Downregulation of cGAS/STING expression in tumor cells by cancer-associated fibroblasts in colorectal cancer

Abstract

The tumor cell-intrinsic cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is critical for activating anti-tumor immunity and enhancing immune checkpoint blockade therapy in colorectal cancer (CRC). Cancer-associated fibroblasts (CAFs), key components of the CRC tumor microenvironment, negatively regulate the anti-tumor immune response. However, their impact on tumor cell-intrinsic cGAS-STING expression remains unclear. In the present study, we investigated whether CAFs can downregulate cGAS-STING expression in CRC. We found that cGAS-STING expression in tumor cells inversely correlated with stromal expression of versican (VCAN), an immunosuppressive CAF marker, in CRC tissues. Co-culture experiments using primary human CAFs derived from CRC tissues revealed that CAFs downregulated cGAS and/or STING expression in CRC cell lines (WiDr, LoVo, HCT116). Furthermore, CAFs expressing VCAN and fibronectin 1 appeared to mediate this suppression. These findings suggest that immunosuppressive CAFs contribute to the downregulation of tumor cell-intrinsic cGAS-STING expression in CRC. Therefore, targeting CAFs to restore cGAS-STING expression may represent a promising strategy to enhance the efficacy of CRC treatment.

Keywords: Cancer-associated fibroblast; Colorectal cancer; Tumor cell-intrinsic cGAS–STING; Versican.

Fig. 1

Association between stromal VCAN expression and tumor cell-intrinsic cGAS–STING expression in CRC. (A) Representative IHC images of VCAN, cGAS, and STING in surgically resected CRC specimens. Scale bars: 500 µm for low-magnification images and 100 µm for high-magnification images. (B, C) Comparison of H-scores for cGAS (B) and STING (C) between stromal VCAN-high and VCAN-low CRC groups. Values are presented as mean ± SD. Statistical significance was determined using the Mann–Whitney U test. *p < 0.05, **p < 0.01.

Fig. 2

Isolation and characterization of human primary CAFs from CRC tissues. (A) Schema of CAF isolation and image of isolated primary CAFs. (B) Flow cytometry gating strategy to define human primary CAFs isolated from CRC tissues. (C) qPCR analysis of the indicated molecules in all cultured CAFs (CAFs1–10). −ΔCT values are shown. (D) ELISA analysis of TGF-β1 levels in the CM of CAFs1–10.

Fig. 3

Downregulation of cGAS–STING expression in CRC cells by co-culture with CAFs. (A) Western blot analysis of cGAS and STING in WiDr, LoVo, and HCT116 cells, with β-actin as a loading control. (B) Western blot analysis of cGAS and STING in WiDr, LoVo, and HCT116 cells co-cultured with CAFs for 72 h. β-actin was used as a loading control. (C, D) The quantification of cGAS and STING was normalized to β-actin, presenting the relative expression levels of cGAS (C) and STING (D) in WiDr, LoVo, and HCT116 cells co-cultured with CAFs compared to the control (CRC cells alone). Values are presented as mean ± SD. Statistical significance was determined using the Mann–Whitney U test. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig. 4

CAF markers highly expressed in CAFs that negatively impact cGAS–STING expression in CRC cells. (A, B) Comparison of CAF marker gene expression, as determined by qPCR, between CAFs that strongly downregulated cGAS (s-down-cGAS CAFs) and those that moderately/not downregulated cGAS (m/n-down-cGAS CAFs) (A), and between CAFs with strongly downregulated STING (s-down-STING CAFs) and those with moderately/not downregulated STING (m/n-down-STING CAFs) (B). Values are presented as mean ± SD. Statistical significance was determined using the Mann–Whitney U test.