Use of campylobacter jejuni and its toxin in preparation of drugs for preventing, diagnosing, predicting, treating colorectal cancer and extra-intestinal tumor metastasis

Abstract

The use of Campylobacter jejuni and its toxin in the preparation of drugs for preventing, diagnosing, predicting, treating colorectal cancer and extra-intestinal tumor metastasis. Campylobacter, especially Campylobacter jejuni and toxin CDT, can be used as a marker to distinguish whether a colorectal cancer patient has metastasis. Experiments have proved that Campylobacter jejuni has the ability to promote metastasis and invasion. Campylobacter jejuni promotes the invasion and metastasis of colon cancer cells by CDT protein and the up-regulated JAK2 / STAT3 / MMP9 signaling pathway activated by CDT protein. When the process is blocked, the tumor metastasis-promoting ability of Campylobacter jejuni is weakened or lost. Campylobacter jejuni also promotes the metastasis of extra-intestinal tumors. Based on drugs or agents that can block this process, the metastasis of colorectal cancer cells and extra-intestinal tumors can be prevented and inhibited. Based on Campylobacter jejuni and its toxin, corresponding reagents or methods are developed for diagnosing or predicting the metastasis of colorectal cancer and extra-intestinal tumors. Through the marker provided in the present application, it is expected to inhibit tumor metastasis and prolong the survival of patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of Campylobacter jejuni and its toxins in the preparation of drugs for the prevention, diagnosis, prediction, and treatment of colorectal cancer and extraintestinal tumor metastasis. Background Technology

[0002] Colorectal cancer (CRC) is the second leading cause of cancer death, with distant metastasis being a major cause of treatment failure and cancer-related death. Approximately one-quarter of CRC patients develop metastatic disease, and 20% experience distant metastasis during their illness, severely impacting prognosis. Currently, there are no effective methods for predicting, diagnosing, or inhibiting colorectal cancer metastasis; therefore, a corresponding and effective prediction, diagnosis, or inhibition strategy is urgently needed. Furthermore, the mechanisms of tumor development and metastasis may differ, and even if existing technologies provide corresponding diagnostic and therapeutic targets for colorectal cancer development and tumor growth, they may not be applicable to the metastatic process.

[0003] In recent years, the role of intratumoral bacteria in tumor development, progression, and prognosis has received increasing attention, and they also play a crucial role in tumor metastasis. For example, *Bacteroides fragilis* appears in breast cancer and stimulates metastasis by secreting *Bacteroides fragilis* toxin (BFT). *Fusobacterium nucleatum* (F. nucleatum) colonizes breast cancer through the lectin Fap2 and attaches to the Gal-GalNAc displayed by tumor cells, thereby promoting tumor proliferation and metastasis. Based on this, this application aims to study microorganisms associated with colorectal cancer metastasis and their pro-metastatic pathways, thereby providing strategies for the prevention, diagnosis, and inhibition of colorectal cancer metastasis based on these microorganisms and related pathways, thus filling the gap in existing technologies regarding the lack of effective diagnostic, inhibitory, and therapeutic methods for colorectal cancer metastasis. Summary of the Invention

[0004] This invention aims to overcome at least one deficiency of the prior art and provide a drug for the preparation of drugs for the prevention, diagnosis, prediction, and treatment of colorectal cancer and extraintestinal tumor metastasis. It is expected to enable timely diagnosis of whether CRC patients have metastasized and whether metastasis is likely, allowing for timely implementation of appropriate treatment strategies and promoting CRC treatment. Furthermore, it is expected to provide corresponding drugs to inhibit colorectal cancer metastasis and / or extraintestinal tumor metastasis, improving the treatment efficacy of colorectal cancer and extraintestinal tumors.

[0005] One object of the present invention is to provide a marker for colorectal cancer metastasis, namely, Campylobacter with relatively abnormal abundance in the intestine. More preferably, it is Campylobacter jejuni with abnormally enriched abundance in the intestine.

[0006] Another object of the present invention is to provide the use of a Campylobacter jejuni detection reagent in the preparation of a medicament for diagnosing or prognosing colorectal cancer metastasis. In one or more embodiments of the present invention, the inventors have found that Campylobacter jejuni accumulates in patients with postoperative metastatic colon cancer and that Campylobacter jejuni promotes the invasion and metastasis of colorectal cancer cells; therefore, the Campylobacter jejuni detection reagent can be used to prepare a medicament for diagnosing colorectal cancer metastasis. Further, the medicament includes a reagent-form medicament.

[0007] Another object of the present invention is to provide the use of Campylobacter jejuni antibiotics in the preparation of medicaments for the prevention and treatment of colorectal cancer and / or extraintestinal tumor metastasis. Further, extraintestinal tumors include lung cancer and breast cancer. In one embodiment of the present invention, after blocking CDT expression, the metastatic and invasive effects of Campylobacter jejuni are rendered ineffective. In addition to directly blocking CDT and its downstream pathway nodes as described below, directly killing or inhibiting Campylobacter jejuni expressing CDT protein should also have the effect of preventing and treating colorectal cancer metastasis. Simultaneously, in one embodiment of the present invention, it has also been found that Campylobacter jejuni promotes the invasion and metastasis of extraintestinal tumors through CDT-derived toxins. Therefore, this antibiotic also facilitates the treatment of extraintestinal tumors, including inhibiting extraintestinal tumor metastasis.

[0008] Another object of the present invention is to provide the use of CDT-JAK / STAT / MMP pathway inhibitors in the preparation of drugs for inhibiting colorectal cancer and / or extraintestinal tumor metastasis. In one or more embodiments of the present invention, it is shown that Campylobacter jejuni upregulates JAK / STAT / MMP pathway genes in tumor cells through the action of CDT (especially cdt B), thereby promoting the invasion and metastasis of colorectal cancer cells. Inhibitors that block this pathway (such as JAK2 inhibitors) can weaken or block the pro-metastatic phenomenon caused by the pathway. Therefore, inhibitors at each stage of the process from the completion of CDT assembly in Campylobacter jejuni to its activation and upregulation of the JAK / STAT / MMP pathway can be used to prepare drugs for inhibiting colorectal cancer metastasis. More preferably, the CDT-JAK / STAT / MMP pathway inhibitor is a CDT-JAK2 / STAT3 / MMP9 pathway inhibitor. Similarly, since the CDT-JAK / STAT / MMP pathway inhibitor blocks downstream of the Campylobacter jejuni CDT, it can also be applied to the treatment and metastasis inhibition of extraintestinal tumors.

[0009] Another object of the present invention is to provide the use of CDT blockers and / or JAK / STAT / MMP pathway inhibitors in the preparation of medicaments for inhibiting colorectal cancer and / or extraintestinal tumor metastasis. Specifically, this involves directly blocking CDT in Campylobacter jejuni or inhibiting JAK / STAT / MMP pathway nodes in tumor cells for use in the preparation of medicaments for inhibiting colorectal cancer and / or extraintestinal tumor metastasis.

[0010] Preferably, the drug includes a drug that inhibits the function of Campylobacter jejuni in promoting colorectal cancer and / or extraintestinal tumor metastasis.

[0011] Preferably, the CDT blocking agent includes a CDT antibody and a CDT subunit blocking agent, wherein the CDT subunit blocking agent includes cdtB, cdtA and / or cdtC blocking agents.

[0012] Preferably, the JAK / STAT / MMP pathway inhibitors include JAK2 / STAT3 / MMP9 pathway inhibitors.

[0013] Preferably, the JAK2 / STAT3 / MMP9 pathway inhibitors include JAK2 inhibitors and MMP9 inhibitors.

