The risk factors for colorectal cancer liver metastasis in a mouse model of Alzheimer's disease

The risk factors for colorectal cancer liver metastasis in a mouse model of Alzheimer's disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The risk factors for colorectal cancer liver metastasis in a mouse model of Alzheimer's disease Mengke Nie, Yiqian Qiao, Bin Wang, Tianjun Wang, Xiaowei Ma, Jie Zhi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4300147/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and Objective: To investigate the occurrence of colorectal cancer (CRC) liver metastasis (CRLM) and the risk factors in mice with Alzheimer's disease (AD). methods: Mice in the AD group (APP/PS1 models of AD) and the control (CON) group (wild-type C57BL/6J mice) were injected with MC38 cells to establish CRLM models. After the experiment, the tumor nodules on liver surface were counted, and the liver weight, volume were measured. 16S rDNA sequencing, enzyme-linked immunosorbent assay, Pearson’s analysis and immunohistochemical technique were showed to investigate the impact of AD on CRLM and its possible mechanism. Results: Compared to the CON group, the AD group exhibited a increase in the number of tumor nodules on the liver surface, with consistent findings in both liver weight and volume measurements correlating with the metastatic count. Analysis of 16S rDNA sequencing revealed distinct alterations in the intestinal microbiota of the AD group. Furthermore, relative to the CON group, the AD group exhibited notably elevated levels of NGF expression in both the colon and liver. Additionally, discernibly elevated concentrations of VEGF and CXCL12 were observed in both serum and liver tissues of the AD group compared to the CON group. The results of Pearson correlation analysis indicated positive correlations between intestinal NGF levels and both hepatic CXCL12 and VEGF levels. The AD group had smaller number of hepatic KCs than that in the CON group. Conclusions: AD accelerates CRLM. The mechanism may be caused by gut flora affecting hepatic KCs, thus linking the brain-gut-liver axis. Alzheimer's disease Colorectal cancer Liver metastasis Brain-gut-liver axis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Colorectal cancer (CRC) ranks as the prevailing form of gastrointestinal malignancy on a global scale [ 1 , 2 ]. While there have been notable improvements in the prognosis of CRC patients due to advancements in diagnostic techniques and treatment modalities, CRC liver metastasis (CRLM) remains a non-negligible problem, wherein around half of CRC patients manifest liver metastases upon initial diagnosis [ 3 – 5 ]. Although a small proportion of CRLM can be removed surgically, the post-resection 5-year survival rate is merely around 36% [ 6 , 7 ]. CRC can occur at any age, but it is most commonly seen in the middle-aged and elderly population. With the aging of population, the prevalence rates of some degenerative diseases such as Alzheimer's disease (AD) are also rising. Therefore, there is an increased probability of coexistence of these two conditions [ 8 – 10 ]. A retrospective analysis revealed a 45% higher likelihood of postoperative mortality in CRC patients with preexisting AD in comparison to those with CRC alone (hazard ratio [HR]: 1.45, 95% confidence interval [CI]: 1.29–1.63) [ 11 ]. Another investigation into the factors contributing to mortality in CRC patients indicated that the probability of death attributed to AD rose from 1.73–10.47% as survival time extended (from 2 to 11 months to more than 180 months) [ 12 ]. In the past few years, a growing body of research has substantiated the prominent role of intestinal microbiota in the pathogenesis of CRLM and AD. It has been found that intestinal flora disorders can induce the release of metastasis-related secretory protein cathepsin K (CTSK), initiating Toll-like Receptor 4 (TLR4)-dependent M2 macrophage polarization, thus promoting CRLM [ 13 ]. In addition, alterations in intestinal flora accelerate the neuronal degeneration, thus promoting AD [ 14 ]. Nerve growth factor (NGF), emanating from enteric glial cells, is involved in both the development of AD and the pathogenesis of CRC. Cholinergic neurons are selectively degraded in AD, and loss of acetylcholine is directly associated with cognitive decline. NGF is the most potent growth factor in improving the survival of these cholinergic neurons [ 15 ]. In addition, NGF regulates intestinal peristaltic function and maintains the integrity of the intestinal mucosal barrier by participating in the intestinal inflammatory response [ 16 ]. Tumor metastasis is a multifaceted process influenced by a multitude of factors. Researches have demonstrated that the activation of the CXCL12/CXCR4 axis is linked to an poor prognosis for individuals with CRLM [ 17 , 18 ]. The CXCL12/CXCR4 axis activation leads to the upregulation of specific microRNAs within CRC cells. Through exosomes, these miRNAs are transferred to macrophages. Consequently, these exosome-mediated miRNAs prompt the M2 polarization of macrophages by modulating PTEN via the activation of the PI3K/Akt signaling pathway. Then the M2-polarized macrophages contribute to the progression of cancer metastasis by bolstering epithelial-mesenchymal transition (EMT) and releasing vascular endothelial growth factor (VEGF) [ 19 ]. VEGF, in turn, supports tumor cell survival and dissemination by attracting myeloid-derived suppressor cells (MDSCs), which suppress the local immune microenvironment. The level of VEGF within tumor tissue can serve as an indicator of the risk of metastasis in colorectal cancer [ 20 , 21 ]. Immune components present in the liver encompass Kupffer cells (KCs), T lymphocytes, natural killer cells, and hepatic sinusoidal endothelial cells, Among which KCs, functioning as macrophages within the liver, hold a significant role in the context of CRLM [ 22 , 23 ]. However, the incidence of CRLM in AD patients remains unclear, and few studies have investigated whether AD can influence the occurrence of CRLM by causing changes in intestinal flora, NGF, and factors related to liver metastasis or whether interventions in intestinal flora can reduce the risk of CRLM. In this particular investigation, mouse models of CRLM were established by intrapleural injection of MC38 colon cancer cells in AD mice in an attempt to investigate the impact of AD on CRLM and its possible mechanism. 1. Materials and methods 1.1 Feeding and grouping of experimental animals Six 5-month-old male SPF-grade APP/PS1 double-transgenic mice were purchased from HFK BioScience (Beijing, China) (animal production license number: [SCXK (Beijing) 2019-0008]), and the inbred strain used was C57BL/6J. Six 5-month-old male SPF-grade wild-type C57BL/6J mice were purchased from Exinvivo (Hebei, China) (animal production license number: [SCXK (Hebei) 2020-002]. The mice were accommodated within the SPF-grade animal facility at the Experimental Center of Hebei General Hospital. Approval for the experiments was obtained from the Experimental Animal Ethics Committee of the Hebei General Hospital (approval number: 202215). The mice were provided housing in compliance with the Laboratory Animal - Requirements of Environment and Housing Facilities issued by the Ministry of Health of the People's Republic of China. This included maintaining a 12:12-hour light-dark cycle and a constant room temperature of 24℃. Adequate food and water were accessible to the animals on an ad libitum basis. Following a one-week adaptation period of feeding, the mice were then categorized into two distinct groups (n = 6 each): the control (CON) group: wild-type C57BL/6J mice, which were used to establish CRLM models; and the AD group: APP/PS1 double-transgenic mice, which were also used to establish CRLM models. 1.2 Cell line culture The murine colon carcinoma cell line MC38 (Cell Bank of Chinese Academy of Sciences, Shanghai, China) was cultured in RPMI 1640 (Gibco, Santa Cruz, CA, USA) medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% double antibodies (100U/mL penicillin and 100µg/mL streptomycin) in a constant temperature incubator containing 5% CO 2 at 37℃. 