Construction method and application of animal model for promoting colorectal cancer liver metastasis by using enterococcus in quail chicken

Abstract

The invention relates to a construction method and application of a colorectal cancer liver metastasis promoting animal model of quail chicken enterococcus, and the construction method specifically comprises the following steps: constructing an in-situ starting colorectal cancer liver metastasis animal model, and carrying out isolated culture and identification on live bacteria in a liver metastasis focus of the model, so as to determine the quail chicken enterococcus as candidate bacteria; clinical sample analysis is further combined, and it is clear that the quail chicken enterococcus can play a role in the colorectal cancer liver metastasis process; and finally, further verifying the function of the quail chicken enterococcus SY2-1 with the preservation number of CCTCC (China Center For Type Culture Collection) NO: M20241929 by using two colorectal cancer liver metastasis models. The experiment result shows that the abundance and the detection positive rate of the quail chicken enterococcus in a clinical sample in the colorectal cancer liver metastasis focus of a patient are obviously higher than those of normal liver tissues, which indicates that the quail chicken enterococcus has the potential of serving as a predictive marker for occurrence and development of the colorectal cancer liver metastasis and serving as a biological intervention target of the colorectal cancer liver metastasis; and the enterococcus quail chicken SY2-1 can promote the construction of a colorectal cancer liver metastasis animal model.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application number CN202411268663.1 filed on September 11, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine technology, and specifically to a method for constructing an animal model of colorectal cancer liver metastasis promoted by Enterococcus gallinarum and its application. The above-mentioned Enterococcus gallinarum can be used to promote colorectal cancer liver metastasis and the construction of related animal models, and can be used as a target for intervention or diagnosis of colorectal cancer liver metastasis, and a strain of Enterococcus gallinarum SY2-1 was isolated and identified. Background Art

[0004] As one of the three most common cancers in the world, the incidence of colorectal cancer has been increasing year by year at a rate of 3.2% since the end of the last century. According to clinical statistics, about 20% of patients newly diagnosed with colorectal cancer have metastases, and another 25% of patients gradually develop colorectal cancer metastasis during subsequent disease progression. The mortality rate of patients caused by colorectal cancer metastasis ranks first among all-cause mortality rates of colorectal cancer, and the 5-year survival rate of patients is less than 20%. Among the distal organs affected by colorectal cancer metastasis, the liver is the most frequently metastatic organ. At present, the treatment options for colorectal cancer liver metastasis are relatively limited, and surgical resection is still the first choice. Only a few patients are suitable for surgical resection, and the 5-year survival rate of patients after resection is only about 48%. The high incidence and mortality make it particularly important and urgent to develop new intervention or prevention targets for colorectal cancer liver metastasis. By exploring the new influencing factors and their mechanisms of action of colorectal cancer liver metastasis, it can provide ideas for the development of intervention targets and new intervention strategies that can be applied in clinical practice.

[0005] As research progresses, multiple factors have been shown to play a role in colorectal cancer liver metastasis, which can be divided into two categories: one is the inherent factors of tumor cells, such as susceptibility gene mutations, chromatin instability, etc., and the other is the microenvironment of distal organs, including immune cells, stromal cells, specific metabolites and even microorganisms (mainly bacteria) in the microenvironment. In the past decade of research, bacteria represented by Fusobacterium nucleatum have been shown to be associated with colorectal cancer liver metastasis. Based on the technology of bacterial flora sequencing, researchers first detected the DNA of Fusobacterium bacteria including Fusobacterium nucleatum in liver metastases of colorectal cancer patients, and isolated active Fusobacterium bacteria from liver metastases of colorectal cancer patients, and Fusobacterium-positive patient tissues were more likely to successfully construct PDX (Patients-derived xenografts) models in mice. This suggests that some bacterial components in colorectal tumors (such as Fusobacterium bacteria) are inherent and key components of the tumor microecology and play an important role in the occurrence and progression of colorectal cancer. In addition to Fusobacterium nucleatum, Escherichia coli C17 strains in colorectal tumors were found to be able to destroy the intestinal vascular barrier by releasing the virulence regulator VirF, causing bacteria to translocate from the original site to the liver through the gut-liver axis, promoting the maturation of the pre-metastatic niche, and thus promoting colorectal cancer liver metastasis. Because the microbiota is closely related to geographical location and residents' living habits, there is a lack of exploration and research on microbial community factors related to colorectal cancer liver metastasis from domestic samples.

[0006] The current microbial research programs for colorectal cancer liver metastasis have the following deficiencies: (1) Research by foreign teams lacks the exploration of microbial community factors related to colorectal cancer liver metastasis from domestic sample sources; (2) Bacteria that have been reported to promote colorectal cancer liver metastasis include Fusobacterium nucleatum and Escherichia coli, both of which have been previously shown to have a promoting effect in colorectal cancer in situ tumors. There has been no research on new functional bacteria in colorectal cancer liver metastasis.

