KR20230113195A - Diagnosis of decreased immunity through intestinal flora analysis of gastric cancer patients and theragnosis composition using intestinal flora - Google Patents

Abstract

The present invention relates to immunocompromised diagnosis through intestinal flora analysis of gastric cancer patients and a Theragnosis composition using the intestinal flora. It was confirmed that there was a difference between healthy adults and patients with benign tumors. In addition, in the gastric cancer patient group, it was confirmed that the immune function was lowered, and the immune function of the patient was lowered as the gastric cancer progressed. In addition, when the patient-derived macrophages were treated with Faecalibacterium prausnitzii strain and its metabolite, butyrate, which was confirmed to be reduced in the gastric cancer patient group, PD-L1 and IL increased due to immunosuppression. It was confirmed that -10 was effectively suppressed. In addition, it was confirmed that apoptosis was effectively induced when gastric cancer cell lines were treated with Faecalibacterium prausnitzii strain and its metabolite butyrate. In addition, it was confirmed that butanoic acid inhibits tumor growth and reduces the expression of tumor markers and immunocompromised markers in an avatar animal model of a gastric cancer patient in which the patient's immune status is reflected and a gastric cancer cell line is transplanted. Therefore, it was confirmed that personalized medicine can be realized by diagnosing the intestinal flora colony collapse and immunosuppression in the gastric cancer patient group, and confirming the effect of increasing immunity and restoring the intestinal flora according to the treatment with the effective substance of the present invention.

Description

Diagnosis of decreased immunity through intestinal flora analysis of gastric cancer patients and theragnosis composition using intestinal flora}

The present invention relates to immunocompromised diagnosis through intestinal flora analysis of gastric cancer patients and to a theragnosis composition using the intestinal flora.

In order to treat cancer, researchers around the world are conducting numerous studies on various topics such as intracellular signaling mechanisms and metastasis of cancer cells, side effects of drugs, etc., and new drugs are developed every year, which makes many cancer patients expect a therapeutic effect. Clinical trials are being conducted. In addition to tumor tissue removal by surgery and radiation therapy, current anticancer drugs used for chemotherapy are applied differently depending on each type of cancer. In general, chemotherapeutic agents prescribed to cancer patients target cancer cells and have an effect of killing cancer cells, but also affect normal cells, resulting in side effects such as hair loss, fever, and reduced immunity in patients. Afterwards, based on genetic research on cancer, targeted anticancer drugs targeting genetic mutations occurring in each cancer type were developed, and side effects caused by existing chemotherapy drugs were improved, but cancer cells adapt very quickly to the environment. In order to escape from the attack of the target anticancer agent, resistance to the anticancer agent is induced, and there is a problem in that a 100% continuous cancer treatment effect by the targeted anticancer agent cannot be expected. Recently, studies on anticancer agents and tumor microenvironments have been actively conducted, and immunotherapeutic agents for various immune checkpoint inhibitors that control the immunity of patients while maintaining anticancer effects have been developed and are being used as therapeutic agents for patients. Among them, treatment for PD-1/PD-L1 is known to show a high therapeutic response in patients with skin cancer, lung cancer, and the like. These immuno-anticancer agents have functions of inhibiting the proliferation of cancer cells and increasing the activity of immune cells by regulating the function of immune cells in the tumor microenvironment. However, immuno-anticancer drugs also do not show the same anti-cancer effect in all patients, biomarkers for immuno-anticancer drugs are not clearly identified, anti-cancer drug resistance occurs due to JAK-STAT gene mutation, or autoimmune diseases due to the characteristics of antibody composites. There are problems such as the occurrence of expensive treatment costs.

On the other hand, the microbiome refers to microorganisms living in the human body, and includes the microorganisms in the intestine. The number of microbiome is more than twice the number of pure human cells, and the number of genes is more than 100 times. Therefore, it is also called the Second Genome because it is impossible to discuss human genes without mentioning microorganisms. The microbiome is a field that can be widely used in the development of new drugs and research on treatments for incurable diseases, as it can analyze the relationship between the principle of production of beneficial and harmful bacteria and diseases. The microbiome is also used in the development of food, cosmetics, and therapeutics.

As the next generation sequencing (NGS) technology advances, the importance of intestinal microbes in physiology and immune regulation of the human body is emerging, probiotics that can directly control the structure of the human intestinal microbiome. The importance of probiotics is emerging. Recently, probiotics have been known to be deeply related to the human microbiome through many studies, and their function as a regulator that changes the intestinal environment is attracting attention. Intake of probiotics is known not only to promote digestion through microbiome regulation, but also to inhibit inflammatory bowel disease, infectious diseases, and harmful bacteria. It has been found to be effective in various immune-related diseases and cancer, including cancer.

