Membrane vesicles sourced from coprophilobacter rodentium as well as preparation method and application of membrane vesicles

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a membrane vesicle derived from coprophilobacter rodent in preparation of a medicine for preventing, treating or relieving breast cancer. The membrane vesicles derived from the coprophilobacter rodent are separated from bacterial culture supernatant of the membrane vesicles through an ultracentrifugation method; experiments show that the membrane vesicles sourced from the coprophilobacter rodent can effectively influence the biological function of a breast cancer cell line and inhibit the growth of breast cancer tumors; the research on the action mechanism of the membrane vesicles shows that the membrane vesicles derived from the coprophilobacter rodent can promote the maturation of dendritic cells in a tumor microenvironment and obviously increase the infiltration of CD8 + T cells, so that the anti-tumor immune response of a body is effectively activated; the safety evaluation shows that the membrane vesicle treatment from the coprophilobacter rodent has no toxic or side effect on the body; in conclusion, as a novel bacterium-derived immunotherapy preparation, the membrane vesicles derived from the coprobacterium rodent show a good application prospect in prevention and treatment of the breast cancer.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and microbiology, specifically to the application of a membrane vesicle (F-MVs) derived from *F. rodentii* in the preparation of products for the prevention, treatment, or relief of breast cancer. Background Technology

[0002] In 2020, there were 2.26 million new cases of breast cancer (BC) in women worldwide, making it the fifth leading cause of cancer death globally. Among women, breast cancer accounts for one-quarter of all cancer cases and one-sixth of all cancer deaths, making it the most common cancer and a leading cause of cancer death among women aged 20-59. Statistics released by the National Cancer Center in 2019 show that in 2015, there were approximately 304,000 new cases of breast cancer in Chinese women, ranking first among female malignant tumors and becoming the most common malignant tumor among Chinese women. Continuous advancements in medical technology have propelled breast cancer treatment into a new stage, with the application of diverse treatment methods making comprehensive treatment a core principle. Currently, commonly used clinical treatments include surgery, chemotherapy, radiotherapy, endocrine therapy, and targeted therapy. Among surgical treatments, mastectomy and breast-conserving surgery are the main choices; in the treatment of advanced breast cancer, chemotherapy remains a crucial core method in clinical practice.

[0003] Extracellular vesicles (EVs) are nanoscale membrane-bound particles released from cells into the extracellular environment, containing biological substances such as proteins, nucleic acids, and lipids. EVs play crucial roles in a variety of biological processes, including intercellular communication, immune regulation, and tissue homeostasis; therefore, research on their medical applications has expanded significantly. Because EVs can mimic the characteristics of their source cells and have the potential for drug delivery through engineering, they show great promise in disease treatment. For example, EVs derived from immune cells such as natural killer cells can exert potent anti-tumor effects by modulating the immune response against cancer cells.

[0004] Probiotic-derived extracellular vesicles retain many beneficial properties of their parent cells and show potential in cancer treatment. Current strategies include modulating gut microbiota to enhance immune responses, exerting direct antitumor effects, and using them in combination with conventional therapies for synergistic effects. Compared to traditional cell-derived extracellular vesicles, probiotic-derived extracellular vesicles have several advantages: 1) they are easy to obtain, purify, and scale up for production, making them cost-effective and suitable for clinical applications; 2) because they are derived from microorganisms, they are less likely to carry harmful carcinogens or immunosuppressive substances; 3) these vesicles are naturally able to survive in the harsh gastrointestinal environment, making them ideal for oral administration, a less invasive route that improves patient compliance and quality of life.

[0005] Breast cancer, a major malignant tumor threatening women's health, faces numerous challenges in clinical treatment despite advancements in surgery, chemotherapy, targeted therapy, and endocrine therapy, as well as breakthroughs in precision medicine. These challenges include: the high heterogeneity of tumors makes it difficult to uniformly adapt treatment plans; widespread drug resistance in chemotherapy, targeted therapy, and endocrine therapy significantly limits treatment efficacy, and effective treatment options are limited after drug resistance develops; treatment-related side effects not only reduce patients' quality of life but may also affect treatment adherence; the risk of tumor metastasis and recurrence, incomplete coverage of precision treatment, and a lack of predictive biomarkers further increase the difficulty of treatment; and the difficulty in achieving a cure for advanced-stage patients, with significant room for improvement in long-term efficacy and survival benefits. These existing deficiencies highlight the necessity and urgency of continuously exploring new treatment strategies and optimizing existing protocols.

