Mechanism for promoting colorectal cancer by Streptococcus gallyticus metabolite through macrophage polarization and IL-17 pathway and application of Streptococcus gallyticus metabolite

Abstract

The invention discloses a mechanism for promoting colorectal cancer through macrophage polarization and an IL-17 (interleukin-17) pathway by Streptococcus gallyticus streptococcus metabolite and application of the Streptococcus gallyticus streptococcus metabolite, and relates to the technical field of microorganism-tumor immunoregulation. According to the invention, specific metabolites, namely inosine-5 '-monophosphate (IMP), methionine, uridine and creatine, in a Streptococcus gallyticus supernatant promote CRC (cyclic redundancy check) through the following two paths: M2 polarization of macrophages: TAM infiltration of tumor-related macrophages is remarkably increased, an immunosuppressive microenvironment is induced, and tumor growth is promoted; and IL-17 signal channel activation: RNA-seq shows that the IL-17 channel is the most significant enrichment channel, the expression of IL-17F and IL-22 is specifically up-regulated, and inflammation and tumor progression are promoted. The invention reveals that the Sg metabolite is a key driving factor of CRC instead of bacteria for the first time, reveals a mechanism that the Sg metabolite activates and promotes colorectal cancer through macrophage M2 polarization and IL-17 pathway, provides an application value of the Sg metabolite in CRC diagnostic marker screening and targeted therapeutic drug development, and provides a new idea for CRC diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial-tumor immune regulation, and specifically to the mechanism and application of Streptococcus gallolyticus metabolites in promoting colorectal cancer through macrophage polarization and IL-17 pathway. Background Art

[0002] Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths worldwide. People with inflammatory bowel disease (IBD) have a 2-3 times higher risk of developing CRC than those without IBD. Recent studies have shown that the gut microbiome plays a key role in the progression from colitis to CRC. Among the various bacterial species present in the intestine, certain species, such as Streptococcus gallolyticus (Sg), have been found to be associated with CRC.

[0003] Sg is a Gram-positive bacterium that inhabits the human gastrointestinal tract as a commensal organism. The association between Sg and CRC was first identified in 1951. Initially, it was observed that individuals infected with Sg had a significantly increased risk of developing CRC in the following years, despite the absence of any intestinal symptoms during that time. A comprehensive 12-year study showed that 75% of patients with Sg infective endocarditis subsequently developed colonic malignancies, while a 40-year literature review revealed that 60% of patients with Sg infection developed colorectal adenomas or cancer. Another independent 24-year study demonstrated a significantly higher incidence of colon tumors in patients with Sg bacteremia (70%) compared with controls (32%). Furthermore, studies have shown a significant increase in the detection rate of Sg in the fecal tissues of patients diagnosed with CRC. Specifically, the prevalence of Sg fecal carriage in patients with CRC was approximately fivefold and threefold higher compared with patients with IBD and healthy controls, respectively. Furthermore, the presence of Sg in tumorous colon tissue was approximately ten times higher than in normal colon tissue. Interestingly, approximately 74% of tumor tissues and 47% of adjacent normal tissues from CRC patients tested positive for Sg. Furthermore, another investigation determined that Sg was detected in the intestines of 99 healthy volunteers in 62.5%. Taken together, these studies strongly suggest a complex link between Sg and CRC, making Sg a potential pathogenic bacterium in the development of CRC. Furthermore, the seroprevalence of Sg-specific IgG antibodies was 68% in patients with CRC, 78% in patients with adenomas, and 16.66% in healthy controls. This observation suggests that the prevalence of Sg is higher in early-stage adenomas than in advanced cancers, implicating Sg in the early stages of colorectal carcinogenesis.

[0004] Although the exact mechanism of Sg affecting CRC is not clear, it is speculated that Sg culture supernatant contains specific molecular substances that can actively promote the development of CRC. Bacteria can secrete a variety of substances, including growth factors, proteases, cytokines, various other proteins and a variety of metabolites, which promote complex interactions between the host immune system and cancer cells. Certain metabolites, such as lactic acid, provide nutrients for cancer cells and promote cancer progression, while others, such as butyric acid, inhibit pro-inflammatory genes and hinder tumor growth. There are a variety of components in the supernatant of Sg, such as proteins, lipids and metabolites, which have important potential for the development of CRC. There is controversy in the current study about the potential of Sg supernatant to promote CRC cell proliferation. Some studies suggest that direct contact between Sg and cells is a necessary condition for its effectiveness, while others believe that Sg supernatant contains specific substances that can promote the development of CRC.

