Gut Microbiota Alteration Influences Colorectal Cancer Metastasis to the Liver by Remodeling the Liver Immune Microenvironment

Abstract

Background/aims: This study aimed to explore the effect of gut microbiota-regulated Kupffer cells (KCs) on colorectal cancer (CRC) liver metastasis.

Methods: A series of in vivo and in vitro researches were showed to demonstrate the gut microbiota and its possible mechanism in CRC liver metastasis.

Results: Fewer liver metastases were identified in the ampicillin-streptomycin-colistin and colistin groups. Increased proportions of Parabacteroides goldsteinii, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Bacteroides uniformis were observed in the colistin group. The significant expansion of KCs was identified in the ampicillin-streptomycin-colistin and colistin groups. B. vulgatus levels were positively correlated with KC levels. More liver metastases were observed in the vancomycin group. An increased abundance of Parabacteroides distasonis and Proteus mirabilis and an obvious reduction of KCs were noted in the vancomycin group. P. mirabilis levels were negatively related to KC levels. The number of liver metastatic nodules was increased in the P. mirabilis group and decreased in the B. vulgatus group. The number of KCs decreased in the P. mirabilis group and increased in the B. vulgatus group. In vitro, as P. mirabilis or B. vulgatus doses increased, there was an opposite effect on KC proliferation in dose- and time-dependent manners. P. mirabilis induced CT26 cell migration by controlling KC proliferation, whereas B. vulgatus prevented this migration.

Conclusions: An increased abundance of P. mirabilis and decreased amount of B. vulgatus play key roles in CRC liver metastasis, which might be related to KC reductions in the liver.

Keywords: Colorectal neoplasms; Gastrointestinal microbiome; Kupffer cells; Liver metastasis.

Fig. 1

Changes in gut microbiota and colorectal cancer (CRC) liver metastasis induced by different antibiotics. (A) Schematic diagram of the mouse experimental process of CRC liver metastasis with different antibiotics (upper arrow: CRC liver metastasis model, as established by colon 26 [CT26] splenic injection; lower arrow: antibiotic treatment). (B) Image of CRC liver metastases in animals treated with different antibiotics at the end of the experiment. (C) Number of CRC liver metastases and liver volume in different groups (n=15 per group; a mouse in the Vanc group and a mouse in the ASC group were lost during the experiment due to emaciation and intestinal obstruction). (D) Venn diagram of the total number of species among the control, Vanc, and Coli groups (n=6 per group). (E) Principal coordinates analysis (PCA) of operational taxonomic units in the control, Vanc, and Coli groups. (F) Alpha diversity analysis of the gut microbiomes among the control, Vanc, and Coli groups. Alpha diversity includes the Shannon, Chao1, and Simpson indices. (G) Beta diversity, reflected by the weighted Unifrac distance. Control group, untreated; Vanc group, vancomycin; Coli group, colistin; ASC group, a mix of ampicillin, colistin and streptomycin. *p<0.05, ^†p<0.01.

Fig. 2

Comparison of gut microbiota composition among three groups. (A) Linear discriminant analysis effect size (LEfSe) for differentially abundant taxa from the three groups. p=0.05 using the Wilcoxon rank-sum test, with linear discriminant analysis (LDA) score >4. (B) Taxonomic cladogram from the LEfSe showing differences in fecal taxa. Dot sizes are proportional to the abundance of the taxon (n=6 per group). (C) Compositional differences at the species level in the gut microbiome between the control and Coli groups. (D) Compositional differences at the species level in the gut microbiome between the control and Vanc groups. Control group, untreated; Vanc group, vancomycin; Coli group, colistin; ASC group, a mix of ampicillin, colistin, and streptomycin.

Fig. 3

Tax4Fun functional prediction analysis of differential bacteria associated with hepatic Kupffer cell (KC) accumulation; tumor inhibition was noted in the three groups. (A) Tax4Fun functional prediction in the control, Vanc, and Coli groups, as shown as a heatmap. (B) Enrichment of immune system, environmental information, and cellular process pathways were compared in the control and Coli groups (dot size symbolizes the p-value). (C) Enrichment of immune system, environmental information, and cellular process pathways were compared in the control and Vanc groups. (D) Hematoxylin and eosin staining images and quantification of hepatic KCs from different gut flora backgrounds (n=5 per group, ×200). (E) Analysis of the correlation between KCs and Proteus mirabilis or Bacteroides vulgatus. Control group, untreated; Vanc group, vancomycin; Coli group, colistin; ASC group, a mix of ampicillin, colistin, and streptomycin. *p<0.05, ^†p<0.01.

Fig. 4

Colorectal cancer (CRC) liver metastasis and hepatic Kupffer cell (KC) induced by Proteus mirabilis or Bacteroides vulgatus. (A) Images of liver metastatic nodules in each group. (B) Numbers of liver metastases in each group. Hematoxylin and eosin staining images (×200) (C) and quantification of hepatic KCs from different bacteria treatment (D). Control group, untreated; Vanc group, vancomycin; Coli group, colistin; ASC group, a mix of ampicillin, colistin, and streptomycin. *p<0.05, ^†p<0.01.

Fig. 5

Differential influences of Proteus mirabilis or Bacteroides vulgatus on Kupffer cell (KC) proliferation and effects of KCs induced by P. mirabilis or B. vulgatus on CT26 cell migration. (A) The difference in KC proliferation between the 103, 104, 105, 106, and 107 CFU/mL P. mirabilis groups and phosphate-buffered saline (PBS) group. (B) Migration of CT26 cells induced by KCs treated with different doses of P. mirabilis (n=3 per group). (C) Differences in KC proliferation between 103, 104, 105, 106, and 107 CFU/mL B. vulgatus groups and the PBS group. (D) Migration of CT26 cells induced by KCs treated with different doses of B. vulgatus (n=3 per group). CFU, colony-forming unit. *p<0.05, ^†p<0.01.