[0006] Therefore, discovering probiotic sources of MVs with tumor-suppressing effects is of great significance for improving the survival rate and quality of life of breast cancer patients. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the present invention provides the application of membrane vesicles (F-MVs) derived from Coccidioides rodentae in the preparation of related products for the prevention, treatment or relief of breast cancer.

[0008] A membrane vesicle derived from *F. rodentae*, wherein the membrane vesicle is derived from *F. rodentae*.

[0009] A method for preparing membrane vesicles derived from *F. rodentii* includes the following steps: centrifuging *F. rodentii* culture to obtain culture supernatant, filtering the supernatant through a 0.22 μm filter membrane for sterilization, ultracentrifuging the filtrate and collecting the precipitate, and then resuspending the precipitate with phosphate buffer to obtain the membrane vesicles.

[0010] The use of a rodent-derived membrane vesicle in the preparation of a pharmaceutical composition for the prevention, treatment or relief of breast cancer.

[0011] Preferably, the membrane vesicles can promote the maturation of dendritic cells in the tumor microenvironment and increase CD8+. + T-cell infiltration activates the body's anti-tumor immune response, thereby playing a role in preventing, treating, or alleviating breast cancer.

[0012] Preferably, the dosage form of the drug is an injectable formulation.

[0013] Preferably, the concentration of membrane vesicles in the drug is not less than 2.66 × 10⁻⁶. 11 Particles / ml

[0014] One beneficial effect of this invention is that it provides the application of *F. coli*-derived membrane vesicles in the preparation of products for the prevention or treatment of breast cancer. In vitro and in vivo experiments have confirmed that the membrane vesicles can effectively inhibit breast cancer cell proliferation and induce apoptosis, and significantly inhibit tumor growth in animal models. Mechanistic studies show that its antitumor effect is not achieved through direct cytotoxicity, but rather through remodeling the tumor immune microenvironment: the membrane vesicles can promote dendritic cell maturation and enhance their antigen-presenting function, thereby activating CD8+ T cells and promoting their infiltration into tumor tissue, thus triggering a specific antitumor immune response. Safety evaluation shows that experimental animals treated with the membrane vesicles did not exhibit significant toxic reactions, and there was no pathological damage to major organs, indicating good biocompatibility at effective doses. In summary, the membrane vesicles, as a novel bacterial immunotherapeutic agent, possess clear antitumor activity, immunomodulatory function, and good safety, and are easy to prepare and widely available, providing a new candidate strategy for breast cancer immunotherapy, and have significant development value and application prospects.

[0015] Other features and advantages of the invention will become clear from the following detailed description of exemplary embodiments of the invention with reference to the accompanying drawings. Attached Figure Description

[0016] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0017] Figure 1 This is a flowchart illustrating the preparation process of F-MVs provided in the embodiments of this application; Figure 2 These are transmission electron microscope (TEM) images of F-MVs provided in the embodiments of this application; Figure 3 These are the F-MVs particle size distribution and Zeta potential diagrams provided in the embodiments of this application; Figure 4 These are SDS-PAGE and Western Blot identification results of F-MVs provided in the embodiments of this application; Figure 5 This is a graph showing the in vitro evaluation results of the effect of F-MVs on breast cancer cell proliferation provided in the embodiments of this application; Figure 6 This is a graph showing the detection results of F-MVs in vitro inducing apoptosis in breast cancer cells, as provided in the embodiments of this application. Figure 7 This is a diagram showing the therapeutic effect of F-MVs on orthotopic breast cancer in mice, as provided in the embodiments of this application. Figure 8 This is a diagram illustrating the preventive effect of F-MVs on mouse breast cancer provided in the embodiments of this application; Figure 9 This is a flow cytometry analysis of tumor immune cell infiltration in a mouse breast cancer mouse, provided in an embodiment of this application. Figure 10 This is a pathological analysis diagram of the main organs of breast cancer mice stained with HE, provided in the embodiments of this application. Detailed Implementation

[0018] To make the inventive purpose, technical solution and beneficial technical effects of this application clearer, the following detailed description of this application is provided in conjunction with specific embodiments. It should be understood that the embodiments described in this specification are merely for explaining this application and are not intended to limit this application.

