Tumor inhibitory escherichia coli 18 and application thereof

Abstract

The invention belongs to the technical field of biological medicines, particularly relates to tumor inhibitory escherichia coli 18 and application thereof, and more particularly relates to escherichia coli HZEc18 and application thereof. According to the present invention, the bacteria in the cervical tissue and the vaginal swab of the cervical cancer patient are subjected to in vitro culture and purification to obtain a plurality of strains having potential influence on the tumor microenvironment, and the Escherichia coli HZEc18 has strong tumor cell inhibition activity and host cell interaction, and can be used for the development of antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a tumor-inhibiting Escherichia coli 18 and use thereof, and more particularly to an Escherichia coli HZ_Ec_18 and use thereof. BACKGROUND

[0002] Cervical cancer is a malignant tumor occurring in the uterine cervix and is the fourth most common malignant tumor in women worldwide. The main clinical manifestations of patients with cervical cancer are massive or abnormal vaginal bleeding, especially in some patients after sexual intercourse, who may have watery, mucus-like or foul-smelling vaginal discharge. Patients in the late stage may have lower extremity edema, flank and pelvic or lower back pain, etc.

[0003] Cervical cancer is mainly divided into squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma. Among them, cervical squamous cell carcinoma is a cancer caused by canceration of squamous epithelial cells; cervical adenocarcinoma is caused by the simultaneous differentiation of reserve cells under the endometrial columnar cells into glandular cells and squamous cells. Adenocarcinoma ranks second in the global incidence of cervical cancer and is increasing year by year, and the patient population is showing a trend of youth. Compared with squamous cell carcinoma, adenocarcinoma has various pathological types, different cell morphologies, lower sensitivity to radiotherapy and chemotherapy, higher recurrence rate, easy occurrence of distant metastasis and obvious adverse reactions of poor prognosis, etc. However, the treatment of adenocarcinoma patients cannot be well guided only according to the pathological type. At present, the treatment plan for cervical adenocarcinoma still refers to that for squamous cell carcinoma, thus leading to various negative effects or complications and functional disorders in some adenocarcinoma patients caused by over-treatment.

[0004] At present, the methods for treating many tumors including cervical cancer mainly include traditional therapies (such as surgery, chemotherapy, radiotherapy), targeted therapy, immunotherapy (such as immune checkpoint inhibitors, CAR-T cell therapy), gene therapy and combined treatment strategies. However, these treatment plans still face many challenges. For example, the limitations of CAR-T therapy: in solid tumors, CAR-T cells are difficult to effectively infiltrate the tumor microenvironment and are susceptible to immune suppression; in addition, the traditional CAR-T preparation is costly and may cause side effects such as cytokine release syndrome (CRS). For another example, the drug resistance of immunotherapy: some patients do not respond to immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors), which may be related to CD8-positive T cell exhaustion or metabolic disorders (such as glucose deprivation) in the tumor microenvironment. For another example, the limited applicability of targeted therapy: KRAS mutations have long been considered as “undruggable” targets. Although new generation inhibitors (such as D3S-001) have made progress, some patients may still develop drug resistance. For another example, the optimization needs of combined therapy: how to balance efficacy and toxicity (such as the potential side effects of iron death inducers combined with CAR-T), and how to accurately screen biomarkers (such as PD-L1, TMB) to predict treatment response, are still unsolved problems.

[0005] Cervical cancer is closely related to persistent infection of high-risk human papillomavirus (HPV), especially HPV16 and HPV18. HPV oncoproteins E6 and E7 cause uncontrolled cell proliferation and genomic instability by targeting p53 and Rb proteins, inducing abnormal DNA methylation or histone modification, etc. It is generally believed that squamous cell carcinoma is highly related to HPV16, while adenocarcinoma is more closely related to HPV18. However, HPV infection alone is not sufficient to cause cancer, and other causes such as immunosuppression (e.g. HIV infection), long-term oral contraceptives, multiple pregnancies, and early sexual behavior can also promote the occurrence of cervical cancer. Therefore, the main treatment options for early-stage cervical cancer patients in current clinical practice include hysterectomy, lymph node dissection, radiotherapy, and chemotherapy. For advanced patients, comprehensive treatment with radiotherapy as the main treatment is supplemented by chemotherapy drugs such as cisplatin and etoposide.

