Inactivated bacteria for treating oral squamous cell carcinoma and use thereof

Abstract

The application discloses inactivated bacteria and application thereof for treating oral squamous cell carcinoma. It is found that heat-inactivated Campylobacter concisus has significant anti-tumor activity on a mouse model of oral squamous cell carcinoma, can significantly inhibit tumor growth, promote the formation of tertiary lymphoid structures in oral squamous cell carcinoma, and has no significant toxicity to other tissues in the whole body. Therefore, the heat-inactivated Campylobacter concisus and a composition thereof can be used for preparing a medicine for treating oral squamous cell carcinoma, increasing an anti-tumor immune response, and relieving the symptoms of cancer, 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 an inactivated strain for treating oral squamous cell carcinoma and application thereof. BACKGROUND

[0002] Oral squamous cell carcinoma (OSCC), also known as oral squamous carcinoma, is one of the most common malignant tumors in the head and neck, mainly occurring in the labial buccal part, tongue, gingiva and other parts. The main risk factors associated with OSCC include alcohol, tobacco, chewing betel nut, radiation and viral infection. Advanced OSCC has a high recurrence rate and metastasis rate, and if the patient relapses in a short period of time, the tumor tissue has a high histopathological grade, is accompanied by cancer invasive growth or lymphatic vessel invasion, the prognosis will also be poor. At present, the treatment scheme for OSCC in clinical practice still focuses on surgical treatment and radiotherapy and chemotherapy, but due to drug transport blockage or intracellular molecular target interference, most OSCC shows limited response to common cytotoxic drugs. The current clinical strategy cannot significantly improve the survival rate of OSCC patients, therefore, it is crucial to explore more effective drugs for treating oral squamous carcinoma.

[0003] Tertiary lymphoid structures (TLSs) are usually formed by inflammation, infection and tumor stimulation in the human body, are more mature immune structures in the tumor microenvironment, and can exist in various tumors. Studies have shown that TLSs are more distributed in OSCC than in other tumors, and have higher maturity. At the same time, it has also been found that the prognosis of patients with TLSs in OSCC is significantly better than that of patients lacking TLSs. Therefore, exploring the formation mechanism of TLSs and finding drugs that can promote the formation of TLSs may be the key to treating OSCC.

[0004] Campylobacter concisus C. concisus C. c) belongs to gram-negative bacteria and is a highly fastidious thermophilic bacteria. Campylobacter concisus It grows under anaerobic and microaerobic conditions, and the presence of oxygen can significantly promote its growth. Campylobacter concisus It was first isolated from the body of a periodontitis patient, and subsequent many studies have mainly focused on its role in intestinal diseases, and it is believed that part of Campylobacter concisus has the ability to cause intestinal diseases. So far, no study has disclosed Campylobacter concisus has the effect of treating OSCC. SUMMARY

[0005] ​To make up for the deficiencies of the prior art, the present application provides an inactivated strain for treating oral squamous carcinoma, and provides a new microbial intervention strategy for treating oral squamous carcinoma, so as to overcome the technical problem that there is an urgent need for an effective drug for treating oral squamous carcinoma in the prior art.

[0006] The above application object of the present application is realized by the following technical scheme:

[0007] The first aspect of the present application provides an inactivated strain for treating oral squamous carcinoma, wherein the inactivated strain is Campylobacter concisus a strain.

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

[0009] Further, the physical inactivation includes heat inactivation, radiation inactivation and pressure inactivation.

[0010] Further, the inactivation is heat inactivation.

[0011] Specifically, the step of heat inactivation is 60 DEG C water bath heating for 30 min.

[0012] Further, the number of bacteria of the inactivated strain is not less than 1x10 6 CFU.

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

[0014] In some embodiments, unless otherwise mentioned, the term "treatment" refers to reversing or alleviating a disease or disorder with which the term is used, or one or more symptoms thereof, inhibiting the progression of the disease or disorder, or one or more symptoms thereof, or preventing the disease or disorder, or one or more symptoms thereof. The term "treatment" used in the present application refers to the "treatment" behavior as defined above. Therefore, the treatment or treatment regimen of a disease in a mammal can include one or more of the following: inhibiting 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; alleviating the symptoms of the disease.

[0015] The second aspect of the present application provides a drug for treating oral squamous carcinoma, wherein the product comprises one or more of the inactivated strain provided in the first aspect of the present application, the culture of the inactivated strain, the lysate of the inactivated strain, the extract of the inactivated strain or the freeze-dried powder of the inactivated strain.

