Campylobacter for treating oral squamous cell carcinoma and application thereof

Abstract

The invention discloses campylobacter for treating oral squamous cell carcinoma and application of the campylobacter. It is found that the Campylobacter showae strain has remarkable anti-tumor activity on oral squamous cell carcinoma model mice, tumor growth can be remarkably inhibited, and formation of a three-level lymph structure in the oral squamous cell carcinoma is promoted. Therefore, the Campylobacter showae strain and the composition thereof can be used for preparing products for prevention, treatment, adjuvant treatment or prognosis nursing of the oral squamous cell carcinoma, and a new microbial intervention strategy is provided for the oral squamous cell carcinoma and related immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of health and wellness, specifically relating to a Campylobacter bacterium for treating oral squamous cell carcinoma and its application. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is a malignant tumor that occurs in the oral mucosal epithelium. It is characterized by high recurrence and metastasis rates, and postoperative maxillofacial defects in most patients severely impact their appearance and psychological well-being. Currently, the main treatments for OSCC include surgery, chemotherapy, and radiotherapy; however, these treatments have not achieved satisfactory results. Many patients also experience serious problems such as postoperative tissue defects, radiation-induced osteonecrosis of the jaw, low bioavailability of chemotherapy drugs, and a high risk of developing radiotherapy and chemotherapy resistance. This underscores the importance of seeking new treatment methods.

[0003] Tertiary lympHoid structures (TLSs) are ectopic lymphoid tissues that form within non-lymphatic organs under pathological conditions such as chronic inflammation or cancer. Their structure is similar to that of lymph nodes, containing immune cell components such as T-cell areas, B-cell follicles, high endothelial venules (HEVs), and dendritic cells (DCs). TLSs are key sites in inducing and maintaining anti-tumor immune responses in the tumor microenvironment, and their formation mechanisms include chronic stimulation induction, cytokine regulation, and chemokine and cytokine mediation. TLSs play roles in initiating anti-tumor immunity and mediating immune cell homing, and are clinically closely related to patient prognosis and immunotherapy response.

[0004] Campylobacter showae (C. showae, Cs) is a Gram-negative, chemoheterotrophic, microaerophilic, motile bacterium belonging to the genus Campylobacter. The typical strain, SU A4, was initially isolated from plaque samples from the gingival crevices of human teeth, but has since been found in colonic tissue and feces. Since its discovery, C. showae has been associated with a variety of diseases due to its pathogenicity, including Crohn's disease, periodontitis, inflammatory bowel disease, and ulcerative colitis. To date, no studies have reported the therapeutic efficacy of Campylobacter showae for OSCC. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a Campylobacter bacterium for treating oral squamous cell carcinoma, offering a new microbial intervention strategy for the treatment of oral squamous cell carcinoma, thereby overcoming the current technical problem of the urgent need for effective drugs to treat oral squamous cell carcinoma.

[0006] The above invention objectives of the present application are achieved by the following technical solutions.

[0007] The first aspect of the present application provides a Campylobacter showae strain for treating oral squamous cell carcinoma.

[0008] Further, the number of the Campylobacter showae strain is not less than 1×10 6 CFU.

[0009] Further, the Campylobacter showae strain has the effect of promoting the formation of oral tertiary lymphoid structures.

[0010] Further, the Campylobacter showae strain includes a culture obtained by inoculating it on a culture medium and culturing.

[0011] Further, the inoculation method includes but is not limited to any one or more of the following: plate streaking method, slant inoculation method, pour culture method, puncture inoculation method, liquid inoculation method.

[0012] Further, the inoculation amount can be 0.1%-20%, and in some cases, it can also be a higher or lower inoculation amount. Specifically, the inoculation amount can be 1%-20%, 2%-20%, 1%-15%, 1%-10%, 1%-5%, 1%-8%, 5%-15%, 5%-10%, 5%-8%, 8%-10%, 8%-15%, 5%-12%, 2%-7%.