[0005] The mechanism of Sg promoting cancer is unknown, and previous studies have focused on controversy between "bacterial direct contact" or "supernatant component effect", lacking systematic research on Sg metabolites regulating tumor immune microenvironment. Therefore, we propose that Streptococcus gallolyticus metabolites promote colorectal cancer through macrophage polarization and IL-17 pathway mechanism and application.

[0006] The above information disclosed in the background section is only intended to increase an understanding of the background of the present application and can include prior art known to those of ordinary skill in the art. SUMMARY

[0007] The purpose of the present application is to provide Streptococcus gallolyticus metabolites promote colorectal cancer through macrophage polarization and IL-17 pathway mechanism and application to solve the problems raised in the background art.

[0008] To achieve the above purpose, the present application provides the following technical solution: Streptococcus gallolyticus metabolites promote colorectal cancer through macrophage polarization and IL-17 pathway mechanism, and the specific metabolites in the supernatant of Streptococcus gallolyticus, inosine-5'-monophosphate (IMP), methionine, uridine and creatine, promote CRC through the following double pathways:

[0009] M2 polarization of macrophages: significantly increase tumor-associated macrophage TAM infiltration, induce immune suppressive microenvironment, and promote tumor growth;

[0010] Activation of the IL-17 signaling pathway: RNA-seq showed that the IL-17 pathway was the most significantly enriched pathway, specifically upregulating the expression of IL-17F and IL-22, promoting inflammation and tumor progression.

[0011] The present invention also provides the use of Streptococcus gallolyticus metabolites in the screening of CRC diagnostic markers and the development of targeted therapeutic drugs.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] This invention reveals for the first time that Sg metabolites are the key driving factors of CRC, rather than the bacteria themselves. It also reveals the mechanism by which Sg metabolites promote colorectal cancer through macrophage M2 polarization and IL-17 pathway activation, and proposes the application value of Sg metabolites in the screening of CRC diagnostic markers and the development of targeted therapeutic drugs, providing new ideas for the diagnosis and treatment of CRC.

[0014] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Analysis of the effects of Sg abundance and Sg supernatant on colon cancer cells: (a) Sg abundance in tumor tissues was significantly higher than that in normal adjacent tissues; (b) Sg supernatant promoted the proliferation of HCT116 and HT29 cells; (c) Volcano plot of differential metabolites between BHI medium and Sg supernatant; (dg) The levels of inosine-5'-monophosphate (IMP), methionine, uridine, and creatine in Sg supernatant were significantly higher than those in blank medium; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant;

[0016] Figure 2 : Analysis of the effect of Sg supernatant on tumor growth and macroscopic parameters of AOM / DSS mice: (a) Colonoscopy of mice; (b) Colon tumor status of mice; (c) Body weight of mice in the first week of DSS administration; (d) Different degrees of tumors in mice induced by AOM / DSS; (e) Number of colon tumors in mice; (f) Colon tumor burden in mice; (gh) Length of the colon of mice; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant;

[0017] Figure 3: Sg supernatant aggravates the inflammation level in AOM / DSS mice: (a) HE pathological assessment; (b) DAI score on the day when mice significantly lost weight; (c) HE pathological inflammation score; (dg) PTGS2 (d), IL-6 (e), IL-1β (f), and IL-8 (g) expression levels in mouse colon tumor tissues were detected by qPCR; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant;

[0018] Figure 4 : Sg supernatant intervention increases TAM infiltration and tends to M2 polarization in mouse colon tumor tissue: (a) Immunohistochemistry of mouse colon (x100); (b) Flow cytometric analysis of mouse colon tumor tissue; (ce) Statistical analysis of macrophages (c), M1-like macrophages (d), and M2-like macrophages (e) in TAMs; (f) M1 / M2 ratio analysis; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant;