[0006] Recent studies have shown that changes in the tumor microenvironment are also a key factor affecting tumor development. Endogenous microorganisms, as a "double-edged sword", are truly involved in the process of tumor occurrence and development. Microbial communities secrete various metabolites such as lactic acid, short-chain fatty acids (butyric acid), secondary bile acids, and even bacterial toxin proteins during their growth, which can change the tumor microenvironment and have multiple effects on tumor cells, such as promoting cancer cell proliferation and metastasis, inhibiting host immune response, and causing tumor cell immune escape. Traditional views believe that the cervix is a sterile environment, but in recent years, researchers have confirmed through high-throughput sequencing technology that there is a unique microbial community in cervical cancer tissue, including bacteria, fungi, and viruses. It has been reported that more than 16% of cancers worldwide (lung cancer, cervical cancer, breast cancer, and pancreatic cancer, etc.) are related to microorganisms in their development or metastasis. Researchers can identify a variety of microorganisms in different types of tumors, and the composition of these microorganisms is also affected by various factors, resulting in significant differences between individuals such as gender, age, physical condition, daily habits (smoking and drinking, etc.). With the development of microbiome and metagenomics, researchers can culture and study endogenous bacteria or fungi in tumor tissue in vitro based on multi-omics, making it possible to explore new targets for cancer treatment and tumor treatment intervention strategies.

[0007] Existing research also shows that by modifying viral vectors, bacterial delivery systems, or natural strains to activate immune responses or direct killing mechanisms, most studies show that microorganisms are used as carriers to inhibit tumor cell activity, and related microbial therapies have been studied in animal and clinical models, also showing significant inhibition of tumor growth. This indicates that microbial intervention (such as antibiotic regulation or probiotic combination therapy) may become an important strategy and intervention means for exploring new therapies for tumors.

[0008] Liu et al. published a study in Cancer Cell that lung cancer tissue-derived Aspergillus sydowiiAspergillus sydowii MDSC infiltration can be induced and host immune response can be suppressed to promote cancer development. Cai et al. published a research in Cell, which revealed that intratumoral microbiota can change the cancer cell cytoskeleton to enhance its tolerance to mechanical shear force in blood, further promoting the metastasis of cancer cells to the lung. Lauren et al. also reported in Cancer Cell that L-lactate-producing Lactobacillus in uterine cancer patient tissues Lactobacillus iners significantly associated with reduced patient survival, the bacteria can induce metabolic rearrangement of uterine cancer cells to enhance their resistance to radiotherapy and chemotherapy. Escherichia coli carrying PKS genes (encoding DNA damage toxins) E. coli After adhering to host cells, the secreted toxin protein causes microsatellite instability and chromosomal damage of host immune cells to drive the development of colorectal cancer. Klebsiella pneumoniae Klebsiella pneumoniae TLR4 can be activated by expressing penicillin-binding protein PBP1B, promoting the proliferation of hepatocellular carcinoma cells and activating pro-cancer signaling pathways, ultimately causing the progression of hepatocellular carcinoma. Barath et al. found that Fusobacterium nucleatum Fusobacterium nucleatum can adhere to pancreatic epithelial cells and stimulate tumor and normal cells to secrete granulocyte-macrophage colony-stimulating factor or chemokines to increase the proliferation and invasion ability of cancer cells, further promoting the development of pancreatic cancer. It can be seen that the human microbiota is of great significance to tumor occurrence and cancer treatment, although the intestinal microbiota has always been the main focus of research in these fields, the understanding of the existence and pathological correlation of intratumoral microbiota has begun to emerge, and it has revealed the important mechanisms of intestinal flora or endogenous microorganisms in promoting cancer development or metastasis and whether the origin of intratumoral microbiota is related to intestinal flora has become an important scientific problem to be solved in cancer treatment. SUMMARY

[0009] The purpose of the present application is to screen a potential functional strain that can be used as a drug or nanobody delivery system, anti-tumor activity, for the treatment of cervical cancer, liver cancer or ovarian cancer. The second purpose of the present application is to provide the application of the above functional strain.

[0010] In order to solve the above problems, the present application provides a tumor inhibitory Escherichia coli 18, the strain number of the Escherichia coli 18 is HZ_Ec_18, and the classification name is Escherichia coli HZ_Ec_18, which has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC No: M20251870.

[0011] The present application also provides the use of the tumor inhibitory Escherichia coli 18 for preparing a drug with at least one of the following effects (1)-(3): (1) a drug for preventing and / or treating a tumor; (2) a drug having anticancer cell activity; (3) a drug having anti-pan-cancer cell activity.

[0012] Specifically, the tumor includes cervical cancer, liver cancer, or ovarian cancer.

[0013] Specifically, the cervical cancer includes at least one of cervical adenocarcinoma, cervical squamous carcinoma, and cervical adenosquamous carcinoma.

[0014] Specifically, the cancer cell includes cervical cancer cell HeLa, cervical cancer cell SiHa, human liver cancer cell HepG2, or human ovarian cancer cell A 2780.

[0015] Specifically, the tumor-inhibiting E. coli 18, i.e., E. coli HZ_Ec_18, exists in the form of a living cell.

