Inactivated thallus for treating oral squamous cell carcinoma and application thereof

Abstract

The invention discloses an inactivated thallus for treating oral squamous cell carcinoma and application of the inactivated thallus. The invention discovers that the thermally inactivated Campylobacter concisus has obvious anti-tumor activity on an oral squamous cell carcinoma model mouse, can obviously inhibit tumor growth and promote formation of a three-level lymphatic structure in the oral squamous cell carcinoma, and has no obvious toxicity on other tissues of the whole body. Therefore, the thermally inactivated Campylobacter concisus and the composition thereof can be used for preparing medicines for treating the oral squamous cell carcinoma and functional products for increasing anti-tumor immune response and relieving cancer symptoms, 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 an inactivated bacterial strain for treating oral squamous cell carcinoma and its application. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the head and neck, primarily occurring in the lips, cheeks, tongue, and gums. Major risk factors associated with OSCC include alcohol, tobacco use, betel nut chewing, radiation exposure, and viral infections. Advanced OSCC has a high recurrence and metastasis rate; the prognosis is poor if the patient experiences recurrence within a short period, has a high tumor histopathological grade, exhibits aggressive tumor growth, or shows lymphovascular invasion. Current clinical treatment for OSCC focuses on surgery and radiotherapy / chemotherapy. However, due to drug transport blockage or interference with intracellular molecular targets, most cases of OSCC show limited response to common cytotoxic drugs. Current clinical strategies do not significantly improve the survival rate of OSCC patients; therefore, exploring more effective drugs for treating oral squamous cell carcinoma is crucial.

[0003] Tertiary lymphoid structures (TLSs) in the human body are typically formed by inflammation, infection, and tumor stimulation. They are more mature immune structures within the tumor microenvironment and can be found in various tumors. Studies have shown that TLSs are more abundant and more mature in OSCC than in other tumors. Furthermore, research has found that patients with OSCC containing TLSs have significantly better prognoses than those lacking TLSs. Therefore, exploring the formation mechanisms of TLSs and identifying drugs that promote their formation may be key to treating OSCC.

[0004] Campylobacter concisus (C. concisus, Cc) is a Gram-negative bacterium, a highly selective thermophilic bacterium. It grows under anaerobic and microaerophilic conditions, and the presence of oxygen significantly promotes its growth. Initially isolated from patients with periodontitis, many subsequent studies have focused on its role in intestinal diseases, suggesting that some Campylobacter concisus species possess the ability to induce intestinal disorders. To date, no studies have demonstrated that Campylobacter concisus has a therapeutic effect on OSCC (Obstructive Syndrome of the Disease). Summary of the Invention

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

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] A first aspect of the present invention provides an inactivated strain for treating oral squamous cell carcinoma, said inactivated strain being Campylobacter concisus.

[0008] Furthermore, the inactivation includes physical inactivation, chemical inactivation, and biological inactivation.

[0009] Furthermore, the physical inactivation includes thermal inactivation, radiation inactivation, and pressure inactivation.

[0010] Furthermore, the inactivation is thermal inactivation.

[0011] Specifically, the heat inactivation step involves heating in a 60°C water bath for 30 minutes.

[0012] Furthermore, the number of inactivated strains is not less than 1×10⁻⁶. 6 CFU.

[0013] Furthermore, the inactivated strain has the effect of promoting the formation of tertiary lymphoid structures in oral squamous cell carcinoma.

[0014] In some embodiments, unless otherwise specified, the term "treatment" means reversing or alleviating a disease or condition or one or more of its symptoms used with the term, suppressing the progression of the disease or condition or one or more of its symptoms, or preventing the disease or condition or one or more of its symptoms. The term "treatment" as used in this invention refers to the act of "treatment" as defined above. Therefore, treatment or treatment regimens for diseases in mammals may include one or more of the following: suppressing the growth of the disease, i.e., inhibiting its development; preventing the spread of the disease; alleviating the disease; preventing the recurrence of the disease; and alleviating the symptoms of the disease.

