Use of b. oleici in the preparation of a drug for preventing or treating colorectal cancer

Abstract

The application discloses application of Bacteroides oleamicus in preparation of drugs for preventing or treating colorectal cancer. The abundance of Bacteroides oleamicus in the present application is significantly reduced in tissues and feces of colorectal cancer patients. In a mouse model, Bacteroides oleamicus can be used for intragastric administration to inhibit the growth of colorectal cancer, and has no significant influence on the weight of mice, the secretion of inflammatory factors in serum and the expression of barrier proteins. The present application proves that Bacteroides oleamicus has important significance for early diagnosis, prevention and treatment of colorectal cancer, and provides a clearer path for clinical application and transformation.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and microbiology, specifically to the application of a type of Bacteroides rich in oleate in the preparation of drugs for the prevention or treatment of colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is a malignant tumor originating from the mucosal epithelium of the colon or rectum. Its global incidence is approximately 6.1%, and its mortality rate is approximately 9.3%. Statistics from 2024 show that there are approximately 2.013 million new cases and 1 million deaths globally each year. In China, there are approximately 556,000 new cases annually, and this number is increasing year by year. Current standard treatments for CRC include surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. However, these therapies have limited effectiveness in treating advanced and metastatic colorectal cancer. Furthermore, there is a lack of effective, low-toxicity preventative interventions for precancerous lesions and high-risk populations. Therefore, effectively preventing and treating CRC is a major public health issue that urgently needs to be addressed.

[0003] Recent studies have shown that gut microbiota dysbiosis is closely related to the occurrence and development of colorectal cancer. (e.g., pks) + Escherichia coli can promote the development of colorectal cancer (CRC) by inducing DNA interstrand cross-linking and double-strand breaks; Fusobacterium nucleatum can promote CRC by inducing chronic inflammation or regulating immunity. Meanwhile, studies have shown that some beneficial bacteria can effectively inhibit CRC development. For example, Bifidobacterium can regulate gut microbiota and immune responses and inhibit the proliferation of colon cancer cells; Lactobacillus can reduce the expression of inflammatory factors and inhibit carcinogen-induced CRC development. Therefore, based on the multiple regulatory effects of gut microbiota on CRC, developing a beneficial bacteria-based drug that can effectively prevent and treat CRC is an urgent clinical need. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides the application of Bacteroides oleate in the preparation of drugs for the prevention or treatment of colorectal cancer, providing a new microbial intervention strategy for the prevention and treatment of colorectal cancer, thereby overcoming the current technical problem in the field of the urgent need for effective drugs for the treatment of colorectal cancer.

[0005] The above-mentioned objective of this invention is achieved through the following technical solutions.

[0006] In a first aspect, the invention provides the use of Bacteroides oleate or its derivatives in the preparation of medicaments for the prevention or treatment of colorectal cancer.

[0007] In this invention, the term "oleic acid-rich Bacteroides" belongs to the phylum Bacteroidetes, class Bacteroidetes, order Bacteroidetes, family Bacteroidetes, and genus Bacteroides. It is a Gram-negative, non-motile, non-spore-forming, obligate anaerobic intestinal commensal bacterium that primarily colonizes the human gut microbiota. This species possesses strong oleic acid accumulation and lipid metabolism capabilities, participating in the breakdown and transformation of fatty acids and carbohydrates in the intestine, as well as the generation of short-chain fatty acids. It plays a crucial physiological role in regulating host intestinal barrier function, lipid metabolism homeostasis, immune microenvironment balance, and intestinal flora remodeling.

[0008] Furthermore, the oleic acid-rich Bacteroides is a live bacterium.

[0009] In this invention, the term "live bacteria" refers to live microbial cells prepared through a specific process that possess complete metabolic activity and reproductive capacity. Their core characteristics lie in maintaining the integrity of their cell structure and the normal functioning of their physiological processes. They are capable of in-situ colonization, proliferation, or temporary survival within the host body, and exert probiotic functions through mechanisms such as occupancy effects, nutrient competition, metabolic product secretion, and immune system interactions.

[0010] Furthermore, the derivatives of the oleic acid-rich Bacteroides include bacterial lysates or structural components thereof, and bacterial ferments.

[0011] In this invention, the term "bacterial lysate" refers to a mixture of non-living bacterial contents obtained after the bacterial cell wall and membrane structure have been completely broken down by physical, chemical, or enzymatic methods and then purified. Its components mainly include cell wall fragments, intracellular proteins, nucleic acids, and organelle remnants, and no longer contain intact live bacteria. Its structural components refer to specific structural substances isolated and purified from the lysate, including cell walls, exosomes, inner membranes, and surface appendages. The core mechanism of action of this lysate or its structural components relies on the pathogen-associated molecular patterns it retains, enabling it to mimic the natural infection process and induce a non-specific immune response by activating host pattern recognition receptors.