In addition, as the field of probiotics has grown significantly, much attention has been focused on prebiotics, synbiotics, and postbiotics. Dietary fiber and oligosaccharides, which serve as food for lactic acid bacteria, are representative examples. Synbiotics are a combination of probiotics and prebiotics. On the other hand, postbiotics, which has recently been attracting attention, is a material containing useful metabolites produced by probiotics and components of microorganisms, and is clearly defined as 'a non-living form of microorganisms beneficial to the host's health or a formulation containing components of the microorganisms' Postbiotics is attracting attention as a new alternative material that can overcome the limitations of safety, function, and stability of existing probiotics materials.

Accordingly, the present inventors identified and selected a microbiome with reduced diversity and taxa in the cancer patient group, confirmed that the selected strains improved the immunity of the cancer patient group, and the metabolites of the strain also improved the immune response. completed the present invention.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, containing butyrate or microbiome as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving cancer, including butyrate or microbiome as an active ingredient.

Another object of the present invention is to administer to a subject a composition containing butyrate or microbiome as an active ingredient; Faecalibacterium sp. ) to provide a method for increasing strains and reducing proteobacteria (Phylum) strains.

Another object of the present invention is to administer to a subject a composition containing butyrate or microbiome as an active ingredient; T cell function and antigen presenting cells (Antigen It is to provide a way to increase the function of Presenting Cell (APC).

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising butyrate or microbiome as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving cancer, including butyrate or microbiome as an active ingredient.

In addition, the present invention, butanoic acid (Butyrate) or the step of administering a composition containing a microbiome as an active ingredient to the subject; including, Cancer patient group Faecalibacterium genus ( Faecalibacterium sp. ) strain It provides a method for increasing and reducing Proteobacteria Phylum strains.

In addition, the present invention, the step of administering to a subject a composition containing butyrate or microbiome as an active ingredient; T cell function and antigen presenting cell (Antigen Presenting Cell) of a cancer patient group, including , APC) provides a method for increasing the function.

The present invention confirmed that the diversity of intestinal flora was reduced in gastric cancer patients, and it was confirmed that the composition ratio of intestinal flora was different from that of healthy adults and patients with benign tumors. In addition, in the gastric cancer patient group, it was confirmed that the immune function was lowered, and the immune function of the patient was lowered as the gastric cancer progressed. In addition, when the patient-derived macrophages were treated with Faecalibacterium prausnitzii strain and its metabolite, butyrate, which was confirmed to be reduced in the gastric cancer patient group, PD-L1 and IL increased due to immunosuppression. It was confirmed that -10 was effectively suppressed. In addition, it was confirmed that apoptosis was effectively induced when gastric cancer cell lines were treated with Faecalibacterium prausnitzii strain and its metabolite butyrate. In addition, it was confirmed that butanoic acid inhibits tumor growth and reduces the expression of tumor markers and immunocompromised markers in an avatar animal model of a gastric cancer patient in which the patient's immune status is reflected and a gastric cancer cell line is transplanted. Therefore, it was confirmed that personalized medicine can be realized by diagnosing the intestinal flora colony collapse and immunosuppression in the gastric cancer patient group, and confirming the effect of increasing immunity and restoring the intestinal flora according to the treatment with the effective substance of the present invention.