[0015] A second aspect of the present invention provides a medicament for treating oral squamous cell carcinoma, the product comprising one or more of the following: an inactivated bacterial strain provided in the first aspect of the present invention, a culture of the inactivated bacterial strain, a lysate of the inactivated bacterial strain, an extract of the inactivated bacterial strain, or a lyophilized powder of the inactivated bacterial strain.

[0016] Furthermore, the culture of the inactivated strain refers to the culture obtained by inoculating the aforementioned Campylobacter concisus strain onto a culture medium and then subjecting it to an inactivation step along with the bacterial cells.

[0017] Furthermore, the inoculation methods include, but are not limited to, any one or more of the following: streak plate inoculation, slant inoculation, pour culture, puncture inoculation, and liquid inoculation.

[0018] Furthermore, the vaccination dose can be 0.1%-20%, and in some cases, it can be higher or lower. Specifically, the vaccination dose 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%.

[0019] Furthermore, the culture medium can be a solid culture medium, a semi-solid culture medium, or a liquid culture medium. It can also be any suitable culture medium type disclosed in the prior art, or a culture medium that is further improved on the culture medium type disclosed in the prior art to improve the performance of the strain. It can also be a culture medium not disclosed in the prior art but that can be used for the culture of the aforementioned Campylobacter concisus strain.

[0020] Furthermore, the lysate of the inactivated strain refers to the product obtained by lysing the aforementioned heat-inactivated Campylobacter concisus strain. The lysis can be physical or chemical. 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, where the enzymatic hydrolysis can be with hydrolases or oxidases. Lysis can also be achieved by increasing intracellular pressure to induce spontaneous cell rupture.

[0021] Furthermore, the extract of the inactivated strain refers to the product obtained by separating and concentrating the lysate of the aforementioned inactivated strain. The separation methods include, but are not limited to, solvent extraction, distillation, precipitation, electrodialysis, chromatography, centrifugation, and membrane separation; the concentration methods include, but are not limited to, evaporation concentration and freeze concentration.

[0022] Furthermore, the lyophilized powder of the inactivated strain refers to the product obtained by lyophilizing the culture of the aforementioned inactivated strain. The lyophilized powder generally also includes a lyophilization protectant. The lyophilization protectant includes one or more of the following: fucose, trehalose, sucrose, lactose, stachyose, sorbitol, glycerol, erythritol, fructooligosaccharides, xylooligosaccharides, galactooligosaccharides, inulin, isomaltooligosaccharides, resistant dextrin, whey protein, collagen, skim milk, xylose, arabinose, ribose, rhamnose, galactose, mannose, fructose, sorbitol, galactitol, xylitol, mannitol, maltitol, lactitol, raffinose, manno-oligosaccharides, maltodextrin, and polydextrose.

[0023] In some embodiments, the medicament of the present invention can be administered to the subject once or more daily. A unit dose refers to a physically discrete unit suitable for unit administration to a patient, and each unit contains a suitable drug carrier and a predetermined amount of the inactivated Campylobacter concisus strain of the present invention to provide therapeutic effect. The dosage varies depending on the severity of the patient's disease and the microorganisms, as well as any adjunctive active ingredients used in conjunction. Additionally, the total daily dose may be divided into several doses and administered continuously as needed.

[0024] In some implementations, the patient to whom the drug for treating oral squamous cell carcinoma may be administered is an animal, preferably a mammal (human and non-human animals), including but not limited to: humans, non-human primates (especially higher primates such as macaques, cynomolgus monkeys, stump-tailed macaques, rhesus monkeys, shrews, golden snub-nosed monkeys, and tree shrews), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, any livestock or pets, etc.

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

[0026] Furthermore, the pharmaceutically acceptable excipients and / or excipients include, but are not limited to, any one or more of fillers, diluents, binders, preservatives, lubricants, antioxidants, and stabilizers.

[0027] In specific implementation schemes, the fillers include, but are not limited to: mannitol, xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, sodium bicarbonate, etc.

[0028] In specific implementation schemes, the diluents include, but are not limited to: lactose, sodium chloride, glucose, urea, starch, water, etc.

[0029] In specific implementation schemes, the adhesive includes, but is not limited to: starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginate, xanthan gum, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc.