[0019] For simplicity, this article only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range. Similarly, any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included in the range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form an unspecified range.

[0020] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0021] The above-described invention content is not intended to describe every disclosed implementation or every method of implementation in this application. Instead, the following description provides more specific examples of exemplary implementation methods. Throughout the application, guidance is provided through a series of embodiments that can be used in various combinations. In each instance, the examples are listed only as representative groups and should not be construed as exhaustive.

[0022] Example 1: Preparation of F-MVs 1. Experimental Methods 1.1 Bacterial Culture Prepare liquid culture medium according to the PYG medium instructions. After reviving *Bacillus rodentii*, culture it under anaerobic conditions at 37°C and plot the bacterial growth curve. Based on the growth curve, when the bacteria reach the logarithmic growth phase (OD600 value of 0.8~2.0), transfer the culture to a sterile centrifuge tube for collection. The logarithmic growth phase is the stage before the bacteria enter the plateau phase.

[0023] 1.2 Obtaining F-MVs from bacterial culture supernatant Collect bacterial supernatant and centrifuge at 4°C at gradients (1000 g, 3000 g, 5000 g, 8000 g) for 20 min each to obtain culture supernatant. Then, further filter using a 0.22 μm filter to remove parent bacterial debris and other contaminants. Concentrate the supernatant further by centrifugation at 4°C, 4000 g for 20 min using a 100 kDa ultrafiltration tube. Then, ultracentrifuge at 4°C, 140000 g for 2 h. Wash once with phosphate-buffered saline (PBS), and resuspend the obtained precipitate in an appropriate amount of PBS; this is the F-MVs. (Flowchart shown). Figure 1 .

[0024] Example 2: Morphological characterization of F-MVs 1. Experimental Methods Take 50 μL of F-MVs suspension and drop it onto a copper grid. Add PBS to the culture dish and use tweezers to hold the copper grid and wash it in PBS. After fixing F-MVs with glutaraldehyde for 5 min, wash with DEPC water for 1 min. After staining with acetaldehyde hydrogen peroxide for 5 min, place the copper grid on a stainless steel ring, absorb excess water with filter paper, dry for 10 min, and observe the shape of F-MVs under a microscope.

[0025] 2. Experimental Results refer to Figure 2 The morphology of F-MVs was observed by transmission electron microscopy. F-MVs have a clear bilayer membrane-like structure and a regular shape.

[0026] Example 3: Particle size and potential characterization of F-MVs 1. Experimental Methods Resuspend the F-MVs in 1 mL of PBS, then add the F-MVs to a cuvette or syringe and analyze the particle size or potential of the F-MVs using the instrument. Follow the instrument's standard operating procedure for specific operations.

[0027] 2. Experimental Results refer to Figure 3 , Figure 3 Figure A shows the particle size results of F-MVs. The average diameter of F-MVs is approximately 122.9 nm, and there are 2.66 x 10⁻⁶ particles per milliliter of liquid. 11 One F-MVs.

[0028] refer to Figure 3 , Figure 3 B shows the Zeta potential results for F-MVs, which are approximately -16mV.

[0029] Example 4: Identification of protein components of F-MVs Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most commonly used protein expression analysis techniques in polyacrylamide gel electrophoresis. The principle of this technique is based on the different molecular weights of proteins in the sample, causing them to separate in the electrophoretic gel. SDS-PAGE is an indispensable procedure in experiments expressing and purifying exogenous proteins in *E. coli*, typically used to detect protein expression status (expression level, expression distribution) and analyze the purity of the target protein.