[0013] Further, the culture medium can be a solid culture medium, a semi-solid culture medium or a liquid culture medium, and can also be any suitable culture medium type disclosed in the prior art, or a culture medium further improved on the basis of the culture medium type disclosed in the prior art to improve the performance of the strain, or a culture medium not disclosed in the prior art but capable of being used for the culture of the aforementioned Campylobacter showae strain.

[0014] In the present application, the prevention refers to various means or measures for preventing the occurrence or development of diseases before the diseases are recognized by clinical standards, including medical, physical or chemical methods, to prevent and reduce the occurrence or development of various symptoms of diseases, and in specific embodiments of the present application, the diseases are preferably oral squamous cell carcinoma.

[0015] In the present application, the treatment refers to preventing and reducing the occurrence or development of the disease, so that the development or aggravation of the disease course is inhibited, suppressed, reduced, improved, slowed down, stopped, delayed or reversed, various indicators of the disease, disorder or pathological state during the maintenance and / or medication are reduced or alleviated, or the disease, disorder or condition is cured or eliminated, in specific embodiments of the present application, the disease is preferably oral squamous cell carcinoma.

[0016] The second aspect of the present application provides a bacterial agent for treating oral squamous cell carcinoma, the bacterial agent comprising one or more of the fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, freeze-dried powder or cell lysate of the Campylobacter showae strain provided in the first aspect of the present application.

[0017] Further, the fermentation broth refers to the liquid after inoculating the bacterial solution into the culture medium and culturing for a period of time, including the fermentation broth supernatant and the fermentation broth precipitate.

[0018] Further, the fermentation broth supernatant refers to the clear liquid on the top after centrifugation of the fermentation broth, which contains rich metabolic products and part of bacterial fragments during bacterial growth and reproduction, acidic substances and bacteriocins secreted by bacteria (which have antagonistic and killing effects on harmful bacteria), amino acids after bacterial decomposition of food, and synthesized vitamins, and also includes enzymes secreted by bacteria which are useful to the human body, and part of bacterial components which also have immune promoting effects on the human body.

[0019] Further, the fermentation broth precipitate refers to the precipitate after centrifugation of the liquid, which includes free proteins, residual bacterial bodies, broken cells and residues of culture medium, including proteins and intracellular matrix.

[0020] Further, the live bacteria, also known as active bacterial flora, can colonize and reproduce in the organism, which is beneficial to increase the number of beneficial bacteria.

[0021] Further, the freeze-dried powder is obtained by freeze-drying the Campylobacter showae strain provided in the first aspect of the present application. The freeze-dried powder generally also includes a freeze-drying protective agent. The freeze-drying protective agent includes but is not limited to: a pH buffer, a filler, a sugar, a non-ionic surfactant, a ligand, etc. The pH buffer includes but is not limited to any one or more of Tris, an amino acid or a salt thereof. The filler includes but is not limited to any one or more of mannitol, glycine, bovine serum albumin. The sugar can be a disaccharide, such as any one or more of sucrose or trehalose. The non-ionic surfactant includes but is not limited to Tween, which can be Tween-20, Tween-60, Tween-80, etc. The freeze-drying protective agent can also include an antioxidant, etc., and the freeze-drying protective agent can also specifically include albumin, polyethylene glycol, etc.

[0022] Further, the cell lysate can be obtained by lysing the bacterial cells of the culture provided in the first aspect of the present application. The lysing can be physical lysing or chemical lysing. The physical lysing includes but is not limited to grinding, ultrasonic disruption, etc. The chemical lysing includes but is not limited to chemical reagent lysing, enzymatic lysing, which can be hydrolytic enzyme or oxidase. The lysing can also be by increasing the intracellular pressure to make the cells self-rupture.

[0023] Further, the bacterial agent can be in solid or liquid form.

[0024] The third aspect of the present application provides a medicament for treating oral squamous carcinoma, which comprises the Campylobacter showae strain or culture provided in the first aspect of the present application.

[0025] Further, the medicament further comprises pharmaceutically acceptable adjuvants and / or excipients.

