cGAS-STING agonist cGAMP enhances natural killer cell-mediated cytotoxicity against gastric cancer cells

doi:10.12122/j.issn.1673-4254.2026.02.21

Abstract

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.

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.

Figures/Tables 7

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

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

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.

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.

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.

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.

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