[0016] Specifically, the number of living bacteria of the tumor-inhibiting E. coli 18, i.e., E. coli HZ_Ec_18, is MOI = 10-60, and the number of living bacteria varies according to different tumor cells, for example, MOI = 10, 20, 40, or 60 can be adjusted.

[0017] Specifically, the tumor-inhibiting E. coli 18, i.e., E. coli HZ_Ec_18, is used as the only active ingredient.

[0018] The application also provides a pharmaceutical preparation comprising a therapeutically effective amount of the tumor-inhibiting E. coli 18, i.e., E. coli HZ_Ec_18.

[0019] Specifically, the pharmaceutical preparation further comprises other pharmaceutically acceptable carriers and / or adjuvants compatible with the E. coli HZ_Ec_18.

[0020] Specifically, the pharmaceutical preparation is in a pharmaceutically acceptable parenteral administration dosage form and / or a gastrointestinal administration dosage form; wherein, the parenteral administration dosage form includes at least one of an injection administration dosage form, a cavity administration dosage form, a mucosa administration dosage form, and a skin administration dosage form; the gastrointestinal administration dosage form includes at least one of a tablet, a granule, a capsule, a solution, a powder, a sustained-release preparation, an emulsion, a suspension, a syrup, and a drop.

[0021] In an embodiment of the application, the pharmaceutical preparation can further comprise at least one of a chemotherapeutic drug, an immune checkpoint inhibitor, an immune cell therapy drug, an iron death inducer, and a KRAS inhibitor.

[0022] In the detailed description of the present application, the chemotherapeutic drug includes at least one of cisplatin, etoposide, paclitaxel, camptothecin, 5-fluorouracil, doxorubicin, mitomycin, epirubicin.

[0023] In the detailed description of the present application, the immune checkpoint inhibitor includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, KLRA.

[0024] In the detailed description of the present application, the immune cell therapy drug includes at least one of T cell therapy drug, tumor infiltrating lymphocyte therapy drug, NK cell therapy drug.

[0025] The present application obtains multiple strains with potential influence on tumor microenvironment by in vitro culture and purification of bacteria in the cervical tissue and vaginal swab of cervical cancer patients. Among them, Escherichia coli HZ_Ec_18 has stronger tumor cell inhibition activity and interaction with host cells compared with other Escherichia coli. It is speculated that its genes may have large-scale recombination or mutation at key gene sites, resulting in obvious difference in activity from other strains. The present application further observes the morphological changes of Escherichia coli HZ_Ec_18 after co-incubation with cervical cancer tumor cells by transmission electron microscopy, showing mitochondrial swelling, lysosome destruction, intracellular material degradation, and cell membrane destruction.

[0026] The present application provides a new perspective of "from microorganism to host" for tumor treatment through the interaction of intratumoral flora and tumor. The complex interaction network of flora and tumor promotes the development of personalized treatment and combination therapy, and reveals that flora plays a key role in tumor occurrence, metastasis and treatment. Therefore, the Escherichia coli HZ_Ec_18 with anti-tumor activity isolated from tumor clinical samples in the present application provides important theoretical and material support for subsequent bacterial-based intervention therapy.

[0027] The present application directly isolates and cultures strains in the tissue of cervical cancer patients, which can be used as a potential functional strain of drug or nanobody delivery system and anti-tumor activity.

[0028] Biological preservation instructions Escherichia coli HZ_Ec_18, the preservation date is August 21, 2025, the preservation number is CCTCC NO: M20251870, and the classification and naming is Escherichia coli HZ_Ec_18, Escherichia coliHZ_Ec_18, the deposit unit name is: China Center for Type Culture Collection, the deposit center address is: Wuhan University, China Center for Type Culture Collection, Wuhan, Wuchang District, Bayi Road, Wuhan, Hubei Province. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed to be used in the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present disclosure, and other drawings can also be obtained by those skilled in the art without creative labor.

