Campylobacter for treating oral squamous cell carcinoma and application thereof

Abstract

The application discloses a Campylobacter for treating oral squamous cell carcinoma and application thereof. It is found by the application that a Campylobacter showae strain has significant anti-tumor activity on a mouse model of oral squamous cell carcinoma, can significantly inhibit tumor growth, and promote the formation of tertiary lymphoid structures in oral squamous cell carcinoma. Therefore, the Campylobacter showae strain and a composition thereof can be used for preparing products for preventing, treating, adjuvant treating or prognosis nursing oral squamous cell carcinoma, and provide a new microbial intervention strategy for oral squamous cell carcinoma and related immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of health, and in particular relates to a Campylobacter bacterium for treating oral squamous cell carcinoma and application thereof. BACKGROUND

[0002] Oral squamous cell carcinoma (OSCC), also known as oral squamous carcinoma, is a malignant tumor occurring in the oral mucosa epithelium, which has the characteristics of high recurrence and metastasis rate, and most patients will have serious impact on their appearance and psychology due to postoperative maxillofacial defects. At present, the main treatment methods for OSCC are surgical treatment, chemotherapy and radiotherapy, but these treatment methods have not achieved satisfactory effects. Most patients also have postoperative tissue defects, radiation-induced osteonecrosis, low bioavailability of chemotherapy drugs, and resistance to radiotherapy and chemotherapy, etc. This highlights the importance of seeking new treatment methods.

[0003] Tertiary lymphoid structures (TLSs) are ectopic lymphoid tissues formed in non-lymphoid organs under pathological conditions such as chronic inflammation or cancer, which have similar structures to lymph nodes and contain T cell zones, B cell follicles, high endothelial venules (HEV) and dendritic cells (DC) and other immune cell components. TLSs are key sites for inducing and maintaining anti-tumor immune responses in the tumor microenvironment, and their formation mechanisms include chronic stimulation induction, cytokine regulation, chemokine and cytokine mediation. TLSs have the effects of initiating anti-tumor immunity and mediating immune cell homing, and are also closely related to patient prognosis and immunotherapy response in clinical practice.

[0004] Campylobacter showae C. showae Campylobacter (C. jejuni, C. s) is a gram-negative, chemo-heterotrophic, microaerophilic and motile bacterium belonging to the genus Campylobacter. The typical strain SU A4 of this species was originally isolated from plaque samples in the gingival crevice of the human oral cavity, but this strain has also been found in colon tissue and feces since its discovery. C. C. showae It is associated with various diseases due to its pathogenic nature, including Crohn's disease, periodontitis, inflammatory bowel disease and ulcerative colitis. So far, no study has disclosed Campylobacter showae has the effect of treating OSCC. SUMMARY

[0005] To make up for the shortcomings of the prior art, the present application provides a Campylobacter bacterium for treating oral squamous cell carcinoma, which provides a new microbial intervention strategy for the treatment of oral squamous cell carcinoma to overcome the technical problem that there is an urgent need for effective drugs for treating oral squamous cell carcinoma in the art.

[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 bacterium for treating oral squamous cell carcinoma, wherein the Campylobacter bacterium is Campylobacter showae a strain.

[0008] Further, the number of bacteria 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 the strain on a culture medium and culturing.

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

[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%, or 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 be any suitable culture medium disclosed in the prior art, or a culture medium further improved on the basis of the culture medium 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 Campylobacter showae strain.

[0014] In the present application, the prevention refers to various means or measures for preventing the occurrence or development of a disease before the disease is clinically recognized, including medical, physical, or chemical methods, to prevent and reduce the occurrence or development of various symptoms of the disease. In specific embodiments of the present application, the disease is preferably oral squamous cell carcinoma.

[0015] In this invention, the treatment refers to the treatment aimed at preventing and reducing the occurrence or development of a disease, thereby inhibiting, suppressing, alleviating, improving, slowing down, stopping, delaying, or reversing the progression or aggravation of the disease. The various indicators of maintaining and / or using the medication include alleviating or reducing symptoms or complications, or curing or eliminating the disease, disorder, or condition. In a specific embodiment of this invention, the disease is preferably oral squamous cell carcinoma.

