Liver metastasis holds significance in the prognosis of CRC patients. However, the exact mechanism of CRLM has not yet been elucidated. Our present study linked AD and CRLM from the perspective of the brain-gut-liver axis and confirmed that AD might promote CRLM. The possible mechanism may be as follows: AD causes changes in intestinal flora and promotes the increase of intestinal NGF content via the brain-intestinal axis, which in turn causes the increase of intrahepatic NGF content and induces the increase of intrahepatic VEGF and CXCL12 expression through the gut-liver axis; in addition, the change of intestinal flora may also reduce the content of intrahepatic KCs, which suppresses the intrahepatic immune microenvironment and ultimately promotes CRLM.
Within this study, a CRLM model was established by intrasplenic injection of MC38 colon cancer cells into APP/PS1 mice, with an attempt to investigate whether AD has an effect on CRLM. It was found that the AD group exhibited noticeably greater quantities of tumor nodules on the liver surface, along with increased liver weight and volume, as compared to the CON group. These findings strongly indicate that AD substantially enhances the development of CRLM. Bi et al. had demonstrated that AD could promote the growth of lung cancer; by establishing a subcutaneous transplantation tumor model, they transplanted fecal microbiota from AD mice into the gut of lung cancer mice; compared with mice transplanted with fecal microbiota from normal mice, animals transplanted with fecal microbiota from APP/PS1 mice had significantly larger subcutaneous tumors [24].
Our results showed that AD led to alterations in the composition and proportional distribution of intestinal microbial species in mice with CRLM, with Lactobacillus spp. being the gut microbiota that exhibited a notable reduction in abundance within the intestines of mice in the AD group, suggesting that gut microbiota may play an important role in the mechanism via which AD promotes CRLM. Lactobacillus is known to be a probiotic. Studies have demonstrated that oral administration of live Lactobacillus rhamnosus GG can enhance the efficacy of programmed death receptor-1 (PD-1) inhibitors by increasing tumor-infiltrating dendritic cells and T cells. In addition, Lactobacillus rhamnosus GG (LGG) in combination with PD-1 inhibitors promoted the enrichment of gut microbiota towards Lactobacillus rhamnosus and Lactobacillus monomorphicus, thereby inducing the activation of dendritic cells and the migration of CD8 + T cells towards tumor tissue [25]. Supplementation with Lactobacillus rotundus MXJ32 also significantly inhibited the growth of CRC as it could increase the expression of tight junction proteins (Occludin, Claudin-1, and ZO-1) and repair the damaged goblet cells, thereby facilitating the restoration of intestinal barrier function, alleviating intestinal inflammation, promoting the growth of beneficial bacteria such as Lactobacillus and Bifidobacterium, suppressing the presence of detrimental bacteria like Desulfovibrio, and effectively reshaping the overall intestinal microenvironment [26]. Therefore, the reduced gut abundance of Lactobacillus spp. due to AD may contribute to CRLM. Our functional enrichment of gut microbiota suggested that the differential flora functions in the AD group were mainly enriched in membrane transport and cellular processes, indicating that the altered intestinal flora due to AD may lead to functional changes in the cells.
In terms of cellular function, we found that AD was associated with increased NGF level in the colon of mice but had little effect on VEGF level, suggesting that NGF may be a key cytokine for the functioning of the intestinal flora. Evidence suggests that the NGF level is elevated in the intestine of mice with disrupted intestinal barriers [27]. In fact, the elevated NGF may be associated with excessive activation of enteric glial cells in the colon. These cells play a pro-tumor role in the early stage of CRC, and their depletion can lead to a significant reduction in tumor burden in AOM/DSS-induced CRC mice [28]. Thus, AD may promote the alterations of intestinal flora via the brain-gut axis, further causing elevated NGF levels in the colon and ultimately promoting liver metastasis of CRC.
We further investigated the mechanism by which colonic NGF promotes CRLM. The intestine-liver axis is a bidirectional link between the intestine and intestinal flora and the liver. The portal vein conveys products originating from the intestines to the liver, where bile and antibodies are produced and subsequently transported into the intestine via the bile ducts [29]. Our present study showed that the levels of NGF, VEGF, and CXCL12 were elevated in the livers of AD mice. Further analysis revealed that the colonic NGF content was positively correlated with the hepatic VEGF and CXCL12 levels. Studies have indicated that the interaction between NGF and its high-affinity receptor, tropomyosin receptor kinase A (TRKA), triggers the secretion of VEGF by tumor cells [30, 31]. The relationship between VEGF and CRLM is definite, and anti-VEGF drugs have been widely used in the treatment of CRC. For example, bevacizumab plus standard chemotherapy improves survival in patients with unresectable CRLM [32]. CXCL12 has also been found to be involved in CRLM. Kim et al. found that the expression of CXCL12 receptor CXCR4 is markedly elevated in hepatic metastases compared to primary CRC, suggesting CXCL12 is expressed at high levels in tumor metastatic tissues [33]. In summary, colonic NGF may cause elevated levels of NGF in the liver via the gut-liver axis, which in turn induces elevated levels of hepatic VEGF and CXCL12, thereby promoting the metastasis of CRC to the liver.
The gut microbiota not only influences the liver's cytokine secretion via the gut-liver axis but also maintains a significant connection with the intrahepatic immune microenvironment [34]. In our study, we found that the content of KCs was significantly decreased in the liver of AD mice. KCs, as a special type of macrophages in the liver, are important members of the intrahepatic immune microenvironment and the first line of defense for the liver to play its immune role. Upon the arrival of detrimental substances expelled from the gastrointestinal tract through the portal circulation, KCs serve as the ultimate component of the intestinal barrier, capable of enacting anti-inflammatory responses [35]. For example, intrahepatic KCs are activated when endotoxemia is caused by disturbances in the intestinal flora and disruption of the gut microbiota barrier, thereby inhibiting the spread of inflammation [36]. It has been reported that intrahepatic KCs can phagocytose tumor cells by secreting factors such as IL-6 and reactive oxygen species in the early stages of CRLM [34]. Therefore, the altered gut microbiota caused by AD may be one of the reasons for the decrease of intrahepatic KCs. In addition, reduction in intrahepatic KCs can lead to immunosuppression in the liver, and thus CRC cells may be more likely to metastasize to the liver.
Although our present study provides an experimental basis for finding new strategies to combat CRLM, it had some limitations. First, we did not elucidate the specific roles and mechanisms of NGF, VEGF, and CXCL12 in the process via which AD promotes CRLM; and second, we did not explore the potential correlations of alterations in intestinal flora with NGF and KCs. Nevertheless, our results have suggested that AD may induce changes in intestinal flora through the brain-gut axis, which in turn induces a decrease in the number and function of KCs through the liver-gut axis, ultimately promoting CRLM, although the exact mechanism deserves further investigations.