cGAS-STING激动剂cGAMP可增强自然杀伤细胞的抗胃癌效应

doi:10.12122/j.issn.1673-4254.2026.02.21

摘要/Abstract

摘要：

目的探讨环鸟苷酸-腺苷酸合成酶-干扰素基因刺激蛋白（cGAS-STING）激活剂增强自然杀伤细胞（NK细胞）杀伤胃癌细胞的分子机制。方法使用NK-92系细胞，在X-VIVO15培养基中培养至1×10⁶/mL，放入CO₂培养箱中进行培养；将胃癌细胞系（MGC-803细胞、MKN-45细胞）在RPMI 1640培养基中培养；5、1、0.2 μmol/L的cGAMP激动剂与准备好的NK-92系共培养24 h；通过qPCR、ELISA法检测NK-92系细胞与肿瘤细胞共培养后细胞因子IFN-γ的表达情况；将培养好的NK-92系细胞与用CellTracker™CM-DiI染料标记的肿瘤细胞以不同的比值（1∶1、2∶1和4∶1）进行共培养，通过死细胞染色试剂盒测定肿瘤细胞活力；最后通过流式细胞杀伤术检测NK-92系的杀伤程度。结果 cGAS-STING激动剂cGAMP可剂量依赖性增强NK-92系细胞IFN-γ、TNF-α等促炎细胞因子的mRNA表达与分泌（P<0.01），上调细胞表面活化受体（NKp36、NKp44等）表达，激活STING-TBK1-IRF3信号通路；体外实验中，cGAMP预处理的NK-92系细胞对MGC-803、MKN-45胃癌细胞杀伤率升高，且该效应可被STING阻断剂H-151逆转；小鼠荷瘤体内实验中，cGAMP联合NK细胞治疗使裸鼠胃癌移植瘤质量下降约40%（P<0.05）、生长减缓。结论 cGAS-STING通路可增强NK细胞分泌IFN-γ能力，进而提升其对胃癌细胞的杀伤作用，为NK细胞联合STING激动剂治疗胃癌提供了理论依据。

关键词: cGAS-STING, 胃癌细胞, NK-92系细胞, 肿瘤免疫治疗

Abstract:

Objective To explore the molecular mechanism by which cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) agonists enhance cytotoxicity of natural killer (NK) cells against gastric cancer cells. Methods NK-92 cells were cultured in X-VIVO15 medium to a density of 1×10⁶/mL and treated with 5, 1, or 0.2 μmol/L cGAMP for 24 h before co-culture with gastric cancer MGC-803 and MKN-45 cells in RPMI 1640 medium. The expression of IFN-γ in co-cultured NK-92 cells and tumor cells was detected by qPCR and ELISA. The treated NK-92 cells were then co-cultured with the tumor cells labeled with CellTracker™ CM-DiI at different ratios (1:1, 2:1, and 4:1), and tumor cell viability and cytotoxicity of NK-92 cells were determined using a dead cell staining kit and flow cytometry-based cytotoxicity assay. Results The cGAS-STING agonist cGAMP dose-dependently enhanced mRNA expression and secretion of the pro-inflammatory cytokines (such as IFN-γ and TNF-α) in NK-92 cells, and NK-92 cells treated with 5 μmol/L cGAMP showed approximately 3-fold increase of IFN-γ and TNF-α levels. cGAMP treatment also upregulated the expression of activating receptors (such as NKp36 and NKp44) on the cell surface and activated the STING-TBK1-IRF3 signaling pathway in NK-92 cells. NK-92 cells pretreated with cGAMP showed increased cytotoxicity against MGC-803 and MKN-45 cells, which could be reversed by treatment with H-151 (a STING inhibitor). In tumor-bearing nude mice, combined treatment with cGAMP and NK cells reduced the mass of xenografts by nearly 40% and significantly slowed tumor growth. Conclusion Activating the cGAS-STING pathway can enhance IFN-γ secretion of NK cells to improve their cytotoxicity against gastric cancer cells, suggesting a therapeutic strategy for gastric cancer using NK cells combined with STING agonists.

