State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
Minimally Invasive Interventional Division, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
J Immunother Cancer. 2021 Jun;9(6). doi: 10.1136/jitc-2020-002305.
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.
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.
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.
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.
中性粒细胞在肿瘤发展中扮演着有争议的角色。然而，程序性细胞死亡配体 1（PD-L1）阳性中性粒细胞的功能可能会抑制抗肿瘤免疫的细胞毒性。在这项研究中，我们阐明了肿瘤微环境（TME）中 PD-L1 阳性中性粒细胞的刺激物，并探索了通过抑制肝癌中的 PD-L1 阳性中性粒细胞来增强仑伐替尼效果的最佳组合。
采用 RNA 测序和多色流式细胞术分析在患者样本、皮下和原位小鼠模型中检测仑伐替尼治疗后中性粒细胞的浸润情况。从外周血和肿瘤组织中分离中性粒细胞和 T 细胞，并使用磁珠进行细胞毒性测定进行纯化。使用 Yellow Springs Instrument（YSI）生产的生化分析仪检测代谢物和细胞因子。通过体外筛选途径抑制剂来识别可能减少 PD-L1 阳性中性粒细胞浸润的候选物。进一步进行体内实验验证。
仑伐替尼通过在 TME 中诱导 CXCL2 和 CXCL5 的分泌增加中性粒细胞的募集。进入 TME 后，中性粒细胞向 N2 表型极化。同时上调 PD-L1 的表达。因此，仑伐替尼对肿瘤细胞的疗效受到阻碍。增加的 PD-L1 阳性中性粒细胞与抑制性 T 细胞表型呈正相关。进一步的研究表明，由免疫细胞衍生的干扰素 γ 激活的 JAK/STAT1 途径和由肿瘤衍生的高浓度乳酸激活的 MCT1/NF-κB/COX-2 途径可以诱导 PD-L1 阳性中性粒细胞。后者可以被 COX-2 抑制剂塞来昔布显著抑制。进一步的体内实验验证了塞来昔布降低了乳酸刺激的 PD-L1 阳性中性粒细胞的存活率，并促进了仑伐替尼的抗肿瘤作用。
PD-L1 阳性中性粒细胞降低了 T 细胞的细胞毒性。肿瘤衍生的乳酸通过 MCT1/NF-κB/COX-2 途径诱导中性粒细胞上 PD-L1 的表达。因此，COX-2 抑制剂可以减少 PD-L1 阳性中性粒细胞并恢复 T 细胞的细胞毒性。这可能为仑伐替尼提供一种有效的辅助治疗方法。
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