Application of parabacteroides dielsii SYSU-210 and metabolite thereof in cancer treatment

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to application of parabacteroides dielsii SYSU-210 and metabolites thereof in cancer treatment. The parabacteroides dieldrii is a parabacteroides dieldrii SYSU-210 strain, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32494, and the preservation date is November 04, 2024. According to the application disclosed by the invention, by carrying out anti-tumor treatment on tumor-bearing mice with colon cancer and breast cancer by using the single parabacteroides dieldrii SYSU-210 and a metabolite thereof, the screened SYSU-210 has a good treatment effect on both the colon cancer and the breast cancer, and effector T cell infiltration in tumors of the mice in a treatment group is remarkably increased; and when the compound is combined with an immunotherapy alpha PD-1 inhibitor or a chemotherapeutic drug oxaliplatin for use, the effect of synergistically enhancing the curative effect is shown.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to the application of Parabacteroides distasonis SYSU-210 and its metabolites in the treatment of cancer. Background Art

[0002] Cancer is one of the major public health problems globally, and its incidence and mortality rates continue to rise.

[0003] Immune checkpoints are protective protein molecules on human immune cells, mainly functioning to restrict T cell function and prevent their overactivation from causing damage to the body itself. This mechanism is exploited by tumor cells, which can overexpress immune checkpoint inhibitory molecules to suppress the human immune response, thereby escaping the surveillance and killing of the human immune system and ultimately leading to tumor growth. In response to this process, immune checkpoint inhibitors (ICIs) have been developed. By blocking the binding of immune checkpoints to their ligands, the inhibition of immune function can be relieved, and immune cells can be reactivated to exert anti-tumor effects.

[0004] Inhibitors of programmed cell death protein-1 (PD-1) and its ligand (PD-L1) are immune checkpoint monoclonal drugs, and their responses in terms of breadth, depth, and persistence are very good. The marketed nivolumab, pembrolizumab, and tislelizumab belong to PD-1 inhibitors and are mainly used for the treatment of melanoma and non-small cell lung cancer; the PD-L1 inhibitors atezolizumab, durvalumab, and avelumab have been approved for the treatment of urothelial carcinoma.

[0005] In recent years, in immunotherapy, inhibitors of CTLA-4, PD-1, and PD-L1 have been successfully used in the clinical treatment of various malignant tumors. PD-1 / PD-L1 blockers have shown positive application potential in the treatment of tumors. The application of antibodies against PD-1 / PD-L1 in cancer treatment has made remarkable progress. PD-1 / PD-L1 blockers are widely used in the treatment of various cancers such as non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, mesothelioma, and esophageal squamous cell carcinoma. In the face of refractory tumors, combination therapies with immune checkpoint inhibitors are mostly adopted clinically, including the combination of different immune checkpoint inhibitors, as well as the combination with traditional treatment methods such as chemotherapy and radiotherapy, and emerging immunotherapy means.

[0006] However, despite the significant progress in immunotherapy, challenges still remain, such as treatment resistance, adverse reactions, lack of effective predictive biomarkers, and high treatment costs.

[0007] A major challenge in cancer immunotherapy is the resistance to ICI treatment. The mechanisms of ICI resistance can be classified into tumor-intrinsic factors and tumor-extrinsic factors. Regulatory T cells (Tregs) inhibit the function of effector T cells (Teff) through inhibitory cytokines and direct contact, thereby limiting inflammation and promoting self-tolerance, which may lead to immune therapy resistance.

[0008] In addition, after T cell restriction is lifted, it will not only attack tumor cells but also attack normal self-cells. PD-1 / PD-L1 blockers may increase the risk of drug-related adverse reactions, which usually originate from the skin, gastrointestinal tract, liver, and endocrine system, although other organ systems may also be affected. Immune-related adverse events (irAEs) include cardiotoxicity, cytokine release syndrome, myocarditis, pneumonia, hepatitis, fatigue, rash, and diarrhea, etc.

[0009] Currently, there are still no effective predictive biomarkers to guide clinical precision medicine and clinical trial design. Although tumor mutation burden (TMB) and the expression level of PD-L1 are used as biomarkers to predict the efficacy of PD-1 / PD-L1 therapy, they are not always accurate.

[0010] The cost of immunotherapy is high, and the treatment cost of PD-1 / PD-L1 blockers is relatively high. There are also combination treatment strategies (such as combination with PD-1 inhibitors, chemotherapy, radiotherapy, or targeted therapy), which can improve the efficacy, but at the same time increase the complexity of treatment and potential side effects.

[0011] All in all, not all patients can benefit from it. According to clinical studies, only about 20 - 45% of patients respond to PD-1 / PD-L1 antibody therapy. PD-1 / PD-L1 antibody therapy provides new treatment options for cancer patients, but further research is still needed to optimize treatment strategies and improve treatment effects. Summary of the Invention

[0012] The object of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide an application of Parabacteroides distasonis SYSU-210 and its metabolites in the treatment of cancer. The Parabacteroides distasonis SYSU-210 and its metabolites can enhance the anti-tumor immune response of the body by increasing the infiltration of effector T cells in the tumor, enhancing the activity and function of T cells, and simultaneously reducing inhibitory regulatory T cells, thereby effectively treating tumors.