Key words: cGAS-STING, Gastric cancer cells, NK-92 cells, Tumor Immunotherapy.

王强, 柴志欣, 邓玉露, 张志葳, 龚英, 高圣, 冯平锋. cGAS-STING激动剂cGAMP可增强自然杀伤细胞的抗胃癌效应[J]. 南方医科大学学报, 2026, 46(2): 434-442.

Qiang WANG, Zhixin CHAI, Yulu DENG, Zhiwei ZHANG, Ying GONG, Sheng GAO, Pingfeng FENG. cGAS-STING agonist cGAMP enhances natural killer cell-mediated cytotoxicity against gastric cancer cells[J]. Journal of Southern Medical University, 2026, 46(2): 434-442.

图/表 7

表1 动物实验分组和依据

Tab.1 Experimental Animal Treatment Groups (n=4)

Group

Post-modeling treatment method

Treatment basis

PBS

肿瘤体积达 100 mm³ 后, 隔日腹腔注射 100 μL PBS,

持续至实验终点

作为空白对照,排除注射操作、溶剂(PBS)

对肿瘤生长的影响,明确后续治疗效应的特异性

NK Cell

肿瘤体积达 100 mm³ 后, 尾静脉回输 1×10⁷个 NK细胞;

隔日腹腔注射 20000 IU IL-2, 持续至实验终点

IL-2 是 NK 细胞体外培养和体内存活的关键细胞因子,

可维持 NK 细胞活性,该组用于验证单独 NK

细胞对胃癌移植瘤的杀伤效果

NK Cell+cGAMP

肿瘤体积达 100 mm³ 后, 尾静脉回输 1×10⁷个 NK 细胞;

隔日腹腔注射 20000 IU IL-2; 隔日腹腔注射 1 μg/只 cGAMP,

持续至实验终点

基于前期体外实验中 cGAMP 对 NK 细胞的激活效应,

该组通过联合给药验证 cGAMP 是否能协同增强 NK

细胞的体内抗胃癌活性

表2 引物序列

Tab.2 qPCR primers

Gene	Forward primers (5'to3')	Reverse primers (5'to3')
GAPDH	CTGTTCGACAGTCAGCCGCATC	GCGCCCAATACGACCAAATCCG
IFN-γ	TCGGTAACTGACTTGAATGTCCA	TCGCTTCCCTGTTTTAGCTGC
TNF-α	GAGGCCAAGCCCTGGTATG	CGGGCCGATTGATCTCAGC
GZMB	CCCTGGGAAAACACTCACACA	GCACAACTCAATGGTACTGTCG
IL-2	AACTCCTGTCTTGCATTGCAC	GCTCCAGTTGTAGCTGTGTTT
Perforin	GACTGCCTGACTGTCGAGG	TCCCGGTAGGTTTGGTGGAA
NKG2D	CCTTGACCGAAAGTTACTGTGG	GGCTGGCATTTTGAGACATACAA
TIGIT	TGGTCGCGTTGACTAGAAAGA	GGGCTCCATTCCTCCTGTC

图1 cGAS-STING激动剂促进NK-92细胞促炎细胞因子表达

Fig.1 Effects of cGAS-STING agonist on mRNA expressions in NK-92 cells. Statistical analysis was done using one-way ANOVA with Tukey post-tests (n=3). *P<0.05, **P<0.01.

图2 cGAS-STING激动剂增强NK-92细胞活性具有cGAMP剂量依赖性

Fig.2 Dose-dependent enhancement of NK-92 cell activity by cGAS-STING agonist cGAMP (Mean±SD, n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图3 cGAS-STING激动剂促进NK-92细胞活化