[0030] Western blotting (WB) is a method that separates protein samples by molecular weight using polyacrylamide gel electrophoresis, transfers them to a hybridization membrane (blot), and then uses a primary / secondary antibody complex for specific detection of the target protein. WB is one of the most popular and well-established techniques for protein analysis.

[0031] 1. Experimental Methods 1.1 The specific steps for SDS-PAGE are as follows: 1.1.1 Take out the pre-made gel plate, put the gel plate into the electrophoresis clamp, align and clamp it (short glass slide facing inward), pour the pre-prepared electrophoresis solution into the electrophoresis clamp, and remove the comb (before loading the sample).

[0032] 1.1.2 Sample loading: According to the quantitative results, add the marker and sample to the well in sequence. (When loading the sample, tilt it vertically at 45°.)

[0033] 1.1.3 Electrophoresis: After loading all samples, place the electrophoresis clamp into the electrophoresis tank and add an appropriate amount of electrophoresis buffer. After aligning the positive and negative electrodes, close the electrophoresis apparatus lid, turn on the power, adjust the voltage to 80V, and start the electrophoresis. When the sample bands have just entered the lower separating gel (about 30 minutes), adjust the voltage to 120V. When the sample bands reach about 0.5cm below the gel plate, turn off the power. Open the electrophoresis lid, remove the electrophoresis clamp, and remove the electrophoresis gel plate.

[0034] 1.1.4 After staining and electrophoresis, remove the gel and stain with Coomassie Brilliant Blue. If the staining solution is reused after 10 minutes at room temperature, the staining time needs to be extended.

[0035] 1.1.5 Decolorization Remove the colloid from the staining solution and place it in the destaining solution. Destain repeatedly until the background is clear. The gel can be stored in double-distilled water and the results can be photographed.

[0036] The specific steps of 1.2WB are as follows: The preliminary experimental methods were exactly the same as those used for SDS-PAGE, and will not be repeated here.

[0037] 1.2.1 Cutting the Gel: Pour the pre-prepared transfer solution into a container. Gently pry open the two plates using a gel cutter, remove the long gel plate, cut the gel, and place it in the transfer solution. Immerse the transfer clamp, mesh screen, filter paper, and membrane separately in the transfer solution. Place the transfer clamp black-side down, and then apply the soaked mesh screen, filter paper, gel, and membrane in sequence. Use a plate to remove the gel and place it on the membrane. Then apply the filter paper and mesh screen, close the transfer clamp tightly, and place it in the transfer tank.

[0038] 1.2.2 Transfer: After placing the transfer clamp, put the transfer tank into the ice bath transfer box, pour the transfer buffer into the transfer box, and place the ice box in the box at the same time. After aligning the positive and negative electrodes, close the transfer apparatus lid, plug in the power, adjust the current value to 300mA, and transfer for 45-60 minutes. After the transfer is completed, remove the transfer apparatus lid, take out the transfer clamp, open the clamp and remove the pad, filter paper and gel, and you can see the membrane with the transferred protein. 1.2.3 Blocking: After the transfer, place the membrane in a dish, remove the membrane, put it in a small box, add 5% skim milk powder blocking solution, and place it on a shaker for 2-4 hours.

[0039] 1.2.4 Antibody Incubation: After blocking, take a plastic box, place the cut membrane inside, add the pre-prepared OmpA (Boson) antibody, and incubate at 4°C overnight. After primary antibody incubation, pour in PBST washing buffer to wash the membrane 3-4 times, 10 min each time. After washing, add the pre-prepared secondary antibody and incubate at room temperature for 2 h. After secondary antibody incubation, remove the membrane and place it in a washing box, wash 3 times with PBST, 10 min each time.

[0040] 1.2.5 Development and Exposure: Prepare the luminescent solution and mix well. Add 1 ml of each solution. Place the film horizontally on the luminescent plate, spread the luminescent solution evenly, and start luminescence.

[0041] 2. Experimental Results refer to Figure 4 A. SDS-PAGE was used to detect characteristic proteins of F-MVs, observing flagellate virulence protein B (FliB) and various outer membrane proteins (OMPs) typically expressed on the bacterial outer membrane. (Reference) Figure 4 B. The expression of the outer membrane protein ompA was specifically detected by Western blotting, suggesting that F-MVs originated from rodents.

