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. 2020 Jul 16;18(1):113.
doi: 10.1186/s12964-020-00596-9.

YYFZBJS ameliorates colorectal cancer progression in ApcMin/+ mice by remodeling gut microbiota and inhibiting regulatory T-cell generation

Hua Sui  1 , Lu Zhang  1 , Kaijuan Gu  2 , Ni Chai  3 , Qing Ji  1 , Lihong Zhou  1 , Yan Wang  1 , Junze Ren  4 , Limei Yang  1 , Bimeng Zhang  5 , Jing Hu  6 , Qi Li  7   8
Affiliations

YYFZBJS ameliorates colorectal cancer progression in ApcMin/+ mice by remodeling gut microbiota and inhibiting regulatory T-cell generation

Hua Sui et al. Cell Commun Signal. .

Abstract

Background: Progression of Colorectal cancer (CRC) is influenced by single or compounded environmental factors. Accumulating evidence shows that microbiota can influence the outcome of cancer immunotherapy. T cell, one of the main populations of effector immune cells in antitumor immunity, has been considered as a double-edged sword during the progression of CRC. Our previous studies indicate that traditional Chinese herbs (TCM) have potential anticancer effects in improving quality of life and therapeutic effect. However, little is known about the mechanism of TCM formula in cancer prevention.

Methods: Here, we used C57BL/6 J ApcMin/+ mice, an animal model of human intestinal tumorigenesis, to investigate the gut bacterial diversity and their mechanisms of action in gastrointestinal adenomas, and to evaluate the effects of Yi-Yi-Fu-Zi-Bai-Jiang-San (YYFZBJS) on of colon carcinogenesis in vivo and in vitro. Through human-into-mice fecal microbiota transplantation (FMT) experiments from YYFZBJS volunteers or control donors, we were able to differentially modulate the tumor microbiome and affect tumor growth as well as tumor immune infiltration.

Results: We report herein, YYFZBJS treatment blocked tumor initiation and progression in ApcMin/+ mice with less change of body weight and increased immune function. Moreover, diversity analysis of fecal samples demonstrated that YYFZBJS regulated animal's natural gut flora, including Bacteroides fragilis, Lachnospiraceae and so on. Intestinal tumors from conventional and germ-free mice fed with stool from YYFZBJS volunteers had been decreased. Some inflammation' expression also have been regulated by the gut microbiota mediated immune cells. Intestinal lymphatic, and mesenteric lymph nodes (MLN), accumulated CD4+ CD25+ Foxp3 positive Treg cells were reduced by YYFZBJS treatment in ApcMin/+ mice. Although YYFZBJS had no inhibition on CRC cell proliferation by itself, the altered Tregs mediated by YYFZBJS repressed CRC cancer cell growth, along with reduction of the phosphorylation of β-catenin.

Conclusions: In conclusion, we demonstrated that gut microbiota and Treg were involved in CRC development and progression, and we propose YYFZBJS as a new potential drug option for the treatment of CRC. Video abstract.