图4 cGAS-STING激动剂增强NK-92细胞对胃癌细胞的体外杀伤能力

Fig.4 cGAS-STING agonist enhances cytotoxicity of NK-92 cells against gastric cancer cells in vitro. NK-92 cells pretreated with 1 μmol/L cGAMP showed significantly enhanced cytotoxicity against gastric cancer cells MGC-803 (A) and MKN-45 (B) at effector-to-target ratios of 1:1, 2:1 and 4:1. C: STING antagonist H-151 blockade assay, in which NK-92 cells were pretreated with 1×10-6 mol/L cGAMP alone or combined with 1×10-6 mol/L H-151 for 24 h, then co-cultured with MGC-803 or MKN-45 cells for another 4 h. Dead tumor cells were analyzed by flow cytometry. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图5 cGAS-STING激动剂增强NK-92细胞对胃癌细胞的体内杀伤能力

Fig.5 　cGAS-STING agonist enhances NK-92 cell cytotoxicity against gastric cancer cells in nude mice. A: Process of tumor-bearing mouse model establishment and treatment process. B: Representative images of tumors at experimental endpoint showing obviously reduced tumor size in NK+cGAMP group. C: Bar graph of tumor weight at endpoint showing reduced tumor mass in NK+cGAMP group by 40% relative to that in NK-only group. D: Line graph of tumor volume in each group at endpoint. Data are presented as Mean±SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

