南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (12): 2006-2014.doi: 10.12122/j.issn.1673-4254.2023.12.03

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CD39小分子抑制剂ARL67156增强NK细胞对胃癌细胞的杀伤作用

龚 英，艾丽飞热·艾麦提，何宗忠   

  1. 南方医科大学南方医院检验医学科，广东 广州 510515；南部战区总医院输血医学科，广东 广州 510010
  • 出版日期:2023-12-20 发布日期:2023-12-29

ARL67156, a small-molecule CD39 inhibitor, enhances natural killer cell cytotoxicity against gastric cancer cells in vitro and in nude mice

GONG Ying, AIMAITI Ailifeire, HE Zongzhong   

  1. Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; Department of Transfusion Medicine, Southern Theater Command General Hospital, Guangzhou 510010, China
  • Online:2023-12-20 Published:2023-12-29

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摘要： 目的 探讨CD39小分子抑制剂ARL67156增强NK细胞杀伤胃癌细胞的影响及其机制。方法 采用磁珠抗体的方法外周血分离纯化人原代自然杀伤细胞（NK细胞），用100 μmol/L的ARL67156处理NK细胞24 h后，Western blot蛋白印迹检测NK细胞活化的信号通路，ELISA检测NK细胞与胃癌细胞共培养上清中细胞因子IFN-γ分泌情况，通过流式细胞术检测NK细胞的CD107a脱颗粒；流式细胞术体外检测NK细胞与胃癌细胞共培养后的杀伤能力；将12只小鼠随机分为3组（4只/组）：PBS组、NK细胞组、NK细胞+ARL67156组，建立皮下胃癌荷瘤小鼠模型，尾静脉回输NK细胞，腹腔注射ARL67156，采用裸鼠皮下成瘤模型研究人原代NK细胞杀伤胃癌细胞的疗效。结果 健康人外周血NK细胞表达（25.97±5.69）%的CD39分子；与对照组相比，ARL67156处理后的NK细胞上调vav1、Syk等蛋白（P<0.05），同时促进NKG2D、DAP10、CD57、CD16等活化分子的表达（P<0.05），降低TIGIT、KIR等抑制性受体表达，促进NK细胞中IFN-γ、CD107a脱颗粒的分泌（P<0.05）；体内实验中，与对照组相比，ARL67156联合NK细胞组，MKN-45胃癌瘤体体积更小（P<0.05）。结论 ARL67156通过vav1-Syk信号通路激活NK细胞，ARL67156在体外体内均能够增强NK细胞杀伤胃癌细胞的能力，能够为NK细胞在胃癌肿瘤免疫治疗中提供策略和理论指导。

关键词: 肿瘤免疫治疗；NK细胞；胃肿瘤；CD39；细胞因子

Abstract: Objective To investigate the effect of ARL67156, a small-molecule inhibitor of CD39, on cytotoxicity of natural killer (NK) cells against gastric cancer cells. Methods Human peripheral blood-derived primary NK cells isolated and purified using a magnetic bead antibody method were treated with 100 μmol/L ARL67156 for 24 h, and the signaling pathway of NK cell activation was detected by Western blotting. The level of interferon-γ (IFN-γ) in the supernatant of NK cells co-cultured with gastric cancer cells was detected using ELISA, and NK cell CD107a degranulation was measured with flow cytometry. The cytotoxicity of NK cells against co-cultured gastric cancer cells was evaluated using flow cytometry. In a nude mouse model bearing subcutaneous gastric cancer xenografts, the therapeutic effect of intravenous transfusion of NK cells and intraperitoneal injection of ARL67156 was assessed by measuring the changes in tumor volume. Results (25.97±5.69) % of peripheral blood NK cells from healthy individuals positive for CD39 expression. Treatment with ARL67156 significantly upregulated the activation molecules including NKG2D, DAP10, CD57, and CD16 and reduced the expressions of the inhibitory receptors TIGIT and KIR, thereby promoting the secretion of IFN-γ and CD107a degranulation in NK cells (P<0.05). In both the in vitro and in vivo experiments, ARL67156 significantly enhanced the cytotoxicity of NK cells against gastric cancer cells (P<0.05). Conclusion ARL67156 activates NK cells through the vav1-Syk signaling pathway to enhance their cytotoxicity against gastric cancer cells, which may serve as a new strategy for NK cell immunotherapy for gastric cancer.

Key words: tumor immunotherapy; natural killer cells; gastric cancer; CD39; vav1-Syk signaling pathway