Alterations in the gut microbiota influenced CRC liver metastasis
Spontaneous CRC liver metastasis was induced using an intrasplenic tumor injection in BALB/c mice, as previously described [9] (Fig. 1A). ASC antibiotics were added to sterile drinking water to deplete the gut commensal bacteria [20] (Fig. 1B). Fewer liver metastases were identified in mice in the ASC group (P = 0.003). Colistin alone treatment (Coli group) inhibited the liver metastases of CRC (P = 0.041). Whereas, vancomycin alone treatment (Vanc group) promoted the liver metastases of CRC (P = 0.028). Mice treated with vancomycin targeting gram-positive bacteria had more liver metastases than that of mice treated with colistin targeting gram-negative bacteria (P < 0.01); compared to the Vanc group, a robust decrease of liver metastasis was observed in mice in the ASC group (P < 0.01). Similar results were observed for liver volume, a smaller liver volume was observed in mice of the ASC group, whereas, a larger liver volume was performed in mice of the Vanc group; meanwhile, the liver volume in mice of the Vanc group was larger than that of mice in the Coli group and ASC group (P < 0.05) (Fig. 1B-C).
To better understand the role of the gut microbiome in response to CRC liver metastasis, fecal samples were prospectively collected and the landscape of the gut microbiome was assessed in all available samples via 16S rDNA sequencing, including samples from the Control group, Vanc group, Coli group and ASC group (6 fecal samples per each group). Since ASC treatment efficiently reduced gut commensal bacterial load, it resulted in the complete elimination of gut bacteria. A Venn graph revealed that a total of 395 (Control group), 258 (Vanc group), and 373 (Coli group) species were identified, and a total of 220 species were common among the groups (Fig. 1D). Principal coordinate analysis (PCA) demonstrated that the microbial community structure was clearly separated between the Vanc and Coli groups, while that in the Coli and Control groups was not completely separated (Fig. 1E). Moreover, significant differences were observed in the Alpha diversity of the gut microbiota between the Coli and Vanc groups using several methods such as Chao1, Shannon, and Simpson indexes; while Coli group significantly increased the diversity of the microbiota with Shannon and Simpson diversity (Fig. 1F). From Beta diversity analysis, relative to the Control group, the difference coefficient of gut microbiome diversity in the Coli and Vanc groups was 0.145 and 0.509, respectively. Furthermore, a correlation coefficient of 0.466 in commensal bacteria discrepancy was presented between the Coli and Vanc groups (Fig. 1G). The above results suggested that modulating gut commensal bacteria affected CRC liver metastasis; community richness and diversity increased could depress the liver metastasis of CRC.
Specific gut microbiome is responsible for liver metastasis of CRC
It was determined if differences existed in the gut microbiota among three groups, high-dimensional class comparisons of common bacterial taxa were revealed through LEfSe Bar and Cladogram. At the phylum level, Firmicutes and Bacteroidetes were rich in the Coli group; Proteobacteria was rich in the Vanc group. At the species level, Bacteroides_vulgatus, Bacteroides_uniformis and Helicobacter_mastomyrinus were rich in the Coli group, while Proteus_mirabilis was rich in the Vanc group (Fig. 2A-B). Compared to the Control group based on a Wilcoxon rank-sum test, it was performed that an increased abundance of Parabacteroides_goldsteinii, Bacteroides_vulgatus, Bacteroides_ thetaiotaomicron, Bacteroides_uniformis were enriched in the Coli group, while an increased proportion of Parabacteroides_distasonis, Proteus_mirabilis were enriched in the Vanc group at the species level (P < 0.05, Fig. 2C-D). These results suggested the increasing of Parabacteroides_goldsteinii, Bacteroides_vulgatus, Bacteroides_thetaiotaomicron, Bacteroides _uniformis might inhibit the CRC liver metastasis, the increasing of Parabacteroides_distasonis, Proteus_mirabilis could promote liver metastasis of the CRC.
Function prediction of the differential bacteria among three groups
Whether the underlying mechanism from commensal bacteria was responsible for the observed liver metastasis from the function prediction of Tax4Fun, it was observed that the differential gene functions in the three groups mainly included gene, cell motility, various metabolisms, immune system, and transport (Fig. 3A). Further pair-wise comparisons were performed to uncover the distinction of the immune system, which showed that there were obvious differences in immune system between the Coli and Control groups, as well as both the Vanc and Control groups, the differential functions among three groups contained bacterial motility protein, bacterial secretion system, two-component system, additionally, NOD-like receptor signaling pathway and IL-17 signaling pathway (Fig. 3B-D). The Tax4Fun function prediction specified that gut microbiome alteration affected CRC liver metastasis, which was correlated with IL-17 signaling pathway.
The role of hepatic KCs in liver metastasis of CRC and its correlationship with differential microbiota.
According to Tax4Fun function prediction above, immune signals, in particular, IL-17 signaling pathway was remarkably different in the three groups. To explore the mechanism behind tumor suppression, the number of hepatic KCs in CT26 tumor-bearing mice was examined by immunohistochemistry. A prominent expansion of hepatic KCs were observed in mice in the ASC group and the Coli group (P = 0.001, P = 0.038, respectively). While a remarkable reducing of hepatic KCs was observed in mice in the Vanc group (P = 0.027). Similar to the liver metastasis and liver volume results, accumulation of hepatic KCs was also observed in mice in the ASC and Coli groups compared to that of mice in the Vanc group (P < 0.01, P < 0.01, respectively) (Fig. 4A). These findings suggested that depressing gram-negative bacteria promoted KCs leading to an anti-tumor metastatic function, while depleting gram-positive bacteria reduced KCs resulting in a stronger pro-tumor metastatic function.
To define the exact role of gut microbiota regulating hepatic KCs accumulation, the relationship between differential bacteria of the three groups and KCs contents were tested. There was positive correlation between Bacteroides_vulgatus and KCs contents (P = 0.011, r = 0.705); while there was negative correlation between Proteus_mirabilis and KCs contents (P = 0.028, r = 0.632) (Fig. 4B-C).
Taken together, an increased abundance of Bacteroides_vulgatus and a decreased amount of Proteus_mirabilis might be involved in the regulation of hepatic KCs number, resulting in the different tendency of CRC liver metastasis.