[0030] Figure 1 For the composition analysis results of cervical cancer and healthy women's vaginal flora; among them, A is the LefSeq analysis and the genus level intratumor flora composition analysis results (HC: healthy women cohort, HCRD: cervical cancer patient cohort); B is the KEGG database flora function annotation result; Figure 2 For the species annotation results in the vagina of cervical cancer patients and healthy women; Figure 3 For the correlation analysis results of cervical cancer patient vaginal flora and CRP; among them, A is the comparative analysis result of CRP concentration in the serum of healthy women and cervical cancer patients (Wilcoxon rank sum test, ***P=0.0003); B-E are respectively the correlation analysis (Pearson correlation analysis) results of CRP with Enterobacteriales (B), Enterobacteriaceae (C), Escherichia-Shigella (D) and Staphylococcus (E); Figure 4 For the isolation and identification results of Escherichia coli; among them, A is the SEM observation result of Escherichia coli morphology, the length of the bacillus is about 2 µm, and the bacterial body grows flagella (the scale is 2 µm); B is the phylogenetic tree constructed based on 16S rRNA gene sequence, different strains can be clustered into different gene clusters (neighbor-joining method, bootstrap value=2000); Figure 5 For the influence results of different Escherichia coli strains on cervical cancer cell growth; Figure 6 For the influence of different Escherichia coli on tumor cell activity; among them, A is the influence of No. 18 Escherichia coli on HeLa cells; B is the influence of No. 8 Escherichia coli on SiHa cells; Figure 7 For the influence results of inactivated Escherichia coli and bacterial culture supernatant on cervical cancer cell growth; among them, A-B are the influence of inactivated strains on cervical cancer cell activity; C-D are the influence of bacterial culture supernatant on cervical cancer cell activity; Figure 8The results of the influence of E. coli on different tumor cells; wherein, A-B are the influence of No. 18 E. coli on liver cancer cells (HepG2) and ovarian cancer cells (A2780), respectively; Figure 9 The results of lactic acid concentration detection after E. coli and tumor cells were co-incubated; wherein, A-B are the results of L-lactic acid (A) and D-lactic acid (B) concentration detection in the co-culture system of E. coli and HeLa and SiHa, respectively. DETAILED DESCRIPTION

[0031] The above only describes the embodiments of the present disclosure, and does not limit the present disclosure. Any modification, equivalent replacement, improvement, etc. made within the principles of the present disclosure shall be included in the protection scope of the present disclosure.

[0032] In order to make the purpose, technical solutions and advantages of the present application clearer, the following will further describe the embodiments of the present application in a specific manner with reference to the accompanying drawings.

[0033] In the following examples of the present application, unless otherwise defined, all technical terms used have the same meaning as generally understood by those skilled in the art.

[0034] The present application discloses a strain of E. coli and a pharmaceutical composition and application. Those skilled in the art can refer to the content herein and appropriately improve the process parameters to achieve it. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0035] In the description of the present application, the list of items connected by the term "at least one of" or other similar terms can mean any combination of the listed items. For example, if items A, B are listed, the phrase "at least one of A, B" means only A; only B; or A and B. In another example, if items A, B, C are listed, the phrase "at least one of A, B, C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A can include a single element or multiple elements. Item B can include a single element or multiple elements. Item C can include a single element or multiple elements.

[0036] "comprising" and "including" as used herein are open-ended and also include the other components, but also only the components listed in the specific case. For example, "comprising" and "including" can mean that other components can also be included or can only include the components listed.

[0037] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the properties. The endpoints of the ranges and any numerical values are approximations only and are used to illustrate exemplary ranges. Any numerical value, however, can include smaller or larger values, even if not expressly listed. The endpoints of the ranges and any numerical values are approximations only and are used to illustrate exemplary ranges. The endpoints of the ranges and any numerical values are approximations only and are used to illustrate exemplary ranges.

[0038] The term "strain" refers to a member of a bacterial species that has genetic characteristics that distinguish it from closely related members of the same bacterial species. The genetic characteristics can be the absence of all or part of at least one gene, the absence of all or part of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. The genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In the case where a strain gains or loses antibiotic resistance or gains or loses biosynthetic capability (e.g., an auxotrophic strain) compared to another strain of the same species, the strains or nutrients / metabolites can be distinguished by selection or counter-selection using the antibiotic. It is known in the art that bacterial species can be classified and identified by traditional classification methods and molecular biology methods. Traditional classification methods include, for example, cell morphology observation, Gram staining, flagella staining, various metabolic experiments, etc. Molecular biology methods include ribosomal RNA sequence determination methods, whole genome sequencing-based determination methods, etc.

[0039] The term "immune checkpoint inhibitor" is an antagonist that targets an immune checkpoint protein that enhances a protein that stimulates an immune response or blocks a protein that inhibits an immune response, thereby exhibiting an anticancer effect through an immune response. In some embodiments, the immune checkpoint inhibitor can be a protein or peptide, such as a soluble fusion protein; can be an antibody or antigen-binding fragment thereof that binds to the immune checkpoint protein to be inhibited; can be an inhibitory nucleic acid (e.g., an siRNA molecule, an shRNA molecule, an antisense RNA) that specifically binds to an mRNA encoding the immune checkpoint protein.

[0040] In the following specific examples, the operations involved are carried out under conventional conditions or the conditions recommended by the manufacturer, unless otherwise specified. The raw materials used are conventional products available on the market, unless otherwise specified.