[0012] In this invention, the term "bacterial fermentation product" refers to the cell-free liquid component obtained after fermentation in a specific culture medium, through processes such as centrifugation and filtration to remove bacterial cells. Its functional components are entirely derived from metabolic products secreted extracellularly during bacterial growth, mainly including short-chain fatty acids, organic acids, bacteriocins, enzymes, and quorum sensing signaling molecules, among other bioactive substances. Because it does not contain bacterial cells themselves, its mechanism of action does not depend on the survival and colonization of microorganisms, but rather on the direct, exogenous supplementation of small-molecule metabolites to rapidly intervene in the local microenvironment, thereby achieving the goal of disease intervention.

[0013] Furthermore, the unit dose of the drug is not less than 1.0 × 10⁻⁶. 8 CFU.

[0014] Furthermore, the unit dose of the drug is 1.0 × 10⁻⁶. 8 CFU.

[0015] Furthermore, the dosage forms of the drug include oral liquid, granules, lyophilized powder, tablets, or capsules.

[0016] Furthermore, the dosage form of the drug is an oral liquid.

[0017] In this invention, the term "oral liquid" refers to a liquid preparation made from bacteria and suitable excipients through a dispersion process, specifically for oral administration. Its physical form includes true solutions, colloidal solutions, or suspensions, and it must meet strict standards for microbial limits, pH value, and stability, featuring rapid absorption and flexible dosage.

[0018] In this invention, the term "granule" refers to a dry, granular solid preparation with a specific particle size, made by uniformly mixing live bacteria cells or bacterial derivatives as the main active pharmaceutical ingredient with suitable pharmaceutical excipients, and then processing them through granulation, drying, and sizing processes. This preparation can be taken by dissolving it in warm water. Depending on the prescription and process, it can be made into ordinary granules or enteric-coated granules. It effectively protects the activity and functional structural components of the bacteria. After oral administration into the digestive tract, it disintegrates and disperses, releasing active substances, thereby exerting its effects on intestinal microecological regulation, immune regulation, and corresponding pharmacological functions.

[0019] In this invention, the term "lyophilized powder" refers to a porous, loose solid powder formed by removing moisture from a bacterial liquid suspension through a freeze-drying process under low-temperature vacuum conditions. This dosage form, by immobilizing drug molecules in a glassy matrix, maximizes the protection of the stability of heat-sensitive and easily hydrolyzed drugs, and requires reconstitution with a suitable solvent before use.

[0020] In this invention, the term "tablet" refers to a solid preparation made by using live bacterial cells or bacterial derivatives as the main active pharmaceutical ingredient, combined with suitable pharmaceutical excipients, and processed through formulation processes such as pulverization, compounding, mixing, granulation or non-granulation, and compression molding. Such tablets can be made into ordinary tablets, enteric-coated tablets, chewable tablets, dispersible tablets, etc., according to drug release requirements. They are administered orally and can disintegrate in the stomach or intestines to release the active pharmaceutical ingredient, thereby exerting microecological regulation or corresponding pharmacological effects.

[0021] In this invention, the term "capsule" refers to a solid dosage form made by using live bacterial cells or bacterial derivatives as the active pharmaceutical ingredient, combined with suitable pharmaceutical excipients to form a content, which is then filled into a hollow hard capsule or enteric-coated capsule shell. Depending on the capsule shell material, it can be divided into gastric-soluble and enteric-coated types. Gastric-soluble capsules dissolve and release rapidly in the stomach, while enteric-coated capsules resist gastric acid and dissolve in the pH environment of the intestine. The capsule shell effectively masks unpleasant drug odors, isolates against photo-oxidative degradation, and can achieve colon-targeted delivery through enteric coating. This dosage form has high bioavailability and better gastrointestinal tolerance to irritating drugs than tablets.

[0022] In this invention, the term "drug" refers to a class of biological products that use at least one of live bacteria, bacterial lysates or their structural components, or bacterial ferments as the main pharmaceutical active substance, add other anti-colorectal cancer active ingredients and / or suitable pharmaceutical excipients, and are manufactured by modern pharmaceutical processes, and have the functions of preventing, treating or assisting in improving colorectal cancer-related lesions and regulating the body's physiological functions, and that meet the drug quality standards, prescribed indications and dosage.

[0023] Furthermore, the other active ingredients for treating colorectal cancer include probiotics, chemotherapy drugs, immunomodulators, targeted drugs, or anti-inflammatory drugs.

[0024] In this invention, the term "probiotics" refers to a class of live microorganisms that, when ingested in sufficient doses, can colonize the host's gut and exert beneficial effects on the host's physiological health by regulating the gut microbiota structure and enhancing the intestinal mucosal barrier function.

