WO2020147191A1 - APPLICATION OF AKKERMANSIA MUCINIPHILA OR PREVOTELLA IN PREPARING DRUG FOR INCREASING γδ T CELL ACCUMULATION IN TUMOR MICROENVIRONMENT AND ENHANCING ANTITUMOR IMMUNITY - Google Patents

Abstract

Provided is an application of Akkermansia muciniphila or Prevotella in preparing an antitumor drug for increasing γδ T cell infiltration in a tumor microenvironment. Also provided are a pharmaceutical composition and an edible composition comprising Akkermansia muciniphila or Prevotella.

Description

Application of Akkermania or Prevotella in drugs that increase tumor microenvironment γδT cell accumulation and enhance anti-tumor immune function Technical field

The present invention relates to the field of biomedicine, in particular to the use of Ackerman myxobacter (or Ackerman myxa bacteria) or Prevotella or a composition comprising the use of Ackerman myxobacter or Prevotella to resist tumors and increase γδ T cells (TCR-γδ positive T cells, TCRγδ positive T cells or TCR-γδ+T cells) in the application of drugs for infiltration in the tumor microenvironment.

Background technique

So far, malignant tumors are still the main killer threatening human health. For example, liver cancer is a malignant tumor with a very high degree of malignancy, a very low cure rate, and a poor prognosis. The incidence of liver cancer in the world ranks 5th among men and the second highest mortality rate; among women The rate is 9th, and the death rate is 6th. China is a country with a high incidence of liver cancer, with deaths accounting for more than 50% of the global deaths. Traditional treatments include surgical resection, radiotherapy and chemotherapy, but the prognosis is poor. Breast cancer is one of the most common malignant tumors in women worldwide. The latest World Health Organization's cancer today (CANCER TODAY) epidemiological statistics show that in 2018, there were approximately 2 million new cases of breast cancer worldwide and 600,000 deaths. Although current treatments include surgery, chemotherapy, radiotherapy, targeted therapy, neoadjuvant chemotherapy, and many other comprehensive treatment methods, the current treatment effect is limited, the problems of tumor metastasis and recurrence are still difficult to solve, and the side effects of chemotherapy and radiotherapy are serious. , Severely damage the patient's immune system and quality of life. Therefore, it is urgent to develop more effective therapies that can kill tumor cells to the maximum without harming normal cells.

Tumor immunotherapy refers to a treatment method that stimulates the body to produce a tumor-specific immune response through active or passive means, restores or enhances the activity of the body's immune system, and thereby inhibits and kills tumor functions. At present, it mainly includes blocking of immune checkpoints, adoptive cell infusion, and tumor vaccines. Although γδ T cells are one of the key immune cell subgroups for the body to exert anti-tumor function, the current T cell immunotherapy is not effective, mainly because the infiltration and function of γδ T cells in the tumor microenvironment are inhibited, which is manifested in tumor infiltration The small amount, low activity and poor targeting of γδ T cells result in a considerable number of patients receiving anti-tumor immunotherapy that cannot respond effectively to immunotherapies such as T cell inhibitory molecule blocking antibody therapy, which seriously affects anti-tumor immunotherapy effect. Therefore, solving the problem of insufficient γδT cell infiltration in the tumor microenvironment is the key to solving the challenges faced by tumor immunotherapy.

Akkermansia muciniphila is a gram-negative, oval-shaped intestinal bacteria that colonizes the mucus layer and can specifically degrade mucin, accounting for 1 to 3% of the total intestinal microbes. One of the most abundant single species in the Tao has a certain anaerobic capacity. Current studies have found that the colonization of Ackermann myxobacteria in the human body may be negatively correlated with obesity and type 2 diabetes, and may also have a certain correlation with cardiometabolic disorders, and may play an important role in body metabolism. Prevotella copri is a gram-negative anaerobic bacterium, a symbiotic bacterium in the human intestine. Current studies have found that it may be related to the susceptibility of rheumatoid arthritis, and may also be related to insulin resistance in diabetic patients.

However, there is no report on the use of intestinal bacteria including Akkerman myxobacteria and/or Prevotella to increase the infiltration of γδT cells in the tumor microenvironment to enhance the body's anti-tumor function.