[0013] To achieve the above object, the technical solution adopted by the present invention includes:

[0014] In the first aspect, the present invention provides a Parabacteroides distasonis, which is the Parabacteroides distasonis SYSU-210 strain, deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 32494, the deposit date of November 04, 2024, and the taxonomic name of Parabacteroides distasonis.

[0015] Preferably, the 16S rRNA sequence of the Parabacteroides distasonis SYSU-210 strain is as shown in SEQ ID NO.1.

[0016] In the present invention, the dominant strain Parabacteroides distasonis SYSU-210 (CGMCC No. 32494) is obtained by anaerobic culture, isolation, and purification of a healthy human fecal sample through the serial dilution method, and is identified as Parabacteroides distasonis by Gram staining, microscopic examination, single colony morphology observation, and molecular biology experiments.

[0017] In the second aspect, the present invention provides a Parabacteroides distasonis culture, which is prepared from the Parabacteroides distasonis.

[0018] Preferably, the Lysobacter enzymogenes culture is prepared by at least one of the following methods (1)-(3):

[0019] (1) Scraping the activated Parabacteroides distasonis into a sterile solvent to obtain a Parabacteroides distasonis bacterial suspension, and the bacterial suspension is the Parabacteroides distasonis culture;

[0020] (2) Scraping the activated Parabacteroides distasonis and inoculating it into a liquid medium for culture, centrifuging to obtain the supernatant, and then filtering the supernatant to obtain a fermentation filtrate, and the fermentation filtrate is the Parabacteroides distasonis culture;

[0021] (3) Scraping the activated Parabacteroides distasonis and inoculating it into a liquid medium for culture, centrifuging to obtain the supernatant, then filtering the supernatant to obtain a fermentation filtrate, and separating the metabolites in the fermentation filtrate, and the metabolites are the Parabacteroides distasonis culture.

[0022] Preferably, the metabolites include sebacic acid.

[0023] In the third aspect, the present invention provides the application of the Parabacteroides distasonis, or the Parabacteroides distasonis culture in the preparation of a drug for preventing and / or treating cancer.

[0024] Preferably, the cancer includes colorectal cancer and breast cancer.

[0025] Parabacteroides distasonis (PD) is one of the core members of the human gut microbiota, and its safety is guaranteed. The fact that it is the dominant strain in the corresponding patients undergoing immunotherapy also indicates that Parabacteroides distasonis plays a certain role in tumor treatment. Moreover, in this invention, anti-tumor treatment was carried out on tumor-bearing mice with colon cancer and breast cancer using Parabacteroides distasonis single strain SYSU-210 and its metabolites, and it was found that the SYSU-210 strain screened in this invention had good therapeutic effects on both, significantly increasing the infiltration of effector T cells in the tumors of the treatment group mice. This finding provides a new idea for the immunotherapy of cancer and is expected to become a new adjuvant therapy in future cancer treatment.

[0026] Fourthly, this invention provides the application of the Parabacteroides distasonis as described above, or the Parabacteroides distasonis culture combined with immune checkpoint inhibitors or chemotherapeutic drugs in the preparation of drugs for treating cancer.

[0027] Preferably, the immune checkpoint inhibitors include at least one of antibodies against PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, VISTA, and A2aR; the chemotherapeutic drugs include oxaliplatin.

[0028] Preferably, the immune checkpoint inhibitor is an antibody against PD-1 and / or an antibody against PD-L1.

[0029] Through experimental exploration, it was found that when effector T cells in patients are exhausted and dysfunctional, PD-1 / PD-L1 inhibitors cannot restore them either, while the Parabacteroides distasonis SYSU-210 provided in this invention can enhance the proliferation ability and cytotoxicity of T cells and restore their killing ability against tumors. Therefore, by using the scheme of combining the Parabacteroides distasonis SYSU-210 strain or its metabolites with the immunotherapy αPD-1 inhibitor or the chemotherapeutic drug oxaliplatin, the ability of the immune system to recognize and attack tumor cells can be effectively enhanced, and the growth and metastasis of tumors can be synergistically inhibited, thus significantly improving the immunotherapy effect.

[0030] Fifthly, this invention provides a drug for preventing or treating cancer, which includes immune checkpoint inhibitors, and also includes the Parabacteroides distasonis as described above, and / or the Parabacteroides distasonis culture.