[0025] Furthermore, the pharmaceutical excipients include pharmaceutically acceptable carriers and prebiotics.

[0026] In this invention, the term "pharmaceuticalally acceptable carrier" refers to a class of pharmaceutical excipients that meet pharmaceutical safety standards, have good biocompatibility, have no direct pharmacological therapeutic activity, can carry, disperse, encapsulate or deliver active pharmaceutical ingredients, can participate in formulation formation, maintain drug stability, regulate drug release, and have no obvious toxic side effects or irritation to the body after administration, and do not have adverse compatibility reactions with the active ingredients in the drug.

[0027] In this invention, the term "prebiotic" refers to an inactive food component that can resist digestion in the host's upper digestive tract, reach the colon intact, and be selectively fermented and utilized by beneficial intestinal bacteria, thereby specifically promoting their growth, enhancing their metabolic activity, or improving their community composition.

[0028] Furthermore, the drug described in this invention can be administered via the gastrointestinal tract or by injection. Gastrointestinal administration includes oral administration, sublingual administration, and rectal administration; injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intraperitoneal injection; in some embodiments, oral administration is preferred.

[0029] Furthermore, the colorectal cancers include tubular adenocarcinoma, papillary adenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, undifferentiated carcinoma, adenosquamous carcinoma, medullary carcinoma, micropapillary carcinoma, serrated adenocarcinoma, and cribriform comedo adenocarcinoma.

[0030] In this invention, the term "colorectal cancer" is a general term for colon cancer and rectal cancer, covering the entire large intestine segment from the cecum to the dentate line of the rectum. In clinical epidemiology, due to the high similarity between the two in etiology, pathology, and treatment principles, they are often combined in statistics and studies and collectively referred to as colorectal cancer.

[0031] In this invention, the term "prevention" refers to a series of organized and purposeful interventions conducted before the onset of a disease or in its early stages, through proactive and controllable interventions to eliminate or reduce pathogenic risk factors, block exposure to pathogens, and enhance the body's defense capabilities, thereby preventing the occurrence of the disease, delaying its progression, reducing the frequency of onset, mitigating the severity of the condition, and reducing the risk of complications and death. In some embodiments, the term refers to minimizing the spread or worsening of the disease in its early stages, achieved through the administration of one or more preventative agents to patients with the disease.

[0032] In this invention, the term "treatment" refers to reducing, improving, or eradicating a disease or one or more symptoms related to a disease. In some embodiments, the term refers to minimizing the spread or worsening of a disease due to the administration of one or more therapeutic agents to a patient suffering from the disease. For the purposes of the various aspects and embodiments provided by this invention, treatment includes, but is not limited to, reducing, alleviating, or improving one or more clinical manifestations or side effects of the treated disease or condition, improving one or more clinical outcomes, reducing the severity of the disease, delaying or slowing disease progression, improving, alleviating, or stabilizing a disease state, and other beneficial results described in this invention.

[0033] A second aspect of the present invention provides the use of a reagent for detecting Bacteroides oleate expression in the preparation of colorectal cancer diagnostic products, said reagent being used to detect Bacteroides oleate-specific gene sequences, protein antigens, or metabolites in subject samples.

[0034] Furthermore, the products include reagent kits, biochips, and test strips.

[0035] Furthermore, the biochip includes a gene chip or a protein chip.

[0036] Furthermore, the product includes a storage container and / or an application device.

[0037] Furthermore, the storage container includes storage bottles, storage bags, and storage boxes.

[0038] Furthermore, the application device includes a pre-filled syringe, an automatic injection pen, a needle-free injector, and an enema administration device.

[0039] Furthermore, the reagents include those for detecting Bacteroides oleate-specific gene sequences, protein antigens, or metabolites in subject samples using the following detection methods: qPCR, high-throughput sequencing, fluorescence in situ hybridization, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, protein immunoblotting, mass spectrometry, and chromatography.

[0040] Furthermore, the reagent includes specific primers for detecting Bacteroides oleate 16S rRNA, the nucleotide sequences of which are shown in SEQ ID NO:1-30.

[0041] Furthermore, the reagent is used to specifically detect the protein antigens of the Bacteroides oleate, the protein antigens including surface protein antigens or secretory protein antigens.

[0042] Furthermore, the reagent is used to specifically detect the metabolites of the oleic acid-rich Bacteroides, which include short-chain fatty acids, lipid metabolism derivatives, vitamins, peptides, and sugar metabolism products.

[0043] Furthermore, the subject samples include fecal samples, intestinal mucosal tissue samples, intestinal fluid samples, and peripheral blood samples.

[0044] In some implementations, the subject sample is a fecal sample or a peripheral blood sample.

[0045] Furthermore, the diagnosis includes one or more of the following: auxiliary diagnosis, early screening, prognostic assessment, or efficacy monitoring.