[0007] Regarding the intestinal pathogen Enterococcus gallinarum, existing studies have disclosed that Enterococcus gallinarum differentiates into independent lineages with different niche preferences and pathogenicity through intrahost evolution, one of which is adapted to mucosal colonization and evades immune detection and elimination. The above strains have increased translocation and survival rates in the mesenteric lymph nodes and liver, and induce increased intestinal inflammation and liver inflammation; verification found that Enterococcus gallinarum isolated from feces showed enhanced competitive adaptability in the intestinal cavity, while Enterococcus gallinarum isolated from the liver showed enhanced ability to transfer to the liver (Within-host evolution of a gutpathobiont facilitates liver translocation. Nature. 2022 Jul; 607 (7919): 563-570.); prior art literature also discloses that Enterococcus gallinarum transfers to mesenteric lymph nodes and liver in multiple mouse models of inflammatory diseases. For example, in patients with systemic lupus erythematosus, it was found that the pathogenic commensal Enterococcus gallinarum can be translocated from the small intestine to the liver. However, none of the above studies involved the application of Enterococcus gallinarum in promoting liver metastasis of colorectal cancer. Summary of the invention

[0008] In order to overcome at least one problem existing in the prior art, the present invention isolates and cultures bacteria in colorectal cancer liver metastases to find candidate bacteria that may play a role, further verifies the detection abundance and positive rate of the candidate bacteria in clinical samples, and verifies the function of the candidate bacteria in a colorectal cancer liver metastasis animal model.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention is to provide an application of Enterococcus gallinarum or a microbial agent thereof, which is selected from at least one of the following applications: application of Enterococcus gallinarum or a microbial agent thereof in constructing a colorectal cancer liver metastasis model, application of Enterococcus gallinarum or a microbial agent thereof in screening drugs for treating colorectal cancer liver metastasis, application of Enterococcus gallinarum as a predictive marker for the occurrence and development of colorectal cancer liver metastasis, and application of Enterococcus gallinarum as a target for intervention or diagnosis of colorectal cancer liver metastasis.

[0011] Furthermore, the colorectal cancer liver metastasis model includes a colorectal cancer in situ liver metastasis model and a spleen injection colorectal cancer liver metastasis model.

[0012] Furthermore, the steps of constructing the colorectal cancer-liver metastasis model include:

[0013] Step S1: Adjust the bacterial solution concentration of Enterococcus gallinarum to 10 9 CFU / ml, 100 μL was administered to mice;

[0014] Step S2, the Enterococcus gallinarum liquid was gavaged every other day. After 3 days, the mouse colorectal cancer liver metastasis cell line was injected into the mouse cecal serosa or mouse spleen to construct a colorectal cancer liver metastasis model.

[0015] Furthermore, the mouse intestinal cancer liver metastasis cell line is a mouse colon cancer cell clone CT26-TL-CL5 (Deposit Number: CCTCC NO: C2024307). It is understandable that the above mouse intestinal cancer liver metastasis cell line can also be a cell line conventionally used in the art, such as CT26-FL3, CT26.WT, etc.

[0016] Furthermore, when injected into the cecal serosa of mice, the cell concentration of the cell line CT26-TL-CL5 was 1×10 6 / 15μL, mixed with an equal amount of matrix gel, and 30μL of cell suspension was injected.

[0017] Furthermore, when injected into the spleen of mice, the cell line CT26-TL-CL5 was adjusted to a cell concentration of 10 7 / mL, take 100μL of cell suspension for injection.

[0018] Furthermore, the drug for treating colorectal cancer liver metastasis is a drug that reduces the abundance of Enterococcus gallinarum.

[0019] Furthermore, the drug includes antagonistic bacteria and bacteriophages of Enterococcus gallinarum, and also includes small molecules or macromolecular compounds that can reduce the abundance of Enterococcus gallinarum.

[0020] Furthermore, the dosage form of the drug includes solid, liquid, gel, semi-fluid, aerosol or powder.

[0021] Furthermore, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0022] Furthermore, the abundance of Enterococcus gallinarum SY2-1 in liver metastasis tissues was detected by using reagents to further determine the occurrence and development of liver metastasis of colorectal cancer. The above occurrence and development includes the development process of the disease itself, the development changes before and after the use of drugs, etc.

[0023] The second aspect of the present invention is to provide a strain of Enterococcus gallinarum SY2-1, which is classified and named Enterococcus gallinarum, with a preservation number of CCTCC NO: M20241929, a preservation date of September 6, 2024, and a preservation unit of China Center for Type Culture Collection (abbreviated as CCTCC). The preservation unit address is Wuhan University, Wuhan, China.

[0024] Furthermore, the Enterococcus gallinarum SY2-1 was isolated from liver metastases of colorectal cancer in BALB / c model mice.

