Fig. 6

Neutrophil Arnt^−/−promoted colorectal cancer growth by altering the gut microbiota. WT and Arnt^−/− mice were cohoused and underwent colorectal cancer induction for 80 days after the injection of azoxymethane (AOM) and the addition of dextran sulfate sodium salt (DSS) to the drinking water. The number of colonic tumors (A) and the tumor diameter (B) are summarized. Representative of three independent experiments (A). The graph summarizes data from three independent experiments with twelve mice per group (B). (C-D) Analysis of feces and fecal metabolites in WT and Arnt^−/− mice. Feces were collected at the end of the experiment. Feces were used for DNA isolation and sequencing. (C) Total OTU sequences taxonomically assigned to bacterial genera. Each bar indicates (D) an unweighted UniFrac-based PCoA plot based on all OTUs and represents the mean of the microbiota composition. Representative of two independent experiments with five or six mice per group. (E) Analysis of feces and fecal metabolites in WT and Arnt^−/− mice. Feces were collected at the end of the experiment and used to stimulate HT-29 cells. Metabolites from cancer-induced mice obviously affected HT-29 cell adhesion and growth (green and blue lines), especially metabolites from Arnt^−/− mice, which caused most HT-29 cells to die (blue line). Representative of three independent experiments. (F) Percentage of tumor-infiltrating neutrophils in the colon. (G) Tumor-infiltrating neutrophils were isolated and stimulated with LPS in vitro as indicated to detect NETs. The percentage of NETs-forming cells was quantified. (H) MFI of CXCR2 in tumor-infiltrating neutrophils. Representative of three independent experiments with four mice per group (F-H). (I-K) Colorectal cancer induction for 80 days after the injection of AOM and the addition of DSS to the drinking water and weekly antibiotic injection, as indicated in I. The number of colonic tumors (J) and the tumor diameter (K) are summarized. The graph summarizes data from three independent experiments with fifteen mice per group (J-K). ***P < 0.001, compared with the indicated groups. n.s., not significant