[0016] A second aspect of the present invention provides a bacterial agent for treating oral squamous cell carcinoma, the bacterial agent comprising the bacterial agent provided in the first aspect of the present invention. Campylobacter showae One or more of the following: fermentation broth of the strain, fermentation broth supernatant, fermentation broth precipitate, live bacteria, lyophilized powder, or cell lysate.

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

[0018] Furthermore, the supernatant of the fermentation broth refers to the clear liquid at the top after centrifugation of the fermentation broth. It contains abundant metabolic products and some bacterial fragments during bacterial growth and reproduction, acidic substances and bacteriocins secreted by bacteria (which have antagonistic and bactericidal effects on harmful bacteria), amino acids after bacterial decomposition of food, and synthesized vitamins, as well as enzymes secreted by bacteria that are useful to the human body; and some bacterial components also have immune-boosting effects on the human body.

[0019] Furthermore, the fermentation broth precipitation refers to the precipitation of the liquid after centrifugation, including free proteins, residual bacterial cells, broken cells, and culture medium residues, including proteins and intracellular matrix.

[0020] Furthermore, the live bacteria, also known as active bacterial flora, can colonize and multiply within organisms, which helps increase the number of beneficial bacteria.

[0021] Furthermore, the freeze-dried powder is the product provided in the first aspect of the present invention. Campylobacter showae The strain is obtained by freeze-drying. The freeze-dried powder generally also includes a freeze-drying protectant. The freeze-drying protectant includes, but is not limited to, pH buffers, fillers, sugars, nonionic surfactants, ligands, etc. The pH buffer includes, but is not limited to, Tris, amino acids, or their salts, any one or more. The fillers include, but are not limited to, mannitol, glycine, bovine serum albumin, any one or more. The sugars can be disaccharides, such as sucrose or trehalose, any one or more. The nonionic surfactants include, but are not limited to, Tween, and Tween-20, Tween-60, Tween-80, etc. The freeze-drying protectant may also include antioxidants, and specifically, albumin, polyethylene glycol, etc.

[0022] Furthermore, the cell lysis buffer can be obtained by lysing bacterial cells cultured from the culture provided in the first aspect of the present invention. The lysis can be physical lysis or chemical lysis. Physical lysis includes, but is not limited to, grinding and ultrasonic disruption. Chemical lysis includes, but is not limited to, lysis with chemical reagents and enzymatic hydrolysis, wherein the enzymatic hydrolysis can be hydrolytic enzymes or oxidases. Lysis can also be achieved by increasing intracellular pressure to induce spontaneous cell rupture.

[0023] Furthermore, the microbial agent can be a solid formulation or a liquid formulation.

[0024] A third aspect of the present invention provides a medicament for treating oral squamous cell carcinoma, the medicament comprising the components provided in the first aspect of the present invention. Campylobacter showae Strains or cultures.

[0025] Furthermore, the drug also includes pharmaceutically acceptable excipients and / or excipients.

[0026] In some embodiments, the excipients and / or excipients may 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 croscarmellose sodium (CCNa) and low-substituted hydroxypropyl cellulose (L-HPC); lubricants selected from magnesium stearate, silica, talc; and coating materials selected from gastric / enteric coatings (such as acrylic resin, shellac). Protectants, used to maintain the stability of the microbial agent, are selected from trehalose, skim milk powder, and glycerin; surfactants are selected from polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol; flavoring agents are selected from sodium saccharin, cyclamate, aspartame, acetylsupan-K, glycerin, sorbitol, mannitol, sucrose, simple syrup, and aromatic syrup; solubilizers are 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, glucoheponic acid, 2,5-dihydroxybenzoic acid, and acidic amino acids, etc.

[0027] In some embodiments, the pharmaceutical formulation of the present invention may also contain targeted delivery components, specifically including biodegradable polymers, biodegradable grafts, non-biodegradable grafts, and biodegradable microparticles such as biodegradable microspheres, nanoparticles, etc.

[0028] In specific implementation schemes, the choice of which pharmaceutical excipients to combine with the drug formulation depends on the formulation to be made. When making ordinary formulations, the choice of which pharmaceutical excipients to use is well known to those skilled in the art.

[0029] In some implementations, the term "pharmaceutical acceptable" means a composition that is physiologically acceptable and does not cause allergic reactions such as gastrointestinal symptoms, dizziness, or similar reactions when administered to a human.