[0014] Preferably, the JAK2 inhibitor includes AG490; the MMP9 inhibitor includes MMP9 shRNA. The MMP9 shRNA includes shRNA with target sequences target-1 and / or target-2, wherein the sequence of target-1 is 5'-aaGGACGGCAAATTTGGTTTC-3'; and the sequence of target-2 is 5'-gaCCATCATAACATCACATAC-3'. MMP9 shRNA includes shMMP9-1 and / or shMMP9-2, wherein: the sequence of shMMP9-1 is: 5'-CcggaaGGACGGCAAATTTGGTTTCCTCGAGGAAACCAAATTTGCCGTCCTTTTTTTg-3'; 5'-aattcaaaaaaaGGACGGCAAATTTGGTTTCCTCGAGGAAACCAAATTTGCCGTCCTT-3'; the sequence of shMMP9-2 is: 5'-CcgggaCCATCATAACATCACATACCTCGAGGTATGTGATGTTATGATGGTCTTTTTg-3'; 5'-aattcaaaaagaCCATCATAACATCACATACCTCGAGGTATGTGATGTTATGATGGTC-3'.

[0015] Another object of the present invention is to provide a kit comprising a Campylobacter jejuni detection reagent, a Campylobacter jejuni antibiotic reagent, and / or a CDT-JAK / STAT / MMP pathway inhibitor. The CDT-JAK / STAT / MMP pathway inhibitor includes a CDT blocker and / or a JAK / STAT / MMP pathway inhibitor.

[0016] Another object of the present invention is to provide a tumor metastasis inhibitory drug, comprising Campylobacter jejuni antibiotic and / or CDT-JAK / STAT / MMP pathway inhibitors. Tumor metastasis includes colorectal cancer metastasis and / or extraintestinal tumor metastasis. Extraintestinal tumors include lung cancer and breast cancer. More preferably, the aforementioned colorectal cancer metastasis includes postoperative metastasis after colorectal cancer surgery.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The inventors discovered that Campylobacter jejuni colonizes in CRC tissue and has a higher abundance in the primary tumors of patients with postoperative metastasis. In fact, Campylobacter jejuni can serve as a marker to distinguish whether colorectal cancer patients are likely to metastasize. The abundance of Campylobacter jejuni can be detected to achieve a corresponding diagnostic function. In one or more embodiments of the present invention, in vitro and in vivo experiments demonstrated the metastatic and invasive capabilities of Campylobacter jejuni. Campylobacter jejuni promotes the invasion and metastasis of colon cancer cells through CDT protein and its activated and upregulated JAK2 / STAT3 / MMP9 signaling pathway in tumor cells. Furthermore, by blocking CDT, inhibiting CDT subunits, or using inhibitors of nodes in the JAK2 / STAT3 / MMP9 pathway, the pro-invasive and metastatic functions of Campylobacter jejuni are weakened or eliminated, and the metastasis of colorectal cancer cells is inhibited or eliminated. Therefore, providing Campylobacter jejuni antibiotics or CDT-JAK / STAT / MMP9 pathway inhibitors (especially CDT-JAK2 / STAT3 / MMP9 pathway inhibitors) is beneficial for preventing and inhibiting the metastasis of colorectal cancer cells and can be used to prepare corresponding drugs. Through the biomarkers and uses provided in this application, it is expected to inhibit the metastasis of colorectal cancer, prolong patient survival, and improve prognosis. Furthermore, Campylobacter jejuni also promotes extraintestinal tumor metastasis through intestinal colonization and cdtB; the aforementioned inhibitors can also be beneficial for inhibiting and treating extraintestinal tumor metastasis. Attached Figure Description

[0018] Figure 1The presence of Campylobacter spp. in patients with postoperative metastasis of colorectal cancer (CRC) is shown. (A) Shannon diversity index of primary tumor microbiomes compared with those from CRC patients after metastasis (metastasis group) (n=21) and those without metastasis (non-metastasis group) (n=20). (B) Principal coordinate analysis (PCoA) compared the microbial composition of primary tumors between the metastasis and non-metastasis groups from cohort 1. (C) Differentiating taxa (log10 LDA>2.4) determined by LEfSe between the metastasis and non-metastasis groups from cohort 1. (D) Relative abundance of Campylobacter spp. in CRC patients with or without postoperative metastasis (cohort 1). (E) Relative abundance of Campylobacter spp. in CRC patients with (n=11) and without (n=9) postoperative metastasis (cohort 2). (F) Relative abundance of Campylobacter spp. in CRC patients with and without metastasis (cohort 3). (G) Detection of Campylobacter spp. in CRC tissues from patients in the metastasis and non-metastasis groups (cohort 1) by fluorescence in situ hybridization (FISH). CRC tissue sections were stained with DAPI (blue), Campylobacter-specific probe (red dot), and EUB338 (green dot), scale bar = 10 μm. Unpaired Student's t test was used for statistical analysis between the metastatic and non-metastatic groups. Data are expressed as mean ± SEM, *p < 0.05. LDA (linear discriminant analysis) was also performed.

[0019] Figure 2 Campylobacter jejuni promotes cell migration and invasion in vitro. (A and B) Migration or invasion was assessed by co-culturing four colon cancer cell lines (CT26, MC38, HCT116, SW620) with PBS (control), DH5α (MOI: 10), or Campylobacter jejuni (MOI: 10) for 12 hours. Migrating and invading cells were indicated by staining with 33% acetic acid, and the optical density (570 nm) of each filter was measured. Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01, and ***p<0.001. MOI, infection diversity.