[0025] Furthermore, the BALB / c model mouse colorectal cancer liver metastasis lesions were obtained from a liver metastasis model initiated from the original site by injecting the mouse colorectal cancer liver metastasis cell line CT26-TL-CL5 (preservation number: CCTCC NO: C2024307) into the cecal serosa of BALB / c mice.

[0026] Furthermore, the 16S rRNA sequence of the Enterococcus gallinarum SY2-1 is shown as SEQ ID NO.1.

[0027] Furthermore, the Enterococcus gallinarum SY2-1 appears as milky white round colonies on Columbia blood agar.

[0028] Furthermore, the Enterococcus gallinarum SY2-1 was stored at -70°C using 25% glycerol.

[0029] The third aspect of the present invention is to provide a microbial agent comprising at least one of the bacterial cells, culture fluid, and metabolites of Enterococcus gallinarum SY2-1 described in any one of the second aspects.

[0030] Furthermore, the live bacteria concentration of the microbial agent is 1×10 7 ~1×10 10 CFU / mL. Specifically, it can be: 1×10 7 ~5×10 7 CFU / mL, 5×10 7 ~1×10 8 CFU / mL, 1×10 8 ~5×10 9 CFU / mL, 5×10 9 ~1×10 10 CFU / mL, etc. Preferably, the live bacteria concentration of the microbial agent is 1×10 9 CFU / mL.

[0031] Furthermore, the preparation steps of the microbial inoculant include: streaking the quail and gall Enterococcus SY2-1 on a brain heart infusion plate, picking a single colony to culture in brain heart infusion broth the next day; after culturing for a predetermined time, centrifuging the bacterial solution, discarding the supernatant, resuspending with an appropriate amount of sterile saline, and adjusting the concentration of the bacterial solution. Specifically, streaking the quail and gall Enterococcus SY2-1 strain on a BHI plate, picking a single colony to BHI broth the next day, and placing it in a shaker for culture (37°C, 200rpm) for 12h; after culturing for 12h, taking the bacterial solution to an EP tube, centrifuging (15,000×g, 5min), discarding the supernatant, resuspending with an appropriate amount of sterile saline, and adjusting the concentration of the bacterial solution to 1×109 CFU / mL.

[0032] The fourth aspect of the present invention is to provide the use of any of the Enterococcus gallinarum described in the second aspect or the microbial agent described in the third aspect, wherein the above-mentioned use is any of the uses described in the first aspect.

[0033] The fifth aspect of the present invention is to provide a method for constructing a colorectal cancer-liver metastasis model, and the steps of any construction method involved in the application of the first aspect are the same, specifically: using a bacterial solution of Enterococcus gallinarum SY2-1, the preservation number of Enterococcus gallinarum SY2-1 is CCTCC NO: M20241929.

[0034] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0035] The present invention found that Enterococcus gallinarum is specifically enriched in liver metastases of patients with colorectal cancer and model mice. The positive / negative detection and abundance of Enterococcus gallinarum are expected to assist in the evaluation of the prognosis or malignancy of patients with colorectal cancer liver metastasis. It provides a potential biological target for the clinical treatment of colorectal cancer liver metastasis, and provides targets and biomarkers for the subsequent development of a combination of specific antimicrobial therapy and conventional intervention strategies to treat colorectal cancer liver metastasis.

[0036] The present invention first constructs an in situ initiated colorectal cancer liver metastasis animal model, isolates, cultures and identifies live bacteria in the liver metastasis foci of the model, thereby determining Gallinarum enterococcus as a candidate bacterium; further combined with clinical sample analysis, it is clarified that Gallinarum enterococcus can play a role in the process of colorectal cancer liver metastasis; finally, two animal models - an in situ initiated colorectal cancer liver metastasis model and a spleen injected colorectal cancer liver metastasis model - are used to perform functional verification using the isolated and identified Gallinarum enterococcus SY2-1, and it is found that Gallinarum enterococcus SY2-1 can significantly promote liver metastasis of colorectal cancer mice, thereby developing a new method for constructing an animal model of colorectal cancer liver metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0038] Figure 1 It is a schematic diagram of the cecal in situ tumor and liver metastasis of the intestinal cancer liver metastasis animal model mouse in one embodiment of the present invention; wherein, 3 weeks after modeling, part a represents the formation of in situ tumor in the mouse, and part b represents the occurrence of liver metastasis.

[0039] Figure 21 is a schematic diagram of the results of the abundance and positive rate statistics of Enterococcus gallinarum in clinical FFPE (Formalin Fixed Paraffin Embedded) samples of colorectal cancer liver metastasis in one embodiment of the present invention; wherein, part a is the relative abundance statistics of Enterococcus gallinarum in colorectal cancer liver metastasis (CRLM) and healthy liver (Healthy liver); part b is the ratio statistics of the number of Enterococcus gallinarum positive tissues in colorectal cancer liver metastasis and healthy liver;

[0040] Figure 3 This is a taxonomic relationship diagram of Enterococcus gallinarum SY2-1 isolated from liver metastasis in one embodiment of the present invention; wherein, the diagram is generated after comparison by the BLAST website.