[0041] In the following examples of the present application, 30 healthy women and 63 cervical cancer patients' vaginal secretions were subjected to 16S rRNA sequencing to identify high-abundance strains in cervical cancer. Fresh cervical tissues and vaginal secretions from 32 cervical cancer patients were collected and subjected to in vitro bacterial isolation and culture under anaerobic and aerobic conditions, respectively. By analyzing the biochemical indicators of healthy and patient women, based on the significant increase of C-reactive protein (CRP, an immune-related indicator) in tumor patients, combined with 16S rRNA sequencing data, the relative abundance of strains was analyzed for correlation with CRP (Pearson analysis), and functional strains with potential regulation of host immune signaling pathways in cervical cancer patients were screened.

[0042] In the following examples of the present application, after sample collection, all sample processing, bacterial culture and purification were completed under sterile conditions in the laboratory. Strain identification was completed using 16S rRNA sequencing and evolutionary analysis and observing strain morphology under a scanning electron microscope. The effect of high-abundance cultivable strains on tumor cell activity was detected by CCK-8 kit method. Finally, the concentration of D- / L-type lactic acid in the supernatant after co-culture of strains and tumor cells was quantified by colorimetric method, i.e. the effect of strains on tumor microenvironment.

[0043] After screening, the following examples of the present application obtained an Escherichia coli strain E. coli 18, with strain number HZ_Ec_18, classified as HZ_Ec_18, which has been preserved in the China Center for Type Culture Collection, located at Wuhan University, Biaoyi Road, Wuchang District, Wuhan City, Hubei Province, China, on August 21, 2025, with preservation number CCTCC No: M20251870. Escherichia coli In an embodiment of the present application, the Escherichia coli strain can also be a variant of the Escherichia coli strain with preservation number CCTCC No: M20251870. Any variant, mutant, recombinant or derivative strain obtained from HZ_Ec_18 as starting material by natural mutation, mutagenesis, screening, genetic engineering, gene editing, recombination technology or other conventional molecular biology methods in the art, as long as it substantially retains or can achieve the same or similar tumor inhibitory effect as HZ_Ec_18 and its available uses, is considered to fall within the scope of the present application. For the sake of clarity, the protection of the present application can be determined based on structural characteristics or functional (i.e. maintaining tumor inhibitory activity) identity.

[0044]

[0045] ​It has been verified that the E. coli HZ_Ec_18 has the effect of anti-cancer cell activity, for example, against cervical cancer cells HeLa, cervical cancer cells SiHa, human liver cancer cells HepG2 or human ovarian cancer cells A 2780, all of which show a certain inhibitory effect, that is, anti-tumor activity.

[0046] Similarly, the following examples of the present application further verify that the E. coli HZ_Ec_18 has the effect of anti-cancer cell activity.

[0047] On this basis, the E. coli HZ_Ec_18 has the effect of inhibiting tumors and can be used to prepare drugs for preventing and / or treating tumors, for example, drugs for preventing or treating cervical cancer, liver cancer or ovarian cancer.

[0048] Further, when the E. coli HZ_Ec_18 is used to prepare drugs for preventing and / or treating tumors, the E. coli HZ_Ec_18 exists in the form of living cells. It can be understood that the E. coli HZ_Ec_18 as an active ingredient of an anti-tumor drug can exert a better therapeutic effect when it exists in the form of living cells.

[0049] In some specific application modes, when the E. coli HZ_Ec_18 is used to prepare drugs for preventing and / or treating tumors, the number of viable bacteria of the E. coli HZ_Ec_18 is MOI=10-60, and different MOI values are selected according to the difference of cancer cells, for example, MOI=10, 20, 40 or 60, or a range between any of the above values.

[0050] In some specific application modes, the E. coli HZ_Ec_18 can be used as the only active ingredient of an anti-tumor drug; or other components with anti-tumor activity known in the art can be further compounded. For example, drugs known in the art for preventing or treating cervical cancer, liver cancer or ovarian cancer can be compounded.

[0051] The present application also provides a pharmaceutical preparation comprising a therapeutically effective amount of the E. coli HZ_Ec_18. It should be noted that the term "therapeutically effective amount" refers to the amount of drug required to produce an effective effect, which can be adjusted and changed according to the actual situation and ultimately determined by medical personnel, considering factors including the route of administration and the nature of the preparation, the weight, age and other general conditions of the recipient, and the nature and severity of the disease being treated.

