Fecal Microbiota Transplantation Using Donor Stool Obtained from Exercised Mice Suppresses Colonic Tumor Development Induced by Azoxymethane in High-Fat Diet-Induced Obese Mice

Abstract

The gut microbiota plays an important role in the development of colorectal tumors. However, the underlying mechanisms remain unclear. In this study, we examined the effects of fecal microbiota transplantation (FMT) on azoxymethane (AOM)-induced colorectal tumors in obese mice. We divided the study subjects into the following five groups: high-fat diet (HFD), normal diet (ND), ND+exercise (Ex), HFD+FMT from ND-alone donor (HFD+FMT(ND alone)), and HFD+FMT from ND+Ex donor (HFD+FMT(ND+Ex)). The Ex group performed treadmill exercise for 15 weeks. Thereafter, fecal and colonic mucus samples were extracted for microbiome analysis. The deoxyribonucleic acid sample was collected from the feces and colonic mucosa, and V3-V4 amplicon sequencing analysis of the 16S rRNA gene was performed using MiSeq. The number of polyps was significantly lower in the ND (6.0 ± 1.6) and ND+Ex (1.8 ± 1.3) groups than in the HFD group (11.4 ± 1.5). The ND+Ex group had significantly fewer polyps than the ND group. The HFD+FMT(ND alone) (5.2 ± 0.8) and HFD+FMT(ND+Ex) (2.8 ± 2.6) groups also had significantly fewer polyps than the HFD group. The IL-15 mRNA levels in the colonic tissues were significantly higher in the HFD+FMT(ND alone) group than in the ND group. Fecal ω-muricholic acid concentrations were significantly higher in the HFD+FMT(ND alone) group than in the ND group and in the HFD+FMT(ND+Ex) group than in the ND+Ex group. The ND, ND+Ex, HFD+FMT(ND alone), and HFD+FMT(ND+Ex) groups had a significantly higher abundance of Lacyobacillaceae than the HFD group. In the FMT group, Erysipelotrichaceae and Tannerellaceae were significantly less abundant. Compared with the HFD group, the ND, ND+Ex, HFD+FMT(ND alone), and HFD+FMT(ND+Ex) groups had a significantly higher abundance of Muribaculaceae and a significantly higher abundance of Lactobacillaceae and Rikenellaceae in common among the ND and ND+Ex groups. The common and significantly less common species were Bacteroidaceae in the FMT group and Lactobacillaceae and Rikenellaceae in the ND alone and ND+Ex groups. Bacteroidaceae and Lachnospiraceae were significantly less common in the FMT group. We found that FMT inhibited AOM-induced colorectal tumorigenesis in obese mice. Furthermore, the fecal concentrations of short-chain fatty acids, bile acids, microbiota, and mucosa-associated microbiota differed between the FMT and diet/EX groups, suggesting that the inhibitory effect of FMT on colorectal tumorigenesis may be due to mechanisms different from those of ND alone and ND+Ex.

Keywords: colorectal cancer; exercise; fecal microbiota transplantation (FMT); high-fat diet; obesity.

Figure 1

Study design. Five groups: ‘high-fat’ diet (HFD), normal diet (ND), ND+exercise (Ex), HFD+ fecal microbiome transplantation (FMT) using ND mice feces (ND alone), and HFD+FMT using ND+Ex feces (FMT(ND+Ex)). ip, intraperitoneal injection; AOM, azoxymethane.

Figure 2

Mouse body weight change. At 26 weeks, the highest weight was in the high-fat diet (HFD) group and the lowest in the normal diet (ND) plus exercise (ND+Ex) group. There was no significant difference between HFD, HFD+FMT (ND alone), and HFD+FMT (ND+Ex).

Figure 3

(A) Comparison of number of total colonic polyps; (B) endoscopic findings; (C) comparison of number of colonic polyps and polyp size distribution: The HFD group had the highest number of polyps and the largest polyps. The ND+Ex group had the fewest polyps and the smallest polyp size.

Figure 4

(A) Blood glucose and (B) total cholesterol levels: serum blood glucose levels were higher in the HFD group than in the others and decreased with the addition of FMT.

Figure 5

Fecal SFA levels: (A) succinic acid, (B) lactic acid, (C) acetic acid, (D) propionic acid, and (E) butyric acid. Succinic acid and acetic acid were significantly lower in the ND and ND+Ex groups than in the HFD group. Succinic acid and acetic acid levels were significantly higher in the ND+Ex group than in the HFD group.

Figure 6

Nonconjugated bile acid level in feces. (A) Alpha-MCA, (B) omage MCA, (C) DCA, (D) HDCA, (E) LCA, and (F) oxoDCA. The HFD group had significantly lower TNFα and IL-6 levels in colon tissue than the ND group. Exercise did not affect cytokines, and SPARC was significantly higher only in the ND group.

Figure 7

Cytokine and myokine expression by qPCR in colonic tumors. (A) IL-6, (B) TNF-alpha, (C) IL-15, (D) SPARC, (E) oncostatin M, and (F) irisin. The HFD group had significantly lower TNFα and IL-6 levels in colon tissue than the ND group. Exercise did not affect cytokines, and SPARC was significantly higher only in the ND group.

Figure 8

Fecal microbiota analysis. (A) Taxonomic analysis. (B,C) Comparison of 4 groups: list of bacterial species that showed significant differences between the four groups. (D) Bacterial species that showed significant differences between the two groups of ND and ND+Ex. (E) Bacterial species that showed significant differences between the two groups of HFD and HFD+Ex. In the FMT group, Erysipelotrichaceae and Tannerellaceae were significantly less abundant.

Figure 9

Mucosa-associated microbiota (MAM) analysis. (A) Taxonomic analysis. (B–E) Comparison of 4 groups: list of bacterial species that showed significant differences between the four groups.