[0041] Figure 4 This is a colony morphology diagram of the purified Enterococcus gallinarum SY2-1 in one embodiment of the present invention;

[0042] Figure 5 1 is a schematic diagram showing that the intervention of Enterococcus gallinarum SY2-1 in the orthotopic liver metastasis model of colorectal cancer in one embodiment of the present invention can promote liver metastasis; wherein, part a is the bioluminescent signal imaging of the orthotopic tumors of mice in the Enterococcus gallinarum (Eg) group and the normal saline (NS) group 2 and 3 weeks after modeling; part b is the statistics of the bioluminescent signal intensity of the orthotopic tumors of the two groups of mice 2 and 3 weeks after modeling, and there is no significant difference; part c is the bioluminescent signal imaging of the liver metastasis of mice at the end of the experiment; part d is the statistics of the bioluminescent signal of the liver metastasis of mice at the end of the experiment;

[0043] Figure 6 It is a schematic diagram of the results that the intervention of Gallinarum Enterococcus SY2-1 in the spleen injection colorectal cancer liver metastasis model in one embodiment of the present invention can promote liver metastasis; wherein, part a is the bioluminescence signal of the mouse live liver tumor 2 weeks and 3 weeks after modeling, indicating that the tumor cells were successfully injected into the liver and formed liver metastases; part b is the bioluminescence signal of the mouse ex vivo liver tumor 4 weeks after modeling (i.e., the experimental endpoint); part c is the statistical intensity of the bioluminescence signal of the mouse ex vivo liver tumor at the experimental endpoint.

[0044] The above-mentioned Enterococcus gallinarum SY2-1 has been deposited, and its deposit number is CCTCC NO: M20241929. Its classification name is Enterococcus gallinarum. The deposit date is September 6, 2024. The depository is China Center for Type Culture Collection (CCTCC for short), and the depository address is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0046] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0047] The mouse intestinal cancer liver metastasis cell line used in the following examples is the mouse colon cancer cell clone CT26-TL-CL5, whose preservation number is CCTCC NO: C2024307, the preservation date is September 5, 2024, the preservation unit is the China Center for Type Culture Collection (abbreviated as CCTCC), and the preservation unit address is Wuhan University, Wuhan, China.

[0048] The following embodiments are only used to illustrate the technical solutions of the present invention.

[0049] Example 1 - Construction of an animal model of colorectal cancer liver metastasis

[0050] In this example, a mouse colorectal cancer liver metastasis cell line was used to construct an animal model of colorectal cancer liver metastasis. Five 5-week-old BALB / c female mice (Shanghai Xipu-Bikai Experimental Animal Co., Ltd.) were used, and the mouse colorectal cancer liver metastasis cell line CT26-TL-CL5 (deposit number: CCTCC NO: C2024307) was injected into the cecal serosa of BALB / c mice to construct a liver metastasis model starting from the original site. The specific construction process includes the following steps:

[0051] (1) CT26-TL-CL5 cell culture medium contains RPMI-1640 (Gibco, 11875093), 10% fetal bovine serum (Cegrogen, A0500-3010), 1×GlutaMAX TM (Gibco, 35050061) and 100 U / mL penicillin-streptomycin (Yamei, CB010). The cells were inoculated into a 10 cm cell culture dish, and when the cells at the bottom of the dish grew to 60%-80% coverage, the cells were passaged and expanded.

[0052] (2) Digest the cells with 500 μL of trypsin (Gibco, 27250-018) and place in a 37°C incubator for static digestion. After 2 minutes, add culture medium to terminate the digestion. Transfer the cell suspension into a centrifuge tube and centrifuge at 1000 rpm for 3 minutes, then discard the supernatant.

[0053] (3) Add 1 mL of CT26-TL-CL5 cell culture medium to resuspend the cells and passage them at a ratio of 1:3.

[0054] (4) At the same time, take another tube of centrifuged cells, resuspend the cells in an appropriate amount of RPMI-1640 medium, and adjust the cell concentration to 1×10 6 / 15μl, used for the construction of cecal orthotopic CRC liver metastasis mouse model.

[0055] (5) Add an equal amount of Matrigel (Yisheng, 40183ES10) to the cell suspension diluted to an appropriate concentration, mix well and place on ice. Pipette 30 μL of the cell suspension into a 30G insulin syringe and place on ice for later use.