[0042] Example 5: Effects of F-MVs on Breast Cancer Cell Proliferation EdU (5-ethynyl-2-deoxyuridine) is a thymine deoxynucleoside analogue. During DNA synthesis, it can replace thymine deoxynucleoside and be incorporated into the newly synthesized DNA. The ethynyl group on EdU can react with fluorescently labeled small-molecule azide probes (such as FITC Azide, AF series dyes, etc.) under the catalysis of monovalent copper ions to form a stable triazole ring. This reaction is very rapid and is therefore called a click reaction. Through the click reaction, the newly synthesized DNA is labeled with the corresponding fluorescent probe, and then fluorescent detection equipment can be used to detect cell proliferation.

[0043] 1. Experimental Methods Log-phase breast cancer cell line MDA-MB-231 (ATCC:SNL-061) was seeded onto suitable culture plates, and an appropriate amount of EdU working solution was added to each well. Cells were incubated with various concentrations of F-MVs for several hours, fixed for approximately half an hour, and then subjected to permeation treatment. The reaction solution was used for fluorescent labeling, binding the fluorescent dye to EdU. Images were collected using a fluorescence microscope, and the mean fluorescence intensity (MFI) was quantified using ImageJ software.

[0044] 2. Experimental Results refer to Figure 5 , Figure 5 The image shows the EDU staining results of breast cancer cells. The EdU experiment once again verified the effect of different concentrations of F-MVs on the viability of breast cancer cells. The results showed that F-MVs at a concentration of 50 μg / mL could significantly inhibit breast cancer cells.

[0045] Example 6: Detection of F-MVs-induced apoptosis in breast cancer cells in vitro; This embodiment uses the Annexin V apoptosis assay to detect cell apoptosis. Annexin V is a Ca2+-dependent phospholipid-binding protein with a molecular weight of 35-36 kDa that binds specifically to phosphatidylserine (PS) with high affinity. In the early stages of apoptosis, phosphatidylserine (PS), which is only distributed on the inner side of the lipid bilayer of normal cell membranes, turns outward. Annexin V labeled with fluorescein is used as a fluorescent probe. Additionally, 7-AAD provided in this kit can be used to distinguish between viable early-stage cells and necrotic or late-stage apoptotic cells. 7-AAD is a nucleic acid dye with similar fluorescence properties to PI, but its emission spectrum is narrower, causing less interference with other detection channels. It is the best alternative to PI in multicolor fluorescence analysis and can be used in conjunction with Annexin V. This dye cannot penetrate the intact cell membrane of normal cells or early-stage apoptotic cells, but it can penetrate late-stage apoptotic cells or necrotic cells and bind to their DNA. Therefore, when Annexin V-PE is used in combination with 7-AAD, 7-AAD is excluded from live cells (Annexin V- / 7-AAD-) and early apoptotic cells (Annexin V+ / 7-AAD-), while late apoptotic cells and necrotic cells are stained with both Annexin V-PE and 7-AAD and show double positivity (Annexin V+ / 7-AAD+).

[0046] 1. Experimental Methods 1.1 Cell Plating and Intervention: MDA-MB-231 cells in logarithmic growth phase were harvested, digested, washed, and then plated at 2×10⁻⁶ cells / year. 5 Cells were added to 12-well plates and incubated for 4 hours. After cell adhesion, F-MVs were added to 6 wells of each 12-well plate for 48 hours, while the remaining 6 wells were cultured normally for another 48 hours. The normal culture control group was designated as the Control group, and the bacterial vesicle intervention group was designated as the F-MVs group.

[0047] 1.2 Cell collection: Digest cells with EDTA-free digestion solution, collect cells, wash twice with pre-cooled PBS, and resuspend in 100 μL buffer.

[0048] 1.3 Staining and Detection: Add 2.5 μL Annexin V and 2.5 μL 7-AAD, mix gently, and incubate at room temperature in the dark for 15 min. Then, resuspend the cells in 300 μL PBS, detect by flow cytometry, and analyze the results using Flowjo.