[0046] A third aspect of the invention provides the use of Bacteroides oleate in screening drugs for the treatment of colorectal cancer, said drugs being able to increase the abundance or activity of Bacteroides oleate in subject samples.

[0047] A fourth aspect of the present invention provides a method for in vitro screening of therapeutic drugs for colorectal cancer, the method comprising: 1) The drug to be screened was added to the Bacteroides oleate infection model, and a blank control group and a positive control group were set up and incubated together for an appropriate time; 2) Detect changes in at least one of the following indicators before and after drug treatment: abundance, activity, or metabolite level of Bacteroides rich in oleate; 3) Set a screening threshold. If at least one of the indicators increases significantly after drug treatment compared to before treatment, the drug is determined to be a potential treatment for colorectal cancer.

[0048] Furthermore, the drug to be screened is obtained by at least one of the following methods: selection from a compound library, chemical synthesis, or extraction from natural products.

[0049] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: (1) This invention has determined that the abundance of Bacteroides rich in oleate is significantly reduced in colorectal cancer patients, and that Bacteroides rich in oleate can be used as a biomarker for the diagnosis of colorectal cancer.

[0050] (2) This invention has determined that Bacteroides rich in oleate can significantly inhibit tumor growth in both chemically induced and cell transplantation-induced colorectal cancer animal models.

[0051] (3) The present invention has determined that Bacteroides rich in oleate has no significant effect on mouse body weight, secretion of inflammatory factors and expression of barrier proteins. Therefore, Bacteroides rich in oleate has high safety and has broad application potential in the prevention and treatment of colorectal cancer. Attached Figure Description

[0052] Figure 1 This is a comparative analysis of the abundance of Bacteroides rich in oleate in feces and tumor tissues of colorectal cancer patients and healthy individuals. Figure A shows the statistical analysis of the relative expression level of Bacteroides rich in oleate in tissues; Figure B shows a representative fluorescent staining image of Bacteroides rich in oleate in intestinal tissues; and Figure C shows the statistical analysis of the relative expression level of Bacteroides rich in oleate in feces.

[0053] Figure 2 This is a graph showing the effect of Bacteroides rich in oleate on colorectal cancer in an AOM / DSS-induced spontaneous tumorigenesis model of mouse intestinal cancer. A is a flowchart of the AOM / DSS-induced spontaneous tumorigenesis model in mice; B is a photograph of mouse colon tumors; C is a statistical analysis of the number of mouse colon tumors; and D is a statistical analysis of the percentage of mouse colon tumor diameter.

[0054] Figure 3 The diagram shows the effects of Bacteroides oleate on colorectal cancer in a mouse subcutaneous tumor model. A is a flowchart of the mouse subcutaneous tumor modeling process; B is a photograph of the mouse subcutaneous tumor; C is a statistical analysis diagram of the mouse subcutaneous tumor volume; and D is a statistical analysis diagram of the mouse subcutaneous tumor mass.

[0055] Figure 4 The diagram shows the safety assessment results of Bacteroides oleate, where A is the flowchart of Bacteroides oleate gavage treatment; B is the statistical analysis of mouse body weight; C is the statistical analysis of tumor necrosis factor-α expression in mouse serum; D is the statistical analysis of interleukin-6 expression in mouse serum; and E is a representative image of MUC2, ZO-1, and E-Cadherin staining in mouse intestines. Detailed Implementation

[0056] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0057] Example 1: Comparative analysis of the abundance of Bacteroides rich in oleate in the feces and tumor tissues of colorectal cancer patients and healthy individuals. I. Experimental Materials 1.1 Sample Source This study collected 710 colorectal cancer tissue samples, 227 normal mucosal tissue samples, 90 stool samples from colorectal cancer patients, and 90 stool samples from healthy individuals. All samples were collected from outpatients and inpatients at Sir Run Run Shaw Hospital, affiliated with Zhejiang University School of Medicine. The inclusion and exclusion criteria for colorectal cancer patients are as follows.

[0058] Inclusion criteria: 1) Histopathologically confirmed colorectal cancer; 2) Age between 18 and 90 years at the time of surgery or procedure; 3) No history of anticancer treatment prior to sample acquisition; 4) High-quality frozen stool specimens available for DNA extraction and 16S rRNA sequencing in the biobank; 5) Complete and necessary clinical and pathological data in the electronic medical record.

[0059] Exclusion criteria: 1) Use of antibiotics, prebiotics or probiotics within one month prior to the procedure; 2) Diagnosis of other gastrointestinal diseases; 3) History of colorectal resection; 4) Unqualified tissue quality or lack of key clinical data.

[0060] 1.2 Reagents and instruments: as shown in Table 1.