Summary of the invention

The technical problem to be solved by the present invention is to solve the current tumor immunotherapy problems, especially the problem of insufficient infiltration of γδ T cells in the tumor microenvironment, and provide an anti-tumor and a method for increasing the infiltration of γδ T cells in the tumor microenvironment. Specifically, it is the application of Ackerman myxobacteria or Prevotella in drugs that increase tumor microenvironment γδT cell accumulation and enhance anti-tumor immune function.

In order to achieve the above object, the present invention provides the use of Akkermansia muciniphila or Prevotella copri for anti-tumor. The purpose is to increase the infiltration of γδ T cells in the tumor microenvironment to fight tumors. The Ackerman myxobacteria or Prevotella is any one of the following: Ackerman myxobacteria or Prevotella viable cells; after genetic recombination, transformation or modification, attenuation, inactivation, physical treatment or chemical Treated Myxobacteria or Prevotella; Myxobacteria Ackermann or Prevotella lysates, protein extracts, bacterial components, bacterial extracts and/or metabolites; and/or Myxobacteria Ackermann Or Prevotella culture supernatant.

The tumor of the present invention can be various solid tumors, such as but not limited to liver cancer, breast cancer, lung cancer, melanoma tumor, prostate cancer, fibrosarcoma, gastric cancer, esophageal cancer, colorectal cancer, bladder sarcoma, glioma and other entities Tumors, especially tumors of the liver and/or breast.

In certain embodiments, methods of anti-tumor and increasing γδ T cell infiltration in the tumor microenvironment are combined with other treatment techniques. In certain embodiments, the other treatment methods include, but are not limited to, immunotherapy techniques, surgery, chemotherapy, radiation therapy, gene therapy, or a combination thereof.

In order to better achieve the above-mentioned purpose, the present invention also provides the use of Akkerman myxobacteria or Prevotella in the preparation of drugs for anti-tumor and increasing the infiltration of γδ T cells in the tumor microenvironment. The Ackerman myxobacteria or Prevotella is any one of the following: Ackerman myxobacteria or Prevotella viable cells; after genetic recombination, transformation or modification, attenuation, inactivation, physical treatment or chemical Treated Myxobacteria or Prevotella; Myxobacteria Ackermann or Prevotella lysates, protein extracts, bacterial components, bacterial extracts and/or metabolites; and/or Myxobacteria Ackermann Or Prevotella culture supernatant. The tumor is liver and/or breast, or tumors related to lung, skin, cancer, kidney, prostate, nervous system or bladder, especially tumors of liver and/or breast.

The characteristics of the increased infiltration of γδ T cells in the tumor microenvironment include: an increase in the number of γδ T cells relative to the number of all cells in the tumor microenvironment.

The present invention also provides a therapeutic and preventive pharmaceutical composition, which contains Ackerman myxobacterium or Prevotella as an active ingredient of the medicine. In some embodiments, the pharmaceutical composition may also contain other microbial strains. In one aspect, the Akkerman myxobacterium or Prevotella can inhibit tumor growth. In one aspect, the tumor is a solid tumor, including but not limited to solid tumors such as liver cancer, breast cancer, lung cancer, melanoma, prostate cancer, fibrosarcoma, gastric cancer, esophageal cancer, colorectal cancer, bladder sarcoma and glioma . In certain administration schemes, the tumor includes but is not limited to liver cancer and/or breast cancer.

According to one aspect of the present invention, in the above-mentioned pharmaceutical composition, the Ackerman myxobacteria or Prevotella are any one of the following: Ackerman myxobacteria or Prevotella viable cells; after genetic recombination, Modified or modified, attenuated, inactivated, physically or chemically treated Myxobacteria or Prevotella; Myxobacteria or Prevotella lysates, protein extracts, bacterial components, bacterial extracts And/or metabolites; and/or Ackerman myxobacteria or Prevotella culture supernatant.

According to one aspect of the present invention, the drug combination of the present invention increases the infiltration of γδ T cells in the tumor microenvironment to achieve anti-tumor.

According to one aspect of the present invention, the pharmaceutical composition of the present invention includes a pharmaceutically effective dose of Ackerman myxobacterium or Prevotella and a pharmaceutically acceptable carrier thereof. Wherein, the Ackerman myxobacterium or Prevotella is the active ingredient.