[0049] 2. Experimental Results refer to Figure 6 , Figure 6The results of the apoptosis experiment on MDA-MB-231 cells show that the number of apoptotic cells in the MDA-MB-231 cells treated with F-MVs was significantly higher than that in the control group without intervention. This indicates that the apoptosis of MDA-MB-231 cells increased after F-MVs intervention, and that F-MVs can promote the apoptosis of breast cancer cells, suggesting that F-MVs have anti-breast cancer capabilities.

[0050] Example 7: In vivo efficacy evaluation of F-MVs in treating orthotopic breast cancer in mice 1. Laboratory animals SPF-grade female Balbc mice, approximately 6-8 weeks old, were purchased from the Laboratory Animal Center of Ningxia Medical University and housed within the university's barrier enclosure. The feeding environment was SPF-grade, and the enclosure environment was suitable. Drinking water and feed for the mice were provided by the university's Laboratory Animal Center. All animal experiments were conducted in strict accordance with the operating procedures of the university's Laboratory Animal Center and were approved by the Animal Ethics Committee of Ningxia Medical University (Yinchuan, China, Approval No.: IACUC-NYLAC-2024-064). Mice were randomly divided into two groups of five each: a breast cancer control group and an F-MVs intervention group.

[0051] Modeling method: Refer to Figure 7 , Figure 7 A is a schematic diagram of the orthotopic breast cancer treatment model. An appropriate amount of MDA-MB-231 cells were subcutaneously injected on day -5. The treatment continued until the tumor volume reached ≥100 mm². 3 Intervention was carried out.

[0052] Feeding: Reference Figure 7 A. Both groups of mice were fed a normal diet. The F-MVs group mice were injected intratumorally with 15ug of F-MVs once every three days for four consecutive interventions.

[0053] 2. Effect Evaluation Two groups of mice were fed for 3 weeks using the above-mentioned modeling and feeding methods. The mice were weighed and their weight was recorded every two days. The mortality of the mice was recorded daily. At the end of the third week, all mice in the breast cancer control group and the F-MVs group were sacrificed by head-neck separation. The mice were dissected and the tumor size was measured by vernier calipers to calculate the tumor volume.

[0054] 3. Experimental Results refer to Figure 7 C. Comparison of body weight between the two groups of mice showed no statistically significant difference in body weight between the breast cancer mouse control group and the F-MVs group during the experiment, indicating that F-MVs have biosafety.

[0055] refer to Figure 7B shows photographs of breast cancer tumors in the control and F-MVs groups of breast cancer mice; (Reference) Figure 7 D represents the comparison of tumor volume between the control group and the F-MVs group of breast cancer mice. The tumor volume in the F-MVs group was significantly smaller than that in the control group. This indicates that F-MVs can effectively inhibit the tumor volume of breast cancer mice compared with the control group.

[0056] Example 8: In vivo evaluation of the preventive effect of F-MVs on breast cancer in mice 1. Laboratory animals The source of the experimental animals was exactly the same as in Example 7, and will not be repeated here. Mice were randomly divided into two groups of five each: a breast cancer control group and an F-MVs intervention group.

[0057] 2. Modeling method: See Figure 8 , Figure 8 A is a schematic diagram of a breast cancer prevention model. On day 0, an appropriate amount of MDA-MB-231 cells were injected into the mammary fat pads of mice. Twenty-one days after the injection of tumor cells, the mouse tumors were collected for detection and analysis.

[0058] 3. Feeding: See Figure 8 A. Both groups of mice were fed a normal diet. The F-MVs group mice were injected with 15ug of F-MVs via the tail vein once every 7 days for a total of four interventions.

[0059] 4. Evaluation of results: The two groups of mice were fed for 3 weeks using the above modeling and feeding methods. The mice were weighed and their weight was recorded every two days. The mortality of the mice was recorded daily. At the end of the third week, all mice in the breast cancer control group and the F-MVs group were sacrificed by head-neck separation. The mice were dissected and the tumor size was measured by vernier calipers to calculate the tumor volume.

[0060] 5. Experimental Results refer to Figure 8 E. The comparison of body weight between the two groups of mice showed that there was no statistically significant difference in body weight between the breast cancer mouse control group and the F-MVs group during the experiment, indicating that F-MVs have biosafety.

