MSI-H/dMMR tumors specifically upregulate TREX1 to suppress antitumor immunity. (A) 4T1, 4T1-Mlh1^−/− (day 45) cells were collected, and expression of indicated proteins was assayed by western blot. (B) RNA-seq data from The Cancer Genome Atlas (TCGA) COADREAD cohort (n = 340, including 60 MSI-H and 280 MSS cases) were analyzed. Gene expression values were normalized to log₂(TPM+1) scale, and TREX1 expression was compared between groups. (C) 4T1, 4T1-Trex1^−/−, 4T1-Mlh1^−/− (day 65), and 4T1-Mlh1^−/− (day 65) cells were collected, and expression of indicated proteins was assayed by western blot. (D) MC38 cells were collected, and expression of indicated proteins was assayed by western blot. (E) MFC, MFC-Trex1^−/−, MFC-Mlh1^−/− (day 70), and MFC-Trex1^−/−Mlh1^−/− (day 70) were collected for TREX1 and MLH1 expression detection by western blot. (F–H) 4T1, 4T1-Trex1^−/−, 4T1-Mlh1^−/− (day 65), 4T1-Mlh1^−/−Trex1^−/− (day 65) cells, MC38, MC38-Trex1^−/− and MFC, MFC-Trex1^−/−, MFC-Mlh1^−/− (day 73, MFC-Mlh1^−/−Trex1^−/−(day 73) cells (n = 3/group) were seeded to a 96-well plate; then the cell growth rates were analyzed by CCK-8 assay; at each time point, CCK8 was added to cells to measure absorbance at 450 nm to show cell viability. (I) BALB/c mice (n = 4) were transplanted s.c. with 5 × 10⁵ 4T1, 4T1-Trex1^−/−cells. (J) C57BL/6 mice and NSG mice (n = 5) were transplanted s.c. with 2 × 10⁶ MC38-Trex1^−/− cells. (K–O) C57BL/6 mice (n = 5) were transplanted s.c. with 1 × 10⁷ MC38 WT or MC38-Trex1^−/− cells. 15 days later, tumor tissues were collected and analyzed by flow cytometry. The percentage of NK cell (K), CD4⁺ T cell (L), neutrophil (M), macrophage (N), and DC (O) in CD45⁺ immune cells is shown. (P) C57BL6/J mice were transplanted s.c. with 2 × 10⁶ MC38 WT or MC38-Trex1^−/− cells and treated with IgG, anti-CD4+anti-CD8+anti-NK1.1, anti-CD4+anti-NK1.1, anti-CD4+anti-CD8, or anti-CD8 antibodies (200 μg/mouse). Tumor growth is shown. (Q) C57BL6/J mice were transplanted s.c. with 2 × 10⁶ MC38 WT or MC38-Trex1^−/− cells and treated with IgG, anti-CD8, or anti-Ly6G antibodies (200 μg/mouse). Tumor growth is shown. Data indicate mean ± SEM and are representative of two (F–Q) and three independent experiments (A and C–E). The statistical analysis was performed by two-way ANOVA in F–J, P, and Q and unpaired Student’s two-tailed t test in K–O. NS, not significant, *P < 0.05, **P < 0.01, and ****P < 0.0001. Source data are available for this figure: SourceData FS1.