Tumor-derived lactate inhibit the efficacy of lenvatinib through regulating PD-L1 expression on neutrophil in hepatocellular carcinoma

BACKGROUND: Neutrophils play a controversial role in tumor development. The function of programmed cell death-1 ligand (PD-L1(+)) neutrophils, however, may inhibit the cytotoxicity of anti-tumor immunity. In this study, we elucidate the stimulators of PD-L1(+) neutrophils in tumor microenvironment (TME) and explore the optimal combination to enhance the effect of lenvatinib by inhibiting PD-L1(+) neutrophils in hepatocellular carcinoma. METHODS: Neutrophil infiltration after lenvatinib treatment was examined with RNA sequencing and multicolor flow cytometry analysis in patient samples, subcutaneous and orthotopic mouse models. Neutrophils and T cells were isolated from peripheral blood and tumor tissues and purified with magnetic beads for cytotoxicity assay. Metabolites and cytokines were detected by a biochemical analyzer manufactured by Yellow Springs Instrument (YSI) and proteome profiler cytokines array. In vitro screening of pathway inhibitors was used to identify possible candidates that could reduce PD-L1(+) neutrophil infiltration. Further in vivo assays were used for verification. RESULTS: Lenvatinib increased neutrophil recruitment by inducing CXCL2 and CXCL5 secretion in TME. After entering TME, neutrophils polarized toward N2 phenotype. PD-L1 expression was simultaneously upregulated. Thus, lenvatinib efficacy on tumor cells hindered. The increasing PD-L1(+) neutrophils positively corelated with a suppressive T cell phenotype. Further investigation indicated that JAK/STAT1 pathway activated by immune-cell-derived interferon γ and MCT1/NF-kB/COX-2 pathway activated by high concentrations of tumor-derived lactate could induce PD-L1(+) neutrophils. The latter could be significantly inhibited by COX-2 inhibitor celecoxib. Further in vivo assays verified that Celecoxib decreased the survival of lactate-stimulated PD-L1(+) neutrophil and promoted the antitumor effect of lenvatinib. CONCLUSIONS: PD-L1(+) neutrophils decrease T cell cytotoxicity. Tumor-derived lactate induces PD-L1 expression on neutrophils via MCT1/NF-κB/COX-2 pathway. Thus, COX-2 inhibitor could reduce PD-L1(+) neutrophil and restore T cell cytotoxicity. This may provide a potent addition to lenvatinib.