[0026] In some embodiments, the adjuvants and / or excipients can be diluents / fillers selected from microcrystalline cellulose, starch (corn, potato), lactose, mannitol, calcium carbonate; binders selected from hydroxypropyl methylcellulose (HPMC), gelatin, polyvinylpyrrolidone (PVP); disintegrants such as cross-linked sodium carboxymethylcellulose (CCNa), low-substituted hydroxypropyl cellulose (L-HPC); lubricants selected from magnesium stearate, silicon dioxide, talc; coating materials selected from gastroenteric coating (such as acrylic resin, shellac); protective agents for maintaining the stability of the bacterial agent selected from trehalose, skimmed milk powder, glycerol; surfactants selected from polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, stearic acid monoglyceride, cetyl alcohol; flavoring agents selected from sodium saccharin, cyclamate, aspartame, acesulfame-k, glycerol, sorbitol, mannitol, sucrose, monosaccharide syrup, aromatic sugar syrup; co-solvents selected from hydrochloric acid, phosphoric acid, propionic acid, acetic acid, lactic acid, citric acid, tartaric acid, boric acid, glucuronic acid, gluconic acid, lactobionic acid, malic acid, threonic acid, glucoheptonic acid, 2, 5-dihydroxybenzoic acid, acidic amino acids, etc.

[0027] In some embodiments, the pharmaceutical preparation of the present application can further add a targeted delivery component, specifically including biodegradable polymers, biodegradable implants, non-biodegradable implants, biodegradable microparticles such as biodegradable microspheres, nanoparticles, etc.

[0028] In specific embodiments, the choice of pharmaceutical adjuvants and the pharmaceutical preparation is made according to the desired preparation, and the choice of pharmaceutical adjuvants for ordinary preparation is well known to those skilled in the art.

[0029] In some embodiments, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans.

[0030] Further, the dosage form of the drug includes, but is not limited to, injections, tablets, capsules, powders, granules, oral solutions, emulsions.

[0031] Specifically, the injection is the most common parenteral administration dosage form, including solution type injection, suspension type injection, emulsion type injection, and sterile powder for injection, etc. It can be administered by intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, etc. Intravenous injection can make the drug quickly enter the blood circulation, and has the advantages of fast drug effect, which is suitable for emergency and drugs requiring fast effect; the absorption speed of intramuscular injection and subcutaneous injection is relatively slow, but the drug effect is maintained for a long time.

[0032] Specifically, the tablet is a tablet-shaped preparation prepared by mixing the drug with excipients and then compressing. It has the advantages of accurate dosage, good stability, high production efficiency, and convenient taking. It can be divided into ordinary tablets, coated tablets, chewable tablets, dispersible tablets, etc.

[0033] Specifically, the capsule is divided into hard capsules and soft capsules. The hard capsule is prepared by filling the drug powder or granules into the hollow hard capsule; the soft capsule is prepared by dissolving or suspending the drug in a suitable oily or non-oily liquid medium, and then using compression or dripping method. The capsule can mask the bad smell of the drug, improve the stability of the drug, and is convenient to take.

[0034] Specifically, the powder is a dry powder preparation prepared by crushing and uniformly mixing the drug with suitable excipients. The powder has small particle size, large specific surface area, easy dispersion, and fast effect, but the stability is relatively poor.

[0035] Specifically, the granule is a dry granular preparation prepared by mixing the drug with suitable excipients to have a certain particle size. It can be divided into soluble granules, suspension granules, effervescent granules, etc. The granule is easy to dissolve, absorbs quickly, and is convenient to carry and take.

[0036] Specifically, the oral solution is a clear liquid preparation for oral administration prepared by dissolving the drug in a suitable solvent. The drug is uniformly dispersed in the solution, absorbs quickly, and has high bioavailability.

[0037] Specifically, the emulsion is a heterogeneous liquid preparation prepared by emulsifying two mutually insoluble liquids, and the liquid droplets are dispersed in another liquid to form a heterogeneous liquid preparation. Oral emulsion can improve the taste and stability of the drug, and improve the bioavailability of the drug.