[0031] Compared with the prior art, the beneficial effects of this invention are as follows:

[0032] (1) The present invention collects fecal samples from healthy individuals, and isolates and purifies the dominant strain Parabacteroides distasonis SYSU-210 (CGMCC No. 32494) through anaerobic culture by serial dilution method. It is identified as Parabacteroides distasonis by Gram staining, microscopic examination, observation of single colony morphology, and molecular biology experiments;

[0033] (2) The present invention uses the single strain Parabacteroides distasonis SYSU-210 and its metabolites to conduct anti-tumor treatment on tumor-bearing mice with colon cancer and breast cancer, and finds that SYSU-210 screened by the present invention has good therapeutic effects on both. It significantly increases the infiltration of effector T cells in the tumors of the treatment group mice. In addition, it can not only inhibit tumor growth but also inhibit the metastasis of tumor cells; when used in combination with the immunotherapy αPD-1 inhibitor or the drug oxaliplatin, it shows an enhanced therapeutic effect;

[0034] (3) The Parabacteroides distasonis SYSU-210 of the present invention has a protective effect on intestinal epithelial cells (NCM460), can enhance its wound healing ability and reduce its damage to oxidative stress. At the same time, supplementing Parabacteroides distasonis can restore the composition of the intestinal flora and regulate the balance of the intestinal flora, suggesting that the Parabacteroides distasonis SYSU-210 can reduce the side effects of the gastrointestinal tract of patients in immunotherapy. Description of the Drawings

[0035] Figure 1 is the colony map of the Parabacteroides distasonis SYSU-210;

[0036] Figure 2 is the phylogenetic tree map constructed for the Parabacteroides distasonis SYSU-210;

[0037] Figure 3 is the anti-tumor effect diagram of the Parabacteroides distasonis SYSU-210 described in Example 2;

[0038] Figure 4 is the result diagram of the tumor flow cytometry experiment after the anti-tumor experiment of the Parabacteroides distasonis SYSU-210 described in Example 2;

[0039] Figure 5 is the anti-tumor flow chart ( Figure 5 A) and anti-tumor effect diagram ( Figure 5 B) of the combination of the Parabacteroides distasonis SYSU-210 and the αPD-1 inhibitor described in Example 3;

[0040] Figure 6Flow cytometry results of tumors after the anti-tumor experiment of Parabacteroides distasonis SYSU-210 combined with αPD-1 inhibitor in Example 3. Here, PD refers to Parabacteroides distasonis SYSU-210;

[0041] Figure 7 Anti-tumor effect diagram of Parabacteroides distasonis SYSU-210 described in Example 4;

[0042] Figure 8 Serum metabolome results of mice after gavage with Parabacteroides distasonis SYSU-210 described in Example 5;

[0043] Figure 9 Anti-tumor effect diagram of the metabolite SA of Parabacteroides distasonis SYSU-210 described in Example 5;

[0044] Figure 10 Flow cytometry results of tumors after the anti-tumor experiment of the metabolite SA of SYSU-210 described in Example 5;

[0045] Figure 11 Anti-tumor flow chart ( Figure 11 A) and anti-tumor effect diagram ( Figure 11 B-C) of the metabolite SA of SYSU-210 combined with αPD-1 inhibitor or the chemotherapeutic drug oxaliplatin in Example 6;

[0046] Figure 12 Flow cytometry results of tumors after the anti-tumor experiment of the metabolite SA of SYSU-210 combined with αPD-1 inhibitor in Example 6;

[0047] Figure 13 Result diagram of the protective effect of Parabacteroides distasonis SYSU-210 on intestinal epithelial cells (NCM460) in Example 7. Detailed implementation mode

[0048] To make the purpose, technical solution and effects of the present invention clearer and more definite, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] Unless otherwise specified, the reagents and strains used in the examples are all conventional reagents and strains in the art, and can be obtained through commercial channels. The experimental operations not specifically described in the examples are all conventional operations in the art or can be understood or known by those skilled in the art according to the existing technology or common knowledge they have mastered.

[0050] Example 1

[0051] This embodiment provides a method for screening and identifying Bacteroides paradiaconesis SYSU-210 strain, which includes the following steps:

[0052] (1) Fecal collection: Add 20 mL of bacterial cryopreservation solution into a cell culture flask. Use a sterilized cotton swab to collect freshly excreted feces from a healthy person into the cell culture flask, seal the flask in an anaerobic bag, and store and transport it in an ice box.

[0053] (2) Dilution and plating: After vortexing, pipette 100 μL of fecal suspension into 900 μL of PBS buffer. Mix well by pipetting, and then pipette 100 μL of the suspension into 900 μL of PBS buffer again, and so on, successively diluting 10 times in gradient. Take 50 μL of the dilution solution of each gradient and spread it on BHI and Columbia blood agar plates, and culture at 37 °C under anaerobic conditions for 48 - 72 h.

[0054] (3) Single colony enrichment culture: Select a plate with 5 - 100 colonies. Randomly pick single colonies with an inoculation loop into 20 μL of PBS, mix well by pipetting, and then take 15 μL of the bacterial solution and spread it on the same kind of plate, and culture at 37 °C under anaerobic conditions for 48 - 72 h. The remaining 5 μL of the bacterial solution is used for Gram staining identification.