[0030] Furthermore, the dosage forms of the drug include, but are not limited to, injections, tablets, capsules, powders, granules, oral solutions, and emulsions.

[0031] Specifically, the aforementioned injectable preparations are the most common parenteral dosage forms, including solution-type injections, suspension-type injections, emulsion-type injections, and sterile powders for injection. They can be administered via intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection. Intravenous injection allows the drug to rapidly enter the bloodstream, resulting in a fast-acting effect, suitable for emergency situations and drugs requiring rapid onset of action; intramuscular and subcutaneous injections have relatively slower absorption rates, but their effects last longer.

[0032] Specifically, the tablets are tablet-shaped preparations made by mixing drugs and excipients and then compressing them. They have advantages such as accurate dosage, good stability, high production efficiency, and convenient administration. They can be divided into various types, including ordinary tablets, coated tablets, chewable tablets, and dispersible tablets.

[0033] Specifically, the capsules are divided into hard capsules and soft capsules. Hard capsules are made by filling a hollow hard capsule with drug powder or granules; soft capsules are made by dissolving or suspending the drug in a suitable oily or non-oily liquid medium and then using compression or dripping methods. Capsules can mask unpleasant odors of drugs, improve drug stability, and are convenient to take.

[0034] Specifically, the powder is a dry powder preparation made by pulverizing and uniformly mixing a drug with suitable excipients. Powders have small particle size, large specific surface area, are easily dispersed, and have a rapid onset of action, but their stability is relatively poor.

[0035] Specifically, the granules are dry granular preparations made by combining drugs with suitable excipients to form granules of a certain particle size. They can be classified into soluble granules, suspension granules, effervescent granules, etc. Granules are easy to dissolve, absorb quickly, and are convenient to carry and take.

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

[0037] Specifically, the emulsion is a heterogeneous liquid formulation formed by emulsifying two immiscible liquids, dispersing one liquid in droplets within the other. Oral emulsions can improve the taste and stability of drugs and enhance drug bioavailability.

[0038] A fourth aspect of the present invention provides a pharmaceutical raw material for treating oral squamous cell carcinoma, the pharmaceutical raw material comprising the ingredients provided in the first aspect of the present invention. Campylobacter showae Strains or cultures.

[0039] Furthermore, the pharmaceutical raw material may be in solid, liquid, or semi-solid form.

[0040] The fifth aspect of the present invention provides the use of Campylobacter as described in the first aspect of the present invention in the preparation of products for treating oral squamous cell carcinoma, said products including drugs, bacterial agents or pharmaceutical raw materials.

[0041] 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.

[0042] Advantages and beneficial effects of the present invention:

[0043] This invention has discovered that, Campylobacter showae Live bacteria exhibited significant antitumor activity against oral squamous cell carcinoma model mice, significantly inhibiting tumor growth and promoting the formation of tertiary lymphoid structures in oral squamous cell carcinoma. Therefore, Campylobacter C. showae Live bacteria and their compositions can be used to prepare products for the prevention, treatment, adjuvant therapy or prognostic care of oral squamous cell carcinoma, providing a new microbial intervention strategy for oral squamous cell carcinoma and related immunotherapies. Attached Figure Description

[0044] 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.

[0045] Figure 2 for Campylobacter showae Figure 1 shows the experimental results of 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).

[0046] Figure 3 for Campylobacter showae Figure 1 shows the experimental results of 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).

[0047] Figure 4 for Campylobacter Figure showing experimental results that facilitate the formation of TLSs. Detailed Implementation

[0048] As used in this invention, the terms “having,” “comprising,” or “including,” or any grammatical variations thereof, are used in a non-exclusive manner. Thus, these terms can all refer to a situation where no other features exist in the entity described in this context besides the features introduced by these terms, and can also refer to a situation where one or more other features are present.

[0049] Furthermore, as used in this invention, the terms “preferred,” “more preferred,” “most preferred,” “particularly,” “more particularly,” “specifically,” “more specifically,” or similar terms are used in combination with optional features without limiting other possibilities.