[0016] Further, the culture of the inactivated strain refers to the culture of the aforementioned Campylobacter concisus strain in the culture medium, which is subjected to the inactivation step together with the bacterial body after cultivation.

[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. Alternatively, it can be a culture medium not disclosed in the prior art but capable of being used for the aforementioned purposes. Campylobacter concisus Culture medium for culturing bacterial strains.

[0020] Furthermore, the lysate of the inactivated strain refers to the product of the aforementioned heat-inactivated strain. Campylobacter concisus The strain is obtained by lysis. 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 application can be administered to a suitable subject once or more times per day. Unit dose means physically discrete unit suitable for unit administration to a patient, and each unit contains a predetermined quantity of the medicament of the present application in association with a suitable pharmaceutical carrier Campylobacter concisus The dosage varies depending on the severity of the patient's disease and the microorganism and the auxiliary effective components used in conjunction. In addition, the total daily dose can be divided into several times and administered continuously as needed.

[0024] In some embodiments, the patient to which the medicament for treating oral squamous carcinoma can be administered is an animal, preferably a mammal (human and non-human animals), including but not limited to: humans, non-human primates (particularly higher primates such as macaques, cynomolgus monkeys, marmosets, lorises, guenons, 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 pet, etc.

[0025] Further, the medicament also includes a pharmaceutically acceptable excipient and / or excipient.

[0026] Further, the pharmaceutically acceptable excipient and / or excipient includes but is not limited to any one or more of a filler, a diluent, a binder, a preservative, a lubricant, an antioxidant, and a stabilizer.

[0027] In specific embodiments, the filler includes but is not limited to mannitol, xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupled sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.

[0028] In specific embodiments, the diluent includes but is not limited to lactose, sodium chloride, glucose, urea, starch, water, etc.

[0029] In specific embodiments, the binder includes but is not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc.

[0030] In specific embodiments, the preservative includes but is 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 drugs, functional microbial agents, or pharmaceutical raw materials.

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

[0038] Further, the functional bacterial agent can be a solid preparation or a liquid preparation.

[0039] In some embodiments, the medical raw material can be in solid form, liquid form or semi-solid form.

[0040] The fourth aspect of the present application provides the use of the inactivated strain provided in the first aspect of the present application in the preparation of a drug for treating oral squamous cell carcinoma.

[0041] The fifth aspect of the present application provides the use of the inactivated strain provided in the first aspect of the present application in the preparation of a product for increasing anti-tumor immune response, which includes a drug, a functional bacterial agent or a medical raw material.

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

[0043] The present application is based on the finding that a heat-inactivated Campylobacter concisus has good anti-tumor activity on oral squamous cell carcinoma model mice, can significantly inhibit tumor growth, promote the formation of tertiary lymphoid structures in oral squamous cell carcinoma, and has no significant toxicity to other tissues in the body. The strain and its composition can be used to prepare drugs for treating oral squamous cell carcinoma, increase anti-tumor immune response, and relieve cancer symptoms, providing a new microbial intervention strategy for oral squamous cell carcinoma and related immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Figures of 16S sequencing results of cancer tissues and cancer-adjacent tissues of TLSs-positive (TLS+) or TLSs-negative (TLS-) OSCC patients; wherein, A is a bacterial abundance column chart at genus level; B is a bacterial differential contribution chart in tumor tissues of TLSs+ and TLSs- OSCC patients.

[0045] Figure 2 Figures of heat-inactivated Campylobacter concisus Figures of experimental results of treating OSCC (C3h mice-SCC7 cell line); wherein, A is an experimental flow chart; B is tumor volume; C is a representative image of tumor; D is the ratio of mouse body weight (measurement day / first day).

[0046] Figure 3 Figures of heat-inactivated Campylobacter concisus Figures of experimental results of treating OSCC (C57 mice-MOC1 cell line); wherein, A is an experimental flow chart; B is tumor volume; C is a representative image of tumor; D is the ratio of mouse body weight (measurement day / first day).

[0047] Figure 4 Heat-inactivated Campylobacter concisus Figure of experimental results for promoting the formation of TLSs.

[0048] Figure 5 Heat-inactivated Campylobacter concisus Figure of experimental results for toxicity analysis of other tissues in the body; wherein A is a figure of blood routine results; B is a figure of liver and kidney function results; C is a figure of pathological section results of heart, liver, spleen, lung and kidney. DETAILED DESCRIPTION

[0049] As used above, the terms "have", "comprise" or "include" or any grammatical variants thereof are used in a non-exclusive way. Thus, these terms can both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As used above, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically", or similar terms are used in conjunction with optional features, i.e. features which are not indispensable to the technical teaching disclosed in this context. Thus, these terms do not restrict the use of such optional features but merely indicate that these features are optional.