1 is a diagram comparing intestinal flora diversity in gastric cancer patients and healthy adults (A: comparison of intestinal flora diversity, B: comparison of reduced intestinal flora).
Figure 2 is a diagram analyzing the diversity of intestinal flora in gastric cancer patients and healthy adults by α-diversity and β-diversity (A: Observed OTU and Chao1 analysis results, B: PCoA plot and bray curstis distance analysis results).
Figure 3 is a diagram comparing the intestinal flora composition ratio in gastric cancer patients and benign tumor patients (A: intestinal flora composition ratio comparison, B: composition comparison in the genus Faecalibacterium)
Figure 4 is a diagram comparing the expression of T cell immunity deterioration factors in gastric cancer patients and benign tumor patients (A: IL-10 expression confirmed, B: IL-17 expression confirmed, C: INF-γ expression confirmed).
Figure 5 is a diagram comparing the expression of PD-1, a marker for reduced immunity of T cells, in gastric cancer patients and benign tumor patients (A: CD4 + cell comparison, B: CD8 + cell comparison).
FIG. 6 is a diagram comparing the expression of PD-L1, an immunocompromising factor in antigen-presenting cells, in gastric cancer patients and benign tumor patients (A: comparison of CD68 + cells, B: comparison of CD11 + cells).
FIG. 7 is a diagram comparing the expression of IL-10, an immunity-decreasing factor, in antigen-presenting cells in gastric cancer patients and benign tumor patients (A: comparison of CD68 + cells, B: comparison of CD11 + cells).
8 is a diagram comparing the expression of PD-1 and CTLA-4, which are T-cell immunity reducing factors, in early gastric cancer patients and late gastric cancer patients (A: comparison of PD-1 expression, B: comparison of CTLA-4 expression). ).
9 is a diagram comparing the expression of PD-L1 and IL-10, which are immune-lowering factors of antigen-presenting cells, in early gastric cancer patients and late gastric cancer patients (A: comparison of PD-L1 expression, B: expression of IL-10) comparison).
10 is a diagram comparing the expression of PD-L1, an immunity-lowering factor, in antigen-presenting cells according to the course of gastric cancer in gastric cancer patients (A: comparison of CD68 + cells, B: comparison of CD11 + cells).
FIG. 11 is a view showing the expression of PD-L1 and IL-10, which are immune-degrading factors, in gastric cancer tissues according to the course of gastric cancer in gastric cancer patients using a confocal microscope (A: analysis result, B: quantification of analysis result).
12 is a diagram comparing the expression of PD-L1 and IL-10 in macrophages of gastric cancer patients treated with butyrate (A: comparison of PD-L1 expression, B: comparison of IL-10 expression).
13 is a diagram comparing the expression of PD-L1 according to the treatment of Faecalibacterium prausnitzii in macrophages of gastric cancer patients.
Figure 14 is a diagram confirming the expression of immunocompromised markers according to the treatment of butyrate or Faecalibacterium prausnitzii in macrophages of gastric cancer patients (A: quantification of marker expression in macrophages, B: Quantification of marker expression in culture, C: Quantification of marker expression following Faecalibacterium prosniche treatment).
15 is a diagram showing apoptosis according to treatment of gastric cancer cell lines with Faecalibacterium prausnitzii of the present invention by flow cytometry (A: flow cytometry results, B: quantification of analysis results).
16 is a diagram showing apoptosis in gastric cancer cell lines treated with butyrate by flow cytometry (A: flow cytometry results, B: quantification of analysis results).
17 is a diagram confirming cell viability according to treatment of gastric cancer cell lines with butyrate using absorbance.
18 is a diagram confirming tumor growth inhibition according to treatment with butanoic acid in an immunostimulating avatar animal model of a gastric cancer patient (A: tumor growth inhibition result, B: quantification of tumor volume).
19 is a diagram confirming the expression of tumor markers and immunocompromised markers in tumor tissue according to treatment with butanoic acid in an immunosimilar avatar animal model of a gastric cancer patient by immunohistochemical staining (A: staining result, B: staining result quantification) .

The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising butyrate or microbiome as an active ingredient.

As used herein, the term "prevention" refers to any action that suppresses symptoms or delays the progression of a specific disease by administering the composition of the present invention.

The term "treatment" used in the present invention refers to all activities that improve or beneficially change the symptoms of a specific disease by administration of the composition of the present invention.

The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. As long as the adjuvant is known in the art, any one may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the effect.

The pharmaceutical composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers usable in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. .

When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, tween 61, cacao paper, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be variously selected according to the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmacological activity may not appear, and if the concentration exceeds 5,000 μg/ml, toxicity to the human body may be exhibited.

In the present invention, the term "microbiome" refers to the entire genetic information of microorganisms living in the human body or the microorganisms themselves, a compound word of microbiota and genome, which are microorganisms that live and live in the human body. It is known that the number of microbiome in the human body is more than twice as large as the number of pure human cells and the number of genes is more than 100 times greater. Therefore, it refers to a field that can be widely used for research on the microbial environment in the human body, such as new drug development and treatment of incurable diseases.

According to one embodiment of the present invention, the microbiome may be Faecalibacterium prausnitzii .

According to one embodiment of the present invention, the composition may be one that increases immune function, and may be one that inhibits the expression of immune degrading factors IL-10, IL-17 and INF-γ.

According to one embodiment of the present invention, increasing the immune function may be suppressing the expression of programmed cell death-1 (PD-1) or CTLA-4 of T cells.

According to one embodiment of the present invention, increasing the immune function suppresses the expression of Programmed Death-Ligand 1 (PD-L1) and IL-10 in antigen presenting cells (APC) or cancer tissues The antigen-presenting cells may be macrophages or dendritic cells.

"PD-L1" and "PD-1" of the present invention are cancer cell or hematopoietic cell surface proteins and T cell surface proteins, and when PD-1, a T cell surface protein, binds to PD-L1, cancer cells can induce T cell immunity. As a protein that induces avoidance of the reaction, PD-L1 is overexpressed in hematopoietic cells, including cancer cells and antigen-presenting cells, and PD-1 is overexpressed in T cells, increasing the expression of PD-L1 and PD-1 complex. If it does, the cancer cell specificity of T cells is lowered, and as a result, it is a protein that lowers the immune function.