Preferably, in the above-mentioned pharmaceutical composition, the Ackerman myxobacteria or Prevotella are any one of the following: Ackerman myxobacteria or Prevotella viable cells; after genetic recombination, transformation or modification, Attenuated, inactivated, physically treated or chemically treated Ackerman myxobacteria or Prevotella; Ackerman myxobacteria or Prevotella lysates, protein extracts, bacterial components, bacterial extracts and/or metabolism物; and/or Ackerman myxobacteria or Prevotella culture supernatant.

Preferably, in the above-mentioned pharmaceutical composition, the pharmaceutical composition may be any one or more dosage forms that are pharmaceutically feasible, including but not limited to tablets, capsules, oral liquids or freeze-dried powders.

Preferably, in the above-mentioned pharmaceutical composition, the pharmaceutically acceptable carrier is skimmed milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, gum arabic, calcium phosphate, alginate, Gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil One or more mixtures.

In order to better achieve the above object, the present invention also provides an edible composition for anti-tumor and increasing the infiltration of γδT cells in the tumor microenvironment, wherein the edible composition comprises Ackerman myxobacterium or Prevotella . The edible composition includes but is not limited to food, health care products, food additives and the like.

Preferably, in the above-mentioned edible composition, the Ackerman myxobacteria or Prevotella are any one of the following: Ackerman myxobacteria or Prevotella viable cells; after genetic recombination, transformation or modification, Attenuated, inactivated, physically treated or chemically treated Ackerman myxobacteria or Prevotella; Ackerman myxobacteria or Prevotella lysates, protein extracts, bacterial components, bacterial extracts and/or metabolism物; and/or Ackerman myxobacteria or Prevotella culture supernatant.

According to one aspect of the present invention, the edible composition of the present invention achieves anti-tumor function by increasing the infiltration of γδ T cells in the tumor microenvironment.

The present invention establishes a mouse liver cancer model and/or breast cancer model by the transplantation tumor research method, and analyzes, detects and detects the function of Ackerman myxobacterium or Prevotella in the mouse liver cancer model and/or breast cancer model. Identification. The present invention proves through experiments that Ackerman myxobacteria or Prevotella can significantly inhibit the growth of liver tumors and/or breast tumors, and can effectively inhibit the growth of transplanted tumors in mice, indicating that Ackerman myxobacteria or Prevotella It has important development and application value in the clinical treatment of tumors.

BRIEF DESCRIPTION

Figure 1 is a schematic diagram of the experimental procedure for detecting Ackerman myxobacteria or inactivated Ackerman myxobacteria for anti-tumor and increasing γδT cell infiltration in the tumor microenvironment in a mouse liver cancer model. The time for administering bacteria or transplanting tumor cells is expressed in days (Day, d).

Figure 2 is a schematic diagram of an experimental procedure for detecting Plasmodium or inactivated Plasmodium for anti-tumor and increasing the infiltration of γδ T cells in the tumor microenvironment in a mouse liver cancer model. The time for administering bacteria or transplanting tumor cells is expressed in days (Day, d).

Figure 3 is a schematic diagram of the experimental procedure for detecting the effect of Prevotella or inactivated Prevotella on tumor growth inhibition and tumor treatment in a mouse breast cancer model. The time for administering bacteria or transplanting tumor cells is expressed in days (Day, d).

Fig. 4 is a comparison diagram of the size of liver cancer tumors of 4 typical mice in each group after treatment with Ackerman myxobacteria or inactivated Ackerman myxobacteria.

Figure 5 is a statistical analysis diagram of the comparison of the size of mouse liver cancer tumors after treatment with Ackerman myxobacteria or inactivated Ackerman myxobacteria.

Fig. 6 is a comparison diagram of the tumor size of liver cancer in 4 typical mice in each group after treatment with Prevotella or inactivated Prevotella.

Fig. 7 is a statistical analysis diagram of a comparison diagram of the size of mouse liver cancer tumors after treatment with Prevotella or inactivated Prevotella.

Fig. 8 is a comparison diagram of the size of breast cancer tumors of 4 typical mice in each group after treatment with Prevotella or inactivated Prevotella.

Fig. 9 is a statistical analysis diagram of the comparison of the size of mouse breast cancer tumors after treatment with Prevotella or inactivated Prevotella.