[0050] Unless otherwise stated, all figures used in this specification and claims to represent volume, weight, temperature, time, density, parts by weight, technical effect, etc., should in any case be understood to be modified by the terms "about" or "approximately". Therefore, unless indicated to the contrary, the numerical parameters listed in the specification and appended claims are approximate values. Unless otherwise stated, the terminology used herein has its common meaning as understood by one of ordinary skill in the art, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by one of those skilled in the art.

[0051] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0052] Example 1: Screening of intratumoral bacteria in OSCC associated with TLSs

[0053] I. Experimental Methods

[0054] 1. Experimental materials

[0055] A total of 113 samples of tumor tissue and adjacent normal tissue were collected from patients with OSCC (59 tumor tissue samples and 54 adjacent normal tissue samples) for 16S detection of intratumoral bacteria. Air was collected in blank tubes at the same time as a blank control.

[0056] (1) Inclusion criteria:

[0057] Patients with primary OSCC;

[0058] No antibiotics were used in the week prior to surgery;

[0059] No other treatment was administered prior to the surgery;

[0060] Those who undergo radical surgical resection.

[0061] (2) Exclusion criteria:

[0062] The primary lesions were multiple in the patients;

[0063] Patients who do not participate in this study.

[0064] (3) Reagents and antibodies: HostZERO Microbial DNA Kit (Zymo, Cat#D4310), CD3 antibody (Abcam, Cat#ab699), CD20 antibody (Thermo Fisher, Cat#14-0202-82).

[0065] 2. Experimental Procedure

[0066] (1) Grouping:

[0067] Based on the distribution of the tertiary lymphoid structures, patients were divided into two groups: the TLS+ group and the TLS- group.

[0068] The TLS+ group is defined as a TLS aggregation that needs to exceed 7000 μm. 2 It must contain at least 100 cells, otherwise it is defined as a TLS-group.

[0069] (2) 16S rDNA sequencing

[0070] Samples were processed under aseptic conditions after collection and stored at –80°C to prevent changes in the microbial community structure. Bacterial DNA was extracted using the HostZERO Microbial DNA Kit, and the concentration and purity of the DNA were determined using a Qubit 4.0 quantitative PCR instrument, following the manufacturer's instructions. The extracted DNA was used as a template to amplify the V3–V4 variable region of the bacterial 16S rRNA gene, using primers for the 16SV34 region (341F: CCTAYGGGRBGCASCAG (SEQ ID NO:1) and 806R: GGACTACNNGGGTATCTAAT (SEQ ID NO:2)). All PCR mixtures were added to 15 µL of Phusion High-Fidelity PCR Master Mix, 0.2 µM of primers, and 10 ng of genomic DNA template. The mixture was first denatured 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 held at 72°C for 5 minutes. After 2% agarose gel electrophoresis, the amplified products were purified using AMPure XP magnetic beads and subjected to secondary PCR using the Illumina Nextera XT Index Kit to add adapters and dual-index tags. The purified library was quantified using Qubit and fragment length and concentration were determined using Agilent Bioanalyzer. The fragments were then equimolarly mixed to form a library, diluted to appropriate concentrations, and sequenced on the Illumina MiSeq platform (2×250 bp paired-end sequencing mode). The raw sequences obtained from sequencing were subjected to adapter removal and quality control using Cutadapt, followed by denoising, chimera removal, and ASV extraction using the DADA2 algorithm on the QIIME2 platform. Finally, species annotation was performed based on the SILVA 138 database, and community diversity and abundance distribution were further analyzed.

[0071] II. Experimental Results

[0072] 16S sequencing results showed that at the genus level Campylobacter This genus is a bacterial genus that shows significant differences in tumor tissues of the TLS+ and TLS- groups, and Figure 1 The contribution of this genus was the highest in tumor tissues of the TLS+ group. Figure 1 A, Campylobacter B). The above results indicate that Campylobacter showae It may play an important role in the formation of TLSs in OSCC patients, and Campylobacter showae It has the potential to treat OSCC.

[0073] Example 2 Campylobacter showae In vivo study of live bacteria therapy for OSCC

[0074] I. Experimental Methods

[0075] 1. Experimental materials

[0076] (1) Mice: C3h mice and C57 mice, female, 8 weeks old. C3h mice were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd., and C57 mice were purchased from Changsha Slack Jingda Animal Experiment Co., Ltd.