According to one embodiment of the present invention, the composition may increase apoptosis of cancer cells.

According to one embodiment of the present invention, the cancer is gastric cancer, colon cancer, rectal cancer, proximal anal cancer, bone cancer, cerebrospinal tumor, head and neck cancer, thymoma, mesothelioma, esophageal cancer, biliary tract cancer, bladder cancer, testicular cancer, small intestine cancer, germ cell tumor, uterus endometrial cancer, fallopian tube carcinoma, vaginal carcinoma, vulvar carcinoma, multiple myeloma, sarcoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bladder cancer, urethral cancer, pituitary adenoma, renal pelvic carcinoma, spinal cord tumor, multiple myeloma, glioma cancer, central CNS central nervous system tumor, hematopoietic tumor, fibrosarcoma, neuroblastoma, astrocytoma, breast cancer, cervical cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, liver cancer, brain cancer, lung cancer, lymphoma, leukemia, malignant melanoma and It may be selected from the group consisting of skin cancer, preferably gastric cancer, but is not limited thereto.

In addition, the present invention provides a food composition for preventing or improving cancer, including butyrate or microbiome as an active ingredient.

As used herein, the term "improvement" refers to any action that at least reduces a parameter associated with the condition being treated, eg, the severity of a symptom.

In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional food compositions.

Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agents described above, natural flavoring agents (thaumatin), stevia extracts (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. there is

In addition, the food composition, in addition to the active ingredient extract, various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. In addition, the food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages.

The functional food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating cancer. In the present invention, 'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functionality for the human body according to Health Functional Food Act No. 6727, and the structure and function of the human body It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions. The health functional food of the present invention may contain ordinary food additives, and the suitability as a food additive is determined according to the general rules of the Food Additive Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations. For example, a health functional food in the form of a tablet is obtained by granulating a mixture obtained by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives in a conventional manner, and then adding a lubricant or the like to compression molding, or as described above. The mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a flavoring agent and the like as needed. Among health functional foods in the form of capsules, hard capsules can be prepared by filling a mixture in which the active ingredient of the present invention is mixed with additives such as excipients in a normal hard capsule. It can be prepared by filling the mixture mixed with gelatin in a capsule base. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention mixed with an excipient, a binder, a disintegrant, etc. by a conventionally known method, and can be coated with sucrose or other coating agent if necessary, Alternatively, the surface may be coated with a material such as starch or talc. Health functional food in the form of granules can be prepared in granular form by a conventionally known method of mixing the active ingredient of the present invention with excipients, binders, disintegrants, etc., and, if necessary, flavoring agents, flavoring agents, etc. can

In addition, the present invention, butanoic acid (Butyrate) or the step of administering a composition containing a microbiome as an active ingredient to the subject; including, Cancer patient group Faecalibacterium genus ( Faecalibacterium sp. ) strain It provides a method for increasing and reducing Proteobacteria Phylum strains.

In addition, the present invention, the step of administering to a subject a composition containing butyrate or microbiome as an active ingredient; T cell function and antigen presenting cell (Antigen Presenting Cell) of a cancer patient group, including , APC) provides a method for increasing the function.

Hereinafter, the present invention will be described in more detail by examples. These examples are merely for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example 1> Comparison of intestinal flora between gastric cancer patients and healthy adults

<1-1> Comparison of intestinal flora diversity (comparison of gastric cancer patients and normal persons)

Changes in the diversity of the intestinal flora by comparing gastric cancer (GC) patients with healthy controls (HC) and comparing gastric cancer (GC) patients with benign tumor patients (Benign tumor (BT)) I wanted to check if it exists. Specifically, the intestinal microbiota of healthy adults and gastric cancer patients and benign tumor patients and gastric cancer patients were compared. After collection of normal subjects, patients with benign tumors, and gastric cancer patients, fecal samples were stored at -70°C until analysis. For metagenome analysis, bacterial genomic DNA isolation, microbial-specific 16S rRNA gene amplification, next-generation sequencing (NGS) analysis, and gut flora analysis and profiling were analyzed. The isolated gDNA is used to amplify the gene with primers prepared in the region that can contain the V3 and V4 parts of the variant region among the bacteria-specific 16S rRNA gene sequences, and attach an index sequence that can distinguish samples to prepare a library. After performing two rounds of PCR, the nucleotide sequence was read using Illumina's Miseq. The translated sequences were analyzed using Quantitative Insights into Microbiological Ecology 2 (QIIME 2) and Lefse, which are bioinformatic analysis tools, and intestinal flora profiling was performed using Prism and R for statistical processing. Then, in order to confirm the abundance of the flora of each group, α-diversity analysis and β-diversity analysis were performed to analyze the similarity between each group. Observed OTUs and Chao1 analysis were performed for α-diversity analysis, and PCoA plot analysis was performed based on bray curstis distance for β-diversity analysis. After that, the composition ratio of Phylum, Family, and Genus of each group was confirmed.