[0020] Figure 3Campylobacter jejuni stimulates metastasis of colorectal cancer cells. (A) Schematic diagram of the experimental setup. (B and C) Single-cell suspensions extracted from subcutaneous tumors injected with PBS, DH5α, or Campylobacter jejuni were used for migration or invasion assays, and the optical density (570 nm) of each filter was measured. (D and F) After splenic injection of extracted CT26(D) or MC38(F) single-cell suspensions into SPF-grade BALB / C mice and C57BL / 6 mice, respectively, liver metastasis was monitored using bioluminescence imaging (BLI) and liver weight, as shown in the illustration of a representative enlarged liver. (E and G) After intravenous injection of extracted CT26(E) or MC38(G) single-cell suspensions into SPF BALB / C mice or C57BL / 6 mice, lung metastases were monitored using bioluminescence imaging (BLI) and lung weight, showing a representative fixed lung. Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0021] Figure 4 The metastatic ability of Campylobacter jejuni depends on the functional cdt gene. (A) Schematic diagram of the experimental setup. (B and C) Migration or invasion was measured using single-cell suspensions extracted from subcutaneous tumors that were intratumorally injected with Campylobacter jejuni wild-type (WT), cdtB KO, cdtB complement (cdtB comp), or PBS (control), and the optical density (570 nm) of each filter was measured. (D and E) Lung metastases were monitored using bioluminescence imaging (BLI) and lung weight after intravenous injection of extracted CT26 (D) or MC38 (E) single-cell suspensions into SPF BALB / C or C57BL / 6 mice via tail vein, showing representative fixed lungs. Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0022] Figure 5Recombinant Campylobacter jejuni-CDT toxin promotes colorectal cancer cell metastasis. (A) Schematic diagram of the experimental setup. (B and C) Migration or invasion assays were performed using single-cell suspensions extracted from subcutaneous tumors that were intratumorally injected with recombinant Campylobacter jejuni-CDT toxin (Cdt A+B+C) or PBS (control), and the optical density (570 nm) of each filter was measured. (D and E) Lung metastases were monitored using bioluminescence imaging (BLI) and lung weight analysis after intravenous injection of extracted CT26(D) or MC38(E) single-cell suspensions into SPF BALB / C or C57BL / 6 mice, showing representative fixed lungs. Statistical analysis was performed using an unpaired Students't test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0023] Figure 6Recombinant Campylobacter jejuni-CDT toxin promotes colorectal cancer cell metastasis through the JAK2 / STAT3 / MMP9 signaling pathway. (AB) RNA-seq of colorectal cancer cells stimulated for 72 hours with CDT toxin (CdtA+B+C) (5nM) or solvent. Differentially expressed genes in (A) CT26 or (B) MC38 cells were analyzed by gene set enrichment analysis (GSEA). (C) Western blot detection of the JAK2 / STAT3 pathway in CT26 and MC38 cells after 72 hours of stimulation with CDT toxin (CdtA+B+C) (5nM) or solvent. (DE) Quantitative PCR was used to detect the mRNA expression levels of MMP family molecules in (D) CT26 and (E) MC38 cells after 72 hours of stimulation with Cdt A+C (5nM) or Cdt A+B+C (5nM) solvent. (F) Western blot detection of MMP9 expression in tumor cells (CT26, MC38) stimulated with solvent or Cdt A+B+C. (G) Western blotting was used to detect the expression of JAK2 / STAT3 / MMP9 pathway proteins after treatment with CdtA+B+C3 (5 nM), AG490 (50 μM), and AG490+CdtA+B+C for 24 hours. (H and I) Cdt A+B+C (3 days, 5 nM) was used to stimulate shNT, shMMP9#1, and shMMP9#2CT26 or MC38 cells in vitro to detect cell (H) migration and (I) invasion. (J) Western blotting was used to detect MMP9 in shNT, shMMP9#1, and shMMP9#2CT26 and MC38 cells treated with CDT toxin (CDT A+B+C) (5 nM) or solvent for 72 hours. (KL) A lung metastasis model was established in BALB / C mice by intravenous injection of Cdt A+B+C-stimulated shNT, shMMP9#1, and shMMP9#2CT26 cells via tail vein. Representative lung weights were measured. Statistical analysis was performed using one-way ANOVA combined with the M Tukey multiple comparison test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0024] Figure 7 Intestinal colonization by Campylobacter jejuni accelerates cancer metastasis. (A) Schematic diagram of the experimental model: BALB / C or C57BL / 6 mice were given a mixture of antibiotics for 1 week, followed by subcutaneous injection of CRC cells (1×10⁻⁶) on day 0. 6Tumor cells were orally infected with Campylobacter jejuni WT, ΔcdtB, cdtB Comp, or PBS on days 0 and 7. On day 16, tumor cells were extracted for in vitro and in vivo experiments. (BE) Detection of Campylobacter jejuni (B and D) and the cdtB gene (C and E) by PCR analysis in subcutaneous tumor tissues of BALB / C or C57BL / 6 mice with intestinal colonization of Campylobacter jejuni WT, ΔcdtB, cdtB Comp, or PBS control. (F and G) Migration or invasion assays were performed using extracted subcutaneous tumor cells, and the optical density (570 nm) was measured for each filter. (H and I) Lung metastasis was monitored using bioluminescence imaging (BLI) and lung weight after tail vein injection of extracted cancer cells into BALB / C mice (n=8 per group) or C57BL / 6 mice (n=8 per group); representative lungs are shown. (J) Schematic diagram of the experimental model: SPF BALB / C mice were given an antibiotic mixture for 1 week, followed by subcutaneous injection of breast cancer cells (4T1, 1×10⁻⁶). 6 Subcutaneous tumors were surgically removed from BALB / c or C57BL / 6 mice on days 0, 7, and 14, and mice were euthanized on day 30. (K and L) PCR analysis was used to detect Campylobacter jejuni (K) and cdtB genes (L) in subcutaneous tumor tissues of BALB / c or C57BL / 6 mice with intestinal colonization of Campylobacter jejuni (WT), ΔcdtB, cdtB Comp, or PBS control (n=6 per group). (M and N) Lung metastasis after subcutaneous injection of breast cancer cells was monitored in BALB / c mice using lung metastasis nodules (control and ΔcdtB group: n=9; Campylobacter jejuni (WT) and cdtBComp group: n=8). Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01 and ***p<0.001.

[0025] Figure 8 The pathways by which Campylobacter jejuni promotes CRC metastasis are summarized.

[0026] Figure 9Campylobacter jejuni did not promote the growth of CRC. (A and B) Tumor volume of CT26 or MC38 cells subcutaneously seeded after intratumoral injection of Campylobacter jejuni, DH5α, or PBS. (C and D) Mice were dissected on day 16, and tumors were visually identified, collected, and weighed. (E and F) Extracted tumor cells (CT26, MC38) were seeded in 6-well plates (200 cells / well) and grown for 8 days, fixed, and stained with crystal violet. (H) Tumor tissue was homogenized and serially diluted in sterile PBS. Tissue suspensions from the Campylobacter jejuni and PBS groups were inoculated onto Campylobacter selective medium, and the DH5α group was inoculated onto LB plates. CFU / g tissue was calculated for each group in each plate. Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0027] Figure 10 Campylobacter jejuni stimulates CRC cell metastasis. (A and C) Bioluminescence imaging (BLI) was used to monitor liver metastasis after extractive cells (stimulated by Campylobacter jejuni, DH5α, or PBS) were injected into the spleens of BALB / C and C57BL / 6, and all enlarged livers are shown in the images. (B and D) Lung metastasis of BALB / C and C57BL / 6 was monitored using bioluminescence imaging (BLI) after extractive cells were injected via the tail vein, and all fixed lungs are shown in the images.

[0028] Figure 11 CdtB knockout attenuated the promoting effect of Campylobacter jejuni on CRC cell invasion and migration. Migration (A) or invasion (B) were measured in CT26 / MC38 cells co-cultured in vitro with Campylobacter jejuni WT, CdtB KO, CdtB comp, or PBS (control). Optical density (570 nm) was measured for each filter. Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0029] Figure 12cdtB knockout had no effect on tumor cell growth. (A and B) Subcutaneous tumor volume after intratumoral injection of Campylobacter jejuni, DH5α, or PBS. (C and D) Mice were dissected on day 16, and tumors were visually identified, collected, and weighed. (H) Tumor tissue was homogenized and serially diluted in sterile PBS. The tissue suspension was plated on Campylobacter-selective medium. CFU / g tissue was calculated for each group in each plate. Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0030] Figure 13 The metastatic potential of Campylobacter jejuni depends on CDT. (A and B) Bioluminescent imaging (BLI) was used to detect lung metastases following tail vein injection of BALB / C and C57BL / 6 cells (stimulated with Campylobacter jejuni WT, CdtB KO, CdtB comp, or PBS). All fixed lungs are shown.

[0031] Figure 14 In vitro stimulation of CDT protein enhanced the metastatic ability of CRC cells. (A and B) CT26 / MC38 cells were co-cultured with solvent (control), Cdt A, Cdt B, Cdt C, or Cdt A+B+C for migration or invasion experiments, and the optical density (570 nm) of each filter was measured. (C and D) CT26 / MC38 cells were co-cultured with solvent (control), Cdt A+C, or Cdt A+B+C for migration or invasion experiments, and the optical density (570 nm) of each filter was measured. (E) CT26 cells were co-cultured with solvent or Cdt A+B+C, and a lung metastasis model was constructed by tail vein injection of BALB / C, and lung weight was measured. Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0032] Figure 15 CDT protein had no effect on tumor cell growth (A and B). Subcutaneous tumor volume after intratumoral injection of Cdt A+B+C and solvent (control). (C and D) Mice were dissected on day 16, and tumors were visually identified, collected, and weighed. (E and F) Extracted tumor cells were seeded in 6-well plates (200 cells / well) and grown for 8 days, fixed, and stained with crystal violet. Statistical analysis was performed using Students's t test. Data are presented as mean ± SEM, ns, p>0.05.

[0033] Figure 16CDT can stimulate CRC cell metastasis. (A and B) Lung metastases of BALB / C and C57BL / 6 were monitored using bioluminescence imaging (BLI) after tail vein injection of extracted cells (stimulated by Cdt A+B+C or solvent), showing all fixed lungs.