Keywords: ApcMin/+ mice; Colorectal Cancer; Fecal microbiota transplantation; Gut microbiota; Immune; Regulatory T cell; Traditional Chinese herb medicine.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Experimental design and effect of YYFZBJS in intestinal tumorigenesis. a Experimental design indicating the timing of intragastric administration and organization of groups. b Macroscopic view of the representative mouse intestinal shows several polypoid and discoid colonic tumors from different groups of ApcMin/+ mice after treatment with YYFZBJS for 20 weeks. c The number of intestinal polyps in small intestinal from different groups of ApcMin/+ mice after treatment with YYFZBJS for 20 weeks. d The number of intestinal polyps in the colon from different groups of ApcMin/+ mice after treatment with YYFZBJS for 20 weeks. The data are presented as the mean ± SD from at least three experiments. e The tumor size distribution in the intestine was listed and compared with control. f Left: typical adenomatous polyp seen in infected ApcMin/+ mice showing high-grade dysplasia and carcinoma in situ. Middle: adenomatous intestinal polyp with the early invasion of neoplastic glands into the muscular layers often seen in ApcMin/+ mice. Right: minute polyp with remnant dysplastic glands close to the surface epithelium. This typical regressive intestinal cancer morphology is seen throughout the intestine in mice. Red arrows indicated adenocarcinoma cell. Magnification bars, 100 μM. g&h Immunohistochemical staining with an antibody against PCNA, Ki67, BrdU in control group and YYFZBJS treatment group. Magnification bars, 50 μM. Data are given as means ± SD of 8 animals per experimental group, with Welch’s correction, one-tailed t-test. #P < 0.05, ##P < 0.01; *P < 0.05, **P < 0.01, &P < 0.05, $P < 0.05 vs. control
Fig. 2
Fig. 2
YYFZBJS modulates the gut microbiome composition. a Heat map of Genus with relative abundances that are significantly different from their relative abundances at the time of YYFZBJS administration. The differentially enriched bacterial Genus in C57BL/6 J mice receiving N. S and YYFZBJS. The relative abundance between control and treatment mice for the genus was calculated for each time. Blue boxes indicate negative associations (n = 7) and red boxes indicate positive associations (n = 8). b Principle component analysis (PCA) analysis at the genus-level, which was used to study the differences in the composition of bacterial communities in the fecal samples between mice treated with YYFZBJS and the Control group. Samples along PC1 (x-axis) explained 58.11% and PC2 (y-axis) explained 14.7% of variability, respectively. c Bar plot of compositional differences at the genus level in the gut microbiome of mice in the combination YYFZBJS group vs. the control group by the Wilcoxon rank-sum test. Data are expressed as mean ± SD. * 0.01 < P ≤ 0.05, ** 0.001 < P ≤ 0.01, *** P ≤ 0.001, Two-sided Hypotheses. d A stacked bar plot of genus-level phylogenetic classification of 16S rRNA frequencies in stool pellets collected from naive animals (N.S; n = 7), Chinese herb decoction-treated animals (YYFZBJS; n = 8). e Relative fold change of the 10 most abundant bacterial families abundances, which was significantly different between mice treated with YYFZBJS and the Control group. f The gut microbiome has a profound effect on the host immune system, including DCs, naive T cells, Tregs and Th17 cells. The relationship between the five types of bacteria and immune cells is summarized
Fig. 3
Fig. 3
Gut microbiota from YYFZBJS volunteers delay the progression of intestinal tumorigenesis. a Design of stool gavage experiment to ApcMin/+ mice. Mice were treatmented with Abx from week 6, and sacrificed at week 22 (n = 8). b Display of the fecal extracts of the ApcMin/+ mice with FMT treatment for 12 weeks. c Left: typical adenomatous polyp seen in infected ApcMin/+ mice showing high-grade dysplasia and carcinoma in situ. Right: minute polyp with remnant dysplastic glands close to the surface epithelium. Blue arrows indicated adenocarcinoma cell. Magnification bars, 100 μM. Histological analysis of intestinal tumors applyed in the two FMT group mice (n = 8 for each group). d The tumor size distribution in the intestine was listed and compared with control-FMT (n = 8 for each group). Data shown represent means ± SD. *P < 0.05 vs. control-FMT. e Immunohistochemical staining with an antibody against PCNA and Ki67 in control-FMT group and YYFZBJS-FMT treatment group. Magnification bars, 100 μM. Data are given as means ± SD of 8 animals per experimental group, with Welch’s correction, one-tailed t-test. *P < 0.05 vs. control-FMT. f Electron microscopy in the lumen infiltration of control-FMT group mice and YYFZBJS-FMT mice at age of week 22. Both microvilli and goblet cells can also be seen. The black arrow refers to the intestinal microvilli; The red arrow indicates a tight connection. Magnification bars, 500 nM. g Fecal bacterial DNA was prepared from Control-FMT group and YYFZBJS-FMT treatment group. Relative genus abundance was shown as percentage of each OTU in the total OTUs (n = 5/group). Data shown represent means ± SD. *P < 0.05