As a result, it was confirmed that in the gastric cancer patient group (GC), compared to healthy adults (HC), the Proteobacteria phylum in the intestinal flora increased and the Actinobacteria phylum decreased. In the gastric cancer patient group, Faecalibacterium species ) and a significant decrease in the upper family (Family) was also confirmed (Fig. 1).

In addition, α-diversity analysis confirmed that the Observed OTUs level was significantly different between the GC and H groups, and it was confirmed that the Chao1 level of the GC group decreased in the Chao1 analysis (Fig. 2A). In the β-diversity analysis, it was confirmed that the HC and GC groups were significantly different in the permutational multivariate analysis of variance (PERMANOVA), and the difference between HC-GC was greater than the difference within each group between HC and GC when the bray curstis distance was distributed. It was confirmed that significantly increased (Fig. 2B).

<1-2> Comparison of intestinal flora composition (comparison of gastric cancer patients and benign tumor patients)

It was confirmed whether there was a difference in the composition of the intestinal flora between gastric cancer patients and benign tumor patients. In the same manner as in Example 1-1, the difference in the composition of the intestinal flora between the gastric cancer patient group (GC) and the benign tumor patient group (BT) was analyzed up to the phylum and genus level.

As a result, as shown in FIG. 3, it was further confirmed that in the gastric cancer patient group (GC), compared to the benign tumor patient (BT), the Proteobacteria phylum in the intestinal flora increased and the Actinobacteria phylum decreased. It was confirmed that the genus Faecalibacterium was significantly reduced. Based on the results of Examples 1-1 and 1-2, it was confirmed that the intestinal flora was dysbiosis in gastric cancer patients.

<Example 2> Comparison of immunity between gastric cancer patients and benign tumor patients

<2-1> Confirmation of reduced T cell immunity

We wanted to compare whether there was a difference in T cell immunity between gastric cancer patients and benign tumor patients. Specifically, blood was obtained from gastric cancer patients and benign tumor patients, and then the expression of IL-10, IL-17, and IFN-γ associated with reduced immunity was analyzed by ELISA.

As a result, as shown in FIG. 4, in the gastric cancer patient group (GC), compared to the benign tumor patient (BT), IL-10, a molecule associated with reduced immunity, was significantly increased, and IFN-γ was significantly decreased. confirmed that there is

<2-2> Identification of markers for lowering T cell immunity

In order to compare whether there is a difference in T cell immunity between gastric cancer patients and benign tumor patients, we investigated programmed cell death-1 (PD-1), which is expressed in T cells when immunity decreases. Specifically, expression of PD-1 CD4+ cells and PD-1 CD8+ cells was confirmed by flow cytometry.

As a result, as shown in FIG. 5 , it was confirmed that the expression of PD1 CD4+ cells and PD-1 CD8+ cells was significantly increased in the gastric cancer patient group (GC) compared to the benign tumor patients (BT).

<2-3> Identification of immunocompromised markers of antigen-presenting cells

In order to compare the immune status of gastric cancer patients and benign tumor patients, we tried to confirm the expression of Programmed Death-Ligand 1 (PD-L1), which is expressed by reduced immunity, in antigen presenting cells (APC). Specifically, the expression of PD-L1, an immunosuppressive protein, in macrophages and dendritic cells in the blood isolated from the patient was confirmed by immunohistochemical staining. In addition, the expression of IL-10 associated with reduced immunity was confirmed in the same manner as above.

As a result, as shown in FIG. 6 , it was confirmed that the expression of PD-L1 in APC was increased in the gastric cancer patient group (GC) compared to the benign tumor patient group (BT).

In addition, as shown in FIG. 7, it was confirmed that the expression of IL-10 in APC was also increased in the gastric cancer patient group (GC) compared to the benign tumor patient (BT), based on the above results, in the gastric cancer patient group. It was confirmed that APC is in a state of suppressing T cell immune activity.