[0050] As used above, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically", or similar terms are used in conjunction with optional features, i.e. features which are not indispensable to the technical teaching disclosed in this context. Thus, these terms do not restrict the use of such optional features but merely indicate that these features are optional.

[0051] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percent, time, temperature, density, weight percent, technical effects, etc. in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations. Unless otherwise indicated, the terms used in the description and claims herein are to be construed as having their ordinary meaning as understood by those of ordinary skill in the art. Each numerical parameter recited in the description and claims is approximated by the use of the term "about" or "approximately", as these terms are construed by those of ordinary skill in the art. Unless otherwise indicated, the terms used in the description and claims herein are to be construed as having their ordinary meaning as understood by those of ordinary skill in the art.

[0052] The present application is further described in conjunction with the specific examples below, which are intended to be illustrative only and not limiting of the application. Those of ordinary skill in the art will understand that various modifications, changes, substitutions, and alterations can be made without departing from the spirit and scope of the application. The scope of the application is defined by the claims and their equivalents. The experimental materials, reagents and raw materials used in the present application are readily available to those of ordinary skill in the art, and can be obtained commercially unless otherwise specified. The experimental methods of the present application, for which specific conditions are not specified, 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 limit the present application as described in detail in the claims.

[0053] Example 1 Intra-tumoral bacteria screening of TLSs related OSCC

[0054] I. Experimental Methods

[0055] 1. Experimental Materials

[0056] 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 intra-tumoral bacteria. The same time air was collected in a blank tube as a blank control.

[0057] (1) Inclusion criteria:

[0058] Primary OSCC patients;

[0059] No use of antibiotics 1 week before surgery;

[0060] No other treatment before surgery;

[0061] Those who underwent radical surgery.

[0062] (2) Exclusion criteria:

[0063] Patients with multiple primary lesions;

[0064] Patients who did not accept this study.

[0065] (3) Reagents and antibodies: HostZERO Microbial DNA Kit (Zymo, Cat#D4310), CD3 antibody (Abcam, Cat#ab699), CD20 antibody (Thermo Fisher, Cat#14-0202-82).

[0066] 2. Experimental steps

[0067] (1) Grouping:

[0068] According to the distribution of three-level lymphoid structures, the patients were divided into two groups: TLS+ group and TLS- group.

[0069] 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.

[0070] (2) 16S rDNA sequencing

[0071] The samples were handled under sterile 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, following the manufacturer's instructions and determining 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 primers 16SV34 region primers (341F: CCTAYGGGRBGCASCAG (SEQ ID NO: 1) and 806R: GGACTACNNGGGTATCTAAT (SEQ ID NO: 2)). All PCR mixtures contained 15 μΐ of Phusion High-Fidelity PCR Master Mix, 0.2 μΜ of primers, and 10 ng of genomic DNA template, and were subjected to a first denaturation at 98°C for 1 min, followed by 30 cycles of 98°C (10 s), 50°C (30 s), and 72°C (30 s), and finally 72°C for 5 min. The amplified products were detected by 2% agarose gel electrophoresis, purified using 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 their fragment lengths and concentrations were determined by Agilent Bioanalyzer, and then equimolar mixed to form a library, which was 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 using Cutadapt, and then subjected to denoising, chimeric removal, and extraction of ASVs (amplicon sequence variants) 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.

[0072] II. Experimental Results

[0073] 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 Campylobacter The contribution of was the highest in the tumor tissues of the TLS+ group Figure 1 A, Figure 1 B). The above results indicated that Campylobacter may play an important role in the formation of TLS in OSCC patients, and Campylobacter concisus has potential for the treatment of OSCC.

[0074] Example 2 Heat-inactivated Campylobacter concisus In vivo study of treating OSCC

[0075] I. Experimental Methods

[0076] 1. Experimental Materials

[0077] (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.

[0078] (2) Cells: Mouse squamous cell carcinoma cell line (SCC7) was purchased from Changsha Yank Biological Technology 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 should be carried out regularly. Cells for in vivo experiments should be selected from logarithmic growth phase cells to ensure the tumor formation rate of the experiment.

[0079] Mouse squamous cell carcinoma cell line (MOC1) was purchased from Changsha Yank Biological Technology 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 logarithmic growth phase cells to ensure the tumor formation rate of the experiment.