1.3 Establishment of CRLM models After the mice were successfully anesthetized, incisions were made along the mid-axillary line of the abdomen to expose the spleen. Subsequently, 0.1mL of MC38 cell suspension (concentration: 5×10 6 cells/mL) was aspirated with a 29-G insulin syringe and then injected into the spleen along its longitudinal axis via the inferior poleof the spleen. The needle was withdrawn after the spleen peritoneum became pale and swollen. A sterile cotton swab was immediately placed over the injection site to prevent bleeding, and then the incisions were closed in layers. 1.4 Experimental steps 1) After 1 week of adaptive feeding, CRLM models were established in both groups; 2) the mice continued to be routinely fed after modeling; 3) on day 14, the feces were picked up from both groups; and 4) at the end of the experiment on day 15, blood, colon, and liver tissues were collected, the liver weight was weighed, the liver volume was measured, and the tumor nodules on liver surface were counted. 1.5 Counting of tumor nodules and measurement of liver weight and volume 1.5.1 Counting of tumor nodules Individual metastases sized less than 0.2cm were counted as 1, sized 0.2–0.4cm (excluding 0.2cm) as 2, sized 0.4–0.6cm (excluding 0.4cm) as 4, and sized greater than 0.6cm as 6. 1.5.2 Measurement of liver weight and volume A Petri dish was placed in advance in an electronic balance, which was zeroed. The liver sample was placed in the Petri dish, and its weight was read and recorded. A measuring cylinder containing saline was prepared, and the scale corresponding to the liquid level (baseline reading) was read in advance. The liver sample was then gently slid into the measuring cylinder along the edge to allow the saline submerges the entire sample, and the current reading was recorded after the liquid level became stable. The liver volume was calculated using the following formula: Liver volume = Current reading (cm 3 ) - Baseline reading (cm 3 ) 1.6 16S rDNA sequencing The feces of mice were collected for DNA extraction. The polymerase chain reaction (PCR) amplification of the V3 - V4 variable regions was performed based on the gene sequences 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3'). After the target bands were recovered by shearing, library construction and sequencing were performed to analyze the DNA information of intestinal flora. The clean reads were obtained for all samples and clustered into operational taxonomic units (OTUs). 1.7 Enzyme-linked immunosorbent assay (ELISA) Serum, liver tissue, and colon tissue were used for ELISA. After the homogenized liver and colon tissues were centrifuged at 3000rpm for 15min at 4°C, the supernatant was harvested. Then, the supernatant and serum were stored in a -20℃ refrigerator. All the ELISA kits used were purchased from Cloud-Clone (Wuhan, China). After sample addition, incubation, washing, enzyme addition, and color development, the reaction was terminated. The value of optical density (OD) was determined at the wavelength of 450 nm. 1.8 Immunohistochemistry (IHC) staining The liver tissues were fixed in 4% paraformaldehyde for 24h and then routinely embedded in paraffin, sectioned, and dewaxed. After antigen repair, serum blocking, and antibody dilution, the sections were added with primary antibody (rabbit anti-mouse F4/80 monoclonal antibody 1:100; CST, Boston, USA) dropwise and incubated overnight at 4°C, followed by dropwise addition of enzyme-labelled goat anti-rabbit IgG polymer (Zhong Shan -Golden Bridge Biological Technology Co., Ltd., Beijing, China), rinsing in PBS (Gibco), colour development with 3,3'-diaminoben-zidine (DAB; Zhong Shan -Golden Bridge Biological Technology Co., Ltd.), rinsing in tap water, re-staining with hematoxylin for 1 min, dehydration, clearing, air-drying, and mounting. The results were interpreted as follows. Three 400×fields of view were randomly selected for image acquisition for each sample under a light microscope (Nikon, Tokyo, Japan), and positive staining was brownish/yellow in color. KCs were counted using Image J (Ver-1.8.0, National Institutes of Health, Bethesda, MD, USA) software and the cell counts were averaged after obtaining three fields of view for each specimen. 1.9 Statistical analysis The statistical analysis was performed using the SPSS 26.0 (IBM Corp., Armonk, NY, USA) software. The measurement data are expressed as mean ± standard deviations, The normality of data and the homogeneity of variances were analyzed first. Comparisons of the normally distributed data were performed using independent-samples t test, and data without normal distribution were analyzed by rank sum test. Pearson correlation analysis was performed to analyze the relationships of colonic NGF level with hepatic CXCL12 and VEGF levels. A P value of less than 0.05 was considered statistically significant. 2. Results 2.1 CRC liver metastasis in AD mice During the rearing period, two mice from the CON group and one mouse from the AD group experienced mortality. The number of CRLM nodules in the AD group was (232.20 ± 65.28), which was significantly larger than the number of tumor nodules on liver surface in the CON group (43.00 ± 34.75) ( P = 0.001) (Fig. 1 A, 1 B). In the AD group, the changes in liver weight and liver volume were significantly correlated with the number of metastases ( P = 0.035; P = 0.024) (Fig. 1 C, 1 D). Thus, CRLM was more likely to occur in AD mice. 2.2 Change of AD on gut microbiota in mice For investigating the influence of AD on intestinal flora, we employed 16S rDNA sequencing technology to analyze the gut microbiota in mice. 2.2.1 Influence of AD on the overall composition of gut microbiota in mice As shown in the dilution curves (Fig. 2 A), the species count per sample within both the CON and AD groups increased rapidly as the sequence number increased, and the curves flattened out when the sequence number reached a certain value, indicating that the number of sequencing for each sample was sufficient for subsequent data analysis. Among the hierarchical clustering curves, the horizontal curves were wide, indicating the samples were species-rich; in contrast, the vertical curves displayed a consistent and unblemished profile, signifying a relatively gradual slope, which implies a uniform dispersion of sample abundance. Illustrated in Fig. 2 B, both groups exhibited elevated abundance and uniformity within the gut microbiota, suggesting the samples selected were appropriate. Alpha diversity and beta diversity allow statistical analysis of the abundance and diversity of the the abundance and diversity of the intestinal flora. As shown in Figs. 2 C to 2 G, no notable disparity emerged in either the alpha or beta diversity of the intestinal microbiota between the CON and AD groups. This observation suggests a resemblance in the abundance and diversity of the gut microbiota among mice in both CON and AD groups.. We also visualized the OTUs of the intestinal flora on the Venn diagram. By clustering OTUs at 97% similarity, there were 883 common OTUs between the AD group and the CON group, 883 unique OTUs in the CON group, and 762 unique OTUs in the AD group, confirming the different species composition between these two groups (Fig. 2 H). Therefore, although the abundance and diversity of the gut microbiota were generally similar between the AD group and the CON group, the species composition had already became different. 2.2.2 The impacts of AD on the relative abundance of gut microbiota The relative abundance of the intestinal flora was further analyzed in both groups. As shown in the relative abundance histograms for the top 10 species in terms of abundance, at the phylum level, the abundance of Verrucomicrobiota , Deferribacteres , Desulfobacterota , and Campylobacterota was higher in the AD group (compared to the CON group); in contrast, the abundance of Bacteroidota , Firmicutes , and Proteobacteria decreased in the AD group (Fig. 3 A). At the genus level, Dubosiella , Lachnospiraceae_NK4A136_group , Akkermansia , Alloprevotella , Ruminofilibacter , and Mucispirillum were more abundant in the AD group than in the CON group, whereas Ligilactobacillus , Lactobacillus , Alistipes , and Limosilactobacillus had decreased abundance (Fig. 3 B). Among the intestinal flora, Lactobacillus spp. had significantly different abundance between these two groups ( P = 0.026) (Fig. 3 C). The samples were further clustered at the level of functional differences based on their functional annotations and abundance information in the database. It was found that the enriched functions of differential flora were mainly Membrane Transport and Cellular Processes in the AD group, while the differential flora functions of the CON group were mainly enriched in Human_Diseases, Organismal_Systems, Genetic_/Environmental_Information_Processing, and Metabolism (Fig. 3 D). Therefore, the altered intestinal flora due to AD might lead to functional changes in cells. 