<Example 3> Comparison of immunity according to progression of gastric cancer

<3-1> Confirmation of reduced T cell immunity

The purpose of this study was to determine whether immunity decreases according to the progression of gastric cancer in gastric cancer patients. Specifically, in the same manner as in Example 2-2, in T cells of early gastric cancer patients (Early gastric cancer, EGC / stage 0 to 1a) and late gastric cancer patients (Advanced gastric cancer, AGC / stage 1b onwards) The expression of PD-1 and immunocompromised marker CTLA-4 was confirmed.

As a result, as shown in FIG. 8 , compared to early gastric cancer patients (EGC), the expression of PD-1 and CTLA-4, which are immunocompromised markers, was significantly increased in T cells in late stage gastric cancer patients (AGC). Confirmed.

<3-2> Identification of immunocompromised markers of antigen-presenting cells

In order to determine whether immunity decreases according to the progress of gastric cancer in gastric cancer patients, in the same manner as in Example 2-3, early gastric cancer patients (Early gastric cancer, EGC / stage 0 to 1a) and late gastric cancer patients (Advanced gastric cancer) The expression of PD-L1 and IL-10 in APC of gastric cancer (after AGC/stage 1b) was compared.

As a result, as shown in FIG. 9 , compared to early gastric cancer patients (EGC), it was confirmed that PD-L1 and IL-10, which are immunocompromised markers of APC, were increased in late stage gastric cancer patients (AGC).

<3-3> Identification of immunocompromised markers of antigen-presenting cells according to the degree of gastric cancer progression

As the degree of progression of gastric cancer increases in gastric cancer patients, it was investigated whether the expression of immunocompromised markers in antigen-presenting cells increases. In the same manner as in Example 3-2, benign tumor patients (BT), stage 1 gastric cancer patients (stage 1, I), stage 2 gastric cancer patients (stage 2, II), and stage 3 gastric cancer patients (stage 3, III) and stage 4 gastric cancer patients (stage 4, IV) were collected, and the expression of PD-L1 in macrophages and dendritic cells was examined.

As a result, as shown in FIG. 10, compared to benign tumor patients (BT), PD-L1 in both macrophages and dendritic cells increased in the gastric cancer patient group, and the advanced stage of gastric cancer ( As the stage) increased, PD-L1 also increased.

<3-4> Confirmation of immunocompromised marker expression in gastric cancer tissue according to gastric cancer progression

In order to determine whether the immunity of gastric cancer patients is reduced according to the progress of gastric cancer, the expression of immune-decreasing markers in gastric cancer tissues was confirmed by confocal microscopy. Specifically, the expression of PD-L1 and IL-10 was compared in gastric cancer tissues of early gastric cancer patients (Early gastric cancer, EGC/stage 0 to 1a) and late gastric cancer patients (Advanced gastric cancer, AGC/stage 1b onwards).

As a result, as shown in FIG. 11 , compared to early gastric cancer patients (EGC), in late stage gastric cancer patients (AGC), PD-L1 and IL-10, which are immunocompromised markers in gastric cancer tissues, were significantly increased. Confirmed.

<Example 4> Sodium butyrate and Faecalibacterium prausnitzii Confirmation of immunity increase following treatment of

Based on the results of Examples 1 to 3, it was attempted to determine whether butyrate, a metabolite of Faecalibacterium, increases immunity. Specifically, the peripheral mononuclear cells isolated from the patient in Example 2-3 were treated with butanoic acid at concentrations of 0.5 and 1 mM and then cultured for 3 days, expression of PD-L1 and IL-10 associated with reduced immunity. This inhibition was confirmed by flow cytometry. Macrophage markers were stained with the CD68 marker. As a control group, a vehicle group treated with PBS was used for isolated peripheral mononuclear cells. In addition, the change in PD-L1 expression according to the treatment of Faecalibacterium prausnitzii, a strain of the genus Faecalibacterium, which was shown to be reduced in the gastric cancer patient group, was further confirmed. In addition, by comparing the Nil group, which is an untreated control group, and the vehicle group treated with PBS and the group treated with butanoic acid to peripheral mononuclear cells isolated from the patient, the activity of macrophages expressing PD-L1 and IL-10 was analyzed , The expression of IL-10 in the culture medium was further confirmed, and the activities of macrophages expressing PD-L1 and IL-10 according to the treatment of Faecalibacterium prosnich were analyzed.

As a result, as shown in FIG. 12, compared to the Vehicle group, the butyrate-treated group decreased the expression of PD-L1 and IL-10 in CD68-positive cells, which are macrophage markers, in a concentration-dependent manner. confirmed that. In addition, it was confirmed that the expression of PD-L1 in macrophages was significantly reduced by the treatment of Faecalibacterium prausnitzii of the present invention (FIG. 13).