[0019] Figure 5 : The mechanism of action of Sg supernatant in promoting CRC was revealed to be related to the IL17 pathway: (a) volcano plot of differentially expressed genes; (b) cluster heat map of differentially expressed genes; (c) KEGG pathway enrichment analysis of differentially expressed genes; (d) expression level of IL-17A in mouse colon tumor tissues; (e) expression level of IL-17F in mouse colon tumor tissues; (f) expression level of IL-22 in mouse colon tumor tissues; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant. DETAILED DESCRIPTION

[0020] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0021] 1. Verification of Metabolites’ Cancer-Promoting Effects

[0022] 1. Clinical data and specimen collection

[0023] This study involved 46 patients with colorectal cancer (CRC) who underwent surgical treatment at Guangzhou First People's Hospital between 2012 and 2015. Specimen collection strictly adhered to aseptic principles. Tumor tissue and adjacent tissue (>5 cm from the tumor edge) from each patient were collected and immediately frozen in liquid nitrogen for rapid preservation. Subsequently, the specimens were transferred to a -80°C freezer for storage. Patient information, such as age, sex, blood type, tumor size, location, stage, smoking history, alcohol history, 5-year survival rate, metastasis status, and pathological indicators, was collected through the medical record system and telephone follow-up.

[0024] 2. Quantitative real-time PCR (qPCR) of Sg abundance

[0025] Genomic DNA (gDNA) was extracted from tissue samples using the Total DNA / RNA / Protein Kit (Omega, USA). A 20 μL reaction solution was prepared, containing 10 μL SYBR Premix Ex Taq II (2X), 1 μL PCR forward primer (10 μM), 1 μL PCR reverse primer (10 μM), 2 μL gDNA solution, and 6 μL ddH2O. The qPCR protocol was as follows: A. Initial denaturation: 95°C for 30 seconds; B. PCR reaction: 40 cycles of 95°C for 5 seconds and 60°C for 30–60 seconds.

[0026] The specific primer sequences of Sg: Forward: 5'-AACGCGAAGAACCTTACCAG-3', Reverse: 5'-GAGTGCCCAACTGAATGAT G-3'.

[0027] 3. Quantitative reverse transcription PCR (RT-qPCR)

[0028] Mouse colon tissue was collected for RT-qPCR analysis. Total RNA was extracted using the Total DNA / RNA / Protein Kit (Omega, USA). RNA was converted into cDNA using the PrimeScript RT Reagent Kit (Takara, Japan) for reverse transcription. The qPCR steps were as follows: Step 1, pre-denaturation at 95°C for 30 seconds. Steps 2 and 3, denaturation at 95°C for 15 seconds, annealing at 60°C for 1 minute, and 40 cycles. Step 4, rapid denaturation at 95°C for 1 second. Step 5, melting step for 6 seconds. The 20 μL reaction mixture contained SYBR Premix Ex Taq II (2X), PCR forward primer (10 uM), PCR reverse primer (10 uM), cDNA solution, and dH2O. ACTB was used as an internal control. Relative gene expression was calculated using the 2-ΔΔCT method. The primer sequences are shown in the table below.

[0029]

[0030] 4. Cell culture

[0031] HCT116 and HT29 cells were purchased from ATCC and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Cells were maintained in a humidified incubator at 37°C and 5% CO2 to ensure optimal growth conditions. The medium was changed every 48 hours to maintain nutrient levels and remove metabolic waste. When the cell confluence reached 70%-80%, 0.25% trypsin-EDTA was used for passage to ensure continued proliferation and viability for subsequent experiments.

[0032] 5. Preparation of Sg supernatant

[0033] The Sg strain (DSM16831) was obtained from Beina Biotechnology (BNCC). Upon receipt, the strain was aerobically revived and cultured in brain heart infusion (BHI) medium (Huakai Microbial Technology Co., Ltd., China) at 37°C. When the bacteria reached the logarithmic growth phase, the suspension was centrifuged at 3000 rpm for 5 minutes to obtain the bacterial culture supernatant. The supernatant was then filtered through a 0.22 μm membrane to ensure sterility and verify the absence of viable bacteria. The filtered supernatant was confirmed to be free of viable bacteria by inoculation culture. The supernatant was freshly prepared before each experiment.