[0055] (4) Bacterial passage: Scrape the bacteria growing on the plate with an inoculation loop, resuspend it in 100 μL of PBS buffer, and then spread the bacterial solution onto the same kind of plate again, and culture under the same conditions for 48 - 72 h, thus isolating the Bacteroides paradiaconesis SYSU-210 strain described in the present invention. The colony picture is as Figure 1 shown. Its colonies are relatively small, white, round, and have regular edges.

[0056] (5) Bacterial cryopreservation: After the bacteria are enriched and cultured to a sufficient amount, collect the bacteria on the plate with an inoculation loop into a sterile cryopreservation solution composed of fetal bovine serum, BHI medium, and glycerol, mix well by pipetting, and store the bacterial strain at -80 °C.

[0057] (6) Gram staining identification: After appropriately diluting the bacterial solution with PBS, add 5 μL in the center of a glass slide, fix it by intermittent baking with an alcohol lamp, add crystal violet staining solution and cover for 1 min, wash with running water, then add Lugol's iodine solution and iodine stain for 1 min, wash with running water, then cover the surface of the glass slide with decolorizing alcohol for 20 - 30 s, gently shake the glass slide during this period, wash off the alcohol with running water, and then use safranin staining solution for counterstaining for 1 min, wash with running water. After the glass slide is dried, observe the bacterial morphology under an optical microscope. If the colonies are not pure and there are more than one bacterial morphology, perform streak plate inoculation on the sample, and re-pick single colonies after culture. After Gram staining, it shows red and short rod-shaped under the microscope.

[0058] (7) Bacterial genomic DNA extraction: Scrape an appropriate amount of bacteria for the extraction of bacterial genomic DNA. Use a bacterial genomic DNA extraction kit to extract bacterial genomic DNA according to the instructions of TIANGEN Biochemical Technology Co., Ltd., and measure the DNA concentration with NanoDrop2000.

[0059] (8) 16S fragment amplification:

[0060] 1) Use the primer pair 27F / 1492R. The 27F sequence: 3’-AGAGTTTGATCMTGGCTCAG-5’

[0061] The 1492R sequence: 3’-GGTTACCTTGTTACGACTT-5’. The reaction system is shown in Table 1;

[0062] Table 1

[0063] Reagent Volume LA Taq enzyme 15 μL PCR Forward Primer (10 μM) 1.2 μL PCR Reverse Primer (10 μM) 1.2 μL Bacterial genomic DNA 3 μL <![CDATA[ddH2O]]> up to 30 μL

[0064] 2) The PCR amplification program is as follows: Pre-denaturation at 95°C for 60 s; Denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 90 s, with 31 cycles; Extension at 72°C for 7 min, and the final temperature is set at 4°C.

[0065] (9) Agarose gel electrophoresis: Prepare 25 mL of 1×TAE buffer containing 1% (w / v) agarose, melt it using a microwave oven, add 2.5 μL of nucleic acid dye GoldView, mix well and pour it into the gel casting tray, insert the comb, and wait for it to cool. After the agarose gel cools, remove the comb, place it in a horizontal electrophoresis tank, add 5 μL of the amplified DNA sample and 3 μL of DL2000 DNA marker to each well, turn on the power supply, perform electrophoresis at a constant voltage of 110 V for 20 min, and then observe under ultraviolet light whether the amplified band is single and whether the size is around 1500 bp.

[0066] (10) 16S rRNA sequencing and result comparison: Send the DNA 16S fragment sample that has been amplified and confirmed by agarose gel electrophoresis to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After obtaining the sequencing results, use ChromasPro software for sequence splicing, and perform a 16S rRNA sequence blast on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to preliminarily identify the genus and species of the bacteria.

[0067] Using the full sequence analysis of 16S rRNA, the 16S rRNA gene sequence of strain SYSU-210 is shown in SEQ ID NO.1. After alignment, the 16S rRNA sequence homology between strain SYSU-210 and the standard strain of Parabacteroides distasonis (Parabacteroides distasonis strain JCM 5828) is 99.03%. Therefore, it is identified, classified and named as Parabacteroides distasonis, and the corresponding Chinese name is Bacteroides paravulgatus, and the strain is named Bacteroides paravulgatus SYSU-210. This strain has been deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 32494, the deposit date of November 4, 2024, and the address of the deposit unit is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0068] Example 2

[0069] In this example, the anti-tumor effect of Parabacteroides distasonis SYSU-210 screened in Example 1 was tested. Taking colorectal cancer as a representative of tumors, an animal experiment was carried out. The specific method is as follows:

[0070] (1) Twenty 6-week-old female Balb / c mice were selected. After clearing the intestinal bacteria with ABX combined antibiotics (100 mg / kg of metronidazole, vancomycin, ampicillin, and neomycin sulfate each) for one week, they were randomly divided into four groups, with 5 mice in each group;

[0071] (2) Group 1 (CON group) was intragastrically administered 100 μL of PBS every two days, Group 2 (BHI group) was intragastrically administered 100 μL of BHI concentrated solution every two days, Group 3 (SYSU-210 group) was intragastrically administered 100 μL of Parabacteroides distasonis SYSU-210 every two days; Group 4 (CM group) was intragastrically administered 100 μL of the concentrated culture supernatant of Parabacteroides distasonis SYSU-210 every two days. Each group was intragastrically administered until the end of the experiment;