[0038] The fourth aspect of the present application provides a medical material for treating oral squamous cell carcinoma, which comprises the Campylobacter showae strain or culture of the first aspect of the present application.

[0039] Further, the medical material can be in solid form, liquid form or semi-solid form.

[0040] The fifth aspect of the present application provides use of the Campylobacter of the first aspect of the present application in the preparation of a product for treating oral squamous cell carcinoma, which comprises a drug, a bacterial agent or a medical material.

[0041] The sixth aspect of the present application provides use of the Campylobacter of the first aspect of the present application in the preparation of a product for increasing anti-tumor immune response, which comprises a food, a health food, a bacterial agent, a drug or a medical material.

[0042] Further, the food is a food composition comprising the Campylobacter showae strain or culture of the first aspect of the present application and suitable food additives.

[0043] In some embodiments, the food composition further comprises prebiotics selected from the group consisting of fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin, resistant dextrin (promoting strain colonization); nutrients selected from the group consisting of vitamin B, zinc (enhancing immune synergy), dietary fiber (such as polydextrose); flavoring agents / odorants selected from the group consisting of citric acid, steviol glycoside, natural fruit powder (improving palatability); stabilizers selected from the group consisting of pectin, xanthan gum (preventing bacterial sedimentation).

[0044] In some embodiments, the food composition can be made into tea, juice or beverage, or made into granules, capsules or powder as health food. In some embodiments, the food composition of the present disclosure can also be added to beverages (including alcoholic beverages), fruits and their processed products (e.g. canned fruits, bottled fruits, jams), fish, meat and their processed products (e.g. ham, sausage, salted beef), candies and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni), biscuits, Korean toffee, dairy products (e.g. butter, cheese), vegetable oil, margarine, plant protein, dry distillation food, frozen food, condiments (e.g. soybean paste, soy sauce, ketchup, etc.). In some embodiments, the food composition of the present application can contain various flavors, natural carbohydrates, etc. as additional ingredients. Carbohydrates are monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose and sucrose; polysaccharides, such as dextrin and cyclodextrin; sugar alcohols, such as xylitol, sorbitol, erythritol, etc. Sweeteners can be used, including natural sweeteners, such as taurine Martin, stevia extract, etc., and synthetic sweeteners such as saccharin, aspartame, etc.

[0045] Furthermore, the health product includes the Campylobacter showae strain or culture provided in the first aspect of the present invention and nutritionally acceptable nutritional additives.

[0046] Furthermore, the nutritional additives include one or more of the following: dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0047] Furthermore, the dosage forms of the health products include tablets, capsules, granules, pills, gel candies, powders, oral liquids, or drops.

[0048] In some implementations, the product is applicable to animals, preferably mammals (humans and non-human animals), including but not limited to: humans, non-human primates (especially higher primates, such as macaques, cynomolgus monkeys, stump-tailed monkeys, rhesus monkeys, flat-headed monkeys, golden monkeys and tree shrews), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, any livestock or pets, etc.

[0049] Advantages and beneficial effects of the present invention:

[0050] This invention reveals that live Campylobacter showae exhibits significant antitumor activity against oral squamous cell carcinoma in mouse models, significantly inhibiting tumor growth and promoting the formation of tertiary lymphoid structures in oral squamous cell carcinoma. Therefore, live Campylobacter showae and its compositions can be used to prepare products for the prevention, treatment, adjuvant therapy, or prognostic care of oral squamous cell carcinoma, providing a novel microbial intervention strategy for oral squamous cell carcinoma and related immunotherapies. Attached Figure Description

[0051] Figure 1 The image shows the 16S sequencing results of cancerous and adjacent tissues from OSCC patients who are TLSs-positive (TLS+) or TLSs-negative (TLS-). In this image, A is a bar chart of bacterial abundance at the genus level, and B is a graph showing the differential contribution of bacteria in tumor tissues of TLSs+ and TLSs- OSCC patients.

[0052] Figure 2 Figure 1 shows the experimental results of Campylobacter showae in treating OSCC (C3h mice); where A is the experimental flowchart; B is a representative image of the tumor; C is the tumor weight; and D is the mouse body weight ratio (measurement day / first day).