[0034] 6. Cell Proliferation Assay

[0035] Cell proliferation was determined using a cell counting kit-8 (CCK-8) (Biosharp, China), and 1000 cells and 100 μL of cell suspension were added to each well of a 96-well plate. After the cells adhered to the bottom, 10 μL of Sg supernatant, BHI medium, or PBS was added to the wells, respectively. After 8 hours of culture, 10 μL of CCK8 solution was added to each well and incubated for 1 hour in the dark. The absorbance value OD450 was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). At least 3 replicate wells were performed for each intervention. The blank control group refers to the absorbance value of the well containing only culture medium but no cells.

[0036] 7. Non-targeted metabolomics

[0037] Untargeted metabolomics analysis was performed on Sg supernatants and BHI blank medium, and sequencing was performed by Novogene Technology Co., Ltd. (Beijing, China). A 1 mL sample was freeze-dried, mixed with 100 μL of 80% methanol, vortexed, incubated on ice for 5 minutes, and then centrifuged at 15,000 × g for 15 minutes at 4°C. The supernatant was diluted to 53% methanol, centrifuged again, and collected for LC-MS analysis. LC-MS was performed using a Hypersil GOLD C18 column at 40°C at a flow rate of 0.2 mL / min. In positive mode, the mobile phases were (A) 0.1% formic acid in water and (B) methanol; in negative mode, (A) 5 mM ammonium acetate (pH 9.0) and (B) methanol. The gradient elution was from 2% to 100% B over 10 minutes, then returned to 2% B over 2 minutes and held at 100% B. The mass spectrometer scan range was m / z 100–1500, and the ESI source parameters were set to 3.5 kV spray voltage, 35 psi sheath gas flow, 10 L / min auxiliary gas flow, 320°C capillary temperature, 60 S-lens RF level, and 350°C auxiliary gas heater temperature. Raw data were processed using Compound Discoverer 3.1 (CD3.1), and peak extraction used a retention time tolerance of 0.2 min, a mass tolerance of 5 ppm, a signal intensity tolerance of 30%, an S / N ratio of 3, and a minimum intensity threshold. Metabolite identification was based on matching molecular ions and fragments with the mzCloud, mzVault, and MassList databases, excluding data with a coefficient of variation (CV) greater than 30% in quality control samples. Data processing was performed using R and Python on CentOS 6.6, and differential metabolites were identified using thresholds of VIP > 1.0, fold change (FC) > 1.2 or < 0.833, and p-value < 0.05.

[0038] 7. Animal Modeling

[0039] Eight-week-old female Balb / c mice (approximately 25 g) were purchased from the Guangdong Medical Laboratory Animal Center and raised under specific pathogen-free (SPF) conditions. After a one-week adaptation period, an azocyclopentane / dextran sodium sulfate (AOM / DSS) model of colorectal cancer (CRC) was established. Mice received an intraperitoneal injection of 12.5 mg / kg AOM (Sigma-Aldrich, USA) on the first day, followed by the addition of 2% DSS (MP Biomedicals, USA) to drinking water for 5 days, followed by normal drinking water for 14 days; this cycle was repeated three times. The experiment ended on day 80. During this process, 200 μL of intervention was administered by oral gavage every other day, and the water was changed three times a week to keep it fresh. All experimental protocols were approved by the relevant ethics committees and followed established guidelines to reduce suffering, ensure humane treatment and strict monitoring throughout the study.

[0040] 8. Mouse colonoscopy

[0041] An endoscope for mice (KARL STORZ, 26430520-1, Germany) was used, equipped with an IMAGE1SD3-LINK connection module and a 64301AA probe (diameter: 1.9 mm, length: 10 cm), a 495NT optical fiber and a protective probe cover (61029C) with an 8.5 cm working length. The camera system was connected to a display screen for real-time imaging and photo recording. Mice were fasted for 24 hours before surgery, anesthetized with 1% sodium pentobarbital by intraperitoneal injection, and placed on a heating pad to maintain body temperature. The anus was lubricated with glycerol, the endoscope was carefully inserted, and the endoscope tip was coated to reduce trauma to the mucosa. The position of the endoscope was adjusted to ensure clear visualization of the colonic mucosa and to record images of the intestinal lumen. After the examination was completed, the endoscope was gently removed and the perianal area was cleaned. The mice were closely monitored for signs of pain or complications. All procedures were performed by experienced operators to ensure the accuracy and consistency of the results.