[0072] (3) Two weeks after intragastric administration, each mouse was subcutaneously injected with 1*10 6 CT26 cells / 100 mL;

[0073] (4) When the tumor volume reached 50 mm 3 (tumor volume = length * width 2 * 0.5), the tumor size was measured starting from the 0th day of the experiment;

[0074] (5) When the tumor grew to a maximum of 1200 mm 3 , the mice were sacrificed, dissected in a biosafety cabinet, the tumor tissues were removed, and the tumor tissues of each group were weighed and the volume was calculated. The results are shown in Figure 3 .

[0075] Figure 3 The results showed that compared with the control group, intragastric administration of Parabacteroides distasonis SYSU-210 had better anti-tumor effects, and intragastric administration of the concentrated culture supernatant of Parabacteroides distasonis SYSU-210 also had anti-tumor effects compared with the control group, but its effects were slightly worse than those of the live bacteria group.

[0076] In addition, this example also explored the anti-tumor proliferation effect of Parabacteroides distasonis SYSU-210. The specific method is as follows:

[0077] For the flow cytometry experiments, some tumors and spleens were taken from each of the above four groups. The tumors were stained with CD3-FITC and CD8a-PE. After gently cutting the tumors with ophthalmic scissors in a 6-well plate, they were placed on a 200-mesh cell sieve, and the tumor was gently ground with the syringe plunger of a 10 mL syringe. After extrusion, a small amount of DMEM medium (or PBS) was used to rinse the cell sieve, and the cell suspension was rinsed into a 50 mL centrifuge tube and then aliquoted into 1.5 mL centrifuge tubes. The cells were washed twice with PBS solution containing 4% FBS and centrifuged at 3200 rpm for 5 min at 4 °C. After pouring off the supernatant after the second wash, the cells were resuspended with the prepared PBS solution containing 4% FBS + antibody (100 μL for each sample). For tumor staining of CD3+CD8a: The antibody was incubated in a black EP tube in a light-shielded box, and the cells were incubated with the antibody in the darkroom. The dosage of the antibody was according to the instruction manual. For 100 μL, 5 μL of CD3 and 5 μL of CD8a were added, and the staining was carried out for 1 h. After the staining was completed, 1 mL of PBS containing 4% FBS was added, the supernatant was centrifuged off, and then the cells were resuspended with 500 μL of PBS containing 4% FBS and loaded onto the machine. The results are shown in Figure 4 .

[0078] Figure 4 The results showed that after intragastric administration of the Parabacteroides distasonis SYSU-210 or its culture supernatant, the number of CD8+ T cells infiltrating in the tumors of mice increased, that is, the anti-tumor immune ability was enhanced, effectively improving the immune system's ability to recognize and attack tumors, thereby achieving better anti-tumor effects.

[0079] Example 3

[0080] This example tested the anti-tumor effects of Parabacteroides distasonis SYSU-210 combined with the immune checkpoint inhibitor αPD-1. Colorectal cancer was used as a representative of tumors for animal experiments. The specific method is as follows:

[0081] (1) Twenty 6-week-old female Balb / c mice were selected. After clearing the intestinal bacteria with ABX combined with antibiotics (100 mg / kg each of metronidazole, vancomycin, ampicillin, and neomycin sulfate) for one week, they were randomly divided into four groups, with 5 mice in each group;

[0082] (2) Group 1 (PBS group) and Group 2 (αPD-1 group) were gavaged with 100 μL of PBS every two days, and Group 3 (SYSU-210 + αPD-1 group) was gavaged with 100 μL of Parabacteroides distasonis SYSU-210 every two days; Group 4 (CM + αPD-1 group) was gavaged with 100 μL of the concentrated supernatant of Parabacteroides distasonis SYSU-210 culture every two days. Each group was gavaged until the end of the experiment;

[0083] (3) Two weeks after gavage, 1 × 10 6 CT26 cells / 100 mL were subcutaneously injected into each mouse. When the tumor volume reached 50 mm 3 (tumor volume = length × width² × 0.5), the measurement of tumor size was started on the 0th day of the experiment;

[0084] (4) From the 9th day after tumor inoculation, Groups 2 - 4 were intraperitoneally injected with the αPD-1 inhibitor (BioXcell, RMP1-14) every 3 days, with a dose of 10 mg / kg, for a total of 3 injections. The specific operation process is as Figure 5 shown in A.

[0085] (5) Body weight was recorded and fresh feces were collected on the 0th, 4th, 8th, 12th, 16th, 20th, and 24th days, and stored at -80 °C for subsequent sequencing.

[0086] (6) When the tumor grew to a maximum of 1200 mm 3 , the mice were sacrificed. Dissection was performed in a biosafety cabinet, and the tumor tissue was removed. The tumor tissue was weighed and its volume was calculated. The results are shown in Figure 5 B.