[0061] Table 1 Experimental Reagents

[0062] II. Experimental Methods 2.1 Sample DNA Extraction DNA was extracted from tissue and fecal samples using the QIAamp PowerFecal (Pro) DNA kit. Weigh 0.25 g of fecal sample or 0.1 g of tissue sample, add 0.7 mL of Buffer CD1, and vortex for 1 min. Place the sample tube in a tissue homogenizer and homogenize at 6.0 m / s for 40 s. Centrifuge (15000×g, 1 min), and transfer the supernatant to a new tube. Add Buffer CD2, vortex for 5 s, incubate at 4℃ for 5 min, and centrifuge. Add Buffer CD3 to the supernatant, mix well, and transfer to an MB Spin Column. Centrifuge (15000×g, 1 min). Wash sequentially with Buffer CD4 and C5, and finally elute the DNA with 50 μL of Buffer C6. DNA concentration and purity were measured using a spectrophotometer and stored at -20℃ for later use.

[0063] 2.2 DNA sample qPCR amplification qPCR was used to quantitatively detect the 16S fragment in the sample DNA. Primer sequences are shown in Table 2. Reaction system (20 μL): 10 μL SYBR Green Master Mix, 0.5 μL each primer, 2 μL template DNA, and ddH2O to a final volume of 20 μL. Reaction program: 95 ℃ for 3 min; 95 ℃ for 15 s, 60 ℃ for 30 s, 72 ℃ for 30 s, for a total of 40 cycles; Melting curve analysis: 95 ℃ for 15 s, 60 ℃ for 1 min, 95 ℃ for 15 s. A standard curve was prepared using standards, and the 16S copy number in the sample was calculated.

[0064] Table 2 Primer Sequences

[0065] 2.3 DNA enrichment and library construction: 1) Samples that tested negative by qPCR were removed, and the remaining samples were enriched and amplified with the 16S V4 fragment. Synthetic biotin-labeled 16S V4 primers were used; the primer sequences are shown in Table 3 (515F.1-4 and 805R.1-24 were used in combination). PCR amplification was performed for 25-30 cycles using the kit required by the Illumina MiSeq sequencer (Nextera XT Index Kit), depending on the bacterial content in the tissue. Reaction system: 25 μL of 2×KAPA HiFi HotStart ReadyMix, 1.25 μL of each primer, 10 μL of template DNA (approximately 50 ng), and water to a final volume of 50 μL. Cycling parameters: 95 ℃ for 3 min; 98 ℃ for 20 s, 55 ℃ for 30 s, 72 ℃ for 30 s, 28 cycles; 72 ℃ for 5 min.

[0066] Table 3 Primer Sequences

[0067] 2) Enrichment and precipitation of biotin-labeled amplified fragments were performed using Dynabeads MyOne Streptavidin C1 Beads. 50 μL of magnetic bead suspension was washed twice with 100 μL of 1×B&W buffer and resuspended in 50 μL of 2×B&W buffer. 50 μL of PCR product was added, mixed, and incubated at room temperature for 15 min. The mixture was then placed on a magnetic rack for 1 min, and the supernatant was discarded. The mixture was washed three times with 200 μL of 1×B&W buffer, and then once with 200 μL of TE buffer. Finally, the magnetic bead-DNA complex was resuspended in 20 μL of sterile water. The enriched and precipitated fragments (without eluting the magnetic beads) were further used for sequencing library construction.

[0068] 3) Add 25 μL of 2×KAPA HiFi ReadyMix, 5 μL of NexteraXT Index primers (N7xx and S5xx), and 20 μL of water to the above magnetic bead-DNA complex, and perform 8 cycles of PCR amplification (98℃ for 20 s, 55℃ for 30 s, and 72℃ for 30 s). After completion, purify the library with Agencourt AMPure XP magnetic beads (1:1 volume ratio), quantify using Qubit, detect fragment size (approximately 390 bp) using an Agilent 2100, and finally perform paired-end 250 bp sequencing on the Illumina MiSeq platform.

[0069] 2.4 Fluorescence in situ hybridization Tissue sections (4 μm) were dewaxed (xylene, 10 min × 2 times), rehydrated with a gradient of ethanol (100%, 95%, 75%, 5 min each), and washed with PBS. They were then treated with proteinase K (10 μg / mL) at 37°C for 15 min, followed by washing with PBS. Hybridization buffer containing 5 ng / μL of a 5'-Cy3-labeled specific probe was added, covered with a coverslip, and denatured at 80°C for 5 min, followed by overnight hybridization in a humidified chamber at 37°C. The next day, the sections were washed twice at 48°C for 15 min each with pre-warmed washing buffer (20 mM Tris-HCl, 0.1 M NaCl, 0.01% SDS), followed by washing with PBS. DAPI staining was performed for 5 min, and the sections were mounted with anti-quenching mounting medium. The sections were observed and photographed under a fluorescence microscope. Five high-power fields were randomly selected from each section, and the proportion of Cy3 fluorescence signal area to the total nuclear area was calculated for comparative analysis of Bacteroides oleate abundance.