[0056] (6) Anesthesia was induced and maintained in mice with pre-depilated abdomen using 1-3% isoflurane. The epidermis and peritoneum were cut open about 1 cm along the linea alba about 1.5 cm above the mouse anus.

[0057] (7) Use a cotton swab moistened with saline to gently pick out the cecum and place it on a sterile gauze moistened with saline to prepare for cell injection.

[0058] (8) Gently press one side of the cecum with a cotton swab moistened with saline, and place the prepared insulin needle tip with the cell suspension facing upwards. Carefully pierce the subserosa of the cecum and inject the cell suspension into the subserosa. After successful injection, a transparent bubble-like structure will bulge on the surface of the serosa. Allow the needle tip to remain in the subserosa for about 20 seconds, remove the needle, and immediately glue the needle hole with a small amount of medical glue.

[0059] (9) The wound of the mouse was sutured and the mouse was placed on a 37°C heating pad to keep warm. After the mouse recovered, it was placed in a sterile cage, marked with the date of surgery, and returned to a ventilated cage for breeding.

[0060] After 3 weeks of modeling, the mice were euthanized. The liver and cecum were removed after laparotomy. Figure 1 As shown, the above method successfully constructed an animal model of colorectal cancer liver metastasis, which formed in situ tumors in mice and liver metastasis occurred.

[0061] Example 2 - Culture and identification of bacterial flora in liver metastases

[0062] In this example, the liver metastasis obtained in Example 1 was cultured and identified, and the specific steps included:

[0063] 1. Bacterial flora culture and identification;

[0064] (1) Remove the liver metastasis and place it in a sterile 1.5 mL EP tube. Rinse with sterile PBS three times and cut the liver metastasis into 1 mm pieces using autoclaved surgical scissors. 3 Add appropriate amount of collagenase (type II) (Yisheng, 40508ES60) to a final concentration of 1 mg / mL, and incubate at 37°C for 1 h for digestion.

[0065] (2) Use a 200 μL pipette to gently blow the digested liver metastasis tissue to fully disperse the metastatic tumor cells.

[0066] (3) Add 1 mL of HEPE buffer (Gibco, 15630130) to rinse the cells, centrifuge (1000 rpm, 5 min), discard the supernatant, and repeat 3 times. After the last wash, discard the supernatant.

[0067] (4) Add Triton X100 (Biyuntian, P0096-100 mL) to the cell pellet and let it stand at room temperature for 20 min to fully lyse the cells and release possible intracellular bacteria.

[0068] (5) The lysed solution was divided into two equal parts, spread on two Columbia blood plates, and cultured overnight in a normal bacterial incubator (37°C) and an anaerobic incubator (37°C, N 2 :H 2 :CO 2 =8:1:1) and cultured for 2 days.

[0069] (6) Pick a single colony grown on the plate, streak it on a new Columbia blood agar plate (CHROMagar) and further purify the single bacterium.

[0070] (7) Colony PCR was performed on the purified single clone to amplify the full-length sequence of the bacterial 16S rRNA gene.

[0071] The above colony PCR process is briefly described as follows:

[0072] 1) First, prepare bacterial lysate to release bacterial DNA. Take several 1.5mL EP tubes and add 20μL ddH 2 O, pick an appropriate amount of monoclonal clones and add them to the corresponding EP tubes, heat at 95℃ for 20min, and then quickly place on ice to cool down.

[0073] 2) Use the above bacterial lysate for PCR identification. 25 μL reaction system contains: 1 μL each of primers 27F / 1492R (10 μM), 2.5 μL template (bacterial lysate), 12.5 μL 2×Hieff PCR Master Mix (Yisheng, 10102ES), ddH 2 O 8 μL. The PCR reaction conditions were: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1.5 min, 72°C extension for 10 min, 8°C. Steps 2-4 were set for 30 cycles.

[0074] (8) After the PCR reaction was completed, the reaction products were sent to Liuhe BGI Technology Co., Ltd. for first-generation sequencing. After sequencing, the sequences were compared by Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to identify the bacterial species. The identification results are shown in Table 1a and Table 1b.

[0075] Table 1a. Bacterial species and colony numbers isolated and cultured from liver metastases of mice with colorectal cancer liver metastasis model constructed by CT26-TL-CL5 (aerobic culture)

[0076]

[0077] Table 1b. Bacterial species and colony numbers isolated and cultured from liver metastases of mice with colorectal cancer liver metastasis model constructed by CT26-TL-CL5 (anaerobic culture)

[0078]

[0079]

[0080] From the results of Table 1a and Table 1b, it can be seen that according to the number of identified colonies, under aerobic or anaerobic culture conditions, Enterococcus gallinarum was the most abundant bacterium detected in the metastatic lesions, so it was selected as a candidate bacterium for further study of its function.