Fig. 4
Fig. 4
Effect of YYFZBJS on the immunity of ApcMin/+ mice. a The difference between the two groups in inflammatory cytokines as assessed by cytokine antibody array. b Top common genes that are regulated significantly after treatment with YYFZBJS. c IL-6, IL-10, IL-17A and TNF-α levels in PBMC of ApcMin/+ were evaluated using ELISA. d Representative pictures of spleens from ApcMin/+ mice that were orally treated with YYFZBJS for 20 weeks. Spleen weight of ApcMin/+ mice was assessed on the right. The data are presented as the mean ± SD from at least three experiments. *P < 0.05, **P < 0.01 vs. control
Fig. 5
Fig. 5
Effect of YYFZBJS on mRNA expression of TH cell in the spleen, MLN and LPL of ApcMin/+ mice. a Foxp3, Gata3, ROR-γt, and T-bet expression in polyp were evaluated using RT-PCR. b Representative flow cytometry plots of intestinal lamina propria cells (LPCs) showing the viable lymphocyte gate on CD4 + T cells. Representative plots showed the frequency of CD25+ Foxp3+ T cells after drug intervention for 20 weeks in the intestinal lamina propria of ApcMin/+, as determined by flow cytometric analysis. The data are presented as the mean ± SD from at least three experiments. *P < 0.05, **P < 0.01 vs. control
Fig. 6
Fig. 6
YYFZBJS inhibited tumor cell proliferation through regulating Treg in vitro.a Left: YYFZBJS extracts samples, 25.66 mg/ml; Right: the mix standard solution; Stationary phase: ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm); mobile phase: acetonitrile (a) and aqueous 0.1% formic acid (b) in gradient (time, min/B%: 0/95, 12/5,14/5,14.1/95,16/5); flow rate: 0.3 ml/min); column temperature:45 °C]. R:6.56 min liquiritigenin ([M-H]-,255.09518 m/z); 6.63 min, luteolin ([M-H]-,285.03936 m/z); 7.12 min mesaconine ([M + H]-,632.3065 m/z); 7.55 min, aconitine ([M + H]-,632.3065 m/z); hypaconitine 7.59 min ([M + H]-,632.3065 m/z). b Interaction network diagram between the active ingredients of YYFZBJS and their targets using prediction software of Cytoscape 3.6.1. c Experimental design indicating CD4 + CD25 + Foxp3+ T cells (Treg) were isolated from spleens of ApcMin/+ mice treated with or without ETBF and/or YYFSBJS in 62.5 μg/ml for 4 h. The ratio of cell to bacterial was 1:10. Then the primed Treg was collected and were assigned to MC-38 cells as 10:1 ratio. d Foxp3 mRNA expression was analyzed by real-time polymerase chain reaction analysis in Treg cells. The data are presented as the mean ± SD from at least three experiments. *P < 0.05, **P < 0.01 vs. ETBF group. e The decrease in ETBF count was observed in Treg incubated with ETBF and YYFZBJS (62.5 μg/ml) group. The representative gut bacteria also had higher colony-forming unit per milliliter as observed from agar plates. f MC-38 cells proliferation was assayed at 0, 12, 24, 36, and 48 h after co-culture with the primed Treg. The data are presented as the mean ± SD from at least three experiments. *P < 0.05, **P < 0.01 vs. MC-38 + Treg+ETBF. g The heatmap displays relative fold changes in expression levels normalized to the mean expression in the Control, Treg incubated with MC-38 cells (Treg: MC-38 = 10:1), ETBF primed Treg incubated with MC-38 cells, and YYFZBJS (62.5 μg/ml) combined with ETBF primed Treg incubated with MC-38 cells for each indicated mRNA of MC-38 cell. The color brightness of each unit is associated with differences. Blue color represents high expression and white color represents low expression. Not all the mRNAs in the figure were labeled. *P < 0.05, **P < 0.01 vs. MC-38 + Treg; ##P < 0.01 vs. MC-38 + Treg+ETBF. h Western blot and quantitative assay of β-catenin (nuclear, cytoplasm) in MC-38 cells. β-actin as loading control The data are presented as the mean ± SD from at least three experiments. **P < 0.01 vs. MC-38 + Treg; #P < 0.05, ##P < 0.01 vs. MC-38 + Treg+ETBF
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