[0061] refer to Figure 8 Image B shows photographs of tumors in the control group and the F-MVs intervention group of breast cancer model mice; for reference. Figure 8 C represents a bar chart showing the average tumor weight in the two groups of mice, for reference. Figure 8 D represents the comparison of tumor volume between the two groups of mice. The experimental results showed that the tumor volume and weight in the F-MVs intervention group were significantly smaller than those in the control group. These results confirm that, compared with the control group, F-MVs can effectively inhibit tumor growth in a breast cancer model mouse.

[0062] Example 9: Regulatory Effect of F-MVs on the Immune Microenvironment of Mouse Breast Cancer 1. Experimental Methods Analysis of immune cells in the immune microenvironment via FCM: Tumor tissues from different groups of mice were collected at specific time points according to different model designs. Mononuclear cells were isolated from the tissues and stained with antibodies using flow cytometry. 1.1 Add cell surface antibody: Add 0.35ul of antibody to each 100ul resuspended cell in a 1.5ml EP tube, gently pipette and mix with a 200ul pipette, vortex for 15s; 1.2 Incubation: Incubate at 4℃ in the dark for 30 min; 1.3 Washing: After 30 min, add 350 μL of PBS containing 2% FBS serum to each tube, mix well and vortex; 1.4 Centrifugation: 4℃, 1500rpm, 5min, discard supernatant; 1.5 Preparation: Resuspend in 350 μL of PBS with 2% FBS, filter through a 200-mesh nylon sieve, and then proceed with the preparation. 1.6 Cell membrane rupture: Following step 4 of the surface staining procedure, discard the supernatant, add 200 μL of membrane rupture buffer to each tube, and mix well; 1.7 Incubation: Incubate at 4℃ in the dark for 30 min; 1.8 Washing: After 30 min, add 350 μL of buffer to each tube, mix well, centrifuge at 1500 rpm and 4 °C for 5 min, and discard the supernatant; 1.9 Add intracellular antibody staining: Resuspend the cells in 100ul buffer to form a 100ul system per tube, and add 1ul of the corresponding intracellular antibody; 1.10 Incubation: Incubate at 4℃ in the dark for 35 minutes; 1.11 Washing: After 35 minutes, add 350 μL of buffer to each tube and mix well; 1.12 Centrifugation: 4℃, 1500rpm, 5min, discard the supernatant after centrifugation; 1.13 Resuspending cells: Add 350 μL of PBS containing 2% FBS to each tube, mix well by pipetting and resuspending the cells; 1.14 The percentage of various immune cells was determined by flow cytometry; 1.15 The percentage of various immune cells was determined by flow cytometry. IFN-rCD8 was analyzed using Flow.Jo software (FlowJo_v10.8.1). + T, CD86 + CD11b ++ The proportion of cells in tumor tissue was analyzed.

[0063] 2. Experimental Results refer to Figure 9 The results are obtained by flow cytometry analysis of immune cell infiltration in tumor tissues of mice in the control and F-MVs treatment groups. Figure 9 It can be seen that, compared with the control group, F-MVs treatment can significantly increase IFN-γCD8 in tumor tissue. + T cells, CD86 + CD11b ++ The proportion of dendritic cells.

[0064] Example 10: Safety Evaluation of F-MVs Hematoxylin-eosin staining, or HE staining for short, is a method where hematoxylin (H) is a basic dye that stains the cell nucleus and ribosomes blue-purple, and structures stained by this basic dye are basophilic. Eosin (E) is an acidic dye that stains the cytoplasm red or pale red, and structures stained by this acidic dye are eosinophilic. This example uses HE staining to observe the morphological characteristics of the heart, liver, spleen, lungs, and kidneys in two groups of mice, including their tissue structure, tissue type, and cellular layers.

[0065] The source of experimental animals and the modeling method are exactly the same as in Example 7, and will not be repeated here.