Figure 10 is a flow cytometric analysis diagram of typical γδ T cells of one mouse in each group after administration of Ackerman myxobacteria or inactivated Ackerman myxobacteria in mice transplanted with liver cancer cells, the right quadrant is γδ T cells, and the right The numbers in the side quadrants show the percentage of γδT cells in the total cells in the liver tumor microenvironment.

Figure 11 is a statistical analysis diagram of the percentage of γδT cells in the total cells in the tumor microenvironment after administration of Ackerman myxobacteria or inactivated Ackerman myxobacteria in mice transplanted with liver cancer cells.

Figure 12 is a flow cytometric analysis diagram of typical γδT cells of one mouse in each group after the mice transplanted with liver cancer cells are administered with Plasmodium or inactivated Plasmodium. The right quadrant is the γδT cell, and the numbers in the right quadrant Shown is the percentage of γδT cells in the total cells in the liver tumor microenvironment.

Figure 13 is a statistical analysis diagram of the percentage of γδ T cells in the total cells in the tumor microenvironment after administering or inactivated Prevotella in mice transplanted with liver cancer cells.

detailed description

The present invention will be further described below in conjunction with specific embodiments. It should be pointed out that the anti-tumor Ackerman myxobacter or Prevotella or the pharmaceutical composition, food, health care products and food additives containing the Ackerman myxobacter or Prevotella of the present invention After being administered to a subject, it can be applied to the above-mentioned indications and exhibit the above-mentioned functions. All dosage forms within the scope of the present invention have been tested. Hereinafter, it is only for illustration. A small part of them is described in the examples, but they should not be construed as limiting the present invention.

The Ackerman myxobacteria or Prevotella referred to in the present invention include but are not limited to any of the following: Ackerman myxobacteria or Prevotella viable cells; after genetic recombination, modification or modification, attenuation, sterilization Live, physically or chemically treated Ackerman myxobacteria or Prevotella; Ackerman myxobacteria or Prevotella lysates, protein extracts, cell components, cell extracts and/or metabolites; and/ Or Ackerman myxobacteria or Prevotella culture supernatant.

The tumor is a solid tumor, such as, but not limited to, liver cancer, breast cancer, melanoma, lung cancer, prostate cancer, fibrosarcoma, gastric cancer, esophageal cancer, bladder sarcoma, and glioma. In certain embodiments, the tumor includes, but is not limited to: liver cancer and/or breast cancer.

The pharmaceutical composition for anti-tumor provided by the present invention includes a pharmacologically effective dose of Akkerman myxobacterium or Prevotella. Wherein, the range of the so-called "pharmaceutical effective dose" is 10 ⁶ to 10 ¹⁰ CFU, preferably 10 ⁹ CFU.

The pharmaceutical composition includes, but is not limited to, tablets, capsules, oral liquids or freeze-dried powders. The pharmaceutically acceptable carrier includes, but is not limited to, skim milk, lactose, glucose, sucrose, sorbitol, acacia, mannose, starch, trehalose, calcium phosphate, alginate, gelatin, calcium silicate, One or more of fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil.

The Ackerman myxobacteria or Prevotella of the present invention can also be made into foods, health products or food additives. The foods, health products or food additives all contain Ackerman myxobacteria or Prevotella viable cells, genetic recombination, transformation or modification, attenuation, inactivation, physical treatment or chemical treatment of Ackerman myxobacteria or general Lysates, protein extracts, cell components, cell extracts and/or metabolites, and/or Ackerman myxobacter or Prevotella culture supernatant Any kind of. These foods, health products or food additives can be used for anti-tumor.

Example 1 Culture of Ackermann Myxobacteria

Cultivation method

Step 1: Take a freeze-dried Akkermansia muciniphila strain (purchased from ATCC), add 200μL TSB medium to dissolve it, take 200μl blood plate to streak, and place it in an incubator under anaerobic conditions. Cultivate for 48 hours at ℃;

Step 2: Pick a single colony and dissolve it in 10 ml TSB medium, and cultivate it under anaerobic conditions at 37°C for 48 hours;

Step 3: Take 500 ml of TSB medium, insert the strain at 1% (v/v), and cultivate for 48 hours under anaerobic conditions at 37°C;

Step 4: Collect the bacterial liquid and centrifuge at 6000 rpm for 10 minutes. After washing twice with normal saline, the bacterium mud was reconstituted for later use and the viable bacteria counted.