[0034] Figure 17 CDT activates the JAK2 / STAT3 / MMP9 signaling pathway to promote metastasis. (A) Differential gene expression between CT26 cells stimulated in vivo by Campylobacter jejuni or PBS using GSEA. (B and C) Transcriptional levels of the MMP family in CT26 (B) and MC38 (C) after in vitro stimulation by Campylobacter jejuni or PBS. (D and E) Migration or invasion assays were performed on CT26 or MC38 cells co-cultured with Cdt A+B+C, AG490, CdtA+B+C+AG490, or solvent (control), and the optical density (570 nm) of each filter was measured. (F) Expression of MMP9 in CT26 and MC38 cells transfected with shNT, shMMP9#1, or shMMP9#2 was detected by qRT-PCR. (G) Western blot analysis of MMP9 in CT26 and MC38 cells transfected with shNT, shMMP9#1, or shMMP9#1. Statistical analysis was performed using unpaired Student's t-test or one-way ANOVA combined with Tukey's multiple comparison test. Data are presented as mean ± SEM, ns. p>0.05, *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] The present invention will now be further illustrated with specific examples. The following embodiments are only for explaining the present invention and do not constitute a limitation thereof. The test samples and test procedures used in the following embodiments include the following (if the specific experimental conditions are not specified in the embodiments, they are usually performed according to conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments can be obtained commercially unless otherwise specified). Example 1

[0038] 1. Cell lines and bacterial strains

[0039] Colon adenocarcinoma cell lines HCT116, SW620, MC38, and CT26 were purchased from the American Type Culture Collection (ATCC) and stored at 37°C, 5% CO2, and 95% humidity. MC38-Luciferase and CT26-Luciferase cells were also included. All cell lines were cultured in DMEM medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA) and 1% penicillin-streptomycin (P / S, Gibco, CA, USA).

[0040] Campylobacter jejuni 81–176 wild-type (from ATCC), CdtB knockout staining, and complement staining were cultured on blood agar plates containing tryptone soybean agar (TSA) supplemented with 5% (vol / vol) sheep blood (solarbio, Beijing, China) and Campylobacter selective supplement (purchased from Oxoid, MA, USA) at 37°C for 48 h in a microaerophilic chamber. DH5α (Sinosoft, Nanjing, China) was cultured on Luria-Bertani (LB) plates at 37°C for 12–16 h.

[0041] 2. Patient and tissue samples

[0042] All fresh frozen tissue and formalin-fixed paraffin-embedded (FFPE) CRC tumor samples were obtained from the Sixth Affiliated Hospital of Sun Yat-sen University. A cohort of CRC patients (stages I-IIIN0M0) with or without distant metastasis within 3 years post-surgery was established. The metastatic and non-metastatic groups were matched based on sex, age, pathological stage, tumor location, excluding patients who had used antibiotics preoperatively or had high-risk factors (intestinal obstruction, perineural invasion, signet ring cell carcinoma, positive surgical margins, indeterminate or positive margins, mucinous adenocarcinoma, <12 lymph nodes examined, local perforation or closure, lymphovascular / vascular invasion). Cohort 2 was then established. Paraffin-embedded slides from cohort 1 were used for FISH. CRC staging was performed according to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM staging system. The protocols for human sample use and informed consent were approved by the Ethics Review Committee of the Sixth Affiliated Hospital of Sun Yat-sen University.

[0043] 3. Mouse experiment

[0044] According to guidelines approved by the International Association for the Protection and Use of Animals (IACUC) of Sun Yat-sen University, mice (C57BL / 6 and BALB / C), aged 6–8 weeks, were purchased from Jicui Pharmaceutical and Beijing Huafukang Biotechnology Co., Ltd., and were subjected to intertumoral injection in a nonspecific pathogen-free (SPF) site. Liver and lung metastases were constructed at the nonspecific pathogen-free (SPF) site of the Sixth Affiliated Hospital of Sun Yat-sen University.

[0045] Mouse colon cancer cells (1×10) 6 (Cells / mouse) Subcutaneous injection into the right axilla of BALB / C or C57BL / 6 mice, followed by multiple intertumoral injections at 5*10 sites on days 7, 10, and 13. 7 CFU-associated bacteria (C. jejuni WT, C. jejuni KO, C. jejuni Comp, or DH5α) or PBS. In in vivo CDT protein experiments, 50 μl (10 μmol / L) of protein was injected as previously described. Mice were sacrificed on day 16, and tumor cells were aseptically extracted (see Subcutaneous Tumor Cell Extraction for details).

[0046] In a liver metastasis model, mice were anesthetized with isoflurane. A 0.5cm incision was then made in the left abdomen to expose the spleen, and 1*102 insulin was administered via injection within 2 minutes. 6Tumor cells were slowly injected into the lower layer. The injection site was then pressed with a sterile cotton swab for 3 minutes. Finally, on days 2 and 3, the spleen was returned to the splenic fossa, the abdomen was sutured, and iodine disinfection was performed. On day 12, 200 μl (15 mg / ml) of d-luciferin (Promega, USA) was injected intraperitoneally, and luciferase images were acquired 10 minutes later using an IVIS system. The mice were then euthanized, the liver was dissected, and fixed in Bouin's Fixative solution (Phygene, Fuzhou, China). In the lung metastasis model, 1*10 cells were slowly injected via the tail vein. 6 One tumor cell was extracted and pressed onto the injection site with a sterile cotton swab for 15 seconds. Other procedures were the same as those for constructing the liver metastasis model.

[0047] 4. Extraction of subcutaneous tumor cells

[0048] All the following procedures are performed under sterile conditions. First, cut the tissue into 1-2mm pieces. 3 The small cubes were washed twice with PBS and then transferred to a 50ml tube containing 15ml of 0.1% trypsin. The 50ml tube was incubated at 37°C with shaking for 1 hour, and then filtered through a 70μm cell filter (Corning, NY, USA) into a new 50ml tube. The cells were centrifuged for 5 minutes (1200 rpm) and the supernatant was discarded. The cells were washed twice with PBS and then transferred to 10cm culture dishes.

[0049] 5. Cell stimulation

[0050] Cells were digested, centrifuged, resuspended, counted, and then seeded in 6-well plates (1.5*10⁻⁶). 6 (cells / well). Bacterial stimulation: After culturing in DMEM containing 1% P / S without FBS for 12 hours, the medium was replaced with DMEM containing 1% P / S without FBS. The bacteria were resuspended in PBS at a multiplicity of infection (MOI) of 10:1 for 12 hours. The cell wells were then rinsed with PBS and starved for 8 hours in DMEM containing 1% P / S without FBS, followed by transwell assays. Protein stimulation: After seeding for 12 hours, the medium was replaced with CDT protein (10 nmol / L) or solvent, and the following experiments were performed after 3 days.

[0051] AG490 stimulation: AG490 was diluted in dimethyl sulfoxide (DMSO). The addition of AG490 (50 μmol / L) inhibited the activation of JAK2 / STAT3, while the control group was treated with DMSO to eliminate the effect of the solvent.

[0052] 6. Migration and Intrusion Detection

[0053] Use a matrix with or without a matrix membrane ( Migration and matrix gel invasion assays were performed in the Transwell chamber of the Basement Membrane Matrix (Corning, NY, USA). Cells (CT26 and MC38: 1 x 10⁻⁶) were used. 5 Holes / hole, SW620 and HCT116: 2*10 5 Cells per well were seeded in the upper chamber filled with 200 μl of DMEM (FBS-free), while the lower chamber was filled with 600 μl of 10% FBS DMEM. After incubation for 12 h (CT26 and MC38) or 24 h (SW620 and HCT116), the cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.2% crystal violet for 30 min. The density of outer membrane-migrating cells was observed under an Olympus microscope. The crystal violet on the migrating cells was stained with 33% acetic acid, and the optical density (OD) of each filter was measured at A570 nm.

[0054] 7. Cell proliferation assay

[0055] Digest, centrifuge, and resuspend the cells, adding them to 6-well plates at 200 cells / well. Culture the cells in DMEM medium, changing the medium every three days. Once cell clusters have formed, fix the cells with 4% paraformaldehyde, stain with crystal violet, and photograph.