[0066] 1. Experimental Methods Two groups of mice were fed separately for three weeks. In the third week, all mice in the breast cancer control group and the F-MVs group were sacrificed by head-neck separation. The heart, liver, spleen, lungs and kidneys of the two groups of mice were obtained by dissection.

[0067] 1.1 Fixation: The heart, liver, spleen, lungs and kidneys of mice were cleaned with PBS and then fixed in 4% paraformaldehyde at 4°C for 48 hours to prevent autolysis or bacterial decomposition after cell death, thereby maintaining the original morphology and structure of the cells.

[0068] 1.2 Dehydration, embedding, and sectioning: The fixed tumor tissue was placed in an embedding cassette and thoroughly dehydrated with a gradient of ethanol from low to high concentrations. After passing through xylene, it was immersed in paraffin at 56°C for 2 hours. After tissue fixation, sections were prepared with a thickness of 3.5 μm.

[0069] 1.3 Dewaxing and Hydration: Place the slides in a 60°C oven and bake for 2 hours before dewaxing and hydration. The procedure is as follows: xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 2 min, 90% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min, and then rinse with tap water.

[0070] 1.4 Staining: Tissue sections were stained with hematoxylin and eosin. Specifically, the slides were blotted dry, hematoxylin staining solution was added, and staining was performed for 5 minutes. The hematoxylin staining solution was rinsed off with running water, and the staining degree was observed under a microscope. Differentiation was performed with 1% hydrochloric acid alcohol for 1-3 seconds, followed by rinsing with water and immersion in tap water for 5-10 minutes to regain blue color. The color was observed under a microscope. If the color was not deep enough, hematoxylin staining solution could be added directly for 1 minute, followed by further differentiation and immediate rinsing with water. If the color was too deep, the differentiation time was increased appropriately. Afterward, 1 drop of 0.5% eosin staining solution was added, and staining was performed for 10-15 seconds. The sections were rinsed with distilled water and observed under a microscope.

[0071] 1.5 Dehydration, mounting, and microscopic examination: The slides were sequentially immersed in the following reagents for dehydration: 50% ethanol for 1 min, 75% ethanol for 1 min, 80% ethanol for 1 min, 85% ethanol for 1 min, 90% ethanol for 1 min, 95% ethanol for 1 min, xylene II for 2 min, and xylene I for 2 min. The tissue sections were removed from the last container, allowed to dry, mounted with neutral resin, and air-dried in a fume hood before microscopic examination.

[0072] 2. Experimental Results refer to Figure 10 , Figure 10 This image shows the histological changes in the heart, liver, spleen, lungs, and kidneys of mice in the breast cancer control group and the F-MVs group. Figure 10 As can be seen, no significant abnormalities were observed in the tissues of the F-MVs group mice compared to the control group, indicating that F-MVs intervention in breast cancer mice does not cause significant damage to the heart, liver, spleen, lungs, and kidneys. Therefore, F-MVs have a certain degree of safety when used to treat, prevent, or alleviate breast cancer, and do not produce toxic side effects.

Claims

1. A membrane vesicle derived from *F. coli*, characterized in that, The membrane vesicles are derived from rodentobacter.

2. A method for preparing membrane vesicles derived from *F. rodentii* as described in claim 1, characterized in that, The bacterial culture of *Bacillus foetida* was centrifuged to obtain the culture supernatant. The supernatant was then filtered through a 0.22 μm filter membrane for sterilization. The filtrate was then ultracentrifuged and the precipitate was collected. The precipitate was then resuspended in phosphate buffer to obtain the membrane vesicles.

3. The use of the rodent-derived membrane vesicles as described in claim 1 in the preparation of pharmaceutical compositions for the prevention, treatment or relief of breast cancer.

4. The application according to claim 3, characterized in that, The membrane vesicles can promote the maturation of dendritic cells in the tumor microenvironment and increase CD8. + T-cell infiltration activates the body's anti-tumor immune response, thereby playing a role in preventing, treating, or alleviating breast cancer.

5. The application according to claim 3, characterized in that, The drug is in the form of an injectable preparation.

6. The application according to claim 3, characterized in that, The concentration of the membrane vesicles in the drug is not less than 2.66 × 10⁻⁶. 11 Particles / ml.

Images