Example 2 Prevotella culture

Cultivation method

Step 1: Take a lyophilized Prevotella copri strain (purchased from ATCC), add 200μL PYG medium to dissolve it, take 200μl blood plate streak, and cultivate in an incubator at 37℃ under anaerobic conditions 48 hours;

Step 2: Pick a single colony and dissolve it in 10 ml TSB medium, and cultivate it under anaerobic conditions at 37°C for 48 hours;

Step 3: Take 500 ml of PYG medium, insert the strain at 1% (v/v), and cultivate for 48 hours under anaerobic conditions at 37°C;

Step 4: Collect the bacterial liquid and centrifuge at 6000 rpm for 10 minutes. After washing twice with normal saline, the bacterium mud was reconstituted for later use and the viable bacteria were counted.

Example 3 Experiment of Ackerman myxobacteria enhancing γδT cell infiltration in tumor microenvironment and treating tumor (liver cancer)

Figure 1 is a schematic diagram of the experimental procedure for detecting Ackerman myxobacteria or inactivated Ackerman myxobacteria to increase the infiltration and treatment of γδ T cells in the tumor microenvironment.

1. Training method

The culture method of Ackermann myxobacteria is the same as that of Example 1.

2. Sample preparation

1) Preparation of live bacteria of Ackerman myxobacteria

Step 1: Take a freeze-dried Akkermansia muciniphila strain (purchased from ATCC), add 200μL TSB medium to dissolve it, take 200μl blood plate to streak, and place it in an incubator under anaerobic conditions. Cultivate for 48 hours at ℃;

Step 2: Pick a single colony and dissolve it in 10 ml TSB medium, and cultivate it under anaerobic conditions at 37°C for 48 hours;

Step 3: Take 500 ml of TSB medium, insert the strain at 1% (v/v), and cultivate for 48 hours under anaerobic conditions at 37°C;

Step 4: Collect the bacterial liquid and centrifuge at 6000 rpm for 10 minutes. After washing twice with normal saline, the bacterium mud was reconstituted for later use and the viable bacteria were counted.

2) Ackerman myxobacteria inactivated bacteria

After the live bacteria count, the Ackerman myxobacter bacteria liquid was heated in a 70°C water bath for 30 minutes to obtain an inactivated bacterial liquid.

3) Ackerman myxobacteria lysate

Ackerman myxobacteria culture broth was treated by ultrasonic disintegration, ultrasonic for 2 seconds, 5 seconds off, and a total of 20 minutes to obtain Ackerman myxobacteria lysate.

4) Ackerman myxobacteria culture supernatant

The Ackerman myxobacteria culture broth was centrifuged at 6000 rpm for 10 minutes, and the supernatant was the Ackerman myxobacteria culture supernatant after centrifugation.

3. Mouse experiment on the effect of Ackerman myxobacteria on tumor prevention

Experimental animals: 27 C57BL/6 mice purchased from the Experimental Animal Center of Sun Yat-Sen University at 3 to 4 weeks in good mental state. The mice were randomly divided into 3 groups, each with 9 mice. The sex and age of the groups were matched. They were the control group, the live bacteria gavage group, and the inactivated bacteria gavage group. The three groups of mice were given physiological saline and Ake Manmyxobacteria and inactivated Ackerman myxobacteria were given by gavage for 4 consecutive treatments. After mouse tumor (liver cancer) cells HEPA1-6 grow to the logarithmic phase, digest the cells with trypsin, add medium for neutralization, collect the cells after centrifugation, wash twice with DPBS to remove residual serum, and finally resuspend the cells with DPBS . Counting of tumor cells, the ¹⁰⁶ cells were inoculated subcutaneously to the right armpit of each mouse. The mice were treated with intragastric gavage for 5 times. Two weeks later, the tumor-bearing mice were sacrificed. After dissection, the size of the subcutaneous transplanted tumor was measured, tumor cells were collected in situ, and the content of γδT cells in the tumor microenvironment was analyzed by flow cytometry.

Example 4 Prevotella promotes the infiltration of γδT cells in the tumor microenvironment and treats tumors (liver cancer) experiments

Fig. 2 is a schematic diagram of the experimental procedure for detecting Plasmodium or inactivated Plasmodium to increase the infiltration of γδ T cells in the tumor microenvironment and to treat tumors.