In addition, as shown in FIG. 14, compared to the Nil group, the expression of PD-L1 and IL-10, which are immunocompromised markers, in macrophages increased in the Vehicle group, and IL-10 in the culture medium increased significantly. , It was confirmed that PD-L1 and IL-10, which were increased in the group treated with butyrate, and IL-10 in the culture medium were significantly decreased (FIGS. 14A and B). In addition, it was confirmed that the increased expression of PD-L1 and IL-10 was significantly decreased by the treatment of Faecalibacterium prosnich (FIG. 14C), so that the butanoic acid or Faecalibacterium prosnich of the present invention It was confirmed that the treatment increased the immunity of macrophages of gastric cancer patients.

<Example 5> Faecalibacterium prosnich ( Faecalibacterium prausnitzii ) and metabolites to confirm gastric cancer apoptosis effect

<5-1> Faecalibacterium prosnich ( Faecalibacterium prausnitzii ) Confirmation of the apoptotic effect of

Faecalibacterium prausnitzii , which was confirmed to be reduced in gastric cancer patients in the present invention, was intended to determine whether apoptosis was induced in gastric cancer cell lines. Specifically, AGS cells, which are gastric cancer cell lines, were cultured, and the cultured AGS cells were treated with Faecalibacterium prausnitzii of the present invention at a concentration of 10 μg/ml, and apoptosis was induced in the cells by flow cytometry analysis. analyzed. As a control group, a vehicle group treated with the same amount of PBS was used.

As a result, as shown in FIG. 15, compared to the Vehicle group, it was confirmed that the number of apoptosis cells increased in the group treated with Faecalibacterium prausnitzii .

<5-2> Faecalibacterium prosnich ( Faecalibacterium prausnitzii ) Confirmation of the apoptotic effect of metabolites of

Faecalibacterium prausnitzii metabolite butyrate of the present invention, Faecalibacterium prausnitzii ) It was attempted to determine whether apoptosis is induced in the same way as Faecalibacterium prausnitzii . Specifically, in the same manner as in Example 5-1, AGS cell lines were treated with butanoic acid at concentrations of 0.5, 1, and 1.5 mM, and cells inducing apoptosis were analyzed by flow cytometry. In addition, in gastric cancer cell lines, the cell killing effect of butyrate was confirmed by measuring the absorbance at a wavelength of 450 nm for cell viability.

As a result, as shown in FIG. 16, it was confirmed that apoptosis was induced in a concentration-dependent manner in the group treated with butyrate compared to the vehicle group.

In addition, as shown in FIG. 17, compared to the vehicle group, it was confirmed that the absorbance decreased in a concentration-dependent manner in the butyrate-treated group, confirming that apoptosis of the gastric cancer cell line was induced.

<Example 6> Confirmation of tumor suppression of butanoic acid in simulated avatar model of gastric cancer patient

<6-1> Construction of a simulated avatar model for gastric cancer patients and confirmation of tumor growth inhibition by butanoic acid

In order to confirm whether butyrate, a metabolite of Faecalibacterium prausnitzii of the present invention, suppresses tumors, a simulated avatar model of a gastric cancer patient was created. Specifically, 6-8 week old immunodeficient mice (NSG) were intravascularly injected with 5x10 ⁶ /mice of PBMC (Peripheral blood mononuclear cells) derived from gastric cancer patients. One week after PBMC injection, gastric cancer cell line AGS cell line 5x10 ⁶ was subcutaneously injected to create a simulated stomach cancer patient avatar model (AGS+PBMC, Vehicel (PBMC)). In addition, in order to confirm the effect of gastric cancer patient-derived PBMC injection in the engraftment of gastric cancer cell lines, a group administered with only AGS (AGS only) was used as a control group. After 3 weeks of AGS cell injection, butanoic acid was orally administered daily at a concentration of 200 mg/kg. After AGS cell injection, the size of the tumor was measured three times using calipers every week, and the length of the long axis and short axis of the tumor was substituted into Equation 1 below, and the volume of the tumor ( Tumor volume) was calculated.

[Equation 1]

As a result, as shown in FIG. 18, compared to the AGS only group, the group injected with patient-derived PBMC significantly increased tumor growth and increased tumor volume, confirming that the patient's immune status was reflected. , In the group treated with butyrate, it was confirmed that the increased tumor volume significantly decreased, confirming the tumor inhibitory effect of butyrate.

<6-2> Verification of the expression of allogeneic markers and immunocompromised markers in simulated avatar models of gastric cancer patients

It was confirmed whether butyrate, a metabolite of Faecalibacterium prausnitzii of the present invention, regulates the expression of tumor markers and immunocompromised markers. Specifically, mice of each group of Example 6-1 were humanely sacrificed, and transplanted tumor tissues were obtained. Thereafter, the obtained tissues were sectioned, and the expression of tumor markers NF-κb and STAT3 and immunocompromised markers PD-L1 and IL-10 were confirmed by immunohistochemical staining.