[0087] Figure 5 The results in B showed that, compared with the blank control group, the live Parabacteroides distasonis SYSU-210 or its culture supernatant of the present invention combined with the αPD-1 inhibitor could significantly enhance the anti-tumor effect.

[0088] In addition, this example also explored the anti-tumor proliferative effect of Parabacteroides distasonis SYSU-210 combined with the αPD-1 inhibitor. The specific method is as follows:

[0089] For each of the above four groups, a part of the spleen was taken for flow cytometry experiments. The tumors were stained with CD3-FITC, CD8a-PE, and GZMB-APC. After gently cutting the tumors with ophthalmic scissors in a 6-well plate, they were placed on a 200-mesh cell sieve. The tumors were gently ground with the plunger of a 10 mL syringe. After extrusion, a small amount of DMEM medium (or PBS) was aspirated to wash the cell sieve, and the cell suspension was washed into a 50 mL centrifuge tube and then aliquoted into 1.5 mL centrifuge tubes. The cells were washed twice with PBS (the second time with PBS solution containing 4% FBS), centrifuged at 3200 rpm at 4 °C for 3 min. After the second wash, the supernatant was discarded, and the cells were resuspended with the prepared PBS solution containing 4% FBS + antibody (100 μL for each sample).

[0090] The results are as Figure 6 shown. After intragastric administration of Parabacteroides distasonis SYSU-210, it can further promote the activation of CD8+ T cells by αPD-1 inhibitor, increase the number of CD8+ T cells secreting GZMB, and further enhance the anti-tumor immunity of mice.

[0091] Example 4

[0092] In this example, the anti-tumor effect of Parabacteroides distasonis SYSU-210 screened in Example 1 was tested, and breast cancer was used as a representative of tumors for animal experiments. The specific method is as follows:

[0093] (1) Ten 6-week-old female Balb / c mice were selected. After clearing the intestinal bacteria with ABX combined with antibiotics (100 mg / kg each of metronidazole, vancomycin, ampicillin, and neomycin sulfate) for one week, they were randomly divided into two groups, with 5 mice in each group;

[0094] (2) Group 1 (PBS group) was intragastrically administered 100 μL of PBS every two days, and Group 2 (SYSU-210 group) was intragastrically administered 100 mL of Parabacteroides distasonis SYSU-210 every two days. Each group was intragastrically administered until the end of the experiment;

[0095] (3) Two weeks after intragastric administration, each mouse was subcutaneously injected with 1*10 6 4T1 cells / 100 μL;

[0096] (4) When the tumor volume reached 50 mm 3 (tumor volume = length * width2 * 0.5), the tumor size was measured starting from the 0th day of the experiment;

[0097] (5) When the tumor grew to a maximum of 1200 mm 3 , the mice were sacrificed, dissected in a biosafety cabinet, the tumor tissues were removed, and the tumor tissues were weighed and the volume was calculated. The results are shown in Figure 7 .

[0098] Figure 7 The results showed that compared with the control group, intragastric administration of Parabacteroides distasonis SYSU-210 also had good anti-tumor effects against breast cancer.

[0099] Example 5

[0100] In this example, the anti-tumor effects of the metabolites of Parabacteroides distasonis SYSU-210 were explored. The sera of the mice in the CON group and SYSU-210 group after dissection in Example 2 were taken, and the supernatants were subjected to spectroscopy with the CON group as the control. Use a pipette to aspirate 100 μL of the sample and place it in a 1.5 mL EPP tube; add 4 times the amount of acetonitrile:methanol (1:1), vortex for 30 s, sonicate in an ice bath for 10 min; place in a -20 °C refrigerator for 1 h; centrifuge at 4 °C and 12,000 rpm for 15 min; take the supernatant and inject it into an AB SCIEX high-resolution Q-TOF liquid chromatography-mass spectrometry instrument for analysis.

[0101] The results of serum metabolomics showed ( Figure 8 ), after intragastric administration with SYSU-210, the metabolite sebacic acid (SA) changed greatly. Therefore, SA was selected in the following of this example to verify its anti-tumor effect, and the experimental method was as follows:

[0102] (1) Select 15 6-week-old female Balb / c mice, randomly divide them into 3 groups with 5 mice in each group; inject 1×10 6 CT26 cells per mouse / 100 μL;

[0103] (2) Starting 14 days before tumor implantation, group 1 (Con group) was intraperitoneally injected with the vehicle (10% DMSO + 5% tween-20 + 40% PEG300 + 45% saline) every two days, group 2 was intraperitoneally injected with SA 10 mg / kg dissolved in the vehicle every two days, and group 3 was intraperitoneally injected with SA 5 mg / kg dissolved in the vehicle every two days;

[0104] (3) When the tumor volume reached 50 mm 3 (tumor volume = length × width2 × 0.5), the tumor size was measured starting from day 0 of the experiment;

[0105] (4) When the tumor grew to a maximum of 1200 mm 3 , the mice were sacrificed, dissected in a biosafety cabinet, the tumor tissues were taken out, and the tumor tissues were weighed and the volume was calculated. The results are shown in Figure 9 .