2.3 The expression of NGF, CXCL12, and VEGF in mice with AD We measured the levels of NGF and VEGF in serum, colon, and liver and the levels of CXCL12 in serum and liver in both groups by ELISA. The results showed that the NGF levels in the colon and liver of mice in the AD group were significantly higher than those in the CON group ( P = 0.029; P = 0.012; Fig. 4 A and 4 B), whereas the NGF level in serum showed no such significant difference (Fig. 4 C). The VEGF levels in the serum and liver of mice in the AD group were significantly higher than those in the CON group ( P = 0.040; P = 0.042; Figs. 4 D and 4 E); however, the VEGF level in colon tissue showed no significant difference (Fig. 4 F). In addition, the levels of CXCL12 in serum and liver were significantly higher in the AD group than in the CON group ( P = 0.031; P = 0.029; Fig. 4 G, 4 H). 2.4 The number of intrahepatic KCs in mice with AD Detection of intrahepatic KCs using the IHC technique revealed that the color development of KCs was concentrated in the cell membrane, which was mainly distributed around the liver sinusoids and the confluent area of the liver lobules, with irregular morphology. The number of intrahepatic KCs in the AD group (32.80 ± 2.63) was significantly smaller than that in the CON group (52.50 ± 3.97) ( P = 0.004) (Fig. 5 ). 3. Discussion 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. Declarations CONFLICT OF INTEREST The authors have no conflict of interest to report. FUNDING This work was supported by Natural Science Foundation of Hebei Province (grant number: H2022307056). Author Contribution Mengke. Nie.: Subject facilitator, Drafting of the manuscript; YiaoQian. Qiao.: Data acquisition, Data analysis and interpretation;Bin.Wang./XiaoWei. Ma.: Substantive review of the manuscript for significant intellectual contributions;Jie. Zhi./TianJun. Wang.: Study supervision; YiTao. Jia. (corresponding author): Study concept and design, Administrative, technical, and material support. ACKNOWLEDGEMENT I wish to pay regards to all who have contributed in writing this paper. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global cancer statistics 2020: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries [J]. CA Cancer J Clin, 2021, 71(3): 209-249. Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and mortality rates and trends--an update [J]. Cancer Epidemiol Biomarkers Prev, 2016, 25(1): 16-27. Millikan KW, Staren ED, Doolas A. Invasive therapy of metastatic colorectal cancer to the liver [J]. Surg Clin North Am, 1997, 77(1): 27-48. Alberts SR, Wagman LD. Chemotherapy for colorectal cancer liver metastases [J]. Oncologist, 2008, 13(10): 1063-1073. Konopke R, Kersting S, Distler M, Dietrich J, Gastmeier J, Heller A, Kulisch E, Saeger HD. Prognostic factors and evaluation of a clinical score for predicting survival after resection of colorectal liver metastases [J]. Liver Int, 2009, 29(1): 89-102. Rees M, Tekkis PP, Welsh FK, O'Rourke T, John TG. Evaluation of long-term survival after hepatic resection for metastatic colorectal cancer: a multifactorial model of 929 patients [J]. Ann Surg, 2008, 247(1): 125-135. Nordlinger B, Guiguet M, Vaillant JC, Balladur P, Boudjema K, Bachellier P, Jaeck D. Surgical resection of colorectal carcinoma metastases to the liver. A prognostic scoring system to improve case selection, based on 1568 patients. Association française de Chirurgie [J]. Cancer, 1996, 77(7): 1254-1262. Cui L, Hou NN, Wu HM, Zuo X, Lian YZ, Zhang CN, Wang ZF, Zhang X, Zhu JH. Prevalence of Alzheimer's disease and Parkinson's disease in China: An updated systematical analysis [J]. Front Aging Neurosci, 2020, 12: 603854. Valentine D, Teerlink CC, Farnham JM, Rowe K, Kaddas H, Tschanz J, Kauwe JSK, Cannon-Albright LA. Comorbidity and cancer disease rates among those at high-risk for Alzheimer's disease: A population database analysis [J]. Int J Environ Res Public Health, 2022, 19(24):16419. Lv G, Wang X, Jiang X, Li M, Lu K. Impact of Alzheimer's disease and related dementias on colorectal cancer screening utilization, knowledge, and associated health disparities [J]. Front Pharmacol, 2022, 13: 872702. Chen Y, Cress RD, Stewart SL, Semrad TJ, Harvey D, Tencredi DJ, Beckett L. Mediating effect of postsurgical chemotherapy on presence of dementia and survival among patients 65 and older with stage III colon cancer [J]. Cancer Epidemiol Biomarkers Prev, 2017, 26(10): 1558-1563. Lu L, Ma L, Zhang X, Susanne Mullins C, Linnebacher M. Analyzing non-cancer causes of death of colorectal carcinoma patients in the us population for the years 2000-2016 [J]. Cancer Med, 2021, 10(8): 2740-2751. Li R, Zhou R, Wang H, Li W, Pan M, Yao X, Zhan W, Yang S, Xu L, Ding Y, Zhao L. Gut microbiota-stimulated cathepsin K secretion mediates TLR4-dependent M2 macrophage polarization and promotes tumor metastasis in colorectal cancer [J]. Cell Death Differ, 2019, 26(11): 2447-2463. Dai CL, Liu F, Iqbal K, Gong CX. Gut microbiota and immunotherapy for Alzheimer's disease [J]. Int J Mol Sci, 2022, 23(23):15230. Humpel C. NGF released from blood cells or collagen hydrogels as a therapeutic target in Alzheimer's disease? [J]. Adv Exp Med Biol, 2021, 1331: 193-202. von Boyen GB, Steinkamp M, Reinshagen M, Schäfer KH, Adler G, Kirsch J. Nerve growth factor secretion in cultured enteric glia cells is modulated by proinflammatory cytokines [J]. J Neuroendocrinol, 2006, 18(11): 820-825. Amara S, Chaar I, Khiari M, Ounissi D, Weslati M, Boughriba R, Hmida AB, Bouraoui S. Stromal cell derived factor-1 and CXCR4 expression in colorectal cancer promote liver metastasis [J]. Cancer Biomark, 2015, 15(6): 869-879. Kim J, Takeuchi H, Lam ST, Turner RR, Wang HJ, Kuo C, Foshag L, Bilchik AJ, Hoon DS. Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk for recurrence and for poor survival [J]. J Clin Oncol, 2005, 23(12): 2744-2753. Wang D, Wang X, Si M, Yang J, Sun S, Wu H, Cui S, Qu X, Yu X. Exosome-encapsulated miRNAs contribute to CXCL12/CXCR4-induced liver metastasis of colorectal cancer by enhancing M2 polarization of macrophages [J]. Cancer Lett, 2020, 474: 36-52. Horikawa N, Abiko K, Matsumura N, Hamanishi J, Baba T, Yamaguchi K, Yoshioka Y, Koshiyama M, Konishi I. Expression of vascular endothelial growth factor in ovarian cancer inhibits tumor immunity through the accumulation of myeloid-derived suppressor cells [J]. Clin Cancer Res, 2017, 23(2): 587-599. Takahashi Y, Kitadai Y, Bucana CD, Cleary KR, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer [J]. Cancer Res, 1995, 55(18): 3964-3968. Paget S. The distribution of secondary growths in cancer of the breast. 1889 [J]. Cancer Metastasis Rev, 1989, 8(2): 98-101. Doak GR, Schwertfeger KL, Wood DK. Distant relations: Macrophage functions in the metastatic niche [J]. Trends Cancer, 2018, 4(6): 445-459. Bi W, Cai S, Hang Z, Lei T, Wang D, Wang L, Du H. Transplantation of feces from mice with Alzheimer's disease promoted lung cancer growth [J]. Biochem Biophys Res Commun, 2022, 600: 67-74. Si W, Liang H, Bugno J, Xu Q, Ding X, Yang K, Fu Y, Weichselbaum RR, Zhao X, Wang L. Lactobacillus rhamnosus GG induces CGAS/sting- dependent type I interferon and improves response to immune checkpoint blockade [J]. Gut, 2022, 71(3): 521-533. Wang T, Zhang L, Wang P, Liu Y, Wang G, Shan Y, Yi Y, Zhou Y, Liu B, Wang X, Lü X. Lactobacillus coryniformis MXJ32 administration ameliorates azoxymethane/dextran sulfate sodium-induced colitis-associated colorectal cancer via reshaping intestinal microenvironment and alleviating inflammatory response [J]. Eur J Nutr, 2022, 61(1): 85-99. Xu XJ, Liu L, Yao SK. Nerve growth factor and diarrhea-predominant irritable bowel syndrome (IBS-D): A potential therapeutic target? [J]. J Zhejiang Univ Sci B, 2016, 17(1): 1-9. Yuan R, Bhattacharya N, Kenkel JA, Shen J, DiMaio MA, Bagchi S, Prestwood TR, Habtezion A, Engleman EG. Enteric glia play a critical role in promoting the development of colorectal cancer [J]. Front Oncol, 2020, 10: 595892. Albillos A, de Gottardi A, Rescigno M. The gut-liver axis in liver disease: Pathophysiological basis for therapy [J]. J Hepatol, 2020, 72(3): 558-577. Garrido MP, Salvatierra R, Valenzuela-Valderrama M, Vallejos C, Bruneau N, Hernández A, Vega M, Selman A, Quest AFG, Romero C. Metformin reduces NGF-induced tumour promoter effects in epithelial ovarian cancer cells [J]. Pharmaceuticals 2020, 13(10): 315-325. Romon R, Adriaenssens E, Lagadec C, Germain E, Hondermarck H, Le Bourhis X. Nerve growth factor promotes breast cancer angiogenesis by activating multiple pathways [J]. Mol Cancer, 2010, 9: 157-168. Rigault E, Lacas B, Glehen O, Smith D, Dupont-Bierre E, Guimbaud R, Malka D, Boige V, Fuerea A, Pignon JP, Ducreux M. Intra-arterial hepatic bevacizumab and systemic chemotherapy in hepatic metastasis of colorectal cancer: A phase II multicentric trial in second-line treatment [J]. Cancer Treat Res Commun, 2023, 34: 100674. Kim J, Takeuchi H, Lam ST, Turner RR, Wang HJ, Kuo C, Foshag L, Bilchik AJ, Hoon DS. Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk for recurrence and for poor survival [J]. J Clin Oncol, 2005, 23(12): 2744-2753. Zeng X, Ward SE, Zhou J, Cheng ASL. Liver immune microenvironment and metastasis from colorectal cancer-pathogenesis and therapeutic perspectives [J]. Cancers, 2021, 13(10):2418-2430. Dixon LJ, Barnes M, Tang H, Pritchard MT, Nagy LE. Kupffer cells in the liver [J]. Compr Physiol, 2013, 3(2): 785-797. Malaguarnera G, Giordano M, Nunnari G, Bertino G, Malaguarnera M. Gut microbiota in alcoholic liver disease: Pathogenetic role and therapeutic perspectives [J]. World J Gastroenterol, 2014, 20(44): 16639-16648. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4300147","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":293831084,"identity":"7529d4d0-f340-4f93-bf18-39110b13a217","order_by":0,"name":"Mengke Nie","email":"","orcid":"","institution":"Huaihe Hospital of Henan University","correspondingAuthor":false,"prefix":"","firstName":"Mengke","middleName":"","lastName":"Nie","suffix":""},{"id":293831086,"identity":"70913e9c-d1b9-4a73-9b55-ec10c119c05a","order_by":1,"name":"Yiqian Qiao","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yiqian","middleName":"","lastName":"Qiao","suffix":""},{"id":293831088,"identity":"e6de17eb-d418-47ab-9158-725342ab1721","order_by":2,"name":"Bin Wang","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Wang","suffix":""},{"id":293831089,"identity":"646d9d12-5e9d-48c9-95ec-94be5fca3ace","order_by":3,"name":"Tianjun Wang","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tianjun","middleName":"","lastName":"Wang","suffix":""},{"id":293831090,"identity":"380fa175-78f6-4ccf-ac36-f9a69788b5c5","order_by":4,"name":"Xiaowei Ma","email":"","orcid":"","institution":"The First Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Ma","suffix":""},{"id":293831091,"identity":"f613a77e-c48c-4fea-a089-7ef2c2ab05f9","order_by":5,"name":"Jie Zhi","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhi","suffix":""},{"id":293831092,"identity":"a32a96a0-81f5-435c-82ff-eeded2ce3578","order_by":6,"name":"Yitao Jia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACxmYgkQDE/OzNBw58+EG0FqAeyZ5jiQdn9hBtF1CLwY0c48McbEQoZm5nfibx8IedHFDLh8MMPAzy/GIHCDmMzUwiISHZWPLM2w2HCywYDGfOTiCkhQGkhTmx73juhsMzeBgSDG4T1ML+DailPrHhQM6DwzxsRGnhAdlyOHHCiRwGorUUWySkHTcGBrIBMJAlCPvFsP/4xps/bKrlgFH5+MOHHzby/NKEtDQwsEgg8SVwqoQDeWDUfCCsbBSMglEwCkY0AADMhUgXEQptWAAAAABJRU5ErkJggg==","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yitao","middleName":"","lastName":"Jia","suffix":""}],"badges":[],"createdAt":"2024-04-21 09:26:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4300147/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4300147/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55236727,"identity":"3026ec90-31c7-46d6-9bc0-771f9fa2a89c","added_by":"auto","created_at":"2024-04-24 14:01:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98314,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the number of tumor nodules on liver surface and the liver weight and volume between the two groups. A: Difference plot of liver metastases in the two groups; B: Comparison of the number of tumor nodules on liver surface between the two groups; C: Comparison of liver weight between the two groups; D: Comparison of liver volume between the two groups; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4300147/v1/33cb48f6d3eab0d24d14be82.jpg"},{"id":55236729,"identity":"8cde818b-9bf3-446a-b9a4-4ce36a502382","added_by":"auto","created_at":"2024-04-24 14:01:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":370047,"visible":true,"origin":"","legend":"\u003cp\u003eImpacts of Alzheimer's disease on the whole intestinal flora in the two groups. A: Dilution curves of individual samples in the two groups; B: hierarchical clustering curves of fecal samples in the two groups; C to F: Alpha diversity of mouse intestinal flora; G: Beta diversity of mouse intestinal flora; H: Venn diagram of differences in abundance of mouse intestinal flora.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4300147/v1/6533b64de6dd158236d4e2ce.jpg"},{"id":55237308,"identity":"c62b92b5-ef1b-41f9-9fac-636978de93b4","added_by":"auto","created_at":"2024-04-24 14:09:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":222572,"visible":true,"origin":"","legend":"\u003cp\u003eImpacts of Alzheimer's disease on the relative abundance of intestinal flora.\u003c/p\u003e\n\u003cp\u003eA: Histogram of relative abundance of mouse intestinal flora at the phylum level; B: histogram of relative abundance of mouse intestinal flora at the genus level; C: Difference plot of mouse intestinal flora between two groups at the genus level; D: Predictive analysis of Tax4Fun function of mouse intestinal differential flora.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4300147/v1/17a420bfa458efe2359c0c32.jpg"},{"id":55236731,"identity":"873f534e-4947-48ee-8587-a77cea70a982","added_by":"auto","created_at":"2024-04-24 14:01:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":205011,"visible":true,"origin":"","legend":"\u003cp\u003eImpacts of AD on NGF, CXCL12, and VEGF in mice. A-C: Levels of NGF in mouse colon, liver, and serum; D-F: Levels of VEGF in mouse serum, liver, and colon; G, H: Levels of CXCL12 in mouse serum and liver; I: Correlation between the colonic NGF level with liver CXCL12; J: correlation between the content of NGF in colon and VEGF in liver; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4300147/v1/d89c18b2f63ca8ee06127e97.jpg"},{"id":55236728,"identity":"b8494f8d-dbf5-45c0-a4c6-a0df34a41ab9","added_by":"auto","created_at":"2024-04-24 14:01:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":77149,"visible":true,"origin":"","legend":"\u003cp\u003eContent of intrahepatic KCs in the two groups of mice (IHC, ×400, bar=50μm). A: Distribution of intrahepatic KCs in the CON group; B: Distribution of intrahepatic KCs in the AD group; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4300147/v1/0ade037633064bb2428801b9.jpg"},{"id":58349056,"identity":"e0556edf-c90d-4285-aa9f-7a4196285afd","added_by":"auto","created_at":"2024-06-14 08:34:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1510758,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4300147/v1/2535111e-4292-45df-b030-4073906dd5ba.