[0053] Figure 3 Figure 1 shows the experimental results of Campylobacter showae in treating OSCC (C57 mice); where A is the experimental flowchart; B is a representative image of the tumor; C is the tumor weight; and D is the mouse body weight ratio (measurement day / first day).

[0054] Figure 4 Figure of experimental results showing that Campylobacter showae promotes the formation of TLSs. DETAILED DESCRIPTION

[0055] As used herein, the terms "have", "comprise", "include" or "contain" or any grammatical variations thereof are used in a non-exclusive sense. Thus, these terms can mean that, in addition to the features introduced by these terms, the entity described in the context can also include other features.

[0056] Furthermore, as used herein, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, but do not exclude the possibility of using such features.

[0057] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as temperature, time, and the like as used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless otherwise indicated, the numerical parameters listed in the specification and claims are approximations. The numerical parameters set forth in the specification and claims have the term "about" associated with them, unless otherwise indicated. Each of the numerical parameters can concretely recite a value that contains error and / or tolerances {+ / - 10% of the stated value).

[0058] The present application is further described in conjunction with the specific examples below, which are intended to illustrate the application and not to limit the same. It will be understood by those skilled in the art that various modifications, changes, substitutions and alterations can be made therein without departing from the spirit and scope of the present application which should be limited only by the scope of the appended claims and equivalents thereof. The experimental materials, reagents and raw materials used in the present application are readily available to those skilled in the art, and can be obtained commercially unless otherwise specified. The experimental methods of the present application not specifically described are generally carried out according to the conventional conditions or according to the conditions recommended by the manufacturers. In particular, the following examples are only used to illustrate the present application and should not in any way limit the scope of the present application. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present application, and should not and will not limit the present application described in detail in the claims.

[0059] Example 1 TLSs-related OSCC intratumoral bacteria screening

[0060] I. Experimental methods

[0061] 1. Experimental materials

[0062] A total of 113 samples of tumor tissues and para-cancer tissues (59 tumor tissues and 54 para-cancer tissues) from OSCC patients were collected for 16S detection of intratumoral bacteria, and the same time air was collected in a blank tube as a blank control.

[0063] (1) Inclusion criteria:

[0064] Primary OSCC patients;

[0065] No use of antibiotics 1 week before surgery;

[0066] No other treatment before surgery;

[0067] Underwent radical surgery.

[0068] (2) Exclusion criteria:

[0069] Patients with multiple primary lesions;

[0070] Patients who did not accept this study.

[0071] (3) Reagents and antibodies: HostZERO Microbial DNA Kit (Zymo, Cat#D4310), CD3 antibody (Abcam, Cat#ab699), CD20 antibody (Thermo Fisher, Cat#14-0202-82).

[0072] 2. Experimental steps

[0073] (1) Grouping:

[0074] According to the distribution of three-level lymphatic structure, the patients were divided into two groups: TLS+ group and TLS- group.

[0075] Among them, the TLS+ group was defined as the TLS aggregate needed to exceed 7000 μm 2 , containing at least 100 cells, otherwise it was defined as the TLS- group.

