Activation of STING by cGAMP Regulates MDSCs to Suppress Tumor Metastasis via Reversing Epithelial-Mesenchymal Transition

Abstract

The role of cGAMP stimulating cGAS-cGAMP-STING-IRF3 pathway to inhibit tumor growth was well-established. Herein, the efficiency and pharmacological mechanism of cGAMP on regulating tumor metastasis was investigated. The effects of cGAMP regulating CD8⁺ T cells and myeloid-derived suppressor cells (MDSCs) in tumor microenvironment was explored. In this study, we found that cGAMP boosted STING signaling pathway to activate the production of IFN-γ from CD8⁺ T cells, and decreased the population of MDSCs in vivo. The metastasis in CT26 tumor bearing mice was inhibited by cGAMP via regulating EMT process. cGAMP played an important role in suppressing the production of reactive oxygen species (ROS) and nitric oxide (NO) from MDSCs, abolished the suppressive function of MDSCs to the T cells. All in all, the results indicated that the STING agonist cGAMP activated the production of IFN-γ from CD8⁺ T cells to suppress MDSCs in vivo.

Keywords: STING; cGAMP; epithelial-mesenchymal transition; metastasis; myeloid-derived suppressor cells.

Figure 1

STING deficiency increases the growth of B16 tumors and reduces infiltrating immune cells. C57 and STING^−/− mice were injected with B16 cells. (A) Tumor volumes of B16 tumor bearing mice were detected in C57 and STING deficiency mice. (B) T cells (CD3⁺ cells and CD3⁺CD8⁺ cells) and IFN-γ released from CD3⁺CD8⁺ cells were detected by flow cytometry. (C) The quantification of CD45⁺ cells, T cells, and IFN-γ were showed in spleen. (D) The quantification of CD45⁺ cells, T cells, and IFN-γ were showed in tumor tissues. (E) The expression of CD8 was detected in tumor tissues as shown by immunofluorescence. (F) The expression level of CD8 in (E) was showed. The values are expressed as mean ± SD. (n = 6; *P < 0.05 **P < 0.01, vs. the control group).

Figure 2

CD11b⁺Gr-1⁺ MDSCs are significantly increased in STING-deficient mice. C57 and STING^−/− mice were injected with B16 cells. (A) The population of MDSCs in spleens was detected by flow cytometry. (B) The population of MDSCs in tumor tissues was detected by flow cytometry. (C) Quantification of the population of MDSCs, PMN-MDSCs and M-MDSCs in spleens. (D) Quantification of the population of MDSCs, PMN-MDSCs, and M-MDSCs in tumor tissues. (E,F) Representative images of the PMN-MDSCs markers, CD11b, and Ly6G, detected by IF in tumor tissues. (G) Ki67 was detected by IF to indicate the proliferation of tumor cells. The values are expressed as mean ± SD. (n = 6; *P < 0.05 vs. the control group).

Figure 3

cGAMP inhibits tumor growth and metastasis in vivo. Mice were treated with cGAMP (20 mg/kg, i.v.) daily until the end of the experiments, 3 d prior to injecting tumor cells. CT26 or B16 cells were injected into the right flank of the mice, while for the metastasis model, CT26 cells were injected into the spleen of mice and B16F10 cells were injected into the tail vein. Mice were sacrificed and harvested the main organs at day 28. (A) cGAMP decreased the tumor volumes of CT26 tumor-bearing mice. (B) CT26-luc cells were used to measure tumor volumes in tumor-bearing mice; cGAMP decreased the radiance of CT26-luc mice. (C) cGAMP decreased the metastasis of CT26 tumor cells in liver tissues. (D) The weight of spleen was detected as shown. (E) cGAMP decreased the tumor volumes of B16 tumor-bearing mice. (F) Lung tissues of B16F10 metastasis tumor mice are shown; cGAMP significantly decreased the metastatic nodes in tumor bearing mice. The values are expressed as mean ± SD. (n = 6; *P < 0.05 **P < 0.01 vs. the control group).

Figure 4

cGAMP inhibits tumor growth and metastasis via regulating MDSCs in vivo. Mice were treated with cGAMP (20 mg/kg, i.v.) daily 3 d before B16 cells were injected into their right flank. (A) CD3⁺ T cells, CD3⁺CD8⁺ T cells, and IFN-γ released from CD3⁺CD8⁺ T cells were detected by flow cytometry. (B) cGAMP increased the population of CD3⁺ T cells, CD3⁺CD8⁺ T cells, and IFN-γ in the spleen. (C) cGAMP increased the population of CD3⁺ T cells, CD3⁺CD8⁺ T cells, and IFN-γ in the tumor tissues. (D) Diagram of the flow cytometry analyses of MDSCs (18). The population of total MDSCs and its sub-population in the spleens (E) and tumor tissues (F) were detected by flow cytometry. (G,H) The expression of CD11b and Ly6G were detected by IF in tumor tissues. The values are expressed as mean ± SD. (n = 6; *P < 0.05 **P < 0.01 vs. the control group).

Figure 5

cGAMP delayed the suppression effects of MDSCs in vitro. MDSCs and T cells were isolated with the Kit. (A) T cell suppression assay was performed to detect the immune suppression effects of MDSCs in each group. The ratio of T cells: MDSCs were 2:1. Representative data of three independent experiments are shown as mean ± SD. (n = 3; **P < 0.01 vs. the CT26 group; ^##P < 0.01 vs. the negative control group). (B) The concentration of ROS was determined with the flow cytometry. (C) The concentration of NO was determined with the flow cytometry. Representative data of three independent experiments are shown as mean ± SD. (n = 3; *P < 0.05, **P < 0.01 vs. the control group).

Figure 6

STING deficiency increased the metastasis of MC38 colon cancer cells. Mice were treated with cGAMP (20 mg/kg, i.v.) daily 3 d before MC38 cells were injected into their spleens. (A) H&E staining of liver tissues to detect metastatic MC38 cancer cells on days 14, respectively. (B) H&E staining of liver tissues to detect metastatic MC38 cancer cells on days 22. (C) IF staining of liver tissues for E-cadherin and Vimentin on day 14. (D) IF staining of liver tissues for E-cadherin and Vimentin on day 22. (E) The quantification of E-cadherin was measured as shown. (F) The quantification of Vimentin was measured as shown. (G) RT-PCR analysis of N-cadherin, Twist, Snail, Vimentin, and E-cadherin. Representative data of three independent experiments are shown as mean ± SD; (n = 6; *P < 0.05, **P < 0.01 vs. the control group).

Figure 7

cGAMP inhibits tumor metastasis by regulating EMT-related genes in the Wnt/β-catenin signaling pathway in vivo. Mice were treated with cGAMP (20 mg/kg, i.v.) daily, 3 d before CT26 cells were injected into their spleens. (A,B) IF staining for Vimentin and E-cadherin in liver tissues. (C) RT-PCR analysis of E-cadherin, Vimentin, N-cadherin, and Twist expression in liver tissues of CT26 tumor bearing mice. (D) CDX2 expression was analyzed by immunohistochemistry. (E) Western blot analysis of Wnt-β/catenin signal pathway in liver tissues. The values are expressed as mean ± SD. (n = 6; *P < 0.05 **P < 0.01, and ^##P < 0.01, vs. the control group).