[0077] (2) Cells: Mouse squamous cell carcinoma cell line (SCC7) was purchased from Changsha Yanke Biotechnology Co., Ltd. Upon arrival, the cells were thawed and revived under sterile conditions and cultured in a humidified incubator at 37°C and 5% CO2. The culture medium used was 1640, supplemented with 15% fetal bovine serum (FBS) and a 1% penicillin / streptomycin mixture. When the cells reached approximately 80–90% confluence, they were passaged using 0.25% trypsin-EDTA digestion, with a passage ratio generally between 1:3 and 1:4. To maintain cell characteristics and avoid over-passaging, mycoplasma testing was performed regularly. Cells in the logarithmic growth phase were selected for in vivo experiments to ensure tumorigenesis rates.

[0078] Mouse squamous cell carcinoma cell line (MOC1) was purchased from Changsha Yanke Biotechnology Co., Ltd. Upon arrival, the cells were thawed and revived under sterile conditions and cultured in a humidified incubator at 37°C with 5% CO2. 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 reached approximately 80–90% confluence, they were passaged using 0.25% trypsin-EDTA digestion, with a passage ratio generally between 1:3 and 1:4. To maintain cell characteristics and avoid over-passaging, cells in the logarithmic growth phase were selected for in vivo experiments to ensure tumorigenesis rates.

[0079] (3) Strains: The strains used in this invention were purchased from Mingzhou Biotechnology, B269393: Campylobacter showae bacteria. Campylobacter showae The bacteria were cultured on Columbia blood agar plates at 37°C in a constant temperature incubator.

[0080] (4) Reagents and antibodies: 1640 medium (Gibco, Cat#C11875500BT), IMDM medium (CytivaHyClone, 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), and penicillin-streptomycin mixture (Gibco, Cat#15140122) were used for cell culture. 0.25% trypsin-EDTA solution (Gibco, Cat#25200056) was used as the digestion medium. Columbia blood agar plate medium (Solarbio, Cat#CM0035) was also used. CD3 antibody (Abcam, Cat#ab16669), CD19 antibody (Abcam, Cat#ab245235), PNAD antibody (Biolegend, Cat#120801).

[0081] 2. Experimental Procedure

[0082] (1) Campylobacter showae Bacterial heat inactivation: Heat in a 60℃ water bath for 30 min.

[0083] (2) Tumor formation experiment in mice

[0084] Thirty 8-week-old C3h mice were subcutaneously injected with 5×10⁻⁶ ppm of the drug into their backs. 5 One SCC7 mouse-derived OSCC cell was used until the tumor volume reached 100 mm. 3 The mice were randomly divided into three groups: a control group, a group receiving live bacteria Cs (…). Campylobacter showae ) group and heat-inactivated Cs ( Campylobacter Group 10 animals per group, at this time different interventions were started. The control group received intratumoral injection of 50 μl of sterile PBS solution, twice a day; the live bacteria Cs group received intratumoral injection of live bacteria resuspended in PBS solution. C. showae Campylobacter showae 50 μl of bacterial culture (1×10) 6 CFU), twice a day; heat-inactivated Cs group: intratumoral injection of PBS solution for resuscitation and heat inactivation. Campylobacter showae 50 μl of bacterial culture (1×10) 6 CFU (carnitine nitrate), twice a day. At the end of the experiment, the tumor weight, body weight, and other indicators of the mice were measured.

[0085] Thirty 8-week-old C57 mice were subcutaneously injected with 2×10⁻⁶ ppm of the drug into their backs.6 MOC1 mouse OSCC cells were used until the tumor volume reached 100 mm. 3 Mice were randomly divided into three groups: a control group, a live bacteria Cs group, and a heat-inactivated Cs group, with 10 mice per group. Different interventions were then initiated. The control group received intratumoral injection of 50 μl of sterile PBS solution every 3 days; the live bacteria Cs group received intratumoral injection of live bacteria resuspended in PBS solution. Campylobacter showae 50 μl of bacterial culture (1×10) 6 CFU), 3 times / day; heat-inactivated Cs group: intratumoral injection of PBS solution for resuscitation and heat inactivation. Campylobacter showae 50 μl of bacterial culture (1×10) 6 CFU), 3 times a day. At the end of the experiment, the tumor weight, body weight, and other indicators of the mice were measured.