[0106] Figure 9The results showed that, compared with the control group, the metabolite SA of SYSU-210 had better anti-tumor effects. Especially when the concentration of SA was 10 mg / kg, the anti-tumor effect was the best.

[0107] In addition, this example also explored the anti-tumor proliferation effect of the metabolite SA of SYSU-210. The specific method was as follows:

[0108] For the flow cytometry experiments, a part of the tumors from each of the above two groups was taken. The tumors were stained with CD3-FITC, CD8a-PE, and GZMB-APC. After gently cutting the tumors with ophthalmic scissors in a 6-well plate, they were placed on a 200-mesh cell strainer. The tumor was gently ground with the plunger of a 10 mL syringe. After extrusion, a small amount of DMEM medium (or PBS) was used to rinse the cell strainer, and the cell suspension was rinsed into a 50 mL centrifuge tube and then aliquoted into 1.5 mL centrifuge tubes. The cells were washed twice with PBS (the second wash was with PBS solution containing 4% FBS), centrifuged at 3200 rpm at 4°C for 3 min. After the second wash, the supernatant was discarded, and the cells were resuspended with the prepared PBS solution containing 4% FBS + antibody (100 μL for each sample). The results are shown in Figure 10 .

[0109] Figure 10 The results showed that in the mice of the SA 10 mg / kg intraperitoneal injection group, there were more CD8+ T cells infiltrating in the tumor than in the control group, and there were also more CD8+ T cells secreting GZMB, indicating that the metabolite SA of SYSU-210 could increase the infiltration of CD8+ T cells in the tumor and enhance anti-tumor immunity.

[0110] Example 6

[0111] This example tested the anti-tumor effect of the metabolite SA of SYSU-210 combined with the immune checkpoint inhibitor αPD-1. Taking colorectal cancer as a representative of the tumor, an animal experiment was carried out. The specific method was as follows:

[0112] (1) Thirty 6-week-old female Balb / c mice were selected and randomly divided into 6 groups, with 5 mice in each group; each mouse was subcutaneously injected with 1*10 6 CT26 cells / 100 mL.

[0113] (2) Starting 14 days before tumor inoculation, groups 1, 3, and 5 were intraperitoneally injected with the vehicle (10% DMSO + 5% tween-20 + 40% PEG300 + 45% saline) every two days, and groups 2, 4, and 6 were intraperitoneally injected with SA 10 mg / kg dissolved in the vehicle every two days, and gavaged until the end of the experiment;

[0114] (3) When the tumor volume reached 50 mm 3(Tumor volume = length * width2 * 0.5) was used to measure the tumor size starting from day 0 of the experiment;

[0115] (4) Starting from the 9th day after tumor inoculation, in groups 3 - 4, the αPD-1 inhibitor was intraperitoneally injected every 3 days at a dose of 10 mg / kg, and in groups 5 - 6, the chemotherapy drug oxaliplatin (OXA) dissolved in the vehicle was intraperitoneally injected every 3 days at a dose of 5 mg / kg, for a total of 3 injections. The specific operation process is as Figure 11 shown in A;

[0116] (5) When the tumor grew to a maximum of 1200 mm 3 the mice were sacrificed, dissected in a biosafety cabinet, the tumor tissues were removed, and the tumor tissues were weighed and their volumes were calculated. The results are shown in Figure 11 (B - C).

[0117] Figure 11 (B - C) The results showed that compared with the blank control group, the SYSU-210 metabolite SA combined with the αPD-1 inhibitor or the chemotherapy drug oxaliplatin could significantly enhance the anti-tumor effect, and its effect was significantly better than that of using SA, the αPD-1 inhibitor or oxaliplatin alone.

[0118] In addition, this example also explored the anti-tumor proliferative effect of the SYSU-210 metabolite SA combined with the αPD-1 inhibitor. The specific method is as follows:

[0119] For each group in groups 1 - 4 above, a part of the tumor was taken for flow cytometry experiments. The tumors were stained with CD3-FITC, CD8a-PE, and GZMB-APC. After gently cutting the tumors with ophthalmic scissors in a 6-well plate, they were placed on a 200-mesh cell sieve, and the tumors were gently ground with the syringe plunger of a 10 mL syringe. After extrusion, a small amount of DMEM medium (or PBS) was used to rinse the cell sieve, and the cell suspension was rinsed into a 50 mL centrifuge tube, and then aliquoted into 1.5 mL centrifuge tubes. They were washed twice with PBS (the second time with PBS solution containing 4% FBS), centrifuged at 3200 rpm at 4 °C for 3 min. After the second wash, the supernatant was poured off, and then resuspended with the prepared PBS solution containing 4% FBS + antibody (100 μL for each sample).