1. Training method

The method of cultivating Prevotella is the same as in Example 2.

2. Sample preparation

1) Preparation of Prevotella viable cells

Step 1: Take a lyophilized Prevotella copri strain (purchased from ATCC), dissolve it in 200μL PYG medium, take 200μl blood plate streak, and incubate in an incubator at 37°C for 48 hours under anaerobic conditions ; Pick a single colony and dissolve it in 10 ml of TSB medium and culture it under anaerobic conditions at 37°C for 48 hours;

Step 3: Take 500 ml of PYG medium, insert the strain at 1% (v/v), and cultivate for 48 hours under anaerobic conditions at 37°C;

Step 4: Collect the bacterial liquid and centrifuge at 6000 rpm for 10 minutes. After washing twice with normal saline, the bacterium mud was reconstituted for later use and the viable bacteria counted.

2) Prevotella inactivated bacteria

After the live bacteria count, the Ackerman myxobacter bacteria liquid was heated in a 70°C water bath for 30 minutes to obtain an inactivated bacterial liquid.

3) Prevotella lysate

The Prevotella culture broth was processed by ultrasonic disintegration, ultrasonic for 2 seconds, stopped for 5 seconds, and lasted for a total of 20 minutes, to obtain the Prevotella lysate.

4) Prevotella culture supernatant

The Prevotella culture broth was centrifuged at 6000 rpm for 10 min to obtain the Prevotella culture supernatant.

3. Mouse experiment on the effect of Prevotella on tumor prevention

Experimental animals: 27 C57BL/6 mice purchased from the Experimental Animal Center of Sun Yat-Sen University at 3 to 4 weeks in good mental state. The mice were randomly divided into 3 groups, 9 in each group (matching the sex and age of the mice between the groups), namely the control group, the live bacteria gavage group, and the inactivated bacteria gavage group. The 3 groups of mice were given physiological saline. , 10 ⁹ CFU of Prevotella and inactivated Prevotella of the same CFU by gavage, 4 consecutive treatments. After mouse tumor (liver cancer) cells HEPA1-6 grow to the logarithmic phase, digest the cells with trypsin, add medium for neutralization, collect the cells after centrifugation, wash twice with DPBS to remove residual serum, and finally resuspend the cells with DPBS . Counting of tumor cells, the ¹⁰⁶ cells were inoculated subcutaneously to the right armpit of each mouse. The mice were treated with intragastric gavage for 5 times. Two weeks later, the tumor-bearing mice were sacrificed. After dissection, the size of the subcutaneous transplanted tumor was measured, tumor cells were collected in situ, and the content of γδT cells in the tumor microenvironment was analyzed by flow cytometry.

Example 5 Prevotella inhibits tumor (breast cancer) growth experiment

Figure 3 is a schematic diagram of the experimental process of detecting the growth of Prevotella or inactivated Prevotella tumors and treating tumors (breast cancer).

1. Training method

The method of cultivating Prevotella is the same as in Example 2.

2. Sample preparation

1) Preparation of Prevotella viable cells

Step 1: Take a lyophilized Prevotella copri strain (purchased from ATCC), dissolve it in 200μL PYG medium, take 200μl blood plate streak, and incubate in an incubator at 37°C for 48 hours under anaerobic conditions ; Pick a single colony and dissolve it in 10 ml of TSB medium and culture it under anaerobic conditions at 37°C for 48 hours;

Step 3: Take 500 ml of PYG medium, insert the strain at 1% (v/v), and cultivate it under anaerobic conditions at 37°C for 48 hours;

Step 4: Collect the bacterial liquid and centrifuge at 6000 rpm for 10 minutes. After washing twice with normal saline, the bacterium mud was reconstituted for later use and the viable bacteria were counted.

2) Prevotella inactivated bacteria

After the live bacteria count, the Ackerman myxobacter bacteria liquid was heated in a 70°C water bath for 30 minutes to obtain an inactivated bacterial liquid.

3) Prevotella lysate

The Prevotella culture broth was processed by ultrasonic disintegration, ultrasonic for 2 seconds, stopped for 5 seconds, and lasted for a total of 20 minutes, to obtain the Prevotella lysate.