[0056] 8. Fluorescence in situ hybridization (FISH)

[0057] FISH assays were performed as described above. Briefly, a specific probe (5'-GCCCTAAGCGTCCTTCCA-3') conjugated to Campylobacter Alexa Fluor Cy3 (50) was labeled with Spectrum-red (Exon biotechnology, Guangzhou, China). A FITC-bound universal bacterial probe (5'-GCTGCCTCCCGTAGGAGT-3') was used as a positive control and labeled with Spectrum-green (Exon biotechnology, Guangzhou, China). 5 μm thick paraffin sections were dewaxed, incubated in lysozyme solution, washed, hybridized with FISH probes (35% hybridization buffer), incubated for 24 h, washed, stained with DAPI, and then imaged using a fluorescence microscopy system.

[0058] 9. MMP9 gene silencing

[0059] The lentiviral vectors shMMP9-1 (74302-11), shMMP9-2 (74304-1), and shNT (shGFP; CON077) were purchased from Genechem Co., LTD. In this embodiment, the target sequence target-1 of shMMP9-1 is 5'-aaGGACGGCAAATTTGGTTTC-3', and the sequence of shMMP9-1 is: 5'-CcggaaGGACGGCAAATTTGGTTTCCTCGAGGAAACCAAATTTGCCGTCCTTTTTTTg-3'; 5'-aattcaaaaaaaGGACGGCAAATTTGGTTTCCTCGAGGAAACCAAATTTGCCGTCCTT-3'. The target sequence of shMMP9-2, target-2, is 5'-gaCCATCATAACATCACATAC-3'. The sequence of shMMP9-2 is: 5'-CcgggaCCATCATAACATCACATACCTCGAGGTATGTGATGTTATGATGGTCTTTTTg-3' (sense strand); 5'-aattcaaaaagaCCATCATAACATCACATACCTCGAGGTATGTGATGTTATGATGGTC-3' (antisense strand). Lentiviral cells were generated by co-transfecting HEK293T cells with packaging plasmids (psPAX2 and pMD2.G) using PEI MAX 40K (Polyscience, ILLINOIS, USA). The culture medium was changed 24 hours after transfection, and the virus was collected 48 hours later, filtered, and then used to infect CT26 and MC38 cells for 48 hours in the presence of 1 μg / ml polybrene. The selection of resistant colonies was achieved by using 24 μg / ml (CT26) or 8 μg / ml (MC38) puromycin for 36 hours.

[0060] 10. Determination and identification of Campylobacter jejuni in tumors

[0061] Tumor tissues were homogenized and serially diluted in sterile PBS. Tumor tissues from the Campylobacter jejuni and PBS groups were resuspended on selective medium under microaerobic conditions and incubated at 37°C for 48 hours. Tumor tissues from the DH5α group were resuspended on LB agar plates and incubated at 37°C for 12–16 hours. Colonies were visually examined and counted.

[0062] Bacterial DNA was extracted from single colonies, and the V1-V9 regions of the 16S rRNA gene were Sanger sequenced to identify individual isolates. Amplification was performed using the following primer set: 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-GGTTACCTTGTTACGACTT-3'. Whole-genome sequencing was performed using the Illumina MiSeq (500v2) platform. High-quality 150 bp paired-end reads were assembled into contigs using SPAdes 3.13.0 software, and the assembled contigs were annotated using RAST. BLAST identification was performed using the rpoB gene sequence.

[0063] 11. RNA and Real-time Quantitative PCR

[0064] Total RNA was extracted from colon cancer cell lines using a total RNA kit (R323-01, Vazyme, China). RNA quality and concentration were then assessed using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Following the manufacturer's protocol, 1 μg of total RNA was reverse transcribed using Hiscript@III RTSuper Mix with a gDNA wiper (R323-01, Vazyme, China). qPCR was performed on an Applied Biosystems 7500 Real-time PCR system using a SYBR Green Real-time PCR master mix (QPK-201, Toyobo, Japan). Ct values ​​were compared between different samples using the 2-ΔΔCt method. GAPDH was used as an internal control gene.

[0065] 12. Protein blotting

[0066] Cell lysis buffer was prepared using RIPA lysis buffer containing a 10% mixture of protease and phosphatase inhibitors (Beyotime, Shanghai, China). Cell lysis buffer was collected, sonicated (10 min), centrifuged (15000 rpm, 15 min), and the supernatant was collected. Quantification was performed using a BCA protein assay kit (Thermofisher, MA, USA). 40 μg of protein was subjected to 12% SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. The membrane was blocked for one hour in 5% skim milk in TBST buffer (50 mmol / L Tris-HCl, 150 mmol / L NaCl, 0.1% Tween-20, pH 7.4) and then incubated overnight at 4°C with primary antibody. Secondary antibody was labeled with HRP, and the signal was detected using an ECL kit. Images were subsequently analyzed using ImageJ 1.43 software. GAPDH antibody was used as a control. MM9 antibody was purchased from Abcam (Cambridge, UK). p-JAK2, JAK2, STAT3, p-STAT, and GAPHD antibodies were purchased from Cell Signaling Technology (CST, MA, US).

[0067] 13. Construction of cdtB knockout Campylobacter jejuni (cdtB KO) and cdtB complement Campylobacter jejuni (cdtB Comp)

[0068] The construction of the Campylobacter jejuni 81-176 deletion mutant mainly includes the following steps: To construct the ΔcdtB deletion mutant vector, the upstream 759bp and downstream 744bp cdtB coding sequences were amplified by PCR, including its own 237bp to avoid affecting the transcription of downstream genes. Two PCR fragments from plasmid PG0127 and a kanamycin resistance cassette were ligated into pBluescript II SK using the Gibson assembly protocol. It is worth noting that, since cdtABC may reside in the same ORF, the kanamycin resistance cassette used for the ΔcdtB mutant (from plasmid PG0385 PCR) is based on the nonpolar design of plasmid pACH1, which contains a 3×stop codon at its 5' end and an RBS region at its 3' end.

[0069] The cdtB complement-modifying Campylobacter jejuni strain was obtained by introducing the wild-type gene at the hsdM locus. Here, CdtB was cloned into plasmid PG0125. To investigate the function of CdtB after Campylobacter jejuni infection of the host, a FLAG tag was attached to the C-terminus of CdtB during complement modification. All plasmids were homologously recombinated into the Campylobacter jejuni chromosomes 81-176 via natural transformation.

[0070] 14. CDT Expression and Purification

[0071] Expression and purification of Campylobacter jejuni CDT protein. The Campylobacter jejuni cdtA fragment was inserted into pCold TF (Takara) using XhoI and BamHI (Thermo Fisher, MA, US), and the cdtB or cdtC fragment was cloned into pET21b (Takara, Japan) using NheI and XhoI (Thermo Fisher, MA, USA). The cdt gene plasmid was transformed into Escherichia coli BL21(DE3) (Tiangen, Beijing, China), and the nucleotide sequence was verified. The recombinant strains were cultured separately in LB broth supplemented with ampicillin (100 μg / ml) overnight at 37°C, and then transferred to 1L of fresh LB broth inoculated with 1% ampicillin and cultured until the OD600 reached 0.5. The expression of toxins CdtA, CdtB, and CdtC was induced by adding 1 mM IPTG (isopropyl-bD-galactopyranoside), and the culture was incubated overnight at 30°C to express the recombinant protein. Bacteria were harvested by centrifugation, washed twice with PBS, and resuspended in 20 ml of lysis buffer (20 mM phosphate buffer, pH 7.8, 10 mM imidazole, 0.5 M NaCl). The mixture was sonicated and centrifuged, and the soluble fraction was incubated with Ni-NTA resin (Smat-Life Sciences) for 1 hour and rotated at 4°C. The His-tagged recombinant protein bound to the resin and was washed away with elution buffer (20 mM phosphate buffer, pH 7.8, 250 mM imidazole, 0.5 M NaCl). The purified protein was obtained by desalting and concentrating via ultrafiltration (10 K molecular weight cutoff (MWCO), Millipore).