[0120] The results are as Figure 12 shown. In the mice in the group intraperitoneally injected with SA at 10 mg / kg combined with the αPD-1 inhibitor group or the combined OXA group, there were more CD8+ T cells secreting GZMB infiltrating in the tumors than in the single-drug groups, further indicating that SA can cooperate with the αPD-1 inhibitor or OXA to increase the infiltration of CD8+ T cells in the tumors and enhance anti-tumor immunity.

[0121] Example 7

[0122] In this example, the effect of Parabacteroides distasonis SYSU-210 on reducing LPS-induced apoptosis of NCM460 cells was explored to verify the protective effect of SYSU-210 on intestinal epithelial cells. The specific method is as follows:

[0123] (1) The experiment was divided into 4 groups: experimental group 1 (Con group), experimental group 2 (LPS group), experimental group 3 (CM + LPS group), and experimental group 4 (SYSU-210 + LPS group);

[0124] (2) After 2*10 5 / well of NCM460 was cultured overnight in a 6-well plate, 15 μL of CM (filtered SYSU-210 culture supernatant with a pH of 7.4) was added to experimental group 3, and 15 μL of SYSU-210 bacterial suspension resuspended in 10 9 CFU / mL PBS was added to experimental group 4. After the other two groups were cultured with an equal volume of PBS for 8 h, LPS at 100 μg / mL was added to the other 3 groups, and an equal volume of PBS was added to the Con group;

[0125] (3) After 48 h of culture, samples were collected (the medium was discarded, the cells were washed with PBS, digested with 500 μL of trypsin for 2 min, and then terminated with 500 μL of DMEM medium; centrifuged at 1000 rpm for 5 min), and the Annexin V-FITC apoptosis detection kit from Beyotime was used;

[0126] (4) Approximately 1 mL of PBS pre-cooled at 4°C was added to resuspend the cells, and the cells were centrifuged again to precipitate. After carefully aspirating the supernatant, the cells were resuspended with 1× binding buffer, and the concentration was adjusted to 1×10 6 / mL; 100 μL of the cell suspension was taken into a 5 mL flow tube, 5 μL of Annexin V-APC and 8 μL of PI were added, and after mixing, it was incubated at room temperature in the dark for 10 - 15 min. After the incubation, 400 μL of 1× AnnexinV binding buffer was added, mixed well, and flow cytometry was performed; AnnexinV-APC was excited by 633 nm, and PI was excited by 488 nm. The results are shown in Figure 13 .

[0127] Figure 13 The results showed that the co-culture of SYSU-210 could effectively reduce LPS-induced apoptosis of NCM460 cells and had a protective effect on intestinal epithelial cells.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A Parabacteroides distasonis, characterized in that, The Parabacteroides distasonis is the Parabacteroides distasonis SYSU-210 strain, which is deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No. 32494, and the deposit date of November 4, 2024.

2. The Parabacteroides distasonis according to claim 1, characterized in that, The 16S rRNA sequence of the Parabacteroides distasonis SYSU-210 strain is as shown in SEQ ID NO.

1.

3. A Parabacteroides distasonis culture, characterized in that, The Parabacteroides distasonis culture is prepared from the Parabacteroides distasonis described in claim 1 or 2.

4. The Lysobacter enzymogenes culture according to claim 3, wherein The Lysobacter enzymogenes culture is prepared by at least one of the following methods (1)-(3): (1) Scraping the activated Parabacteroides distasonis into a sterile solvent to obtain a Parabacteroides distasonis suspension, and the suspension is the Parabacteroides distasonis culture; (2) Scraping the activated Parabacteroides distasonis and inoculating it into a liquid medium for culture, centrifuging to obtain the supernatant, and then filtering the supernatant to obtain a fermentation filtrate, and the fermentation filtrate is the Parabacteroides distasonis culture; (3) Scraping the activated Parabacteroides distasonis and inoculating it into a liquid medium for culture, centrifuging to obtain the supernatant, then filtering the supernatant to obtain a fermentation filtrate, and separating the metabolites in the fermentation filtrate, and the metabolites are the Parabacteroides distasonis culture.

5. The Lysobacter enzymogenes culture according to claim 4, wherein The metabolites include sebacic acid.

6. Use of the Parabacteroides distasonis described in claim 1 or 2, or the Parabacteroides distasonis culture described in any one of claims 3-5 in the preparation of a drug for preventing and / or treating cancer.

7. The application according to claim 6, characterized in that, The cancer includes colorectal cancer and breast cancer.

8. Use of the Parabacteroides distasonis described in claim 1 or 2, or the Parabacteroides distasonis culture described in any one of claims 3-5 in combination with an immune checkpoint inhibitor or a chemotherapeutic drug in the preparation of a drug for treating cancer.

9. The application according to claim 5, wherein The immune checkpoint inhibitor includes at least one of PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, VISTA and A2aR antibodies; the chemotherapeutic drug includes oxaliplatin.

10. A drug for preventing or treating cancer, characterized in that, The drug includes an immune checkpoint inhibitor, and also includes the Parabacteroides distasonis described in claim 1 or 2, and / or the Parabacteroides distasonis culture described in any one of claims 3-5.

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