As a result, as shown in FIG. 19, compared to the AGS only group, it was confirmed that the expression of NF-κb, STAT3, PD-L1, and IL-10, which are tumor markers and immunocompromised markers, increased significantly in the Vehicle group, but In the group treated with carbonic acid (Butyrate), it was confirmed that the increased tumor markers and immunocompromised markers were significantly reduced, and it was confirmed that butyrate had tumor suppression and immunoregulatory effects even in the avatar model reflecting the patient's immune status. .

Therefore, the present invention confirmed that the diversity of intestinal flora was reduced in gastric cancer patients, and it was confirmed that the composition ratio of intestinal flora was different from that of healthy adults and patients with benign tumors. In addition, in the gastric cancer patient group, it was confirmed that the immune function was lowered, and the immune function of the patient was lowered as the gastric cancer progressed. In addition, when the patient-derived macrophages were treated with Faecalibacterium prausnitzii strain and its metabolite, butyrate, which was confirmed to be reduced in the gastric cancer patient group, PD-L1 and IL increased due to immunosuppression. It was confirmed that -10 was effectively suppressed. In addition, it was confirmed that apoptosis was effectively induced when gastric cancer cell lines were treated with Faecalibacterium prausnitzii strain and its metabolite butyrate. In addition, it was confirmed that butanoic acid inhibits tumor growth and reduces the expression of tumor markers and immunocompromised markers in an avatar animal model of a gastric cancer patient in which the patient's immune status is reflected and a gastric cancer cell line is transplanted. Therefore, it was confirmed that personalized medicine can be realized by diagnosing the intestinal flora colony collapse and immunosuppression in the gastric cancer patient group, and confirming the effect of increasing immunity and restoring the intestinal flora according to the treatment with the effective substance of the present invention.

Claims (15)

A pharmaceutical composition for preventing or treating cancer, comprising butyrate or microbiome as an active ingredient. According to claim 1,
The microbiome, Faecalibacterium prausnitzii , the composition of Faecalibacterium prausnitzii . According to claim 1,
The composition is to increase the immune function, the composition. According to claim 1,
To increase the immune function, to suppress the expression of the immunocompromising factors IL-10, IL-17 and INF-γ composition. According to claim 4,
Increasing the immune function is to suppress the expression of programmed cell death-1 (PD-1) or CTLA-4 of T cells, the composition. According to claim 4,
Increasing the immune function is to suppress the expression of Programmed Death-Ligand 1 (PD-L1) and IL-10 in antigen presenting cells (Antigen Presenting Cell, APC) or cancer tissues, the composition. According to claim 6,
The antigen-presenting cells are macrophages or dendritic cells, the composition. According to claim 1,
The composition, to increase the apoptosis (Apoptosis) of cancer cells, the composition. According to claim 1,
The cancers include gastric cancer, colon cancer, rectal cancer, perianal cancer, bone cancer, cerebrospinal tumor, head and neck cancer, thymoma, mesothelioma, esophageal cancer, biliary tract cancer, bladder cancer, testicular cancer, small intestine cancer, germ cell tumor, endometrial cancer, fallopian tube carcinoma, vaginal carcinoma, vulvar carcinoma, multiple myeloma, sarcoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bladder cancer, urethral cancer, pituitary adenoma, renal pelvic carcinoma, spinal cord tumor, multiple myeloma, glioma cancer, CNS central nervous system tumor, hematopoiesis selected from the group consisting of tumor, fibrosarcoma, neuroblastoma, astrocytoma, breast cancer, cervical cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, liver cancer, brain cancer, lung cancer, lymphoma, leukemia, malignant melanoma and skin cancer, composition. A food composition for preventing or improving cancer, containing butyrate or microbiome as an active ingredient. According to claim 10,
The microbiome, Faecalibacterium prausnitzii , the composition of Faecalibacterium prausnitzii . Administering a composition containing butyrate or microbiome as an active ingredient to an individual; increasing Faecalibacterium sp. strains of cancer patient groups, including; A method of reducing bacterial (Proteobacteria) Phylum strains. Administering a composition containing butyrate or microbiome as an active ingredient to a subject; including, T cell function and antigen presenting cell (APC) function of cancer patient group how to increase it. According to claim 13,
The increase in the function of the T cell is to suppress the expression of programmed cell death-1 (PD-1) or CTLA-4 of the T cell, the method. According to claim 13,
Increasing the function of antigen-presenting cells is to inhibit the expression of Programmed Death-Ligand 1 (PD-L1) and IL-10 in antigen-presenting cells.