGX-523). The stained cells were collected, probed with PE-Cy7-conjugated anti-CD3, BV421-conjugated anti-CD45, and APC-conjugated anti-CD8 antibodies, and analyzed using flow cytometry. Data are presented as mean ± SEM of at least four independent experiments. *p < 0.05 and ***p < 0.001