4) Prevotella culture supernatant

The Prevotella culture broth was centrifuged at 6000 rpm for 10 min to obtain the Prevotella culture supernatant.

3. Mouse experiment of Prevotella's anti-tumor effect

Experimental animals: 27 BALB/c mice purchased from the Experimental Animal Center of Sun Yat-Sen University, 3 to 4 weeks old, in good mental state. The mice were randomly divided into 3 groups, 9 in each group (matching the sex and age of the mice between the groups). They were the control group, the live bacteria gavage group, and the inactivated bacteria gavage group. They were given saline and 10 ⁹ CFU of Prevotella or inactivated Prevotella of the same CFU by intragastric administration for 4 consecutive times. After the mouse tumor (breast cancer) cell 4T1 grows to the logarithmic phase, the cells are digested with trypsin, then neutralized with the medium, the cells are collected after centrifugation, washed twice with DPBS to remove residual serum, and finally resuspended in DPBS cell. After cell counting, the ¹⁰⁶ cells were seeded subcutaneously into the right axilla of each mouse. The mice were given intragastric administration for 5 times, and the tumor-bearing mice were sacrificed 2 weeks later, and the size of the subcutaneous transplanted tumor was measured and recorded after dissection.

Result analysis:

The experimental procedure of administering Ackerman myxobacteria and inactivated Ackerman myxobacteria to mice after subcutaneous transplantation of hepatocellular carcinoma cells (HEPA1-6) is shown in Figure 1. The application of Prevotella and inactivated Prevotella The experimental flowchart is shown in Figure 2. Figure 3 is an experimental flow chart of administering Prevotella and inactivated Prevotella to mice after subcutaneously transplanted breast cancer cells (4T1). Figures 4 and 6 are photos of typical liver tumors transplanted into subcutaneous liver cancer cells in mice after administration of Ackerman myxobacteria, inactivated Ackerman myxobacteria, Prevotella, and inactivated Prevotella. Figures 5 and 7 show the statistical analysis results of the size and volume of liver tumors after administration of Ackerman myxobacteria, inactivated Ackerman myxobacteria, Prevotella, and inactivated Prevotella. Fig. 8 is a photo of a typical breast tumor transplanted with subcutaneous breast cancer cells in mice after the administration of Prevotella and inactivated Prevotella. Figure 9 shows the results of statistical analysis of the size and volume of breast tumors after the administration of Prevotella and inactivated Prevotella. The experimental results of Figure 4, Figure 5, Figure 6, and Figure 7 can clearly be observed that after administration of Ackerman myxobacteria, inactivated Ackerman myxobacteria, Prevotella and inactivated Prevotella, liver tumors The volume is greatly reduced by about 2 to 4 times. The experimental results in Figure 8 and Figure 9 show that the volume of breast tumors is significantly reduced by more than 20% after the application of Plasmodium and inactivated Plasmodium, and the reduction is statistically significant Sexual meaning. These experimental results indicate that the administration of Ackerman myxobacteria, inactivated Ackerman myxobacteria, Prevotella or inactivated Prevotella can significantly inhibit tumor growth.

Figure 10 is a flow cytometric analysis diagram of the number of γδT cells in a typical liver tumor microenvironment of one mouse in each group. The numbers in the right quadrant show the percentage of γδT cells in the tumor microenvironment. It can be seen from the flow cytometry diagram that the administration of Ackerman myxobacteria or inactivated Ackerman myxobacteria increased the relative amount of γδT cells in the liver tumor microenvironment by 5 to 8 times compared with the normal saline control group. Figure 11 is a statistical analysis diagram of the percentage of γδT cells in tumor microenvironment cells after administration of Ackerman myxobacteria or inactivated Ackerman myxobacteria in mice transplanted with liver cancer cells. It can be seen from the statistical graph that compared with the saline control group, Ackerman myxobacteria or inactivated Ackerman myxobacteria significantly increased the number of γδT cells in the tumor microenvironment, and these increases were statistically significant. Figure 12 is a flow cytometric analysis diagram of the number of γδT cells in a typical liver tumor microenvironment of one mouse in each group. The numbers in the right quadrant show the percentage of γδT cells in the tumor microenvironment. Similarly, it can be seen from the flow cytometry chart that compared with the normal saline control group, Prevotella and inactivated Prevotella increased the relative amount of γδT cells in the liver tumor microenvironment by about 5-8 times. Figure 13 is a statistical analysis diagram of the percentage of γδ T cells in all cells in the tumor microenvironment after administering Prevotella and inactivated Prevotella in mice transplanted with liver cancer cells. It can be seen from the statistical graph that compared with the normal saline control group, Prevotella and inactivated Prevotella significantly increased the number of γδT cells in the tumor microenvironment, and these increases were statistically significant.