[0076] (2) 16S rDNA sequencing

[0077] The samples were handled under sterile conditions after collection and stored at -80°C to prevent changes in the structure of the microbial community. Bacterial DNA was extracted using the HostZERO Microbial DNA Kit, following the instructions and measuring the DNA concentration and purity by Qubit 4.0 fluorometer. The extracted DNA was used as a template to amplify the V3-V4 variable region of the bacterial 16S rRNA gene, using the 16S V34 region primers (341F: CCTAYGGGRBGCASCAG (SEQ ID NO: 1) and 806R: GGACTACNNGGGTATCTAAT (SEQ ID NO: 2)). All PCR mixtures were added with 15 μΐ, of Phusion High-Fidelity PCR Master Mix, 0.2 μΜ primers and 10 ng of genomic DNA template, and subjected to a first denaturation at 98°C for 1 minute, followed by 30 cycles at 98°C (10 s), 50°C (30 s) and 72°C (30 s), and finally at 72°C for 5 minutes. The amplified products were detected by 2% agarose gel electrophoresis, purified with AMPure XP magnetic beads, and subjected to a second PCR using the Illumina Nextera XT Index Kit to add adapters and dual-index tags. The purified library products were quantified by Qubit and detected for fragment length and concentration by Agilent Bioanalyzer, and then equimolarly mixed to form a library, diluted to an appropriate concentration, and sequenced on the Illumina MiSeq platform (2 x 250 bp paired-end sequencing mode). The raw sequences obtained by sequencing were de-adapted and quality-controlled by Cutadapt, and then subjected to denoising, de-chimerization and feature sequence (ASV) extraction using the DADA2 algorithm of the QIIME2 platform. Finally, species annotation was performed based on the SILVA 138 database, and further analysis of community diversity and abundance distribution was performed.

[0078] II. Experimental Results

[0079] The 16S sequencing results showed that, at the genus level, Campylobacter was a significantly different bacterial genus in the tumor tissues of the TLS+ group and the TLS- group, and the contribution of Campylobacter was the highest in the tumor tissues of the TLS+ group (Fig. 2A, Fig. 2B). Figure 1 A, Figure 1 B). The above results indicated that Campylobacter may play an important role in the formation of TLSs in OSCC patients, and Campylobacter showae has the potential to treat OSCC.

[0080] Example 2 In vivo study of live Campylobacter showae treatment of OSCC

[0081] I. Experimental method

[0082] 1. Experimental materials

[0083] (1) Mice: C3h mice and C57 mice, female, 8 weeks old, C3h mice were purchased from Sanye (Suzhou) Biotechnology Co., Ltd., and C57 mice were purchased from Changsha Slaik Jingda Animal Experiment Co., Ltd.

[0084] (2) Cells: Mouse squamous cell carcinoma cell line (SCC7) was purchased from Changsha Yanko Biotechnology Co., Ltd. After arrival, the cells were thawed and recovered under sterile conditions, and cultured in a 37°C, 5% CO2, humidified incubator. The culture medium was 1640 supplemented with 15% fetal bovine serum (FBS) and 1% penicillin / streptomycin mixture. When the cells grew to about 80-90% confluence, they were digested and passaged with 0.25% trypsin-EDTA, and the passage ratio was generally 1:3 to 1:4. In order to maintain the characteristics of the cells, avoid excessive passage, and at the same time, mycoplasma detection needs to be carried out regularly. Cells for in vivo experiments should be selected from cells in the logarithmic growth phase to ensure the tumor formation rate of the experiment.

[0085] Mouse squamous cell carcinoma cell line (MOC1) was purchased from Changsha Yanko Biotechnology Co., Ltd. After arrival, the cells were thawed and recovered under sterile conditions, and cultured in a 37°C, 5% CO2, humidified incubator. The culture medium was IMDM:F12 (2:1) supplemented with 5% FBS, 1% penicillin / streptomycin mixture, insulin (final concentration 5 μg / mL), hydrocortisone (final concentration 0.04 μg / mL) and epidermal growth factor (final concentration 0.005 μg / mL). When the cells grew to about 80-90% confluence, they were digested and passaged with 0.25% trypsin-EDTA, and the passage ratio was generally 1:3 to 1:4. In order to maintain the characteristics of the cells, avoid excessive passage. Cells for in vivo experiments should be selected from cells in the logarithmic growth phase to ensure the tumor formation rate of the experiment.

[0086] (3) Strain: The strain used in the present application was purchased from Mingzhou Biotechnology, B269393: Campylobacter showae. Campylobacter showae was cultured in Columbia blood agar medium at 37°C in a constant temperature incubator.