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The risk factors for colorectal cancer liver metastasis in a mouse model of Alzheimer's disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) ranks as the prevailing form of gastrointestinal malignancy on a global scale [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While there have been notable improvements in the prognosis of CRC patients due to advancements in diagnostic techniques and treatment modalities, CRC liver metastasis (CRLM) remains a non-negligible problem, wherein around half of CRC patients manifest liver metastases upon initial diagnosis [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although a small proportion of CRLM can be removed surgically, the post-resection 5-year survival rate is merely around 36% [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCRC can occur at any age, but it is most commonly seen in the middle-aged and elderly population. With the aging of population, the prevalence rates of some degenerative diseases such as Alzheimer's disease (AD) are also rising. Therefore, there is an increased probability of coexistence of these two conditions [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A retrospective analysis revealed a 45% higher likelihood of postoperative mortality in CRC patients with preexisting AD in comparison to those with CRC alone (hazard ratio [HR]: 1.45, 95% confidence interval [CI]: 1.29\u0026ndash;1.63) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Another investigation into the factors contributing to mortality in CRC patients indicated that the probability of death attributed to AD rose from 1.73\u0026ndash;10.47% as survival time extended (from 2 to 11 months to more than 180 months) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the past few years, a growing body of research has substantiated the prominent role of intestinal microbiota in the pathogenesis of CRLM and AD. It has been found that intestinal flora disorders can induce the release of metastasis-related secretory protein cathepsin K (CTSK), initiating Toll-like Receptor 4 (TLR4)-dependent M2 macrophage polarization, thus promoting CRLM [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, alterations in intestinal flora accelerate the neuronal degeneration, thus promoting AD [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Nerve growth factor (NGF), emanating from enteric glial cells, is involved in both the development of AD and the pathogenesis of CRC. Cholinergic neurons are selectively degraded in AD, and loss of acetylcholine is directly associated with cognitive decline. NGF is the most potent growth factor in improving the survival of these cholinergic neurons [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, NGF regulates intestinal peristaltic function and maintains the integrity of the intestinal mucosal barrier by participating in the intestinal inflammatory response [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTumor metastasis is a multifaceted process influenced by a multitude of factors. Researches have demonstrated that the activation of the CXCL12/CXCR4 axis is linked to an poor prognosis for individuals with CRLM [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The CXCL12/CXCR4 axis activation leads to the upregulation of specific microRNAs within CRC cells. Through exosomes, these miRNAs are transferred to macrophages. Consequently, these exosome-mediated miRNAs prompt the M2 polarization of macrophages by modulating PTEN via the activation of the PI3K/Akt signaling pathway. Then the M2-polarized macrophages contribute to the progression of cancer metastasis by bolstering epithelial-mesenchymal transition (EMT) and releasing vascular endothelial growth factor (VEGF) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. VEGF, in turn, supports tumor cell survival and dissemination by attracting myeloid-derived suppressor cells (MDSCs), which suppress the local immune microenvironment. The level of VEGF within tumor tissue can serve as an indicator of the risk of metastasis in colorectal cancer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Immune components present in the liver encompass Kupffer cells (KCs), T lymphocytes, natural killer cells, and hepatic sinusoidal endothelial cells, Among which KCs, functioning as macrophages within the liver, hold a significant role in the context of CRLM [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, the incidence of CRLM in AD patients remains unclear, and few studies have investigated whether AD can influence the occurrence of CRLM by causing changes in intestinal flora, NGF, and factors related to liver metastasis or whether interventions in intestinal flora can reduce the risk of CRLM. In this particular investigation, mouse models of CRLM were established by intrapleural injection of MC38 colon cancer cells in AD mice in an attempt to investigate the impact of AD on CRLM and its possible mechanism.\u003c/p\u003e"},{"header":"1. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Feeding and grouping of experimental animals\u003c/h2\u003e \u003cp\u003eSix 5-month-old male SPF-grade APP/PS1 double-transgenic mice were purchased from HFK BioScience (Beijing, China) (animal production license number: [SCXK (Beijing) 2019-0008]), and the inbred strain used was C57BL/6J. Six 5-month-old male SPF-grade wild-type C57BL/6J mice were purchased from Exinvivo (Hebei, China) (animal production license number: [SCXK (Hebei) 2020-002]. The mice were accommodated within the SPF-grade animal facility at the Experimental Center of Hebei General Hospital. Approval for the experiments was obtained from the Experimental Animal Ethics Committee of the Hebei General Hospital (approval number: 202215). The mice were provided housing in compliance with the \u003cem\u003eLaboratory Animal - Requirements of Environment and Housing Facilities\u003c/em\u003e issued by the Ministry of Health of the People's Republic of China. This included maintaining a 12:12-hour light-dark cycle and a constant room temperature of 24℃. Adequate food and water were accessible to the animals on an ad libitum basis. Following a one-week adaptation period of feeding, the mice were then categorized into two distinct groups (n = 6 each): the control (CON) group: wild-type C57BL/6J mice, which were used to establish CRLM models; and the AD group: APP/PS1 double-transgenic mice, which were also used to establish CRLM models.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Cell line culture\u003c/h2\u003e \u003cp\u003eThe murine colon carcinoma cell line MC38 (Cell Bank of Chinese Academy of Sciences, Shanghai, China) was cultured in RPMI 1640 (Gibco, Santa Cruz, CA, USA) medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% double antibodies (100U/mL penicillin and 100µg/mL streptomycin) in a constant temperature incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1.3 Establishment of CRLM models\u003c/h2\u003e \u003cp\u003eAfter the mice were successfully anesthetized, incisions were made along the mid-axillary line of the abdomen to expose the spleen. Subsequently, 0.1mL of MC38 cell suspension (concentration: 5×10\u003csup\u003e6\u003c/sup\u003e cells/mL) was aspirated with a 29-G insulin syringe and then injected into the spleen along its longitudinal axis via the inferior poleof the spleen. The needle was withdrawn after the spleen peritoneum became pale and swollen. A sterile cotton swab was immediately placed over the injection site to prevent bleeding, and then the incisions were closed in layers.\u003c/p\u003e \u003cp\u003e \u003cb\u003e1.4 Experimental steps\u003c/b\u003e \u003c/p\u003e \u003cp\u003e1) After 1 week of adaptive feeding, CRLM models were established in both groups; 2) the mice continued to be routinely fed after modeling; 3) on day 14, the feces were picked up from both groups; and 4) at the end of the experiment on day 15, blood, colon, and liver tissues were collected, the liver weight was weighed, the liver volume was measured, and the tumor nodules on liver surface were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e1.5 Counting of tumor nodules and measurement of liver weight and volume\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e1.5.1 Counting of tumor nodules\u003c/h2\u003e \u003cp\u003eIndividual metastases sized less than 0.2cm were counted as 1, sized 0.2–0.4cm (excluding 0.2cm) as 2, sized 0.4–0.6cm (excluding 0.4cm) as 4, and sized greater than 0.6cm as 6.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e1.5.2 Measurement of liver weight and volume\u003c/h2\u003e \u003cp\u003eA Petri dish was placed in advance in an electronic balance, which was zeroed. The liver sample was placed in the Petri dish, and its weight was read and recorded. A measuring cylinder containing saline was prepared, and the scale corresponding to the liquid level (baseline reading) was read in advance. The liver sample was then gently slid into the measuring cylinder along the edge to allow the saline submerges the entire sample, and the current reading was recorded after the liquid level became stable. The liver volume was calculated using the following formula: Liver volume = Current reading (cm\u003csup\u003e3\u003c/sup\u003e) - Baseline reading (cm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e1.6 16S rDNA sequencing\u003c/h2\u003e \u003cp\u003eThe feces of mice were collected for DNA extraction. The polymerase chain reaction (PCR) amplification of the V3 - V4 variable regions was performed based on the gene sequences 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3'). After the target bands were recovered by shearing, library construction and sequencing were performed to analyze the DNA information of intestinal flora. The clean reads were obtained for all samples and clustered into operational taxonomic units (OTUs).