[0081] Example 3 - Detection of abundance and positive rate of Enterococcus gallinarum in clinical samples

[0082] This embodiment uses clinical samples to detect the abundance and positive rate of Enterococcus gallinarum, which specifically includes the following steps:

[0083] (1) Detection primers:

[0084] ① Detection of Enterococcus gallinarum (reference: Translocation of a gut pathobiont drives autoimmunity in mice and humans. Science. 2018 Mar 9; 359 (6380): 1156-1161.):

[0085] EG-fwd 5'-TTACTTGCTGATTTTGATTCG-3'

[0086] EG-rev 5'-TGAATTCTTCTTTGAAATCAG-3'

[0087] ②16S rRNA (Reference: Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer. Cell. 2022Apr 14; 185(8):1356-1372.e26.):

[0088] Forward 5'-CGGTGAATACGTTCYCGG-3'

[0089] Reverse 5'-GGWTACCTTGTTACGACTT-3'

[0090] (2) Paraffin-embedded samples of colorectal cancer liver metastases were collected from the sample bank of the Pathology Department of the Tenth People's Hospital of Shanghai. DNA was extracted from the samples after sectioning and then analyzed with ddH 2 The DNA sample concentration was diluted to 50 ng / μL, and the abundance of Enterococcus gallinarum in the sample was detected by qRT-PCR (the reagent was TBGreen Premix DimerEraserTM, TAKARA, RR091Q). 20 μL of reaction system was added to a 96-well PCR reaction plate (Monad, MQ50301S): 10 μL of TB Green Premix DimerEraser (2X), 0.6 μL of each of the upstream and downstream primers (10 μM) of PCR, 2 μL of diluted DNA, and ddH 2 O 6.8 μL. The program was set in the LightCycler 96 (Roche Life Science) qRT-PCR instrument: pre-denaturation, 95°C (30 s); PCR reaction, 95°C (5 s), 55°C (30 s), 72°C (30 s), and 40 cycles.

[0091] The above test results are as follows Figure 2 As shown in Figure 2, compared with normal liver, the abundance of Enterococcus gallinarum in colorectal cancer liver metastases was significantly increased ( Figure 2 Part a), and the positive rate increased significantly ( Figure 2 This suggests that Enterococcus gallinarum has the potential to serve as a predictive marker for the development of colorectal cancer liver metastasis and as a biological intervention target for colorectal cancer liver metastasis.

[0092] Example 4 - Identification and preservation of Enterococcus gallinarum SY2-1

[0093] Based on the research results of Examples 2 and 3, this Example identified and analyzed Enterococcus gallinarum isolated from the liver metastasis in Example 2. The isolation and identification steps were referred to Example 2, and the identification results included:

[0094]

[0095] According to the full-length 16S rRNA sequence, the taxonomic relationship generated by BLAST website is as follows Figure 3 As shown. From the above BLAST analysis, it can be seen that the bacteria is closest to Enterococcus gallinarum. The above 16S rRNA sequence has 100% identity with the 16S rRNA sequence of Enterococcus gallinarum B1 strain, and 100% identity with the 16S rRNA sequence of Enterococcus sp. strain CS-6. The 16S rRNA sequence identity with he23 strain, IraqYaHa19 strain, huang82 strain, HCD7-6 strain and other strains is 99%. Therefore, the above strain was identified as Enterococcus gallinarum and named SY2-1.

[0096] (2) The purified Enterococcus gallinarum SY2-1 was cultured on Columbia blood agar overnight and the morphology was as follows: Figure 4 As shown, it appears as milky white round colonies.

[0097] (3) The Enterococcus gallinarum SY2-1 obtained by the above identification has been deposited in a biological depository, and its taxonomic name is Enterococcus gallinarum, and its deposit number is CCTCC NO: M20241929.

[0098] Storage and culture of the above-mentioned Enterococcus gallinarum SY2-1:

[0099] 1) Preservation of strains: Enterococcus gallinarum SY2-1 was stored at -70°C with 25% glycerol for future use;

[0100] 2) Culture of strains: The culture medium used is brain heart infusion broth (liquid culture), brain heart infusion plate or Columbia blood plate (solid culture), and the culture conditions are pH 7.4 and temperature 37°C. Specifically: streak the SY2-1 strain of Enterococcus gallinarum on a BHI plate, pick a single colony and put it into BHI broth the next day, and culture it in a shaker (37°C, 200rpm) for 12 hours; after 12 hours of culture, take the bacterial solution into an EP tube, centrifuge (15,000×g, 5min), discard the supernatant, resuspend it with an appropriate amount of sterile physiological saline, and adjust the bacterial solution concentration (for example, 10 7～ 10 10 CFU / mL, preferably 10 9 CFU / mL).