[0087] (4) Reagents and antibodies: 1640 medium (Gibco, Cat#C11875500BT), IMDM medium (Cytiva HyClone, Cat#SH30228.02), F12 nutrient medium (Cytiva HyClone, Cat#SH30026.01), FBS (TFB TFBC, Cat#SY-T002), insulin (TargetMol, Cat#TP1125), hydrocortisone (TargetMol, Cat#TP1614), epidermal growth factor (Peprotech, Cat#AF-100-15), penicillin-streptomycin mixture (Gibco, Cat#15140122) for cell culture. Digestion solution uses 0.25% trypsin-EDTA solution (Gibco, Cat#25200056). Columbia blood plate medium (Solarbio, Cat#CM0035). CD3 antibody (Abcam, Cat#ab16669), CD19 antibody (Abcam, Cat#ab245235), PNAD antibody (Biolegend, Cat#120801).

[0088] 2. Experimental procedures

[0089] (1) Campylobacter showae heat inactivation: 60°C water bath heating for 30 min.

[0090] (2) Mouse tumorigenesis experiment

[0091] Take 30 8w-old C3h mice, subcutaneously inject 5×10 5 SCC7 mouse OSCC cells on the back, and when the tumor volume grows to 100 mm 3 Above, randomly divide the mice into 3 groups, into control group, live C.s (Campylobacter showae) group and heat-inactivated C.s (Campylobacter showae) group, 10 / group, at this time, start different interventions. The control group was injected with 50 μl of sterile PBS solution intratumorally, 2 times / week; the live C.s group was injected with 50 μl of live Campylobacter showae bacteria solution (1×10 6 CFU) resuspended in PBS solution intratumorally, 2 times / week; the heat-inactivated C.s group was injected with 50 μl of heat-inactivated Campylobacter showae bacteria solution (1×10 6 CFU) resuspended in PBS solution intratumorally, 2 times / week. At the end of the experiment, the mouse tumor weight, body weight and other indicators were measured.

[0092] Take 30 C57 mice of 8w age, subcutaneously inject 2x10 6 OSCC cells of MOC1 mouse origin on the back, and when the tumor volume grows to 100 mm 3 Above, randomly divide the mice into 3 groups, into control group, live C.s group and heat-inactivated C.s group, 10 / group, at this time start different interventions. The control group is injected with 50 μl of sterile PBS solution intratumorally, 3 times / week; the live C.s group is injected with 50 μl of live Campylobacter showae bacteria solution (1x10 6 CFU) resuspended in PBS solution intratumorally, 3 times / week; the heat-inactivated C.s group is injected with 50 μl of heat-inactivated Campylobacter showae bacteria solution (1x10 6 CFU) resuspended in PBS solution intratumorally, 3 times / week. At the end of the experiment, measure the tumor weight, body weight and other indicators of the mice.

[0093] (3) HE staining: after the tumor grows to 1500 mm 3 , terminate the experiment, collect the tumor tissue, fix and embed the sections, and then perform conventional HE staining to observe the pathological features and immune cell infiltration of the subcutaneously transplanted tumor.

[0094] (4) Immunofluorescence

[0095] After the tumor grows to 1500 mm 3 , terminate the experiment, collect the tumor tissue, fix and embed the sections, deparaffinize and rehydrate, antigen repair, cool to room temperature, remove peroxidase, block with goat serum for 30 min, incubate CD3 primary antibody at 4°C overnight, wash with PBS for 3 times, add secondary antibody (HRP), incubate in the dark at room temperature for 50 min, add TSA fluorescent dye, incubate at room temperature for 5 min, wash for 3 times, re-antigen repair, repeat the previous operation, incubate CD19 antibody, finally wash with DAPI for 3 times, mount the sections and observe CD3+ T cells and CD19+ B cells under a microscope. If T cells and B cells form clusters, it is determined as TLS+, otherwise it is TLS-.