[0072] 15. RNA sequencing and analysis

[0073] use Total RNA was extracted from CT26 and MC38 cell lines by Reagent (Life Technologies, Carlsbad, CA, USA). The RNA was sent to Adaptive Biotechnology (Novogene, China) for sequencing. RNA degradation and contamination were monitored on agarose gels. RNA purity was checked using a spectrophotometer (IMPLEN, CA, USA), and RNA integrity was assessed using the RNA Nano 6000 assay kit on a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total of 1 μg of RNA from each sample was used as input material for RNA sample preparation. UltraTM RNA Library Prep Kit for (NEB, USA) Sequencing libraries (non-strand-specific, paired ends) were generated according to the manufacturer's instructions, and index codes were added to the attribute sequences of each sample. Indexed samples were clustered on the cBot ClusterGeneration System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on the Illumina Novaseq platform, generating 150 bp paired-end reads. Differential expression analysis was performed on the two groups (three biological replicates per condition) using the DESeq2 R package (1.16.1). DESeq2 provides a statistical procedure for determining differential expression in digital gene expression data using a model based on a negative binomial distribution. The p-values ​​of the results were adjusted using the Benjamini and Hochberg method to control for false discovery rates. Genes with adjusted p-values ​​<0.05 identified by DESeq2 were designated as differentially expressed. RNA sequencing data were stored in the NCBI SRA database.

[0074] 16. High-throughput 16S rRNA amplicon sequencing and analysis

[0075] Microbial community DNA was extracted using the NucleoSpin Soil Kit (Macherey-Nagel, Germany). DNA was quantified using a dsDNA BR assay kit (Invitrogen, USA) via a Qubit fluorometer, and quality was checked by running aliquots on a 1% agarose gel. The variable region V4 of the bacterial 16S rRNA gene was amplified using degenerate PCR primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Amplicons were purified using AMPure XP beads (Beckman Coulter) and eluted in elution buffer. The library was identified using an Agilent Technologies 2100 Bioanalyzer. The validated library was sequenced according to Illumina's standard procedures on an Illumina HiSeq 2500 platform (BGI, Shenzhen, China), generating 2 × 250 bp paired-end reads.

[0076] Raw reads were filtered to remove adapters and low-quality or ambiguous bases. Paired-end reads were then added to tags using a fast length adjustment procedure for short read sequences (FLASH, v1.2.11) to obtain tags. Tags were clustered into OTUs with a cutoff value of 97% using UPARSE software (v7.0.1090), and chimeric sequences were compared to the Gold database for detection using UCHIME (v4.2.40). OTU representative sequences were then taxonomically classified using the Ribosome Database Project (RDP) classifier v.2.2 with a minimum confidence threshold of 0.6, trained on the Greengenes database v201305 using QIIME v1.8.0. An OTU abundance statistics table for each sample was obtained by comparing all tags with OTUs using USEARCH_global.

[0077] Alpha and Beta diversity were estimated at the OTU level using MOTHUR (v1.31.2) and QIIME (v1.8.0), respectively. Principal coordinate analysis (PCoA) was performed using QIIME. Sample clustering was performed by QIIME based on UPGMA. Barplots and heatmaps at different taxonomic levels were plotted using R package v3.4.1 and R package “gplots”, respectively, and LEfSe clustering or LDA analysis was performed using LEfSe. Important species were determined using R (v3.4.1) based on the Wilcox or Kruskal test.

[0078] 17. Quantitative and statistical analysis

[0079] Except for the specific statistical analyses described above, all other statistical analyses were performed using GraphPad Prism 8 or IMB SPSS 24. Data are expressed as mean ± SE, and differences between two groups were compared using unpaired Student's t-tests. Comparisons between three or more groups were performed using one-way ANOVA combined with Tukey's multiple comparison test. Patient characteristics were determined using Pearson's chi-square test or Fisher's exact test (as applicable). All p-values ​​were two-tailed, and differences with p-values ​​less than 0.05 were considered significant (*P<0.05, **P<0.01, and ***P<0.001), while ns indicated no significant comparison.

[0080] I. Campylobacter accumulates in the primary tumor of patients with postoperative metastasis.

[0081] Cohort 1 included 41 patients with early-stage CRC (T1-3N0M0, stage I and IIA) who underwent surgery. This included 21 patients who developed distant metastases within 3 years post-surgery (metastasis group) and 20 patients who remained metastatic during follow-up (non-metastasis group) matched for T NM stage, age, sex, BMI, tumor location, stage, CA199, CA125, and tumor differentiation. Clinical indicators were comparable. To assess the role of the microbiota in postoperative metastasis, all primary CRC tissues were evaluated by 16S ribosomal RNA (rRNA) gene sequencing. Although the α-diversity level in the metastasis group was low and did not reach statistical significance (…),… Figure 1 A), but the separation of microbial structures in the two groups was very obvious (e.g. Figure 1 (as shown in B).

[0082] Subsequently, linear discriminant analysis of effect size (LEfSe) was used to detect significant differences in bacterial community dominance between the metastatic and non-metastatic control groups. In the primary tumor lesions of patients in the metastatic group, several genera were significantly enriched (e.g., ...). Figure 1 (As shown in C). It is noteworthy that Campylobacter is one of the genera enriched in the transfer group (e.g., ...). Figure 1 (As shown in D). To validate the clinical significance of Campylobacter, a validation cohort (cohort 2, containing 20 patients, of whom 11 had postoperative metastasis and 9 did not. Clinical indicators were comparable). Testing of cohort 2 revealed a significantly higher relative abundance of Campylobacter in the primary lesion of patients with postoperative metastasis compared to the control group without postoperative metastasis. Figure 1 E). Furthermore, in publicly available microbiome datasets (cohort 3, PRJNA531761) ( Figure 1 Similar results were further confirmed in F), which highlighted the important role of Campylobacter in the metastasis of primary CRC lesions.

[0083] To visualize Campylobacter colonization in CRC lesions, fluorescence in situ hybridization (FISH) was performed under aseptic conditions using a Campylobacter-specific probe and a universal eubacterial probe (EUB338). As expected, the analysis showed an increased presence of Campylobacter in CRC lesions from patients with postoperative metastases. Figure 1 G). Therefore, these results indicate that Campylobacter has clinical significance in CRC metastasis.

[0084] II. Campylobacter jejuni promotes CRC metastasis in vitro and in vivo.

[0085] As mentioned above, Campylobacter accumulates in tumor lesions of colorectal cancer patients who have undergone postoperative metastasis. However, it remains unclear whether Campylobacter plays a role in colorectal cancer metastasis. To further investigate the metastatic potential of Campylobacter, the inventors examined the effects of Campylobacter jejuni on cell migration and invasion in vitro.

[0086] Colorectal cancer cell lines CT26, MC38, HCT116, and SW620 were exposed to human clinical isolate C. jejuni 81–176 (Campylobacter jejuni, multiplicity of infection = 10) for 12 hours. Compared with the PBS control, the Campylobacter jejuni group significantly increased cell migration and invasion in these four colorectal cancer cell lines. Figure 2 AB). When control bacteria DH5α were co-cultured with CT26, MC38, HCT116, and SW620 cells, these migration-promoting and invasion-promoting effects were not observed in control bacteria DH5α. Figure 2 AB).