[0042] 9. Flow cytometry

[0043] The colon was cut open longitudinally and rinsed thoroughly with cold PBS. Tumor tissue in the colon was carefully collected and digested with a digestive enzyme solution for 30 minutes on a shaker at 37°C. The enzyme solution consisted of 0.5 mg / mL collagenase IV (Roche, Switzerland) and 0.5 mg / mL DNaseI (Roche, Switzerland), both dissolved in PBS without calcium and magnesium. The digested cell suspension was filtered through a 70 μm cell sieve and resuspended in 1% bovine serum albumin. A single-cell suspension was prepared by centrifugation at 400 × g for 10 minutes at 4°C and then resuspended in 1% bovine serum albumin. Antibody labeling was performed by adding 1 μL of fluorescent antibodies, including APC anti-mouse CD45 (BioLegend, 103111, USA), APC / Cy7 anti-mouse CD11b (BioLegend, 101226, USA), PE anti-mouse F4 / 80 (BioLegend, 123109, USA), FITC anti-mouse CD86 (BioLegend, 105005, USA) and BV421 anti-mouse CD206 (BioLegend, 141717, USA). The mixture was incubated at 4 ° C in the dark for 30 minutes, and the reaction was terminated by adding 1% BSA, followed by centrifugation and resuspending in PBS. The stained cells were then analyzed by flow cytometry BD FACS Canto II (BD Biotechnology, USA), and the data were processed using FlowJo software.

[0044] 10. Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) staining

[0045] The colon was isolated and cleaned, then embedded in paraffin using a Swiss roll technique and cut into 4-μm-thick sections. HE staining and IHC staining were performed as previously described. Microscopic observations were performed, and image analysis was performed using ImageJ. Blinded histological scoring was performed to assess modeling success. Inflammation was assessed using the MCHI scoring system.

[0046] 11. RNA Sequencing

[0047] Transcriptome analysis of mouse tumor tissue was performed using RNA sequencing (RNA-seq) technology from Suzhou PANOMIX Biomedical Technology Co., Ltd. RNA was first extracted and tested for concentration and purity. mRNA was purified and fragmented into 200-300 bp fragments. Double-stranded cDNA was then synthesized, and sequencing libraries were constructed and amplified. Libraries of approximately 450 bp were selected. After library quality control, paired-end sequencing was performed using the Illumina HiSeq platform. The raw sequencing data were initially filtered to obtain clean data. A reference genome index was constructed using Bowtie2, and clean reads were aligned to the reference genome using Tophat2. Read counts for each gene were calculated using HTSeq, and expression was normalized using FPKM. Differentially expressed genes (DEGs) were analyzed using DESeq, with a |log2FoldChange| > 1 and a P value < 0.05. Subsequently, functional enrichment analysis of the GO and KEGG pathways was performed to identify biological processes, molecular functions, and signaling pathways associated with the DEGs. Data visualization using the R package ggplot2 was performed for heatmaps, volcano plots, and bubble plots.

[0048] 12. Statistical methods

[0049] Data were analyzed using SPSS 25.0 software (IBM Corporation, USA). Descriptive statistics are presented as mean ± standard deviation (±s). Two normally distributed groups were compared using the independent sample t-test, while non-normally distributed data were compared using the Wilcoxon signed-rank test. Multiple group comparisons were performed using one-way analysis of variance. Categorical variables were analyzed using the chi-square test, and P < 0.05 was considered statistically significant. Data analysis and visualization were performed using GraphPad Prism 8 (GraphPad Software, USA) and Adobe Illustrator 2020 (Adobe Inc., USA).