[0070] 2.5 Data Analysis 16S sequence analysis (DADA2 denoising and feature table generation) was performed using QIIME2 software (version 2020.2), and statistical analysis was performed using R software (version 4.1). Comparisons between two groups were performed using the Mann-Whitney U test (for non-normal distribution) or the independent samples t-test (for normal distribution), while comparisons among multiple groups were performed using the Kruskal-Wallis test. Results are expressed as mean ± standard deviation (SD), and p < 0.05 was considered statistically significant. GraphPad Prism 9.0 was used for plotting.

[0071] III. Experimental Results Figure 1 A showed that sequencing results of clinical samples indicated that the relative abundance of Bacteroides rich in oleate was significantly reduced in tissue samples from colorectal cancer patients compared to normal intestinal tissue in the control group. Figure 1 B shows that fluorescence in situ hybridization staining results indicate that the positive area ratio of Bacteroides oleate in colorectal cancer tissue is significantly lower than that in normal tissue. Figure 1 The results showed that, compared to fecal samples from healthy controls, the relative abundance of *Bacteroides oleate* was significantly lower in fecal samples from colorectal cancer patients. These results indicate that the abundance of *Bacteroides oleate* is significantly lower in colorectal cancer patients compared to healthy individuals.

[0072] Example 2: Regulation of disease progression in a mouse model of colorectal cancer by Bacteroides oleate I. Experimental Materials 1.1 Experimental animals: C57BL / 6 mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0073] 1.2 Experimental strain: Bacteroides oleate was purchased from Shanghai Yushao Biotechnology Co., Ltd., strain number YS-2021-001.

[0074] 1.3 Experimental cells: The MC38 cell line was donated by Professor Song Zhangfa's research group at the Sir Run Run Shaw Hospital affiliated with Zhejiang University School of Medicine.

[0075] 1.4 Experimental reagents: as shown in Table 4.

[0076] Table 4 Experimental Reagents

[0077] II. Experimental Methods 2.1 Anaerobic culture of Bacteroides oleate in vitro Bacteroides rich in oleic acid (1×10) 6 CFU was inoculated into modified GAM medium and cultured in an anaerobic incubator at 37°C (85% N2, 10% CO2, 5% H2) for 2 days. After 3 days, the concentration was measured at 1.0 × 10⁻⁶. 9 CFU / mL. The culture medium was centrifuged at 3000 rpm for 10 min at 4℃, the bacterial pellet was collected, washed twice with sterile PBS, and resuspended in PBS for later use (heat-inactivated bacterial cells need to be incubated in a water bath at 80℃ for 30 min).

[0078] 2.2 Animal husbandry Four-week-old male C57BL / 6 mice, weighing 20-25 g, were housed in the animal facility of Sir Run Run Shaw Hospital, affiliated with Zhejiang University School of Medicine. The housing temperature was 22 ± 2℃, and the humidity was 55 ± 5%. The bedding was sterilized with gamma rays and changed twice a week. Five mice were housed per cage, with 12 hours of light and 12 hours of darkness daily, fed standard gamma-ray sterilized feed and acidified water. All mice were housed in the animal facility for one week after purchase to acclimatize before the experiments were conducted.

[0079] 2.3 Establishment of an AOM / DSS-induced mouse model of colorectal cancer Mice were divided into a control group and a colorectal cancer model experimental group, with 14 mice in each group. Ten days prior to the procedure, the intestinal flora was cleared by gavage with antibiotics (ampicillin 1 g / L, metronidazole 1 g / L, dissolved in drinking water) and an equal volume of PBS (200 μL daily). Mice in the experimental group were intraperitoneally injected with azomethine (AOM), and the following day, their drinking water was replaced with 2.5% DSS for one week, followed by two weeks of regular drinking water. This DSS-for-one-week and drinking water-for-two-week feeding regimen was repeated for three cycles. During this period, the experimental group mice were given 1.0 × 10⁻⁶ DSS daily between drinking water intervals. 8CFU of Bacteroides oleate (resuspended in 200 μL PBS) was administered by gavage, while the control group received the same volume of PBS daily during the water-drinking interval. After 70 days of AOM / DSS induction, mice were euthanized by cervical dislocation, and the anal canal to the colonic segment was removed via laparotomy. The intestine was dissected along the longitudinal axis, and after cleaning the feces with PBS, the number of intestinal tumors was counted and the maximum diameter was measured under a dissecting microscope. The colonic tissue was fixed by immersion in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick sections for hematoxylin-eosin (HE) staining.