[0030] In specific implementation schemes, the preservatives include, but are not limited to, phenol, cresol, chlorocresol, thymol, benzoic acid and its salts, sorbic acid and its salts, boric acid and its salts, propionic acid, formaldehyde, glutaraldehyde, benzyl alcohol, phenethyl alcohol, chlorobutanol, chloroform, chlorhexidine, etc.

[0031] In specific implementation schemes, the lubricant includes, but is not limited to: zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.

[0032] In specific implementation schemes, the antioxidants and stabilizers include sulfurous acid, sulfites, bisulfites, metabisulfites, dithionite, thiosulfates, thiourea, glutathione, dimercaprol, mercaptoacetic acid and its salts, thiolactic acid and its salts, thiodipropionic acid and its salts, gallic acid and its salts, caffeic acid or its pharmaceutical salts, ferulic acid or its pharmaceutical salts, di-tert-butyl-p-phenol, 2,5-dihydroxybenzoic acid or its salts, salicylic acid or its salts, ascorbic acid and its salts, isoascorbic acid and its salts, nicotinamide, tartaric acid, phosphates, pharmaceutical salts of acetate, citrates, EDTA and its salts.

[0033] 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.

[0034] Furthermore, the dosage forms of the drug include, but are not limited to, injections, tablets, capsules, powders, granules, oral liquids, and pills.

[0035] A third aspect of the present invention provides a product for increasing anti-tumor immune response, the product comprising at least one of the following: an inactivated strain according to the first aspect of the present invention, a culture of the inactivated strain, a lysate of the inactivated strain, an extract of the inactivated strain, or a lyophilized powder of the inactivated strain.

[0036] Furthermore, the products include pharmaceuticals, food compositions, functional microbial agents, or pharmaceutical raw materials.

[0037] In some embodiments, the functional microbial agent mainly comprises the inactivated bacterial strains described in this invention and optionally includes a culture medium composition, such as yeast extract and soybean peptone. It may further include other probiotics co-cultured to enhance the effect, selected from Clostridium butyricum, Lactobacillus (e.g., L. acidopHilus, L. rhamnosus); Bifidobacterium (e.g., Bifidobacterium infantis, Bifidobacterium longum); and yeast (e.g., Saccharomyces boulardii). Furthermore, the functional microbial agent may also include carrier materials such as diatomaceous earth and corn starch (for solid-state fermentation of microbial agents); and synergists such as short-chain fatty acids (e.g., sodium butyrate) and plant extracts (e.g., curcumin, anti-inflammatory synergists).

[0038] Furthermore, the functional microbial agent can be a solid formulation or a liquid formulation.

[0039] In some implementations, the term "food composition" refers to a natural or artificial product containing at least one nutrient, and more preferably to a product that has become edible through certain processing, generally encompassing all foods, food additives, and health functional foods.

[0040] In some embodiments, the food may include, for example, different types of food, beverages, chewing gum, tea, multivitamins, and functional foods. Additionally, the food of the present invention includes, but is not limited to, special nutritional foods (e.g., flavored dairy products), processed meat products, fish products, tofu, jelly, noodles (e.g., instant noodles, Asian noodles), bread, condiments (e.g., soy sauce, soybean paste, chili sauce, mixed sauces), sauces, biscuits (e.g., snacks), confectionery, ice cream, dairy products (e.g., fermented milk, cheese), pickled foods (e.g., various pickles, preserved vegetables), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages), and natural seasonings (e.g., instant noodles). The food, beverage, or food additive may be prepared in a conventional manner.

[0041] In some implementations, the term "health functional food" refers to a group of foods whose value is enhanced through the use of physical, biochemical, or bioengineering techniques to perform and express their functions for a predetermined purpose, or processed foods designed to fully express the in vivo regulatory functions (e.g., rhythm regulation, disease prevention, and disease treatment) of the relevant food composition. Such functional foods may contain food science-acceptable food supplements and may additionally include suitable carriers, excipients, and diluents typically used in their manufacture.