参考文献 29

[1]	Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-49. doi：10.3322/caac.21660
[2]	Joshi SS, Badgwell BD. Current treatment and recent progress in gastric cancer[J]. CA Cancer J Clin, 2021, 71(3): 264-79. doi：10.3322/caac.21657
[3]	Smyth EC, Nilsson M, Grabsch HI, et al. Gastric cancer[J]. Lancet, 2020, 396(10251): 635-48. doi：10.1016/s0140-6736(20)31288-5
[4]	Wagner AD, Syn NL, Moehler M, et al. Chemotherapy for advanced gastric cancer[J]. Cochrane Database Syst Rev, 2017, 8(8): CD004064. doi：10.1002/14651858.cd004064.pub4
[5]	Fuchs CS, Doi T, Jang RW, et al. Safety and efficacy of pembrolizumab monotherapy in patients with previously treated advanced gastric and gastroesophageal junction cancer: phase 2 clinical KEYNOTE-059 trial[J]. JAMA Oncol, 2018, 4(5): e180013. doi：10.1001/jamaoncol.2018.0013
[6]	Kang YK, Boku N, Satoh T, et al. Nivolumab in patients with advanced gastric or gastro-oesophageal junction cancer refractory to, or intolerant of, at least two previous chemotherapy regimens (ONO-4538-12, ATTRACTION-2): a randomised, double-blind, placebo-controlled, phase 3 trial[J]. Lancet, 2017, 390(10111): 2461-71. doi：10.1016/s0140-6736(17)31827-5
[7]	Shitara K, Özgüroğlu M, Bang YJ, et al. Pembrolizumab versus paclitaxel for previously treated, advanced gastric or gastro-oesophageal junction cancer (KEYNOTE-061): a randomised, open-label, controlled, phase 3 trial[J]. Lancet, 2018, 392(10142): 123-33.
[8]	Vivier E, Tomasello E, Baratin M, et al. Functions of natural killer cells[J]. Nat Immunol, 2008, 9(5): 503-10. doi：10.1038/ni1582
[9]	Myers JA, Miller JS. Exploring the NK cell platform for cancer immunotherapy[J]. Nat Rev Clin Oncol, 2021, 18(2): 85-100. doi：10.1038/s41571-020-0426-7
[10]	Liu SZ, Galat V, Galat Y, et al. NK cell-based cancer immunotherapy: from basic biology to clinical development[J]. J Hematol Oncol, 2021, 14(1): 7. doi：10.1186/s13045-020-01014-w
[11]	Bald T, Krummel MF, Smyth MJ, et al. The NK cell-cancer cycle: advances and new challenges in NK cell-based immunotherapies[J]. Nat Immunol, 2020, 21(8): 835-47. doi：10.1038/s41590-020-0728-z
[12]	Li T, Chen ZJ. The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer[J]. J Exp Med, 2018, 215(5): 1287-99. doi：10.1084/jem.20180139
[13]	Marcus A, Mao AJ, Lensink-Vasan M, et al. Tumor-derived cGAMP triggers a STING-mediated interferon response in non-tumor cells to activate the NK cell response[J]. Immunity, 2018, 49(4): 754-63.e4. doi：10.1016/j.immuni.2018.09.016
[14]	Gong Y, Germeraad WTV, Zhang XL, et al. NKG2A genetic deletion promotes human primary NK cell anti-tumor responses better than an anti-NKG2A monoclonal antibody[J]. Mol Ther, 2024, 32(8): 2711-27. doi：10.1016/j.ymthe.2024.06.034
[15]	Ablasser A, Schmid-Burgk JL, Hemmerling I, et al. Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP[J]. Nature, 2013, 503(7477): 530-4. doi：10.1038/nature12640
[16]	Motwani M, Pesiridis S, Fitzgerald KA. DNA sensing by the cGAS-STING pathway in health and disease[J]. Nat Rev Genet, 2019, 20(11): 657-74. doi：10.1038/s41576-019-0151-1
[17]	Corrales L, McWhirter SM, Dubensky TW Jr, et al. The host STING pathway at the interface of cancer and immunity[J]. J Clin Invest, 2016, 126(7): 2404-11. doi：10.1172/jci86892
[18]	Vivier E, Raulet DH, Moretta A, et al. Innate or adaptive immunity? The example of natural killer cells[J]. Science, 2011, 331(6013): 44-9. doi：10.1126/science.1198687
[19]	Orange JS. Formation and function of the lytic NK-cell immunological synapse[J]. Nat Rev Immunol, 2008, 8(9): 713-25. doi：10.1038/nri2381
[20]	Ashkenazi A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily[J]. Nat Rev Cancer, 2002, 2(6): 420-30. doi：10.1038/nrc821
[21]	龚英, 艾丽飞热·艾麦提, 何宗忠. CD39小分子抑制剂ARL67156增强NK细胞对胃癌细胞的杀伤作用[J]. 南方医科大学学报, 2023, 43(12): 2006-14.
[22]	Lu L, Yang C, Zhou XY, et al. STING signaling promotes NK cell antitumor immunity and maintains a reservoir of TCF-1⁺ NK cells[J]. Cell Rep, 2023, 42(9): 113108. doi：10.1016/j.celrep.2023.113108
[23]	Da YY, Liu YX, Hu Y, et al. STING agonist cGAMP enhances anti-tumor activity of CAR-NK cells against pancreatic cancer[J]. Oncoimmunology, 2022, 11(1): 2054105. doi：10.1080/2162402x.2022.2054105
[24]	Zhang LL, Wei XB, Wang ZM, et al. NF-κB activation enhances STING signaling by altering microtubule-mediated STING trafficking[J]. Cell Rep, 2023, 42(3): 112185. doi：10.1016/j.celrep.2023.112185
[25]	Bakhoum SF, Ngo B, Laughney AM, et al. Chromosomal instability drives metastasis through a cytosolic DNA response[J]. Nature, 2018, 553(7689): 467-72. doi：10.1038/nature25432
[26]	Berger G, Knelson EH, Jimenez-Macias JL, et al. STING activation promotes robust immune response and NK cell-mediated tumor regression in glioblastoma models[J]. Proc Natl Acad Sci USA, 2022, 119(28): e2111003119. doi：10.1073/pnas.2111003119
[27]	Wang QW, Bergholz JS, Ding LY, et al. STING agonism reprograms tumor-associated macrophages and overcomes resistance to PARP inhibition in BRCA1-deficient models of breast cancer[J]. Nat Commun, 2022, 13(1): 3022. doi：10.1038/s41467-022-30568-1
[28]	Baird JR, Feng ZP, Xiao HD, et al. STING expression and response to treatment with STING ligands in premalignant and malignant disease[J]. PLoS One, 2017, 12(11): e0187532. doi：10.1371/journal.pone.0187532
[29]	Chen XN, Meng FC, Xu YT, et al. Chemically programmed STING-activating nano-liposomal vesicles improve anticancer immunity[J]. Nat Commun, 2023, 14(1): 4584. doi：10.1038/s41467-023-40312-y