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e1.7 Enzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eSerum, liver tissue, and colon tissue were used for ELISA. After the homogenized liver and colon tissues were centrifuged at 3000rpm for 15min at 4°C, the supernatant was harvested. Then, the supernatant and serum were stored in a -20℃ refrigerator. All the ELISA kits used were purchased from Cloud-Clone (Wuhan, China). After sample addition, incubation, washing, enzyme addition, and color development, the reaction was terminated. The value of optical density (OD) was determined at the wavelength of 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e1.8 Immunohistochemistry (IHC) staining\u003c/h2\u003e \u003cp\u003eThe liver tissues were fixed in 4% paraformaldehyde for 24h and then routinely embedded in paraffin, sectioned, and dewaxed. After antigen repair, serum blocking, and antibody dilution, the sections were added with primary antibody (rabbit anti-mouse F4/80 monoclonal antibody 1:100; CST, Boston, USA) dropwise and incubated overnight at 4°C, followed by dropwise addition of enzyme-labelled goat anti-rabbit IgG polymer (Zhong Shan -Golden Bridge Biological Technology Co., Ltd., Beijing, China), rinsing in PBS (Gibco), colour development with 3,3'-diaminoben-zidine (DAB; Zhong Shan -Golden Bridge Biological Technology Co., Ltd.), rinsing in tap water, re-staining with hematoxylin for 1 min, dehydration, clearing, air-drying, and mounting.\u003c/p\u003e \u003cp\u003eThe results were interpreted as follows. Three 400×fields of view were randomly selected for image acquisition for each sample under a light microscope (Nikon, Tokyo, Japan), and positive staining was brownish/yellow in color. KCs were counted using Image J (Ver-1.8.0, National Institutes of Health, Bethesda, MD, USA) software and the cell counts were averaged after obtaining three fields of view for each specimen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e1.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was performed using the SPSS 26.0 (IBM Corp., Armonk, NY, USA) software. The measurement data are expressed as mean ± standard deviations, The normality of data and the homogeneity of variances were analyzed first. Comparisons of the normally distributed data were performed using independent-samples \u003cem\u003et\u003c/em\u003e test, and data without normal distribution were analyzed by rank sum test. Pearson correlation analysis was performed to analyze the relationships of colonic NGF level with hepatic CXCL12 and VEGF levels. A \u003cem\u003eP\u003c/em\u003e value of less than 0.05 was considered statistically significant.\u003c/p\u003e "},{"header":"2. Results","content":"\u003ch2\u003e2.1 CRC liver metastasis in AD mice\u003c/h2\u003e\u003cp\u003eDuring the rearing period, two mice from the CON group and one mouse from the AD group experienced mortality. The number of CRLM nodules in the AD group was (232.20 ± 65.28), which was significantly larger than the number of tumor nodules on liver surface in the CON group (43.00 ± 34.75) (\u003cem\u003eP\u003c/em\u003e = 0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In the AD group, the changes in liver weight and liver volume were significantly correlated with the number of metastases (\u003cem\u003eP\u003c/em\u003e = 0.035; \u003cem\u003eP\u003c/em\u003e = 0.024) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Thus, CRLM was more likely to occur in AD mice.\u003c/p\u003e\u003ch2\u003e2.2 Change of AD on gut microbiota in mice\u003c/h2\u003e\u003cp\u003eFor investigating the influence of AD on intestinal flora, we employed 16S rDNA sequencing technology to analyze the gut microbiota in mice.\u003c/p\u003e\u003ch2\u003e2.2.1 Influence of AD on the overall composition of gut microbiota in mice\u003c/h2\u003e\u003cp\u003eAs shown in the dilution curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), the species count per sample within both the CON and AD groups increased rapidly as the sequence number increased, and the curves flattened out when the sequence number reached a certain value, indicating that the number of sequencing for each sample was sufficient for subsequent data analysis. Among the hierarchical clustering curves, the horizontal curves were wide, indicating the samples were species-rich; in contrast, the vertical curves displayed a consistent and unblemished profile, signifying a relatively gradual slope, which implies a uniform dispersion of sample abundance. Illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, both groups exhibited elevated abundance and uniformity within the gut microbiota, suggesting the samples selected were appropriate.\u003c/p\u003e\u003cp\u003eAlpha diversity and beta diversity allow statistical analysis of the abundance and diversity of the the abundance and diversity of the intestinal flora. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eC to \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, no notable disparity emerged in either the alpha or beta diversity of the intestinal microbiota between the CON and AD groups. This observation suggests a resemblance in the abundance and diversity of the gut microbiota among mice in both CON and AD groups.. We also visualized the OTUs of the intestinal flora on the Venn diagram. By clustering OTUs at 97% similarity, there were 883 common OTUs between the AD group and the CON group, 883 unique OTUs in the CON group, and 762 unique OTUs in the AD group, confirming the different species composition between these two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Therefore, although the abundance and diversity of the gut microbiota were generally similar between the AD group and the CON group, the species composition had already became different.\u003c/p\u003e\u003ch2\u003e2.2.2 The impacts of AD on the relative abundance of gut microbiota\u003c/h2\u003e\u003cp\u003eThe relative abundance of the intestinal flora was further analyzed in both groups. As shown in the relative abundance histograms for the top 10 species in terms of abundance, at the phylum level, the abundance of \u003cem\u003eVerrucomicrobiota\u003c/em\u003e, \u003cem\u003eDeferribacteres\u003c/em\u003e, \u003cem\u003eDesulfobacterota\u003c/em\u003e, and \u003cem\u003eCampylobacterota\u003c/em\u003e was higher in the AD group (compared to the CON group); in contrast, the abundance of \u003cem\u003eBacteroidota\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, and \u003cem\u003eProteobacteria\u003c/em\u003e decreased in the AD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). At the genus level, \u003cem\u003eDubosiella\u003c/em\u003e, \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e, \u003cem\u003eAkkermansia\u003c/em\u003e, \u003cem\u003eAlloprevotella\u003c/em\u003e, \u003cem\u003eRuminofilibacter\u003c/em\u003e, and \u003cem\u003eMucispirillum\u003c/em\u003e were more abundant in the AD group than in the CON group, whereas \u003cem\u003eLigilactobacillus\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eAlistipes\u003c/em\u003e, and \u003cem\u003eLimosilactobacillus\u003c/em\u003e had decreased abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Among the intestinal flora, \u003cem\u003eLactobacillus spp.\u003c/em\u003e had significantly different abundance between these two groups (\u003cem\u003eP\u003c/em\u003e = 0.026) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eThe samples were further clustered at the level of functional differences based on their functional annotations and abundance information in the database. It was found that the enriched functions of differential flora were mainly Membrane Transport and Cellular Processes in the AD group, while the differential flora functions of the CON group were mainly enriched in Human_Diseases, Organismal_Systems, Genetic_/Environmental_Information_Processing, and Metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Therefore, the altered intestinal flora due to AD might lead to functional changes in cells.