[0042] In addition to the above, foods containing the food compositions according to the present invention may contain a variety of nutrients, microorganisms, minerals (electrolytes), flavoring agents (e.g., synthetic and natural flavoring agents), coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH control agents, stabilizers, preservatives, glycerin, alcohols, carbonizing agents for carbonized beverages, etc., and each of the above components may be used alone or in combination with each other.

[0043] In some implementations, the pharmaceutical ingredient may be in solid, liquid, or semi-solid form.

[0044] The fourth aspect of the present invention provides the use of the inactivated strain provided in the first aspect of the present invention in the preparation of a medicament for treating oral squamous cell carcinoma.

[0045] The fifth aspect of the present invention provides the use of the inactivated strains provided in the first aspect of the present invention in the preparation of products that enhance anti-tumor immune responses, said products including pharmaceuticals, food compositions, functional microbial agents or pharmaceutical raw materials.

[0046] Advantages and beneficial effects of the present invention:

[0047] This invention reveals that heat-inactivated Campylobacter concisus exhibits excellent antitumor activity against oral squamous cell carcinoma in mouse models, significantly inhibiting tumor growth, promoting the formation of tertiary lymphoid structures in oral squamous cell carcinoma, and showing no significant toxicity to other tissues systemically. This strain and its compositions can be used to prepare drugs for treating oral squamous cell carcinoma, enhance antitumor immune responses, and alleviate cancer symptoms, providing a novel microbial intervention strategy for oral squamous cell carcinoma and related immunotherapies. Attached Figure Description

[0048] 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.

[0049] Figure 2 Figure 1 shows the experimental results of treating OSCC with heat-inactivated Campylobacter concisus (C3h mouse-SCC7 cell line); where A is the experimental flowchart; B is the tumor volume; C is a representative image of the tumor; and D is the mouse body weight ratio (measurement day / first day).

[0050] Figure 3Figure 1 shows the experimental results of heat-inactivated Campylobacter concisus in treating OSCC (C57 mouse-MOC1 cell line); where A is the experimental flowchart; B is the tumor volume; C is a representative image of the tumor; and D is the mouse body weight ratio (measurement day / first day).

[0051] Figure 4 Figure showing experimental results of heat-inactivated Campylobacter concisus promoting TLS formation.

[0052] Figure 5 The figures show the experimental results of the toxicity analysis of heat-inactivated Campylobacter concisus on other tissues throughout the body; where A is the blood routine test result; B is the liver and kidney function test result; and C is the pathological section result of the heart, liver, spleen, lung, and kidney. Detailed Implementation

[0053] As used above, the terms “have,” “include,” or “include,” 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 exist besides the presence of these features.

[0054] Furthermore, as used above, 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.

[0055] 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.

[0056] 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.

[0057] Example 1: Screening of intratumoral bacteria in OSCC associated with TLSs

[0058] I. Experimental Methods

[0059] 1. Experimental materials

[0060] 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.

[0061] (1) Inclusion criteria:

[0062] Patients with primary OSCC;

[0063] No antibiotics were used in the week prior to surgery;

[0064] No other treatment was administered prior to the surgery;

[0065] Those who undergo radical surgical resection.

[0066] (2) Exclusion criteria:

[0067] The primary lesions were multiple in the patients;

[0068] Patients who do not participate in this study.

[0069] (3) Reagents and antibodies: HostZERO Microbial DNA Kit (Zymo, Cat#D4310), CD3 antibody (Abcam, Cat#ab699), CD20 antibody (Thermo Fisher, Cat#14-0202-82).

[0070] 2. Experimental Procedure

[0071] (1) Grouping:

[0072] Based on the distribution of the tertiary lymphoid structures, patients were divided into two groups: the TLS+ group and the TLS- group.

[0073] 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.

[0074] (2) 16S rDNA sequencing

[0075] 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.

[0076] II. Experimental Results

[0077] 16S sequencing results showed that, at the genus level, *Campylobacter* was a bacterial genus that differed significantly between TLS+ and TLS- tumor tissues, and the contribution of *Campylobacter* was the highest in TLS+ tumor tissues. Figure 1 A, Figure 1 B). The above results suggest that Campylobacter may play an important role in the formation of TLS in OSCC patients, and that Campylobacter concisus has the potential to treat OSCC.