In the statistical analysis diagram of the above results, * means student t-test p<0.05, and *** means student t-test p<0.001. p<0.05 has statistical significance. There were 9 mice in each treatment group.

In summary, Ackerman myxobacteria, inactivated Ackerman myxobacteria, Prevotella and/or inactivated Prevotella can all have a significant effect on the formation and growth of mouse tumors (liver cancer, breast cancer) The inhibitory effect of the (Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9). In addition, the experimental results of Figure 10, Figure 11, Figure 12, Figure 13 show that in the control group, mouse tumors (liver cancer), gavage Ackerman myxobacteria, inactivated Ackerman myxobacteria, Prevotella and inactivated Prevotella promotes the infiltration of γδT cells in the tumor microenvironment in vivo, thereby enhancing the anti-tumor function, inhibiting tumor growth, and having a good effect on the prevention and treatment of liver cancer and other tumors.

The above content is a further detailed description of the technical solutions provided in conjunction with the preferred embodiments of the invention. It cannot be considered that the specific implementation of the invention is limited to the above descriptions. For those of ordinary skill in the art to which the invention belongs, Without departing from the inventive concept of the present invention, a number of simple deductions or substitutions can be made, all of which should be regarded as belonging to the protection scope of the present invention.

Claims (9)

Akkermansia muciniphila or Prevotella copri is being prepared for anti-tumor and its increase in γδ T cells (TCR-γδ positive T cells, TCRγδ positive T cells or TCR-γδ + T cells) in tumors Application of infiltrating drugs in the microenvironment. The application according to claim 1, wherein the Ackerman myxobacteria or Prevotella is any one of the following: Ackerman myxobacteria or Prevotella viable cells; after genetic recombination, modification Or modified, attenuated, inactivated, physically or chemically treated Myxobacteria or Prevotella; Myxobacteria or Prevotella lysates, protein extracts, bacterial components, bacterial extracts and / Or metabolites; and / or Ackerman myxobacteria or Prevotella culture supernatant. The application according to claim 1, wherein the tumor includes but is not limited to a solid tumor. The application according to claim 1, wherein the tumor includes, but is not limited to, liver and/or breast solid tumors. The application according to claim 1, wherein the characteristics of increased γδ T cell infiltration in the tumor microenvironment include, but are not limited to: the absolute number of γδ T cells in the tumor microenvironment and/or the number of γδ T cells relative to all cells in the tumor microenvironment Increase in number. A pharmaceutical composition for anti-tumor and to increase the infiltration of γδ T cells in the tumor microenvironment, characterized in that the pharmaceutical composition comprises a pharmacologically effective dose of Akkerman myxobacterium or Prevotella and its pharmaceutical composition Acceptable carrier. The pharmaceutical composition according to claim 6, wherein the Ackerman myxobacterium or Prevotella is any one of the following: Ackerman myxobacterium or Prevotella viable cells; after genetic recombination , Modified or modified, attenuated, inactivated, physically treated or chemically treated Ackerman myxobacteria or Prevotella; Ackerman myxobacteria or Prevotella lysates, protein extracts, bacterial components, bacterial cells Extracts and/or metabolites; and/or Ackerman myxobacteria or Prevotella culture supernatant. An edible composition for anti-tumor and increasing the infiltration of γδ T cells in the tumor microenvironment is characterized in that the edible composition includes Ackerman myxobacterium or Prevotella. The edible composition according to claim 8, wherein the Ackerman myxobacter or Prevotella is any one of the following: Ackerman myxobacter or Prevotella viable cell; Recombinant, altered or modified, attenuated, inactivated, physically or chemically treated Myxobacteria or Prevotella; Myxobacteria or Prevotella lysates, protein extracts, bacterial components, bacterial cells Extracts and/or metabolites; and/or Ackerman myxobacteria or Prevotella culture supernatant.

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