[0052] In some specific application modes, the pharmaceutical preparation provided by the present application further comprises other pharmaceutically acceptable carriers and / or adjuvants compatible with the E. coli HZ_Ec_18. It should be noted that "other pharmaceutically" here refers to drugs compatible with the E. coli HZ_Ec_18, and do not cause inactivation, activity reduction, hydrolysis and other physicochemical reactions of the E. coli HZ_Ec_18, and after synergistic action with the E. coli HZ_Ec_18, can improve the therapeutic effect of the E. coli HZ_Ec_18. The above-mentioned carriers can be conventional drug carriers in the art, such as chitosan, liposomes, alginic acid, agar, fibrin, collagen and synthetic high molecular weight polymer carriers, etc. The above-mentioned adjuvants are physiologically inactive, do not affect the therapeutic effect, content determination and stability of the active pharmaceutical ingredient of the pharmaceutical preparation, and the main purpose is to facilitate the preparation of the preparation and the clinical application of the material. For example, it can be conventional starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, calcium carbonate, dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrant, cross-linked polyvinylpyrrolidone, etc.

[0053] In some specific application modes, the pharmaceutical preparation provided by the present application is a pharmaceutically acceptable oral dosage form. For example, powder, granules, pills, capsules, tablets, ointments, liquid preparations, gels, etc. Accordingly, according to the actual pharmaceutical dosage form, the administration mode of the drug can be selected from inhalation, blowing into the mouth, nasal administration, oral administration, parenteral administration, etc.

[0054] Example 1 This example is based on 30 healthy women and 63 cervical cancer patients to analyze the bacterial 16S rRNA sequencing and species composition diversity in vaginal secretions.

[0055] Bacterial genomic DNA was extracted from samples using a kit-based method, and purity and concentration were detected using Nanodrop 2000. DNA fragmentation was performed using Covaris M 220, and fragments of approximately 400 bp were screened and enriched. Subsequently, PE libraries were constructed using a kit-based method, followed by PCR amplification and 16S rRNA sequencing. The raw sequencing data were quality controlled using Fastp and BWA software and compared with human DNA sequences to remove highly similar contaminating reads. MEGAHIT was used to assemble and splice the filtered sequences, screening contigs longer than 300 bp for ORF prediction. The predicted gene sequences were then clustered using CD-HIT software. Finally, SOA Paligner software was used to enrich and align high-quality reads and non-redundant genes for each sample, and the abundance of each gene in the corresponding samples was calculated. The DIAMOND online analysis tool was used to compare non-redundant gene sequences with the NR database to obtain annotation information for species at different taxonomic levels.

[0056] This embodiment is based on the completed 16S rRNA sequencing and diversity analysis of bacteria in vaginal secretions from 30 healthy women and 63 cervical cancer patients. The results are attached. Figures 1-2 As shown in the figure, the results indicate that the vaginal flora composition is disordered in patients with cervical cancer.

[0057] Figure 1 The Latin translations are shown in the table below.

[0058]

[0059] Figure 2 The Chinese Latin or English definitions are shown in the table below.

[0060]

[0061] As attached Figure 1 The LefSeq analysis results shown in Figure A indicate that Firmicutes, Clostridium, and Bacteroidetes are highly abundant flora in the vagina of cancer patients. At the class level, Staphylococcus, anaerobic cocci, and Corynebacterium are significantly enriched. There are significant differences in bacterial composition between the two groups. The relative abundance of beneficial bacteria such as Lactobacillus and Bifidobacterium is reduced in the tumor group, while the relative abundance of some opportunistic pathogens such as Proteus, Staphylococcus, and Streptococcus is increased. This suggests that tumor lesions cause changes in the vaginal environment, which also indicates that the disordered composition of the intratumoral flora in patients leads to changes in dissolved oxygen and pH in the tumor microenvironment.

[0062] As attached Figure 1KEGG annotation results shown in Table A indicate that the function of intratumoral bacteria is mainly enriched in the biodegradation and metabolism of exogenous substances such as amino acids, carbohydrates and energy metabolism, and membrane transport functions, etc., which is speculated to be related to the supply of energy for tumor cell over-proliferation, invasion or migration.

[0063] As shown in Table A, the KEGG annotation results of cervical cancer and healthy female vaginal species annotation results, 16S rRNA sequencing results can detect different abundance of strains such as Escherichia-Shigella, fusobacterium and corynebacterium, etc. Figure 2

[0064] Example 2 In this example, the correlation analysis of intratumoral bacterial flora and C-reactive protein in cervical cancer patients was carried out. By analyzing the changes of biochemical factors in patients, the Pearson analysis method was used to analyze the correlation between CRP and the relative abundance of each bacterial flora.

[0065] In this example, the biochemical factors in the serum of 63 cervical cancer patients and 30 healthy women were analyzed, as shown in Table A. Figure 3 As shown in Table A, the C-reactive protein of cervical cancer patients was significantly increased.