[0080] 2.4 Construction of a mouse subcutaneous tumor-bearing model induced by MC38 cell inoculation Fourteen C57BL / 6 mice were randomly divided into two groups: half received antibiotics to eliminate gut microbiota 14 days prior to treatment, while the other half did not. Subcutaneous tumors were then inoculated with MC38 cells: MC38 cells in logarithmic growth phase were digested with trypsin, washed with PBS, resuspended, and adjusted to a concentration of 5 × 10⁻⁶. 7 100 μL (5 × 10 cells / mL) was injected subcutaneously into the right axilla of each mouse. 6 Cells). When the subcutaneous tumor grows to approximately 50 mm³ (about 7 days after inoculation), administer live or heat-inactivated Bacteroides oleate via gavage daily. 8 CFU / mouse was administered via gavage for 3 consecutive weeks. The control group was administered an equal volume of PBS via gavage. After 3 weeks, mice were euthanized by cervical dislocation, and the subcutaneous tumors were completely dissected, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick sections for HE staining.

[0081] 2.5 Data Analysis Statistical analysis was performed using SPSS 26.0. One-way ANOVA followed by Tukey's multiple comparison test was used for comparisons among multiple groups. p < 0.05 was considered statistically significant.

[0082] III. Experimental Results The procedure for AOM / DSS-induced spontaneous tumorigenesis modeling of intestinal cancer in mice is as follows: Figure 2 As shown in A, the intestinal flora was cleared with antibiotics 10 days in advance, followed by intraperitoneal injection of AOM and DSS water induction of intestinal cancer in mice. During the DSS water induction period, the experimental group was given Bacteroides rich in oleic acid by gavage every day. After 3 cycles of induction, the mice were sacrificed and samples of colorectal tissue were taken and photographed. Figure 2 B. Figure 2 C Figure 2 D showed that, compared with the control group, the average number of tumors and the average maximum tumor diameter were significantly reduced in the *Bacteroides oleate* treatment group. The procedure for subcutaneous tumor modeling in mice is as follows: Figure 3As shown in Figure A, half of the mice were given antibiotics to clear their intestinal flora 14 days in advance, while the other half were not given intestinal flora. They were then inoculated with subcutaneous tumors. When the subcutaneous tumors grew to 50 mm³, they were given live Bacteroides rich in oleate or heat-inactivated Bacteroides rich in oleate by gavage daily for 3 weeks. The mice were then sacrificed and the results were recorded. Figure 3 B. Figure 3 C Figure 3 The results showed that, compared with the antibiotic clearance group, the tumor volume and weight were significantly reduced in the live Bacteroides oleate treatment group, while no significant changes were observed in the heat-inactivated Bacteroides oleate treatment group. In conclusion, live Bacteroides oleate treatment significantly inhibited tumor progression in both the AOM / DSS-induced spontaneous tumorigenesis model and the MC38 cell injection-induced subcutaneous tumor-bearing model in mice.

[0083] Example 3 Safety assessment of Bacteroides rich in oleate I. Experimental Materials 1.1 Experimental animals: Same as section 1.1 in Example 2.

[0084] 1.2 Experimental bacterial strains: Same as section 1.2 in Example 2.

[0085] 1.3 Experimental reagents: as shown in Table 5.

[0086] Table 5 Experimental Reagents

[0087] II. Experimental Methods 2.1 In vitro culture of Bacteroides rich in oleate The method is the same as in Section 2.1 of Example 2.

[0088] 2.2 Animal husbandry Six-week-old male C57BL / 6 mice, weighing 20-25 g, were housed in the animal facility of Sir Run Run Shaw Hospital, affiliated with Zhejiang University School of Medicine. The housing temperature was 22 ± 2℃, and the humidity was 55 ± 5%. The bedding was sterilized with gamma rays and changed twice a week. Five mice were housed per cage, with 12 hours of light and 12 hours of darkness daily, fed standard gamma-ray sterilized feed and acidified water. All mice were housed in the animal facility for one week after purchase to acclimatize before the experiments were conducted.

[0089] 2.3 Animal handling Twenty mice were randomly divided into a control group and an experimental group, with 10 mice in each group. Each group of mice was housed in two cages, with 5 mice in each cage. The experimental group mice were administered 200 μL of Bacteroides oleate (2 × 10⁻⁶) via gavage daily. 8CFU was administered to mice, while the control group received an equal volume of sterile PBS via gavage. Gavage was continued for 14 days. The weight of each mouse was measured using an electronic balance before gavage began (day 0) and on day 14. After 14 days of gavage, mice were fasted for 12 hours (with free access to water). After anesthesia with isoflurane inhalation, approximately 0.5 mL of blood was collected from the orbital venous plexus, allowed to stand at room temperature for 30 min, and then centrifuged at 3000 rpm for 15 min at 4°C. The serum was separated and stored at -80°C. After blood collection, mice were euthanized by cervical dislocation, and colon tissue (2-5 cm from the anus) was harvested via laparotomy. The contents were rinsed with PBS, and a 1 cm segment was cut and fixed in 4% paraformaldehyde.