[0101] Example 5 - Effect of Enterococcus gallinarum SY2-1 intervention on liver metastasis in the orthotopic liver metastasis model of colorectal cancer

[0102] In this example, Enterococcus gallinarum SY2-1 intervenes in an animal model of colorectal cancer liver metastasis initiated from in situ to verify the role of Enterococcus gallinarum SY2-1 in colorectal cancer liver metastasis, which specifically includes the following steps:

[0103] (1) Streak the SY2-1 strain of Enterococcus gallinarum on a BHI plate, pick a single colony and transfer it to BHI broth the next day, and culture it in a shaking incubator (37°C, 200 rpm) for 12 h.

[0104] (2) The OD600 value of the bacterial solution was measured by UV spectrophotometer. After 10-fold gradient dilution, the bacterial solution with different dilutions was spread on BHI plates. The number of colonies was counted the next day and the bacterial concentration was calculated.

[0105] (3) After culturing Enterococcus gallinarum for 12 h, the bacterial solution was transferred to an EP tube and centrifuged (15,000 × g, 5 min). The supernatant was discarded and the suspension was resuspended in an appropriate amount of sterile saline to adjust the bacterial solution concentration to 10 9 CFU / mL, 100 μL was taken for gavage to mice, and the control group mice were gavaged with an equal amount of normal saline.

[0106] (4) Oral gavage was performed every other day. After 3 days of gavage, the mouse cecal serosa was injected with the mouse colorectal cancer liver metastasis cell line CT26-TL-CL5 to construct a colorectal cancer liver metastasis model. For specific steps, see Example 1.

[0107] (5) In the 2nd / 3rd week after modeling, mice were imaged for in situ tumors, and the in situ signal intensity was recorded and counted.

[0108] (6) The experimental endpoint was 3 weeks after modeling. The mice were injected with D-luciferin potassium salt, and the liver was removed for in vitro tumor signal imaging. The signal intensity was recorded and counted.

[0109] The experimental results are as follows Figure 5 As shown by Figure 5 From the a and b parts, we can see that there is no significant difference in the bioluminescence signal intensity of the in situ tumors of the two groups of mice after 2 weeks, 3 weeks, and 4 weeks of modeling. Figure 5 As can be seen from parts c and d, the bioluminescent signal of liver tumor in mice in the group gavaged with Enterococcus gallinarum SY2-1 was significantly stronger than that in the control group, suggesting that intervention with Enterococcus gallinarum SY2-1 significantly promoted liver metastasis in mice.

[0110] Example 6 - Effect of Splenic Injection of Enterococcus Gallinarum SY2-1 on Liver Metastasis in Colorectal Cancer Liver Metastasis Model

[0111] This example uses Enterococcus gallinarum SY2-1 to intervene in the spleen injection colorectal cancer liver metastasis animal model to verify the role of Enterococcus gallinarum SY2-1 in colorectal cancer liver metastasis, which specifically includes the following steps:

[0112] (1) After culturing Enterococcus gallinarum SY2-1 for 12 h, the bacterial solution was transferred to an EP tube and centrifuged (15,000 × g, 5 min). The supernatant was discarded and the suspension was resuspended in an appropriate amount of sterile saline to adjust the bacterial solution concentration to 10 9 CFU / mL, 100 μL was taken for gavage to mice, and the control group mice were gavaged with an equal amount of normal saline.

[0113] (2) Oral gavage was performed every other day. Three days after gavage, CT26-TL-CL5 was injected into the spleen of mice to construct a colorectal cancer liver metastasis model. The specific experimental steps are as follows:

[0114] 1) Digest CT26-TL-CL5 cells, filter the cells through a 70 μm filter, wash the obtained single cells twice with PBS, and resuspend them in an appropriate volume of PBS after the last centrifugation to adjust the cell concentration to 10 7 / mL;

[0115] 2) 5-week-old BALB / c female mice (Shanghai Xipu-Bikai Experimental Animal Co., Ltd.) were used, and the chest and abdomen were depilated. Anesthesia was induced and maintained with 1-3% isoflurane. The epidermis and peritoneum were cut open about 0.5 cm at the lower edge of the left rib of the mouse with a scalpel;

[0116] 3) Moisten a sterile cotton swab with saline, gently pick out the mouse spleen, and place it on a sterile gauze moistened with saline to prepare for cell injection. Mix the cell suspension thoroughly and draw 100μL of cell suspension with a 29G insulin syringe. Inject the needle into the spleen about 1cm at a straight angle and slowly inject the cell suspension;

[0117] 4) Gently press with a cotton swab, and remove the insulin needle after the blister caused by the injection disappears. Continue to press the needle hole for 2-3 minutes;

[0118] 5) Use No. 4 suture to ligate the blood vessels at both ends of the spleen and remove the spleen;

[0119] 6) The wound of the mouse was sutured, and the mouse was placed on a 37°C heating pad to keep warm and wait for recovery. The recovered mouse was placed in a sterile cage, marked with the date of surgery, and returned to a ventilated cage for breeding.