\u003c/p\u003e\u003ch2\u003e2.3 The expression of NGF, CXCL12, and VEGF in mice with AD\u003c/h2\u003e\u003cp\u003eWe measured the levels of NGF and VEGF in serum, colon, and liver and the levels of CXCL12 in serum and liver in both groups by ELISA. The results showed that the NGF levels in the colon and liver of mice in the AD group were significantly higher than those in the CON group (\u003cem\u003eP\u003c/em\u003e = 0.029; \u003cem\u003eP\u003c/em\u003e = 0.012; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), whereas the NGF level in serum showed no such significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The VEGF levels in the serum and liver of mice in the AD group were significantly higher than those in the CON group (\u003cem\u003eP\u003c/em\u003e = 0.040; \u003cem\u003eP\u003c/em\u003e = 0.042; Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE); however, the VEGF level in colon tissue showed no significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). In addition, the levels of CXCL12 in serum and liver were significantly higher in the AD group than in the CON group (\u003cem\u003eP\u003c/em\u003e = 0.031; \u003cem\u003eP\u003c/em\u003e = 0.029; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e\u003ch2\u003e2.4 The number of intrahepatic KCs in mice with AD\u003c/h2\u003e\u003cp\u003eDetection of intrahepatic KCs using the IHC technique revealed that the color development of KCs was concentrated in the cell membrane, which was mainly distributed around the liver sinusoids and the confluent area of the liver lobules, with irregular morphology. The number of intrahepatic KCs in the AD group (32.80 ± 2.63) was significantly smaller than that in the CON group (52.50 ± 3.97) (\u003cem\u003eP\u003c/em\u003e = 0.004) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eLiver 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.\u003c/p\u003e\u003cp\u003eWithin 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 \u003cem\u003eet al.\u003c/em\u003e 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 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur results showed that AD led to alterations in the composition and proportional distribution of intestinal microbial species in mice with CRLM, with \u003cem\u003eLactobacillus spp.\u003c/em\u003e 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. \u003cem\u003eLactobacillus\u003c/em\u003e is known to be a probiotic. Studies have demonstrated that oral administration of live \u003cem\u003eLactobacillus rhamnosus GG\u003c/em\u003e can enhance the efficacy of programmed death receptor-1 (PD-1) inhibitors by increasing tumor-infiltrating dendritic cells and T cells. In addition, \u003cem\u003eLactobacillus rhamnosus GG\u003c/em\u003e (LGG) in combination with PD-1 inhibitors promoted the enrichment of gut microbiota towards \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and \u003cem\u003eLactobacillus monomorphicus\u003c/em\u003e, thereby inducing the activation of dendritic cells and the migration of CD8 + T cells towards tumor tissue [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Supplementation with \u003cem\u003eLactobacillus rotundus\u003c/em\u003e 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 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, the reduced gut abundance of \u003cem\u003eLactobacillus spp.\u003c/em\u003e 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.\u003c/p\u003e\u003cp\u003eIn 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 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. 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.\u003c/p\u003e\u003cp\u003eWe 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 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. CXCL12 has also been found to be involved in CRLM. Kim \u003cem\u003eet al\u003c/em\u003e. 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 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. 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.\u003c/p\u003e\u003cp\u003eThe 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 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. 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.\u003c/p\u003e\u003cp\u003eAlthough 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors have no conflict of interest to report.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis work was supported by Natural Science Foundation of Hebei Province (grant number: H2022307056).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMengke. Nie.: Subject facilitator, Drafting of the manuscript; YiaoQian. Qiao.: Data acquisition, Data analysis and interpretation;Bin.Wang./XiaoWei. Ma.: Substantive review of the manuscript for significant intellectual contributions;Jie. Zhi./TianJun. Wang.: Study supervision; YiTao. Jia. (corresponding author): Study concept and design, Administrative, technical, and material support.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eI wish to pay regards to all who have contributed in writing this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global cancer statistics 2020: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries [J]. CA Cancer J Clin, 2021, 71(3): 209-249.\u003c/li\u003e\n\u003cli\u003eTorre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and mortality rates and trends--an update [J]. Cancer Epidemiol Biomarkers Prev, 2016, 25(1): 16-27.\u003c/li\u003e\n\u003cli\u003eMillikan KW, Staren ED, Doolas A. Invasive therapy of metastatic colorectal cancer to the liver [J]. Surg Clin North Am, 1997, 77(1): 27-48.\u003c/li\u003e\n\u003cli\u003eAlberts SR, Wagman LD. Chemotherapy for colorectal cancer liver metastases [J]. Oncologist, 2008, 13(10): 1063-1073.\u003c/li\u003e\n\u003cli\u003eKonopke R, Kersting S, Distler M, Dietrich J, Gastmeier J, Heller A, Kulisch E, Saeger HD. Prognostic factors and evaluation of a clinical score for predicting survival after resection of colorectal liver metastases [J]. Liver Int, 2009, 29(1): 89-102.\u003c/li\u003e\n\u003cli\u003eRees M, Tekkis PP, Welsh FK, O\u0026apos;Rourke T, John TG. Evaluation of long-term survival after hepatic resection for metastatic colorectal cancer: a multifactorial model of 929 patients [J]. Ann Surg, 2008, 247(1): 125-135.\u003c/li\u003e\n\u003cli\u003eNordlinger B, Guiguet M, Vaillant JC, Balladur P, Boudjema K, Bachellier P, Jaeck D. Surgical resection of colorectal carcinoma metastases to the liver. A prognostic scoring system to improve case selection, based on 1568 patients. Association fran\u0026ccedil;aise de Chirurgie [J]. Cancer, 1996, 77(7): 1254-1262.\u003c/li\u003e\n\u003cli\u003eCui L, Hou NN, Wu HM, Zuo X, Lian YZ, Zhang CN, Wang ZF, Zhang X, Zhu JH. Prevalence of Alzheimer\u0026apos;s disease and Parkinson\u0026apos;s disease in China: An updated systematical analysis [J]. Front Aging Neurosci, 2020, 12: 603854.\u003c/li\u003e\n\u003cli\u003eValentine D, Teerlink CC, Farnham JM, Rowe K, Kaddas H, Tschanz J, Kauwe JSK, Cannon-Albright LA. Comorbidity and cancer disease rates among those at high-risk for Alzheimer\u0026apos;s disease: A population database analysis [J]. Int J Environ Res Public Health, 2022, 19(24):16419.\u003c/li\u003e\n\u003cli\u003eLv G, Wang X, Jiang X, Li M, Lu K. Impact of Alzheimer\u0026apos;s disease and related dementias on colorectal cancer screening utilization, knowledge, and associated health disparities [J]. Front Pharmacol, 2022, 13: 872702.\u003c/li\u003e\n\u003cli\u003eChen Y, Cress RD, Stewart SL, Semrad TJ, Harvey D, Tencredi DJ, Beckett L. Mediating effect of postsurgical chemotherapy on presence of dementia and survival among patients 65 and older with stage III colon cancer [J]. Cancer Epidemiol Biomarkers Prev, 2017, 26(10): 1558-1563.\u003c/li\u003e\n\u003cli\u003eLu L, Ma L, Zhang X, Susanne Mullins C, Linnebacher M. Analyzing non-cancer causes of death of colorectal carcinoma patients in the us population for the years 2000-2016 [J]. Cancer Med, 2021, 10(8): 2740-2751.\u003c/li\u003e\n\u003cli\u003eLi R, Zhou R, Wang H, Li W, Pan M, Yao X, Zhan W, Yang S, Xu L, Ding Y, Zhao L. Gut microbiota-stimulated cathepsin K secretion mediates TLR4-dependent M2 macrophage polarization and promotes tumor metastasis in colorectal cancer [J]. Cell Death Differ, 2019, 26(11): 2447-2463.