[0086] (3) HE staining: When the tumor grows to 1500 mm 3 Afterwards, the experiment was terminated, tumor tissue was collected, fixed, embedded, sectioned, and routinely stained with hematoxylin and eosin (HE) to observe the pathological features and immune cell infiltration of the subcutaneous xenograft.

[0087] (4) Immunofluorescence

[0088] When the tumor grows to 1500 mm 3 Afterwards, the experiment was terminated, tumor tissue was collected, fixed, embedded, sectioned, dewaxed and rehydrated, antigen was repaired, cooled to room temperature, peroxidase was removed, blocked with goat serum for 30 min, incubated overnight at 4°C with CD3 primary antibody, washed 3 times with PBS, then added secondary antibody (HRP), incubated at room temperature in the dark for 50 min, added TSA fluorescent staining solution, incubated at room temperature for 5 min, washed 3 times, antigen repair was repeated, the previous operation was repeated, incubated with CD19 antibody, finally stained with DAPI, washed 3 times, mounted and observed under a microscope for CD3+ T cells and CD19+ B cells. If T cells and B cells aggregated, they were identified as TLS+, otherwise they were identified as TLS-.

[0089] II. Experimental Results

[0090] 1. Figure 2 In vivo study of live bacteria therapy for OSCC in the SCC7 model

[0091] Figure 2 A is a flowchart of the in vivo experiment. The results show that, from representative tumor images ( Campylobacter showae B) It can be observed that, compared with the control group, the injection Figure 2 The tumors in mice treated with live bacteria shrank significantly, and statistical analysis results indicated that the tumor weight in the Campylobacter showae group was significantly reduced. Campylobacter showae C) A significant decrease; in addition, we also used CampylobacterThe inactivated bacterial solution was subjected to corresponding experiments, and the results showed that heat inactivation... C. showae Figure 2 The bacterial culture had no significant inhibitory effect on tumors. At the experimental endpoint, there was no statistically significant difference in body weight among the groups of mice. Campylobacter showae D).

[0092] 2. Figure 3 In vivo study of live bacteria therapy in the MOC1 model of OSCC

[0093] Figure 3 A is a flowchart of the in vivo experiment. The results show that, from representative tumor images ( Campylobacter showae B) It can be observed that, compared with the control group, the injection Figure 3 The tumors in mice treated with live bacteria shrank significantly, and statistical analysis results indicated that the tumor weight in the Campylobacter showae group was significantly reduced. Campylobacter showae C) A significant decrease. In addition, we also used... Campylobacter The inactivated bacterial solution was subjected to corresponding experiments, and the results showed that heat inactivation... C. showae Figure 3 The bacterial culture had no significant inhibitory effect on tumors. At the experimental endpoint, there was no statistically significant difference in body weight among the groups of mice. Campylobacter showae D).

[0094] 3. Campylobacter showae In vivo study on the regulation of TLS formation by live bacteria

[0095] Next, we will use immunofluorescence detection. Campylobacter showae The effect of live bacteria on the formation of TLSs in the tumor microenvironment of mice. The results also showed that... Campylobacter showae It can increase T cell and B cell infiltration and promote the formation of TLSs-like structures, while heat-inactivated Figure 4 There was no obvious effect. Campylobacter showae ).

[0096] The above results collectively indicate that ​ Live bacteria can effectively inhibit the growth of oral squamous cell carcinoma and promote the formation of TLSs in the tumor microenvironment.

[0097] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The application of Campylobacter in the preparation of products for treating oral squamous cell carcinoma, characterized in that, The product is a drug, microbial agent, or pharmaceutical raw material; The Campylobacter is Campylobacter showae Bacteria ATCC 51146.

2. The application according to claim 1, characterized in that, The number of *Campylobacter showae* bacteria is not less than 1 × 10⁻⁶. 6 CFU.

3. The application according to claim 1, characterized in that, The microbial agent includes one or more of the following: fermentation broth of Campylobacter showae, live bacteria, or freeze-dried powder.

4. The application according to claim 1, characterized in that, The microbial agent can be a solid or liquid formulation.

5. The application according to claim 1, characterized in that, The pharmaceutical raw materials are in solid, liquid, or semi-solid form.

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