[0096] II. Experimental results

[0097] 1. In vivo study of Campylobacter showae live bacteria treatment of SCC7 model of OSCC

[0098] Figure 2 A is the flow chart of in vivo experiment, the results show that from the representative images of the tumor ( Figure 2B) It was observed that the tumors of mice injected with live Campylobacter showae were significantly smaller than the control group, and statistical analysis results showed that the tumor weight of mice in the Campylobacter showae group was significantly lower than that of the control group (P < 0.05) (Figure 2B). Figure 2 C) significantly decreased. In addition, we also used the inactivated bacteria solution of Campylobacter showae to carry out the corresponding experiment, and the results showed that the heat-inactivated Campylobacter showae bacteria solution had no obvious inhibitory effect on the tumor. At the end of the experiment, there was no statistically significant difference in the body weight of mice in each group (P > 0.05) (Figure 2C). Figure 2 D).

[0099] 2. In vivo study of Campylobacter showae live bacteria in the treatment of OSCC MOC1 model

[0100] Figure 3 A is the flow chart of in vivo experiment, the results show that from the representative images of tumors (Figure 3A), it can be observed that the tumors of mice injected with live Campylobacter showae were significantly smaller than the control group, and statistical analysis results showed that the tumor weight of mice in the Campylobacter showae group was significantly lower than that of the control group (P < 0.05) (Figure 3B). Figure 3 B) It was observed that the tumors of mice injected with live Campylobacter showae were significantly smaller than the control group, and statistical analysis results showed that the tumor weight of mice in the Campylobacter showae group was significantly lower than that of the control group (P < 0.05) (Figure 2B). Figure 3 C) significantly decreased. In addition, we also used the inactivated bacteria solution of Campylobacter showae to carry out the corresponding experiment, and the results showed that the heat-inactivated Campylobacter showae bacteria solution had no obvious inhibitory effect on the tumor. At the end of the experiment, there was no statistically significant difference in the body weight of mice in each group (P > 0.05) (Figure 2C). Figure 3 D).

[0101] 3. In vivo study of Campylobacter showae live bacteria in the regulation of TLS formation

[0102] Next, we detected the effect of Campylobacter showae live bacteria on the formation of TLSs in the tumor microenvironment of mice by immunofluorescence. The results also showed that Campylobacter showae could increase T cell and B cell infiltration and promote the formation of TLS-like structures, while heat-inactivated Campylobacter showae had no obvious effect (Figure 4). Figure 4 ).

[0103] The above results collectively show that Campylobacter showae live bacteria can effectively inhibit the growth of oral squamous cell carcinoma and promote the formation of TLSs in the tumor microenvironment.

[0104] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A Campylobacter bacterium for use in the treatment of oral squamous cell carcinoma, characterised in that, The Campylobacter showae strain is a Campylobacter showae strain.

2. The Campylobacter bacterium according to claim 1, characterized in that, The Campylobacter showae strain has a bacterial count of not less than 1 x 10 6 CFU.

3. A bacterial agent for treating oral squamous cell carcinoma, characterized by, The bacterial agent includes the Campylobacter showae strain of claim 1 or 2.

4. The bacterial agent of claim 3, characterized in that, The bacterial agent includes one or more of a fermentation broth, live bacteria, freeze-dried powder, or cell lysate of the Campylobacter showae strain.

5. The bacterial agent of claim 3, wherein The bacterial agent is a solid formulation or a liquid formulation.

6. A medicament for treating oral squamous cell carcinoma, characterized by comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: ###0001### (I) The pharmaceutical includes the Campylobacter showae strain of claim 1 or 2.

7. A pharmaceutical material for treating oral squamous cell carcinoma, characterized by comprising the compound of claim 1 or 2. The pharmaceutical material includes the Campylobacter showae strain of claim 1.

8. The pharmaceutical material according to claim 7, wherein The pharmaceutical material is in a solid form, a liquid form, or a semi-solid form.

9. Use of a Campylobacter bacterium according to claim 1 or 2 for the manufacture of a product for the treatment of oral squamous cell carcinoma, characterized in that, The product includes a pharmaceutical, a bacterial agent, or a pharmaceutical material.

10. Use of a Campylobacter bacterium according to claim 1 or 2 for the preparation of a product for increasing the anti-tumour immune response, characterised in that, The product includes a food, a health product, a bacterial agent, a pharmaceutical, or a pharmaceutical material.

Images