[0087] Furthermore, the inventors sought to demonstrate the metastatic ability of Campylobacter jejuni in vivo. CRC cells were subcutaneously seeded in BALB / c (CT26 cell line) and C57BL / 6 (MC38 cell line) mice. After 7 days of tumor growth, Campylobacter jejuni (5*10) was injected into the tumor tissue every 3 days. 7 CFU / mouse) or DH5α (5*10) 7 Tumor cells were extracted on day 16 using CFU / mouse or an equal volume (50 μl) of PBS for in vitro and in vivo downstream experiments. Figure 3 A). Detection revealed that Campylobacter jejuni had no effect on tumor growth. Figure 9 .AD), but it can successfully colonize tumors ( Figure 9 .G). Then, tumor cells were extracted and subjected to migration, invasion, and colony formation assays. The data showed that Campylobacter jejuni could promote the migration and invasion of colon cancer cells. Figure 3 (BC), consistent with the co-culture experiment; colony formation experiment showed no difference in colony formation among the three groups. Figure 9 .EF).

[0088] In in vivo experiments, extracted tumor cells were injected into the spleen via liver metastasis. The total fluorescence intensity in the *Campylobacter jejuni* group was significantly higher than that in the DH5α and PBS groups, and the liver weight was also significantly increased. Figure 3 D, F; Figure 10 (A, C). Furthermore, extracted tumor cells were injected via tail vein for lung metastasis, and the results were consistent with those of the liver metastasis experiment. Figure 3 E, G; Figure 10 (B, D). These data suggest that Campylobacter jejuni in the tumor microenvironment can promote the metastatic ability of tumor cells.

[0089] III. Campylobacter jejuni-induced CRC metastasis-promoting ability depends on cdtB.

[0090] To elucidate the reason why Campylobacter jejuni induces colorectal cancer metastasis, the cdtB gene was knocked out using homologous recombination technology (cdtB KO), and a complement knockout strain (cdtB comp) was established using a plasmid that can express cdtB.

[0091] CT26 or MC38 cells were co-cultured for 12 hours with Campylobacter jejuni wild-type (WT), cdtB KO, cdtB comp, and PBS control, respectively, at a multiplicity of infection (MOI) of 10. The results showed that under cdtB deficiency, the pro-migration and pro-invasion abilities of Campylobacter jejuni were weakened in vitro, while cdtB compensation could restore these abilities. Figure 11 AB).

[0092] Mice were injected subcutaneously into tumor cells. Seven days later, Campylobacter jejuni WT, cdtB KO, cdtB comp, or PBS were injected into the tumor tissue every three days. On day 16, tumor cells were extracted for in vitro and in vivo experiments. Figure 4 A). It was found that the deficiency of the cdtB gene weakens the ability of Campylobacter jejuni to promote cell migration and invasion. After cdtB gene compensation, the ability of Campylobacter jejuni to promote migration and invasion was restored. Figure 4 BC). Furthermore, the deletion of the cdtB gene in Campylobacter jejuni affects tumor proliferation (BC). Figure 12 AD) and the colonization of Campylobacter jejuni in tumor tissue ( Figure 12 .E) No impact.

[0093] The inventors then examined the metastatic ability of these cells in vivo; through tail vein injection in a lung metastasis model, they found that the metastatic ability of Campylobacter jejuni decreased when the cdtB gene was knocked out, and the ability was restored after cdtB supplementation. Figure 4 DE Figure 13 (AB), which is consistent with in vitro experiments.

[0094] IV. The CdtB subunit of CDT can promote tumor metastasis.

[0095] To further confirm the metastatic effect of CDT, CRC cells were stimulated with recombinant CDT whole toxin, and its metastatic ability was tested in vitro and in vivo. CdtA, CdtB, and CdtC were expressed in BL21 cells, and the peptides were purified and reassembled in the active toxin.

[0096] First, we stimulated CRC cells with solvents (lysis buffer, the solvent for CDT), CdtA, CdtB, CdtC, and Cdt A+B+C, respectively, and examined their migration and invasion abilities. We found that only Cdt A+B+C showed a strong enhancing ability in both cell migration and cell invasion assays, while CdtB only induced a slight enhancing ability. Figure 14 AB).

[0097] The intracellular biological functions of CdtB are primarily achieved through the synergistic action of CdtA and CdtC, a point previously revealed in studies. To further illustrate the role of CdtB, the inventors stimulated CRC cells with Cdt A+B+C and Cdt A+C. Naturally, the loss of the CdtB subunit led to a reduction in the metastatic ability of CDT holotoxin. Figure 14 .CD).

[0098] Furthermore, the group of mice that received Cdt A+B+C via tail vein injection showed a higher metastasis rate compared to the group that received solvent-stimulated cells. Figure 14 E).

[0099] Then, the present invention injects Cdt A+B+C or a solvent into the tumor and extracts tumor cells as described above. Figure 5 A). No difference was found in tumor growth ( Figure 15 AF), but Cdt A+B+C can significantly promote the migration and invasion ability of CRC cell lines (AF). Figure 5 BC). A mouse model of lung metastasis also showed that the Cdt A+B+C combination significantly induced metastatic lesion formation. Figure 5 DE Figure 16 AB).

[0100] V. CdtB stimulates tumor metastasis through the JAK2 / STAT3 / MMP9 signaling pathway

[0101] To elucidate the signaling pathways and mechanisms underlying the biological effects of CdtB, the inventors used RNA-seq to compare transcriptional differences between CRC cell lines (CT26 and MC38) co-cultured with CdtA+B+C or in solvent. Gene set enrichment analysis (GSEA) identified differentially expressed genes, showing that the JAK / STAT signaling pathway was significantly upregulated under CDT influence. Figure 6 AB). In addition, CT26 cells stimulated with Campylobacter jejuni or PBS were extracted in vivo (e.g., AB). Figure 3 (As shown in A) RNA-seq was performed, and the results showed that the JAK / STAT signaling pathway was also upregulated in the subcutaneous model treated with Campylobacter jejuni. Figure 17A). JAK / STAT, especially JAK2 / STAT3 signaling, is crucial for human cancer metastasis. Western blot showed that Cdt A+B+C promoted the phosphorylation of JAK2 and STAT3, with no significant difference in total JAK2 and STAT3 protein expression between CT26 and MC38 cells. Figure 6 C).

[0102] Analysis of target genes in the JAK2 / STAT3 pathway revealed that the matrix metalloproteinase (MMP) family, a key site for tumor metastasis, was significantly upregulated in CDT-treated cells. Therefore, the inventors used real-time quantitative PCR (qPCR) to detect MMPs, of which MMP9 has been defined as significantly elevated in tumor metastasis and regulated by the JAK2 / STAT3 pathway. MMP9 was found to be upregulated in both CDT-treated cell lines. Figure 6 DE), and the protein blotting results further proved ( Figure 6 F).

[0103] Co-culturing CT26 and MC38 cells with Campylobacter jejuni or PBS revealed that the MMP family was also upregulated in Campylobacter jejuni-treated cell lines, with only MMP9 being consistent in both cell lines. Figure 17 BC).

[0104] The inventors then used an effective JAK2 inhibitor, AG490, to inhibit the JAK2 / STAT3 pathway. When AG490 was applied, stimulation with Cdt A+B+C failed to upregulate MMP9 levels. Figure 6 G), and lost its migration and invasion progression in CT26 and MC38CRC cell lines (G), and lost its migration and invasion progression. Figure 17 These results indicate that Cdt A+B+C upregulates MMP9 through the JAK2 / STAT3 pathway. To further illustrate the role of the JAK2 / STAT3 / MMP9 signaling pathway in CDT-induced metastasis, we used short hairpin RNA (shRNA) to inhibit MMP9 expression. qPCR and Western blotting showed that MMP9 expression was downregulated at both the mRNA and protein levels. Figure 17 FG). Simultaneously, when MMP9 is inhibited, stimulation by Cdt A+B+C affects MMP9 expression ( Figure 6 J) and enhanced migratory invasion capabilities ( Figure 6 HI) all lost their regulatory function.

[0105] Finally, the inventors established a lung metastasis model in BALB / c mice by intravenous injection of Cdt A+B+C-stimulated CT26 cells (shNT, shMMP9#1 (i.e., shMMP9-1), or shMMP9#2 (i.e., shMMP9-2)). The results showed that when MMP9 was knocked down, CDT whole toxin lost its metastatic ability, indicating that CDT promotes CRC metastasis through the JAK2 / STAT3 / MMP9 signaling pathway. Figure 6 I).