[0050] 2. Results

[0051] 1. High abundance of Sg was found in CRC tissues by qPCR and was associated with tumor location

[0052] The demographic and clinical characteristics of the patients are shown in Table 1. Sg was detected in 93.5% (43 of 46) of CRC tissues (including tumors and adjacent normal tissues), and only 3 patients (6.5%) had no Sg colonization. Notably, the abundance of Sg in tumor tissues was significantly higher than that in adjacent normal tissues (P < 0.01) ( Figure 1 a). Based on the ΔCT value, the samples were divided into two groups: Sg-positive (high abundance) and Sg-negative (low abundance or negative). In tumor tissues, 22 cases (47.8%) were Sg-positive and 24 cases were negative. In normal tissues adjacent to the tumor, 14 cases (30.4%) were Sg-positive and 32 cases were negative. The chi-square test results showed a significant correlation between Sg abundance in tumors and adjacent normal tissues (P<0.001), as well as a correlation with tumor location (P<0.01). However, no significant association was observed between Sg abundance and other factors such as age, sex, blood type, tumor size, stage, smoking, alcohol consumption, CEA, P53, Ki67 positivity rate, five-year survival rate, or recurrence (P>0.05) (Table 2).

[0053] Table 1. Characteristics of the study population

[0054]

[0055]

[0056] Table 2. Correlation analysis between Sg abundance and clinical information in tumor and peritumoral tissues of CRC patients

[0057]

[0058]

[0059] Note:P<0.05 is considered statistically significant.

[0060] 2. Analysis of the effect of Sg supernatant on CRC cell proliferation and its components

[0061] To explore the potential pathogenic effect of Sg on CRC, the supernatant of Sg culture was extracted for further study. Sg supernatant was added to HCT116 and HT29 cells to evaluate its effect on cell proliferation. The results showed that neither the PBS group nor the BHI (Sg culture medium) group had a significant effect on the proliferation of HCT116 and HT29 cells ( Figure 1 b). However, the proliferation of HCT116 and HT29 cells was significantly increased after administration of Sg supernatant compared with the PBS control group ( Figure 1b). In addition, non-targeted metabolomics analysis of the supernatant showed significant changes in metabolite levels. Specifically, the Sg supernatant contained 851 metabolites that were significantly different from the blank medium, of which 67 metabolites showed upregulation and 13 showed downregulation ( Figure 1 c). Notably, the levels of inosine-5'-monophosphate (IMP), methionine, uridine, and creatine in the Sg supernatant were significantly higher than those in the blank medium ( Figure 1 dg).

[0062] 3. Sg supernatant promotes colon tumor progression, aggravates inflammation, and promotes M2 polarization of macrophages in AOM / DSS mice

[0063] The growth of colon tumors was observed by mouse endoscopy. Healthy mice showed smooth round areas, pale pink intestinal mucosa, normal vascular structure, and a smooth and transparent mucosal surface. In contrast, AOM / DSS-induced CRC mice showed intestinal masses of varying sizes, decreased intestinal wall transparency, and loss of normal vascular structure ( Figure 2 a). At the end of the study, the colons of mice were dissected and it was found that all groups except the normal group had masses (confirmed as tumors by HE staining), and the number and size of colon tumors in the Sg supernatant group were significantly increased ( Figure 2 b). Statistical analysis showed that the number of tumors increased significantly after Sg supernatant intervention ( Figure 2 e) and tumor burden ( Figure 2 f) Compared with the model group, the body weight of mice in the normal group increased steadily, while that in the model group decreased significantly. The Sg supernatant group lost a significant amount of weight in the first week of DSS administration ( Figure 2 c). Colon length was significantly shortened after Sg supernatant intervention ( Figure 2 g and Figure 2 h). HE staining showed that the normal mouse colon was composed of mucosa, submucosa, muscle layer and plasma layer. In the normal group, the glands were neatly arranged, the crypts were intact, and there were goblet cells at the base. Mice treated with AOM / DSS showed varying degrees of dysplasia. Mild dysplasia was characterized by glandular disorder, cell heterogeneity and dark-stained nuclei. Low-grade heterogeneous hyperplasia (early adenoma) showed dark-stained cells with pseudo-complex nuclei and no involvement of deeper glands. High-grade heterogeneous hyperplasia (carcinoma in situ) showed cribriform or fused crypts with complete loss of nuclear polarity. Invasive cancer showed that cancer cells crossed the basement membrane and invaded the subbasement membrane ( Figure 2 a). Regarding inflammatory status, the DAI score on the day when weight loss was most significant showed that the Sg supernatant group had a significantly higher score ( Figure 3 b). In the normal mouse group, the colonic epithelial structure remained intact, with no ulcers and submucosal inflammatory cell infiltration. However, the model group showed an increase in chronic inflammatory cells and lymphoid follicle hyperplasia. The Sg supernatant group showed irregular submucosal crypts and visible ulcers (Figure 3 a). Subsequent histological inflammation scores showed that the degree of inflammation in the Sg supernatant group was increased compared with the model group ( Figure 3 c). In addition, RT-qPCR analysis of mouse colon tumor tissues showed that inflammatory factors, namely PTGS2 ( Figure 3 d) IL-6 ( Figure 3 e), IL-1β ( Figure 3 f) and IL-8 ( Figure 3 g), compared with the model group.

