PCK1 S151 phosphorylation–mediated cGAS-IFN-I axis inhibition promotes breast cancer development by abrogating the activity of CD8 ⁺ T cells and NK cells. (A) Treatment schedule for IFNAR, CD8⁺ T cells, and NK cells depletion in the 4T-1 model. At day 0, 4T-1 luciferase-expressing cells (1 × 10⁶) expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant were orthotopically injected into female BALB/c mice. Tumor-bearing mice were intraperitoneally injected with anti-IFNAR, anti-CD8, anti-Asialo-GM1 (ASGM1) antibodies or IgG isotype control as indicated at day −1, 3, 7, 11, 14, and 18 (n = 6 per group). (B and C) Representative bioluminescence images of the indicated 4T-1 tumor-bearing mice treated with an anti-IFNAR antibody or IgG isotype control from the indicated days (B). The quantification of the bioluminescence imaging is shown (C) (n = 6 per group). (D and E) 4T-1 cells (1 × 10⁶) expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant were injected into the mammary fat pad of female BALB/c mice. Representative flow cytometry plots (D) and quantification (E) for CD4⁺ T cells, CD8⁺ T cells, and NK cells within the indicated xenograft tumors (n = 6 per group). (F) Representative flow cytometry plots of the depletion of indicated cells in tumors from 4T-1 tumor-bearing mice treated with anti-CD8 antibody, anti-ASGM1 antibody, combination of anti-CD8, and anti-ASGM1 antibodies, or IgG isotype control, were shown. (G and H) Representative bioluminescence images of the indicated 4T-1 tumor cells bearing mice treated with anti-CD8 antibody, anti-ASGM1 antibody, combination of anti-CD8 and anti-ASGM1 antibodies, or IgG isotype control from the indicated days (G), The quantification of the bioluminescence imaging is shown (H) (n = 6 per group). (I) 4T-1 cells (1 × 10⁶) expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant were injected into the mammary fat pad of female NCG mice. Tumor volumes were calculated (n = 6 per group). (J and K) Human TNBC samples were analyzed with the indicated antibodies. Representative staining images are shown (J). Scale bar, 50 μm. IHC staining of human TNBC samples with the indicated antibodies was scored, and correlation analyses were performed. A Pearson correlation test was used (two-tailed). Note that the scores for some samples overlap (K). (L and M) IHC analyses of the PCK1 low- or PCK high-expressing human TNBC samples were performed with the indicated antibodies. Representative staining images are shown (L). Scale bar, 50 μm. The IHC-positive cells were quantified in n = 6 microscopic fields (M). (N) Kaplan–Meier plots of the overall survival rates in 84 patients with TNBC grouped according to high (staining score, 4–8) and low (staining score, 0–3) expression of PCK1 pS151. Data are representative of as mean ± SD. Statistical significance was determined by two-tailed Student’s t test (C, E, H, and M), two-way ANOVA, followed by Tukey’s test (I) and log-rank test (N); **P < 0.001 and ***P < 0.0001; NS, not significant.