[0090] 2.4 ELISA Experiment The concentrations of TNF-α, IL-6, and IL-1β in serum were detected using an ELISA kit (R&D Systems). The procedure was as follows: add standard or serum sample to each well, add detection antibody, incubate for 2 h, wash the plate, add HRP substrate, develop color, and immediately read the OD value using a microplate reader (450 nm wavelength) after termination. The concentration was calculated based on the standard curve.

[0091] 2.5 Immunohistochemical staining Fixed tissues were routinely embedded in paraffin and sectioned (4 μm). Sections were dewaxed to water, microwaved with citrate buffer (pH 6.0) for 10 min on medium-high heat, and cooled to room temperature. Endogenous peroxidase was blocked with 3% H2O2 for 10 min. Primary antibodies (ZO-1, E-Cadherin, and MUC2, all diluted 1:200) were added and incubated overnight at 4°C. After washing with PBS, HRP-labeled secondary antibody (1:500) was added and incubated at room temperature for 30 min. DAB staining was performed (2-5 min, controlled under a microscope), followed by hematoxylin counterstaining for 30 s, differentiation with hydrochloric acid ethanol, dehydration and clearing, mounting with neutral resin, and observation and photography under a microscope.

[0092] 2.6 Data Analysis The method is the same as in Section 2.5 of Example 1.

[0093] III. Experimental Results Figure 4 A is a schematic diagram of the mouse treatment method. Healthy C57BL / 6 mice were continuously administered PBS or Bacteroides rich in oleate (2×10⁻⁶) by gavage. 8 (CFU / day) for a total of 14 days. Weighing was performed on days 0 and 14, and blood and colon tissue were collected on day 14 for testing. Figure 4 B shows that statistical analysis of mouse body weight indicates that there was no significant change in mouse body weight before and after treatment with Bacteroides oleate. Figure 4 C Figure 4D showed that the ELISA results indicated that there were no significant changes in the expression levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum of healthy mice after gavage with Bacteroides oleate. Figure 4 E. Immunohistochemical results showed that the expression of barrier proteins such as ZO-1, E-Cadherin, and MUC2 in the intestines of healthy mice treated with *Bacteroides oleate* did not change significantly. In summary, compared with the control group, the mice in the *Bacteroides oleate* treatment group showed no significant differences in body weight, inflammatory factors, and intestinal barrier proteins, indicating no significant adverse reactions. This demonstrates that *Bacteroides oleate* has high safety.

[0094] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of Bacteroides oleate or its derivatives in the preparation of drugs for the prevention or treatment of colorectal cancer.

2. The application according to claim 1, characterized in that, The oleic acid-rich Bacteroides is a live bacterium.

3. The application according to claim 1, characterized in that, The derivatives include bacterial lysates or their structural components, and bacterial ferments.

4. The application according to claim 1, characterized in that, The unit dose of the drug is not less than 1.0 × 10⁻⁶. 8 CFU.

5. The application according to claim 1, characterized in that, The dosage forms of the drug include oral liquid, granules, lyophilized powder, tablets or capsules.

6. The application of a reagent for detecting Bacteroides oleate expression in the preparation of colorectal cancer diagnostic products, characterized in that, The reagent is used to detect Bacteroides rich in oleate-specific gene sequences, protein antigens, or metabolites in subject samples.

7. The application according to claim 6, characterized in that, The reagents include those used to detect Bacteroides oleate-specific gene sequences, protein antigens, or metabolites in subject samples using the following detection methods: qPCR, high-throughput sequencing, fluorescence in situ hybridization, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, protein immunoblotting, mass spectrometry, and chromatography.

8. The application according to claim 6, characterized in that, The reagent includes specific primers for detecting Bacteroides oleate 16S rRNA, the nucleotide sequences of which are shown in SEQ ID NO:1-30.

9. The application of Bacteroides oleate in screening drugs for colorectal cancer treatment, characterized in that, The drug can increase the abundance or activity of Bacteroides rich in oleate in subject samples.

10. A method for in vitro screening of therapeutic drugs for colorectal cancer, the method comprising: 1) The drug to be screened was added to the Bacteroides oleate infection model, and a blank control group and a positive control group were set up and incubated together for an appropriate time; 2) Detect changes in at least one of the following indicators before and after drug treatment: abundance, activity, or metabolite level of Bacteroides rich in oleate; 3) Set a screening threshold. If at least one of the indicators increases significantly after drug treatment compared to before treatment, the drug is determined to be a potential treatment for colorectal cancer.

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