[0106] In the above embodiments, the inventors first demonstrated that Campylobacter infection and its toxin CDT are involved in CRC metastasis. Campylobacter colonizes CRC tissue and has a higher abundance in the primary tumor of patients with postoperative metastasis.

[0107] The inventors also demonstrated the metastatic ability of Campylobacter jejuni through in vitro and in vivo experiments, with CDT playing a crucial role in this effect. In vitro, Campylobacter jejuni upregulated the migration and invasion abilities of various tumor cell lines, and in vivo, consistent results were observed when the bacteria were injected intratumorally. Furthermore, injection of Campylobacter jejuni-stimulated cells via tail vein or spleen injection in mice resulted in more metastatic lesions. Notably, cdtB-KO Campylobacter jejuni had no effect on CRC metastatic progression, indicating that CdtB is essential for the metastatic ability of Campylobacter jejuni. Next, using purified bacterial CDT protein, the inventors found that CDT-stimulated CRC cells exhibited enhanced metastatic ability both in vitro and in vivo. Moreover, while CdtB stimulation alone increased tumor cell metastatic ability, it was far less than the effect of Cdt A+B+C, suggesting that the mechanism of CdtB's metastatic effect extends beyond this. These results indicate that Campylobacter jejuni can promote the metastatic ability of CRC cells through CDT secretion, with the metastatic potential of Campylobacter jejuni and CDT playing a key role in this process.

[0108] Mechanistically, CDT stimulates tumor cell metastasis by upregulating the JAK2 / STAT3 / MMP9 signaling pathway. RNA-seq showed that CDT activates the JAK / STAT pathway, followed by significant upregulation of the MMP family. Then, inhibiting JAK2 / STAT3 activation using AG490 weakened the invasiveness and migration of tumor cells. Simultaneously, knocking down the MMP9 gene in CRC cell lines reduced metastasis in both in vivo and in vitro experiments. Furthermore, published RNA-seq data revealed that JAK2 / STAT3 / MMP9 is upregulated in the mucosa of human clones infected with Campylobacter jejuni, further confirming that Campylobacter jejuni activates JAK2 / STAT3 / MMP9.

[0109] In summary, in this embodiment, the inventors discovered that Campylobacter jejuni promotes colorectal cancer metastasis via CDT through the JAK2 / STAT3 / MMP9 pathway. Figure 7 Microbiome analysis of tumor tissue, especially the abundance of Campylobacter, helps in the timely diagnosis of patients at risk of postoperative metastasis after CPC (Cyclophorus infection) and in the timely implementation of preventive measures to avoid CPC metastasis. Furthermore, this embodiment confirms that the JAK2 / STAT3 / MMP9 pathway is essential in the metastasis-promoting effects of CDT (Cyclophorus infection), indicating that inhibiting nodes in this pathway is beneficial in overcoming the adverse effects of Campylobacter jejuni infection, particularly in the treatment and prevention of CPC invasion and metastasis.

[0110] VI. Intestinal colonization by Campylobacter jejuni accelerates cancer metastasis, and this process is facilitated by cdtB.

[0111] In addition to the direct interaction with intratumoral stimuli, the inventors further investigated whether Campylobacter jejuni intestinal colonization could mediate extraintestinal tumor metastasis. Mice subcutaneously injected with tumors were treated with an antibiotic mixture and then gavaged with Campylobacter jejuni WT, ΔcdtB (i.e., cdtB KO), cdtB-Comp, or PBS. Figure 8 As shown in Figure A. Notably, similar Campylobacter jejuni qPCR signals were detected in the intestines and tumors of mice administered the bacteria via gavage, and the cdtB gene load was significantly reduced in the intestines and tumors of mice administered Campylobacter jejuni ΔcdtB via gavage. Figure 8 BE).

[0112] In vitro assays of isolated tumor cells showed that mice treated with Campylobacter jejuni ΔcdtB had significantly reduced cell migration and invasive potential, but these were restored upon stimulation by the cdtB Comp strain. Figure 8 FG). Similar evidence found in a mouse model of lung metastasis indicated higher lesions in mice gavaged with Campylobacter jejuni WT and cdtB Comp, but remission was observed in mice gavaged with Campylobacter jejuni ΔcdtB. Figure 8 More importantly, in mice gavaged with Campylobacter jejuni WT and cdtB Comp, MMP9 was found to be significantly upregulated in tumor tissue.

[0113] To further investigate the role of CDT in cancer metastasis, a breast cancer model was established via subcutaneous tumor injection (4T1). Mice were treated with an antibiotic mixture for one week, followed by gavage with Campylobacter jejuni WT, ΔcdtB, cdtB-Comp, or PBS. Figure 8 As shown in J. Similar bacterial colonization was observed in mice gavage with bacteria, but detection of the cdtB gene revealed a significant decrease in cdtB gene levels in Campylobacter jejuni ΔcdtB mice. Figure 8KL).

[0114] Experiments showed that stimulation with Campylobacter jejuni WT and cdtB Comp induced a higher degree of invasion and migration of isolated breast cancer cells in vitro, compared to stimulation with Campylobacter jejuni ΔcdtB. Furthermore, compared to Campylobacter jejuni WT and cdtB Comp, mice administered Campylobacter jejuni ΔcdtB by gavage showed significantly lower levels of lung lesions. Figure 8 Similarly, Campylobacter jejuni WT and cdtB-Comp significantly upregulated MMP9 in breast cancer cells compared to Campylobacter jejuni ΔcdtB. These data highlight the role of Campylobacter jejuni-derived toxins, suggesting that Campylobacter jejuni intestinal colonization can regulate extraintestinal tumor metastasis through the secretion of cdtB toxins.

[0115] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. The use of a reagent for detecting Campylobacter jejuni or toxin CDT in the preparation of reagents for diagnosing colorectal cancer, characterized in that, The detection is a quantitative or semi-quantitative determination of the abundance or presence of Campylobacter jejuni or toxin CDT in tumor tissue.

2. The use of CDT-JAK / STAT / MMP pathway inhibitors in the preparation of drugs for inhibiting colorectal cancer, characterized in that, The CDT-JAK / STAT / MMP pathway inhibitor is shMMP9#2; The sequence of shMMP9#2 is: 5'-Ccggg aCCAT CATAA CATCA CATAC CTCGA GGTATGTGAT GTTAT GATGG TCTTT TTg-3', 5'-aattc aaaaa gaCCA TCATA ACATC ACATACCTCG AGGTA TGTGA TGTTA TGATG GTC-3'.

3. A reagent kit, characterized in that, The invention includes reagents for detecting Campylobacter jejuni and its toxin CDT; and / or, CDT-JAK / STAT / MMP pathway inhibitors; the Campylobacter jejuni and toxin CDT detection reagents include primer sets 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-GGTTACCTTGTTACGACTT-3'; the CDT-JAK / STAT / MMP pathway inhibitor is shMMP9#2; The sequence of shMMP9#2 is: 5'-Ccggg aCCAT CATAA CATCA CATAC CTCGA GGTATGTGAT GTTAT GATGG TCTTT TTg-3', 5'-aattc aaaaa gaCCA TCATA ACATC ACATACCTCG AGGTA TGTGA TGTTA TGATG GTC-3'.

4. A tumor metastasis inhibitory drug, characterized in that, The reagent includes Campylobacter jejuni and toxin CDT blocking agent, wherein the Campylobacter jejuni and toxin CDT blocking agent is shMMP9#2; The sequence of shMMP9#2 is: 5'-Ccggg aCCAT CATAA CATCA CATAC CTCGA GGTATGTGAT GTTAT GATGG TCTTT TTg-3', 5'-aattc aaaaa gaCCA TCATA ACATC ACATACCTCG AGGTA TGTGA TGTTA TGATG GTC-3'.

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