[0064] Immunohistochemical staining showed that the expression of macrophage marker F4 / 80 in tumors increased after Sg supernatant intervention compared with the model group. It is worth noting that the expression of M2 polarization marker CD206 increased significantly, while the expression of M1 polarization marker CD86 was relatively low ( Figure 4 a). These findings were further confirmed by additional analysis by flow cytometry, showing that Sg supernatant intervention led to an increase in the proportion of macrophages in mouse tumors, promoted M2 polarization, and subsequently decreased the M1 / M2 ratio, compared with the model group ( Figure 4 b- Figure 4 f).

[0065] 4. The mechanism by which Sg supernatant promotes CRC is revealed to be related to the IL17 pathway

[0066] To investigate the specific ways in which Sg supernatant promotes CRC development, RNA-seq sequencing analysis was performed on tumor tissues obtained from mice. The results showed that 68 genes were upregulated and 47 genes were downregulated compared with the model group ( Figure 5 a and Figure 5 b). In addition, KEGG pathway enrichment analysis indicated that the IL-17 signaling pathway was the most significantly enriched ( Figure 5 c). To further validate the RNA-seq data, the expression levels of IL-17 pathway-related factors were evaluated. These evaluations showed no statistically significant differences in the expression levels of IL-17A among the three groups of mice ( Figure 5 d). However, after Sg supernatant intervention, IL-17F ( Figure 5 e) and IL-22( Figure 5 f) The expression of WT and WT groups was significantly increased compared with the model group.

[0067] In summary, these results indicate that Sg metabolites are key drivers of CRC and reveal the mechanism by which Sg metabolites promote colorectal cancer through macrophage M2 polarization and IL-17 pathway activation, which provides new insights into the diagnosis and treatment of CRC.

[0068] Diagnostic application: Metabolite combination markers: Detection of IMP, methionine, uridine, and creatine concentrations in biological samples (serum, feces, and tissues). Concentrations above the threshold indicate CRC risk; gene expression markers: Combined detection of CD206, IL-17F, and IL-22 expression levels for early CRC screening.

[0069] Therapeutic applications: Development of targeted inhibitors: targeting Sg metabolites or their downstream pathways such as IL-17F / IL-22 receptor antagonists, M2 polarization blockers; probiotic / antibiotic therapy: selectively clearing intestinal Sg to reduce the level of carcinogenic metabolites.

[0070] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present invention. Any changes, modifications, replacements and variations of the above embodiments by ordinary technicians in this field within the scope of the present invention should be included in the scope of protection of the present invention.

Claims

1. The mechanism by which Streptococcus gallolyticus metabolites promote colorectal cancer through macrophage polarization and the IL-17 pathway is characterized by: The specific metabolites inosine-5'-monophosphate (IMP), methionine, uridine, and creatine in the supernatant of Streptococcus gallolyticus promote CRC through the following two pathways: Macrophage M2 polarization: significantly increases tumor-associated macrophage (TAM) infiltration, induces an immunosuppressive microenvironment, and promotes tumor growth; Activation of the IL-17 signaling pathway: RNA-seq showed that the IL-17 pathway was the most significantly enriched pathway, specifically upregulating the expression of IL-17F and IL-22, promoting inflammation and tumor progression.

2. Application of Streptococcus gallolyticus metabolites in the screening of CRC diagnostic markers and the development of targeted therapeutic drugs.

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