[0080] (3) Strains: The strains used in the present application were purchased from Mingzhou Biological Technology Co., Ltd. B259806: Campylobacter concisus B269393: Campylobacter showae B269393: Campylobacter concisus B269393 and Campylobacter showae B269393 were cultured in Columbia blood agar medium at 37°C in a constant temperature incubator.

[0081] (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).

[0082] 2. Experimental Procedure

[0083] (1) Campylobacter concisus and Campylobacter showae Bacterial heat inactivation: Heat in a 60℃ water bath for 30 min.

[0084] (2) Tumor formation experiment in mice

[0085] 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 heat-inactivated Cc group, and a control group. Campylobacter concisus ) group and heat-inactivated Cs ( Campylobacter showae 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 heat-inactivated Cc group received intratumoral injection of heat-inactivated Cc solution resuspended in PBS solution. Campylobacter concisus 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. During this period, tumor growth, body weight, and other indicators in mice were measured, and growth curves were plotted.

[0086] 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 heat-inactivated Cs solution resuspended in PBS solution. Campylobacter concisus 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 (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.

[0087] (3) 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 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-.

[0089] (4) Histopathological examination

[0090] 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.

[0091] (5) Complete blood count and liver and kidney function tests

[0092] 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.

[0093] 2) Hematological tests: Tumors in the control group grew to 1500 mm 3Afterwards, 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.

[0094] II. Experimental Results

[0095] 1. Heat inactivation Campylobacter concisus In vivo study of SCC7 model for treating OSCC

[0096] Figure 2 A is the in vivo experimental flowchart. The results show that, compared with the control group, injection heat inactivation... Campylobacter concisus mouse tumor volume ( Figure 2 B) all decreased significantly, as seen in representative tumor images ( Figure 2 C) It can be seen more intuitively that heat inactivation Campylobacter concisus The tumors in the group of mice shrank significantly. Furthermore, we also used... Campylobacter showae The inactivated bacterial solution was subjected to corresponding experiments, and the results showed that heat inactivation... Campylobacter showae The 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).

[0097] 2. Heat inactivation Campylobacter concisus In vivo study of MOC1 model for treating OSCC

[0098] Figure 3 A is the in vivo experimental flowchart. The results show that, compared with the control group, injection heat inactivation... Campylobacter concisus mouse tumor volume ( Figure 3 B) all decreased significantly, as seen in representative tumor images ( Figure 3 C) It can be seen more intuitively that heat inactivation Campylobacter concisus The tumors in the group of mice shrank significantly. Furthermore, we also used... Campylobacter showae The inactivated bacterial solution was subjected to corresponding experiments, and the results showed that heat inactivation... Campylobacter showae The 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).

[0099] 3. Heat inactivation Campylobacter concisus In vivo studies on the regulation of TLS formation

[0100] Next, we will use immunofluorescence to detect heat-inactivated [cells / particles].Campylobacter concisus The effect of bacterial solution on the formation of TLSs in the microenvironment of mouse tumor. The results also showed that heat-inactivated Campylobacter concisus can increase T cell and B cell infiltration, promote the formation of TLSs-like structure, while heat-inactivated Campylobacter showae have no obvious effect ( Figure 4 ).

[0101] 4, heat-inactivated Campylobacter concisus Toxicity study on other tissues of the whole body

[0102] Blood routine ( Figure 5 A) and liver and kidney function ( Figure 5 B) indicators show that the local injection of heat-inactivated Campylobacter concisus bacterial solution has almost no obvious effect on the whole body of mice, and the results of pathological histology also show that the heart, liver, spleen, lung and kidney tissues of the heat-inactivated Campylobacter concisus group have no obvious difference compared with the control group ( Figure 5 C).

[0103] The above results collectively show that heat-inactivated Campylobacter concisus can effectively inhibit the growth of oral squamous cell carcinoma and promote the formation of TLSs in the microenvironment of the tumor, and has no obvious effect on other tissues of the whole body of mice.

[0104] The above examples are 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, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. The application of an inactivated bacterial strain in the preparation of a drug for treating oral squamous cell carcinoma, characterized in that, The inactivated strain is inactivated. Campylobacter concisus ATCC 33237.

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

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

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

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

6. The application according to claim 1, characterized in that, The drug comprises at least one of the following: as described in claim 1 Campylobacter concisus The inactivated strain of ATCC 33237, the aforementioned Campylobacter concisus Cultures of inactivated strains of ATCC 33237 or the aforementioned Campylobacter concisus Lyophilized powder of inactivated strain ATCC 33237.

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