[0066] In this example, the Pearson analysis method was used to analyze the correlation between the relative abundance of the bacterial flora with higher relative abundance in the two cohorts and the CRP concentration. As shown in Table B, C, D and E, high concentration of CRP is positively correlated with the relative abundance of Enterobacteriaceae bacteria in the vagina of cervical cancer patients (R=0.2, P<0.1) and negatively correlated with Staphylococcus (R=-0.099). Due to the small sample size, the sample size will be increased in the later stage for correlation analysis, and it is speculated that Enterobacteriaceae (Escherichia-Shigella) may activate the host immune response or regulate the tumor microenvironment to affect cell activity. Figure 3

[0067] Example 3 In this example, Escherichia coli was isolated and identified based on the intratumoral bacterial flora of cervical cancer.

[0068] In this example, cervical tissue and vaginal secretions of cervical cancer patients were collected and pretreated under sterile conditions in the laboratory. The tissue was washed with sterile normal saline for 5 times, then homogenized and plated on brain heart infusion (BHI) agar medium. The vaginal swab was directly plated on the above-mentioned medium and incubated at 37°C for 24 hours under anaerobic conditions. The sample was treated in the same way. The strain was purified by continuous culture for 3-4 times, and the bacteria were monoclone to BHI liquid medium for culture to logarithmic phase for preservation and storage at-80°C. After the recovered bacteria were cultured, the genomic DNA was extracted, the 16S rRNA gene was amplified using 27F and 1492R primers and sequenced, and the phylogenetic analysis was completed using NCBI tools and MEGA software to preliminarily identify the strain.

[0069] ​​This embodiment is directed to the collected 32 cases of cervical cancer patients with vaginal discharge and cervical tissue bacterial in vitro culture, a total of 18 strains of E. coli were isolated and cultured.

[0070] The No. 8 E. coli of the 18 strains of E. coli was selected and cultured in BHI liquid medium for 3-4 hours (37°C, 180 rpm) to the logarithmic growth phase (OD600 value of 0.5), and then the bacterial bodies were recovered (5000g, 3min), and then the supernatant medium was discarded and the sample was washed three times with phosphate buffer (PBS), and then fixed with 2.5% glutaraldehyde solution at 4°C overnight, and then dehydrated by alcohol gradient, critical point drying, gold spraying and other treatments, and then observed under a scanning electron microscope. The morphology of bacteria, all samples were imaged and observed using a field emission scanning electron microscope (JOEL).

[0071] This embodiment uses SEM to observe the morphology of No. 8 E. coli, and the bacteria have flagella, and the length of the E. coli is 1-2 µm (such as Figure 4 The 16S rRNA sequencing and evolutionary analysis were used to identify E. coli, and the results showed that there were multiple copies of 16S rRNA genes in the E. coli genome (such as Figure 4 The 18 strains of E. coli screened in this embodiment mainly clustered into 4 clusters, and had high consistency with the genes of the E. coli reference strains in the NCBI database. Based on the above method, the isolation, purification and identification of E. coli were completed.

[0072] Example 4 This embodiment further detects the in vitro anti-tumor activity of the 18 strains of E. coli screened (for 2 kinds of cervical cancer tumor cells).

[0073] The E. coli (18 strains of E. coli and DH5α control strain) frozen at -80°C were streaked onto BHI agar medium and placed in a 37°C constant temperature incubator for 16-18h for standby, and single colonies were inoculated into BHI broth medium and cultured to the logarithmic growth phase (37°C, 180 rpm). Then the bacterial bodies were recovered by centrifugation (7000g, 5min), washed with sterile PBS solution for 3 times, and then resuspended in sterile PBS solution for subsequent co-culture with cells.

[0074] On the day before the experiment, 1×10 4HeLa and SiHa cells were seeded into 96-well plates at a cell / mL ratio, and the cell supernatant was discarded. 100 μL of DMEM medium was added to each well. The effect of the bacterial strain on tumor cell viability was initially assessed at MOI=50. The effect of bacterial infection dose on tumor cell viability (dose-dependent) was verified at MOIs of 10, 20, 40, and 60, with a mock control, PBS group, and bacterial control group (DH5α Escherichia coli) established. After 1 h in a cell culture incubator, the cells were washed three times with PBS buffer, and 100 μL of DMEM medium containing 2% FBS and 2% PS was added to each well. The cells were incubated for 24 h, then the medium was discarded, and 100 μL of fresh cell culture medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 1-2 h, and the absorbance at 450 nm was measured to detect the effect of the bacteria on tumor cell viability. Cell viability was calculated and analyzed (CCK-8 method).

[0075] like Figure 5 The results show the effects of co-culturing 18 viable *E. coli* strains selected in this invention with SiHa. Specifically, the 18 viable *E. coli* strains were co-cultured with SiHa (MOI=50), and the cell viability after treatment was detected using the CCK-8 assay. The results indicate that *E. coli* strains 8, 11, 15, and 18 can inhibit the activity of SiHa. Strain 18 showed the strongest inhibition of tumor cell activity, with an inhibition rate of 90%-100%. Strains 11 and 15 inhibited SiHa survival by approximately 50%.