[0120] (3) In the 2nd / 3rd week after modeling, mice were imaged for in situ tumors and the signal intensity of in vivo liver metastases was recorded and counted.

[0121] (4) The experimental endpoint was 4 weeks after modeling, and the mice were injected with D-luciferin potassium salt, and the liver was removed for in vitro tumor signal imaging. The signal intensity was recorded and counted.

[0122] The experimental results are as follows Figure 6As shown in the spleen injection colorectal cancer liver metastasis model, tumor cells were successfully injected into the liver and formed liver metastases. The bioluminescence signal of the liver tumor in the mice gavage with Enterococcus gallinarum SY2-1 was significantly stronger than that in the control group. The results showed that intervention with Enterococcus gallinarum SY2-1 can significantly promote liver metastasis of colorectal cancer in mice.

[0123] As can be seen from the above examples, the present invention, based on the culture method, found that Enterococcus gallinarum was enriched in the liver metastasis of colorectal cancer, which proved that the active Enterococcus gallinarum was colonized in the liver metastasis, suggesting that it may have certain biological functions, so it was further explored as a candidate bacterium. The present invention also proved in clinical samples that the abundance and positive detection rate of Enterococcus gallinarum in the liver metastasis of colorectal cancer in patients were significantly higher than those in normal liver tissue, suggesting that Enterococcus gallinarum has the potential to be a biological intervention target for liver metastasis of colorectal cancer.

[0124] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. An application of Enterococcus gallinarum or a microbial agent thereof, characterized in that: The application is selected from at least one of the following applications: application of Enterococcus gallinarum or its microbial agent in constructing a colorectal cancer liver metastasis model, application of Enterococcus gallinarum or its microbial agent in screening drugs for treating colorectal cancer liver metastasis, application of Enterococcus gallinarum as a predictive marker for the occurrence and development of colorectal cancer liver metastasis, and application of Enterococcus gallinarum as a target for intervention or diagnosis of colorectal cancer liver metastasis.

2. The use according to claim 1, characterized in that: The colorectal cancer liver metastasis model includes a colorectal cancer in situ liver metastasis model and a spleen injection colorectal cancer liver metastasis model; and / or, The drug for treating liver metastasis of colorectal cancer is a drug for reducing the abundance of Enterococcus gallinarum.

3. The use according to claim 1, characterized in that: The steps of constructing the colorectal cancer-liver metastasis model include: Step S1: Adjust the bacterial solution concentration of Enterococcus gallinarum to 10 9 CFU / ml, 100 μL was gavaged into mice; Step S2, the Enterococcus gallinarum liquid was gavaged every other day. After 3 days, the mouse colorectal cancer liver metastasis cell line was injected into the mouse cecal serosa or mouse spleen to construct a colorectal cancer liver metastasis model.

4. A strain of Enterococcus gallinarum SY2-1, characterized in that: The Enterococcus gallinarum is deposited in China Center for Type Culture Collection, and its deposit number is CCTCC NO: M20241929.

5. The Enterococcus gallinarum according to claim 4, characterized in that The Enterococcus gallinarum SY2-1 is isolated from the liver metastasis of colorectal cancer in BALB / c model mice, and its 16S rRNA sequence is shown in SEQ ID NO.

1.

6. A microbial agent, characterized in that: The microbial agent comprises at least one of the bacterial cells, culture solution, and metabolites of Enterococcus gallinarum SY2-1 as described in any one of claims 4 to 5.

7. The microbial agent according to claim 6, characterized in that: The live bacteria concentration of the microbial agent is 1×10 7 ~1×10 10 CFU / mL.

8. The microbial agent according to claim 6, characterized in that: The preparation steps of the microbial agent include: streaking and reviving Enterococcus gallinarum SY2-1 on a brain heart infusion plate, picking a single colony and culturing it in a brain heart infusion broth the next day; after culturing for a predetermined time, centrifuging the bacterial solution, discarding the supernatant, and resuspending it with an appropriate amount of sterile physiological saline to adjust the concentration of the bacterial solution.

9. A use of the Enterococcus gallinarum according to any one of claims 4 to 5, or the microbial agent according to any one of claims 6 to 8, characterized in that: The application is the application described in any one of claims 1 to 3.

10. A method for constructing a colorectal cancer-liver metastasis model, characterized in that: Includes steps: Step A: Adjust the bacterial solution concentration of Enterococcus gallinarum SY2-1 to 10 9 CFU / ml, 100 μL was gavaged into mice; Step B, the bacterial solution of Enterococcus gallinarum SY2-1 was gavaged every other day. After 3 days of gavage, the mouse colorectal cancer liver metastasis cell line was injected into the mouse cecal serosa or mouse spleen to construct a colorectal cancer liver metastasis model; The deposit number of the Enterococcus gallinarum SY2-1 is CCTCC NO: M20241929.

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