\u003c/li\u003e\n\u003cli\u003eDai CL, Liu F, Iqbal K, Gong CX. Gut microbiota and immunotherapy for Alzheimer\u0026apos;s disease [J]. Int J Mol Sci, 2022, 23(23):15230.\u003c/li\u003e\n\u003cli\u003eHumpel C. NGF released from blood cells or collagen hydrogels as a therapeutic target in Alzheimer\u0026apos;s disease? [J]. Adv Exp Med Biol, 2021, 1331: 193-202.\u003c/li\u003e\n\u003cli\u003evon Boyen GB, Steinkamp M, Reinshagen M, Sch\u0026auml;fer KH, Adler G, Kirsch J. Nerve growth factor secretion in cultured enteric glia cells is modulated by proinflammatory cytokines [J]. J Neuroendocrinol, 2006, 18(11): 820-825.\u003c/li\u003e\n\u003cli\u003eAmara S, Chaar I, Khiari M, Ounissi D, Weslati M, Boughriba R, Hmida AB, Bouraoui S. Stromal cell derived factor-1 and CXCR4 expression in colorectal cancer promote liver metastasis [J]. Cancer Biomark, 2015, 15(6): 869-879.\u003c/li\u003e\n\u003cli\u003eKim J, Takeuchi H, Lam ST, Turner RR, Wang HJ, Kuo C, Foshag L, Bilchik AJ, Hoon DS. Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk for recurrence and for poor survival [J]. J Clin Oncol, 2005, 23(12): 2744-2753.\u003c/li\u003e\n\u003cli\u003eWang D, Wang X, Si M, Yang J, Sun S, Wu H, Cui S, Qu X, Yu X. Exosome-encapsulated miRNAs contribute to CXCL12/CXCR4-induced liver metastasis of colorectal cancer by enhancing M2 polarization of macrophages [J]. Cancer Lett, 2020, 474: 36-52.\u003c/li\u003e\n\u003cli\u003eHorikawa N, Abiko K, Matsumura N, Hamanishi J, Baba T, Yamaguchi K, Yoshioka Y, Koshiyama M, Konishi I. Expression of vascular endothelial growth factor in ovarian cancer inhibits tumor immunity through the accumulation of myeloid-derived suppressor cells [J]. Clin Cancer Res, 2017, 23(2): 587-599.\u003c/li\u003e\n\u003cli\u003eTakahashi Y, Kitadai Y, Bucana CD, Cleary KR, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer [J]. Cancer Res, 1995, 55(18): 3964-3968.\u003c/li\u003e\n\u003cli\u003ePaget S. The distribution of secondary growths in cancer of the breast. 1889 [J]. Cancer Metastasis Rev, 1989, 8(2): 98-101.\u003c/li\u003e\n\u003cli\u003eDoak GR, Schwertfeger KL, Wood DK. Distant relations: Macrophage functions in the metastatic niche [J]. Trends Cancer, 2018, 4(6): 445-459.\u003c/li\u003e\n\u003cli\u003eBi W, Cai S, Hang Z, Lei T, Wang D, Wang L, Du H. Transplantation of feces from mice with Alzheimer\u0026apos;s disease promoted lung cancer growth [J]. Biochem Biophys Res Commun, 2022, 600: 67-74.\u003c/li\u003e\n\u003cli\u003eSi W, Liang H, Bugno J, Xu Q, Ding X, Yang K, Fu Y, Weichselbaum RR, Zhao X, Wang L. \u003cem\u003eLactobacillus rhamnosus GG\u003c/em\u003e induces CGAS/sting- dependent type I interferon and improves response to immune checkpoint blockade [J]. Gut, 2022, 71(3): 521-533.\u003c/li\u003e\n\u003cli\u003eWang T, Zhang L, Wang P, Liu Y, Wang G, Shan Y, Yi Y, Zhou Y, Liu B, Wang X, L\u0026uuml; X. \u003cem\u003eLactobacillus\u003c/em\u003e coryniformis MXJ32 administration ameliorates azoxymethane/dextran sulfate sodium-induced colitis-associated colorectal cancer via reshaping intestinal microenvironment and alleviating inflammatory response [J]. Eur J Nutr, 2022, 61(1): 85-99.\u003c/li\u003e\n\u003cli\u003eXu XJ, Liu L, Yao SK. Nerve growth factor and diarrhea-predominant irritable bowel syndrome (IBS-D): A potential therapeutic target? [J]. J Zhejiang Univ Sci B, 2016, 17(1): 1-9.\u003c/li\u003e\n\u003cli\u003eYuan R, Bhattacharya N, Kenkel JA, Shen J, DiMaio MA, Bagchi S, Prestwood TR, Habtezion A, Engleman EG. Enteric glia play a critical role in promoting the development of colorectal cancer [J]. Front Oncol, 2020, 10: 595892.\u003c/li\u003e\n\u003cli\u003eAlbillos A, de Gottardi A, Rescigno M. The gut-liver axis in liver disease: Pathophysiological basis for therapy [J]. J Hepatol, 2020, 72(3): 558-577.\u003c/li\u003e\n\u003cli\u003eGarrido MP, Salvatierra R, Valenzuela-Valderrama M, Vallejos C, Bruneau N, Hern\u0026aacute;ndez A, Vega M, Selman A, Quest AFG, Romero C. Metformin reduces NGF-induced tumour promoter effects in epithelial ovarian cancer cells [J]. Pharmaceuticals 2020, 13(10): 315-325.\u003c/li\u003e\n\u003cli\u003eRomon R, Adriaenssens E, Lagadec C, Germain E, Hondermarck H, Le Bourhis X. Nerve growth factor promotes breast cancer angiogenesis by activating multiple pathways [J]. Mol Cancer, 2010, 9: 157-168.\u003c/li\u003e\n\u003cli\u003eRigault E, Lacas B, Glehen O, Smith D, Dupont-Bierre E, Guimbaud R, Malka D, Boige V, Fuerea A, Pignon JP, Ducreux M. Intra-arterial hepatic bevacizumab and systemic chemotherapy in hepatic metastasis of colorectal cancer: A phase II multicentric trial in second-line treatment [J]. Cancer Treat Res Commun, 2023, 34: 100674.\u003c/li\u003e\n\u003cli\u003eKim J, Takeuchi H, Lam ST, Turner RR, Wang HJ, Kuo C, Foshag L, Bilchik AJ, Hoon DS. Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk for recurrence and for poor survival [J]. J Clin Oncol, 2005, 23(12): 2744-2753.\u003c/li\u003e\n\u003cli\u003eZeng X, Ward SE, Zhou J, Cheng ASL. Liver immune microenvironment and metastasis from colorectal cancer-pathogenesis and therapeutic perspectives [J]. Cancers, 2021, 13(10):2418-2430.\u003c/li\u003e\n\u003cli\u003eDixon LJ, Barnes M, Tang H, Pritchard MT, Nagy LE. Kupffer cells in the liver [J]. Compr Physiol, 2013, 3(2): 785-797.\u003c/li\u003e\n\u003cli\u003eMalaguarnera G, Giordano M, Nunnari G, Bertino G, Malaguarnera M. Gut microbiota in alcoholic liver disease: Pathogenetic role and therapeutic perspectives [J]. World J Gastroenterol, 2014, 20(44): 16639-16648.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer's disease, Colorectal cancer, Liver metastasis, Brain-gut-liver axis","lastPublishedDoi":"10.21203/rs.3.rs-4300147/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4300147/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and Objective:\u003c/strong\u003e To investigate the occurrence of colorectal cancer (CRC) liver metastasis (CRLM) and the risk factors in mice with Alzheimer's disease (AD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethods:\u003c/strong\u003e Mice in the AD group (APP/PS1 models of AD) and the control (CON) group (wild-type C57BL/6J mice) were injected with MC38 cells to establish CRLM models. After the experiment, the tumor nodules on liver surface were counted, and the liver weight, volume were measured. 16S rDNA sequencing, enzyme-linked immunosorbent assay, Pearson’s analysis and immunohistochemical technique were showed to investigate the impact of AD on CRLM and its possible mechanism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Compared to the CON group, the AD group exhibited a increase in the number of tumor nodules on the liver surface, with consistent findings in both liver weight and volume measurements correlating with the metastatic count. Analysis of 16S rDNA sequencing revealed distinct alterations in the intestinal microbiota of the AD group. Furthermore, relative to the CON group, the AD group exhibited notably elevated levels of NGF expression in both the colon and liver. Additionally, discernibly elevated concentrations of VEGF and CXCL12 were observed in both serum and liver tissues of the AD group compared to the CON group. The results of Pearson correlation analysis indicated positive correlations between intestinal NGF levels and both hepatic CXCL12 and VEGF levels. The AD group had smaller number of hepatic KCs than that in the CON group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e AD accelerates CRLM. The mechanism may be caused by gut flora affecting hepatic KCs, thus linking the brain-gut-liver axis.\u003c/p\u003e","manuscriptTitle":"The risk factors for colorectal cancer liver metastasis in a mouse model of Alzheimer's disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-24 14:01:45","doi":"10.21203/rs.3.rs-4300147/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8531a0ca-98c3-4fa1-8ffb-946a294e7372","owner":[],"postedDate":"April 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-14T08:26:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-24 14:01:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4300147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4300147","identity":"rs-4300147","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}