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. 2022 Nov;71(11):2765-2776.
doi: 10.1007/s00262-022-03200-w. Epub 2022 Apr 16.

Role of the cGAS-STING pathway in regulating the tumor-immune microenvironment in dMMR/MSI colorectal cancer

Akinao Kaneta  1 , Shotaro Nakajima  2 , Hirokazu Okayama  1 , Takuro Matsumoto  1 , Katsuharu Saito  1 , Tomohiro Kikuchi  1 , Eisei Endo  1 , Misato Ito  1 , Kosaku Mimura  1   3 , Yasuyuki Kanke  1 , Motonobu Saito  1 , Zenichiro Saze  1 , Shotaro Fujita  1 , Wataru Sakamoto  1 , Hisashi Onozawa  1 , Tomoyuki Momma  1 , Shinji Ohki  1 , Koji Kono  1
Affiliations

Role of the cGAS-STING pathway in regulating the tumor-immune microenvironment in dMMR/MSI colorectal cancer

Akinao Kaneta et al. Cancer Immunol Immunother. 2022 Nov.

Abstract

Deficient mismatch repair (dMMR)/microsatellite instability (MSI) colorectal cancer (CRC) has high immunogenicity and better prognosis compared with proficient MMR (pMMR)/microsatellite stable (MSS) CRC. Although the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has been considered to contribute to the high number of CD8+ TILs, its role in dMMR/MSI CRC is largely unknown. In this study, to examine the role of the cGAS-STING pathway on the recruitment of CD8+ TILs in dMMR/MSI CRC, we used public datasets and clinical tissue samples in our cohorts to evaluate the expression of cGAS, STING, and CD8+ TILs in pMMR/MSS and dMMR/MSI CRCs. According to the analysis of public datasets, the expression of cGAS-STING, CD8 effector gene signature, and CXCL10-CCL5, chemoattractants for CD8+ TILs which regulated by the cGAS-STING pathway, was significantly upregulated in dMMR/MSI CRC, and the expression of cGAS-STING was significantly associated with the expression of CD8 effector gene signature. Immunohistochemistry staining of the clinical tissue samples (n = 283) revealed that cGAS-STING was highly expressed in tumor cells of dMMR CRC, and higher expression of cGAS-STING in tumor cells was significantly associated with the increased number of CD8+ TILs. Moreover, we demonstrated that the downregulation of MMR gene in human CRC cell lines enhanced the activation of the cGAS-STING pathway. Taken together, for the first time, we found that dMMR/MSI CRC has maintained a high level of cGAS-STING expression in tumor cells, which might contribute to abundant CD8+ TILs and immune-active TME.

Keywords: CD8+ TIL; Colorectal cancer; cGAS-STING; dMMR/MSI; pMMR/MSS.

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Conflict of interest statement

No potential conflicts of interest relevant to this article were reported.

Figures

Fig. 1
Fig. 1
MSI CRC has a higher expression of cGAS-STING and CD8 effector gene signature a, b mRNA expression levels of MB21D1 (cGAS) and TMEM173 (STING) in 450 MSS CRCs and 76 MSI CRCs in TCGA (a), and in 459 MSS CRCs and 77 MSI CRCs in GSE39582 (b). c–h The expression of CD8 effector gene signature (c and d), CXCL10 (e and f), and CCL5 g and h in CRC samples in TCGA and GSE39582, respectively (left). Comparison of the expression of CD8 effector gene (c and d), CXCL10 (e and f), and CCL5 g and h between cGAS-low and cGAS-high CRCs (middle), or STING-low and STING-high CRCs (right). Median values were used to differentiate cGAS-low and cGAS-high CRCs, and STING-low and STING-high CRCs. Values are shown as means ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. not significant
Fig. 2
Fig. 2
The expression of cGAS-STING is upregulated in tumor cells of dMMR CRC and associated with the number of CD8+ TILs a Representative IHC staining images for cGAS (upper) and STING (lower) in surgically resected CRC specimens. Scale bars, 100 μm; original magnification × 200. b The percentage of IHC staining intensity of cGAS (left) and STING (right) in pMMR CRC and dMMR CRC tissues. c and d Comparison of H-scores of cGAS (c) and STING (d) between pMMR CRC and dMMR CRC. e Representative IHC staining images of CD8 and CD4 in pMMR and dMMR CRCs. Scale bars, 100 μm; original magnification × 200. f Comparison of the number of CD8+ or CD4+ TILs between pMMR and dMMR CRCs. g Comparison of the number of CD8+ TILs between cGAS-low and cGAS-high CRCs (left), or STING-low and STING-high CRCs (right). Median values were used to differentiate cGAS-low and cGAS-high CRCs, and STING-low and STING-high CRCs. Values are shown as means ± SD. Blue dots; pMMR, red dots; dMMR. **p < 0.01, ****p < 0.0001, n.s. not significant
Fig. 3
Fig. 3
The downregulation of MMR genes induces the activation of the cGAS-STING pathway in human CRC cell lines a Flow cytometry analysis of PBMC and CD8+ cells isolated from PBMC (left). Cell migration analysis of CD8+ cells using CM of SW480 cells or CM of STIGN-activated (2’,3’-cGAMP-stimulated) SW480 cells (n = 3) (right). b qPCR analysis of MLH1 in SNU81 and SW480 cells transfected with MLH1-specific siRNA (n = 3). c Immunoblot analysis of MLH1 and phosphorylated IRF3 (p-IRF3) in SNU81 and SW480 cells transfected with siRNA for MLH1 and stimulated with 5 μg/ml 2’,3’-cGAMP for 6 h. β-actin was used as a loading control. d Immunoblot analysis of STING and MLH1 in SNU81 and SW480 cells transfected with MLH1-specific siRNA together with or without STING-specific siRNA was shown (left). qPCR analysis of CXCL10 and CCL5 in SNU81 and SW480 cells transfected with MLH1-specific siRNA together with or without STING-specific siRNA and stimulated with 5 μg/ml 2’,3’-cGAMP for 18 h (n = 3) (right). The mRNA expression level was normalized to GAPDH. Values are shown as means ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001
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