Fig 2: Fruquintinib enhances the effect of sintilimab to reprogram the immune microenvironment and enhance antitumor immunity in CRC. (A) Percentages of CD8+T cells, CD8+TNFa+T cells,CD8+IFN?+ T cells in tumor tissues determined by FACS in CT26 tumor-bearing mice treated with indicated drugs (shown as fractions of CD45+ cells) in tumors treated with control CMC-Na, anti-PD-1, Fru2.5, arnti-PD-1 and Fru 2.5, measured by flow cytometry at day 14. (B) Representative images of the FACS plots of CD8+ and CD8+IFN?+ CTLs. (C) IHC images for CD8 staining of tumor sections on day14. Scale bar, 200um. (D) Percentage of MDSCs (CD11b+Gr1+) and TAM (CD11b+F4/80+) in each groups on day 14. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Genetic ablation of HCK reduces tumor immunosuppression and augments antitumor responses to aPD1 immunotherapy.(A) Mass of individual subcutaneous MC38 tumors from WT and HckKO hosts. Where indicated, mice were treated with aPD1 (200 µg, once every 3 days) or an isotype-matched IgG control for 10 days. Representative tumors are depicted above the graphs. Scale bar, 1 cm. Each symbol represents an individual mouse. (B) Flow cytometry quantification of the indicated myeloid cell populations from subcutaneous MC38 tumors of WT and HckKO hosts treated as described in (A). Each symbol represents an individual mouse. (C) Quantitative polymerase chain reaction (qPCR) analysis on fluorescence-activated cell sorter (FACS)–purified CD45+CD11b+F4/80HighLy6C-Ly6G- TAMs and CD45+CD11c+F4/80-MHCII+ dendritic cells for markers associated with immune cell activation. Cells were isolated from subcutaneous MC38 tumors of WT and HckKO hosts treated as described in (A). n = 4 mice per group. (D) Mass of individual subcutaneous MC38 tumors from WT and HckKO hosts. Where indicated, mice were treated with aIL-12, aCXCR3, or an isotype-matched IgG control once every 3 days (total of three treatments) before subcutaneous MC38 tumor cell injection and continued until the experimental end point. Once palpable tumors formed, mice were treated with aPD1 for 10 days. Each symbol represents an individual mouse. (E) Mass of individual subcutaneous MC38 tumors from WT bone marrow chimeras reconstituted with cDC1-deficient (cDC1KO) or cDC1-proficient (WT) bone marrow. To deplete TAMs, half of each cohort were treated with aCSF1R before MC38 tumor cell injection and continued until the experimental end point. Once palpable tumors formed, all hosts were treated with aPD1 in the presence or absence of the small-molecule HCK inhibitor RK20449 for 20 days. Each symbol represents an individual mouse. Data represent mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001, with statistical significance determined by one-way ANOVA followed by Tukey’s multiple comparison test.

Fig 4: SR-driven immunosuppression accelerates local tumor progression.a Schematic illustration of surgical resection (SR) treatment. 1×106 CT26 cells were subcutaneously injected into the right flank of BALB/c mice. SR or sham operation was conducted on the right tumor or left skin, respectively, on day 10 postinoculation. b Residual tumor growth kinetics of mice in Untreated, SR and Sham operation groups (n = 6 mice). c Weight of the excised tumor on day 20 after varied treatments (n = 6 mice). d, e Cluster analysis (d) and Venn diagram (e) of differential expression genes in RNAseq between untreated and SR-treated tumors three days post treatment (n = 3 mice). Red and blue colors represent upregulated or downregulated genes, respectively. f Significant enrichment in gene ontology (GO) terms (top 30, n = 3 mice). g Heatmap of differentially expressed genes associated with tumor progression and immunosuppression (n = 3 mice). Red and blue colors represent upregulated or downregulated genes, respectively. h Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment histogram of differentially expressed genes (Statistical difference was calculated using Fisher’s exact test, n = 3 mice). i Representative polychromatic immunofluorescent staining images of tumors from three biologically independent samples showing CD45+ (red), CD11b+ (green), Ki67+ (purple) and HIF-1a+ (orange) cells infiltration for Untreated and SR tumors three days post-treatment. j–o Representative flow cytometric images and the corresponding quantification of MDSCs (CD11b+Gr-1+CD45+) (j, m), TAMs-M2 (CD206hiCD11b+F4/80+CD45+) (k, n) and CTLs (CD8+CD3+CD45+) (l, o). p The ratio of CD8+ cells to CD11b+ MDSCs in tumors. q The ratio of M1 to M2 in tumors. SR, surgical resection; MDSCs, myeloid-derived suppressor cells; TAMs-M2, M2-like macrophages; TAMs-M1, M1-like macrophages; CTLs, cytotoxic T lymphocytes. Data were expressed as means ± SD (n = 3 biologically independent samples). Statistical difference was calculated using two-tailed unpaired student’s t-test. ns, not significant, *P < 0.05, **P < 0.01 and ***P < 0.001. The experiments were repeated three times. Source data are provided as a Source Data file.

Fig 5: Impaired leukocyte recruitment in response to TNF-a–induced peritonitis in STING–/– mice compared with WT mice.(A–H) WT and STING–/– mice received PBS, TNF-a 4 hours (h), or TNF-a 24 h (A–D) or thioglycollate 3 days (E–H) via i.p. injection, and the peritoneal lavage was analyzed by FACS. (A) Representative flow cytometric panels of CD45+CD4+ cells recruited to the peritoneal cavity at 24 h and of CD45+Gr1+ at 4 h. (B–D) Quantification of total CD45+ (B), CD45+CD4+ (C), and CD45+Gr1+ (D) recruited to the peritoneal cavity. n = 3 (control); n = 12 (TNF-a 24 h); n = 3-4 (TNF-a 4 h). (E) Representative flow cytometric panels of CD45+CD11b+ cells recruited to the peritoneal cavity in response to thioglycolate of 3 independent experiments. (F–H) Quantification of CD45+ (F), CD45+CD11b+ (G), and CD45+CD4+ (H) recruited cells to the peritoneal cavity. n = 3 (control); n = 8 (thioglycollate). Data are shown as mean ± SEM values. *P < 0.05 and **P < 0.01; 2-way ANOVA (B–D) and 1-way ANOVA (F–H).