[0078] Example 2: In vivo study of heat-inactivated Campylobacter concisus for the treatment of OSCC

[0079] I. Experimental Methods

[0080] 1. Experimental materials

[0081] (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.

[0082] (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.

[0083] 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.

[0084] (3) Strains: The strains used in this invention were purchased from Mingzhou Biotechnology, B259806: Campylobacter concisus; B269393: Campylobacter showae. Campylobacter concisus and Campylobacter showae were cultured on Columbia blood agar plates in a 37°C incubator.

[0085] (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).

[0086] 2. Experimental Procedure

[0087] (1) Heat inactivation of Campylobacter concisus and Campylobacter showae: heating in a water bath at 60℃ for 30 min.

[0088] (2) Tumor formation experiment in mice

[0089] 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. 3Mice were randomly divided into three groups: a control group, a heat-inactivated Cc (Campylobacter concisus) group, and a heat-inactivated Cs (Campylobacter showae) group, with 10 mice per group. Different interventions were then initiated. The control group received intratumoral injection of 50 μl of sterile PBS solution twice daily; the heat-inactivated Cc group received intratumoral injection of 50 μl (1×10⁻⁶) heat-inactivated Campylobacter concisus bacterial suspension resuspended in PBS solution (1×10⁻⁶). 6 CFU), 2 days / time; 50 μl (1×10⁻⁶) of heat-inactivated Campylobacter showae bacterial culture resuspended in PBS solution was injected intratumorally into the Cs group. 6 CFU (carnitine nitrate), twice a day. During this period, tumor growth, body weight, and other indicators in mice were measured, and growth curves were plotted.

[0090] 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 heat-inactivated Cc 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 heat-inactivated Cc group received intratumoral injection of 50 μl (1×10⁻⁶) heat-inactivated Campylobacter concisus bacterial suspension resuspended in PBS solution. 6 CFU), 3 days / time; 50 μl (1×10⁻⁶) of heat-inactivated Campylobacter showae bacterial culture resuspended in PBS solution was injected intratumorally into the Cs group. 6 CFU (carnitine ferrous sulfate), 3 times a day. During this period, the growth of tumors and body weight of mice were measured, and growth curves were plotted.

[0091] (3) Immunofluorescence

[0092] 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 incubated with secondary antibody (HRP) at room temperature in the dark for 50 min, TSA fluorescent staining solution was added, incubated at room temperature for 5 min, washed 3 times, antigen repair was repeated, and the previous operation was repeated, incubated with CD19, CD3 and other antibodies respectively, and finally DAPI staining was performed, 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-.

[0093] (4) Histopathological examination

[0094] 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.

[0095] (5) Complete blood count and liver and kidney function tests

[0096] 1) Histopathological examination: The tumor in the control group grew to 1500 mm. 3 Afterwards, the experiment was terminated, and the hearts, livers, spleens, lungs, and kidneys of mice from different groups were collected, dehydrated, fixed, embedded, sectioned, and stained with hematoxylin and eosin (HE) to assess the histological changes of the internal organs.

[0097] 2) Hematological tests: Tumors in the control group grew to 1500 mm 3 Afterwards, the experiment was terminated, and venous blood was collected from the eyeballs to detect the red blood cell count, white blood cell count, white blood cell differential count (neutrophil percentage, lymphocyte percentage, monocyte percentage, eosinophil percentage, basophil percentage), and platelet count. Serum was extracted by centrifugation, and the levels of alanine aminotransferase, aspartate aminotransferase, creatinine, uric acid, and blood urea nitrogen were detected to assess the biosafety of heat-inactivated intratumoral bacteria agent for OSCC treatment.

[0098] II. Experimental Results

[0099] 1. In vivo study of SCC7 model of OSCC treated with heat-inactivated Campylobacter concisus.

[0100] Figure 2 A is the in vivo experimental flowchart. The results show that, compared with the control group, the tumor volume of mice injected with heat-inactivated Campylobacter concisus was significantly reduced. Figure 2 B) all decreased significantly, as seen in representative tumor images ( Figure 2 (C) This more clearly shows that the tumors in the mice in the heat-inactivated Campylobacter concisus group were significantly shrunk. Furthermore, we also conducted corresponding experiments using inactivated Campylobacter showae bacterial solution, and the results showed that the heat-inactivated Campylobacter showae bacterial solution had no significant inhibitory effect on tumors. There was also no statistically significant difference in body weight gain among the groups of mice. Figure 2 D).

[0101] 2. In vivo study of heat-inactivated Campylobacter concisus in treating OSCC in a MOC1 model

[0102] Figure 3 A is the in vivo experimental flowchart. The results show that, compared with the control group, the tumor volume of mice injected with heat-inactivated Campylobacter concisus was significantly reduced. Figure 3 B) all decreased significantly, as seen in representative tumor images ( Figure 3 (C) This more clearly shows that the tumors in the mice in the heat-inactivated Campylobacter concisus group were significantly shrunk. Furthermore, we also conducted corresponding experiments using inactivated Campylobacter showae bacterial solution, and the results showed that the heat-inactivated Campylobacter showae bacterial solution had no significant inhibitory effect on tumors. There was also no statistically significant difference in body weight gain among the groups of mice. Figure 3 D).

[0103] 3. In vivo study on the regulation of TLS formation by heat-inactivated Campylobacter concisus

[0104] Below, we used immunofluorescence to detect the effect of heat-inactivated Campylobacter concisus bacterial suspension on the formation of TLSs in the mouse tumor microenvironment. The results also showed that heat-inactivated Campylobacter concisus increased T cell and B cell infiltration and promoted the formation of TLS-like structures, while heat-inactivated Campylobacter showae had no significant effect. Figure 4 ).

[0105] 4. Toxicity studies of heat-inactivated Campylobacter concisus on other tissues throughout the body.

[0106] Complete blood count (CBC) Figure 5 A) and liver and kidney function ( Figure 5 (B) All indicators showed that local injection of heat-inactivated Campylobacter concisus bacterial solution had almost no significant effect on the whole body of mice. Pathological histological results also showed that the heart, liver, spleen, lung, and kidney tissues of the heat-inactivated Campylobacter concisus group were not significantly different from those of the control group. Figure 5 C).

[0107] The above results collectively indicate that heat-inactivated Campylobacter concisus can effectively inhibit the growth of oral squamous cell carcinoma, promote the formation of TLSs in the tumor microenvironment, and have no significant effect on other tissues in mice.

[0108] 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. An inactivated bacterial strain for treating oral squamous cell carcinoma, characterized in that, The inactivated strain is an inactivated Campylobacterconcisus strain.

2. The inactivated strain according to claim 1, characterized in that, The inactivation includes physical inactivation, chemical inactivation, and biological inactivation.

3. The inactivated strain according to claim 2, characterized in that, The physical inactivation includes thermal inactivation, radiation inactivation, and pressure inactivation.

4. The inactivated strain according to claim 1, characterized in that, The inactivation is thermal inactivation.

5. The inactivated strain according to claim 1, characterized in that, The number of inactivated strains is not less than 1×10⁻⁶. 6 CFU.

6. A drug for treating oral squamous cell carcinoma, characterized in that, The drug comprises at least one of the following: an inactivated strain according to any one of claims 1-5, a culture of the inactivated strain, a lysate of the inactivated strain, an extract of the inactivated strain, or a lyophilized powder of the inactivated strain.

7. A product that enhances anti-tumor immune response, characterized in that, The product comprises at least one of the following: an inactivated strain according to any one of claims 1-5, a culture of the inactivated strain, a lysate of the inactivated strain, an extract of the inactivated strain, or a lyophilized powder of the inactivated strain.

8. The product according to claim 7, characterized in that, The products include pharmaceuticals, food compositions, functional microbial agents, or pharmaceutical raw materials.

9. The use of the inactivated strain according to any one of claims 1-5 in the preparation of a medicament for treating oral squamous cell carcinoma.

10. The use of the inactivated strain according to any one of claims 1-5 in the preparation of a product that enhances antitumor immune response, characterized in that, The products include pharmaceuticals, food compositions, functional microbial agents, or pharmaceutical raw materials.

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