[0076] Further analysis of strain 18 was conducted to determine its antitumor activity and its effects on HeLa and SiHa cells. Different bacterial infection doses of 10 CFU, 20 CFU, 40 CFU, and 60 CFU were used. (See attached image) Figure 6 The results shown in Figures A and B indicate that the antitumor activity of Escherichia coli No. 18 was dose-dependent on the bacterial infection dose.

[0077] This embodiment further tested the effect of inactivated Escherichia coli and bacterial culture supernatant on the activity of cervical cancer tumor cells. The CCK8 assay was used to determine the activity of inactivated Escherichia coli (… E. coli _1、 E. coli _5、 E. coli _8、 E. coli _11、 E. coli The effects of _18) and the supernatant after bacterial culture on the growth of cervical cancer cells (HeLa and SiHa) were as follows: Figure 7 As shown. It can be seen that using inactivated strains (such as...) Figure 7 (A and B) and bacterial culture supernatant (e.g.) Figure 7 C and D) do not affect tumor cell activity.

[0078] Example 5 This example further tests the influence of the screened E. coli E. coli colony to BHI agar medium and placed in a 37°C constant temperature incubator for 16-18h for standby, and single colonies were inoculated in BHI broth culture medium to the logarithmic growth phase (37°C, 180rpm), and then the bacterial bodies were recovered by centrifugation (7000g, centrifugation for 5min), washed 3 times with sterile PBS solution, and resuspended in sterile PBS solution for subsequent co-culture with cells.

[0079] On the day before the experiment, human hepatoma HepG2 cells and human ovarian cancer A2780 cells were inoculated at a ratio of 1x10 4 cells / mL into a 96-well plate, the cell supernatant was discarded, and 100μL of DMEM medium was added to each well. The preliminary detection of the influence of the strain on the activity of tumor cells was performed according to MOI=50; the verification of the influence of the infection dose of the strain on the activity of tumor cells (dose-dependent) was performed according to MOI=10, 20, 40, 60, and a blank group (mock), a PBS group, and a strain control group (DH5α E. coli) were set up, and placed in a cell incubator for 1h, washed 3 times with PBS buffer, and then 100μL of DMEM medium containing 2% FBS and 2% PS was added to each well, and placed in a cell incubator for 24h, and then the culture medium was aspirated and 100μL of fresh CCK8-containing cell culture medium was added, incubated at 37°C for 1-2h, and then the absorbance at 450nm was detected to detect the influence of the bacteria on the activity of tumor cells, and the cell viability was calculated and analyzed (CCK-8 method).

[0080] The CCK8 method was used to determine the influence of E. coli E. coli 18 on the growth of human hepatoma cells (HepG2) and human ovarian cancer cells (A2780), and the results are shown in Figure 8 .

[0081] It can be seen that the E. coli E. coli 18 has an inhibitory effect on the activity of HepG2 cells, and the inhibition rate reaches a maximum of 35% (P=0.0001) when the MOI is 60; E. coli 18 can also significantly inhibit the activity of A 2780 cells in a dose-dependent manner, and the inhibition rate reaches a maximum of 45% (P<0.0001) when the MOI is 60.

[0082] The above results show that the E. coli E. coli_18 has potential anti-cancer cell activity and has a wider application scenario.

[0083] The foregoing screening of the E. coli in this embodiment E. coli _18, which has a strain number of HZ_Ec_18 and a classification name of Escherichia coli HZ_Ec_18, has been preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Baoyi Road, Wuchang District, Wuhan City, Hubei Province, the preservation date is August 21, 2025, and the preservation number is CCTCC No: M20251870.

[0084] Example 6 During the co-culture of E. coli and tumor cells, the supernatant of the cell culture medium turned dark yellow, which was speculated to be the change of the metabolic process of bacteria or cells during the co-culture process and the secretion of acidic substances. It was found in the literature that D-lactic acid and L-lactic acid played an important role in the development of tumors. In this embodiment, the lactic acid content was detected based on the co-culture system of the screened E. coli and tumor cells.

[0085] Eighteen strains of E. coli selected and DH5α E. coli were selected to infect HeLa and SiHa cells at MOI = 50, and blank (mock) and PBS groups were set. After 5 hpe, the supernatant was collected, and the lactic acid content in the supernatant was detected using L-lactic acid assay kit (AAT Bioquest) and D-lactic acid assay kit (AAT Bioquest), according to the instructions of the reagent manufacturer to complete the standard curve preparation, and the samples were treated, and Varioskan LUX enzyme label instrument (Thermo) was used for detection.

[0086] The lactic acid content in the supernatant of the tumor cells co-cultured with the 18 strains of E. coli and DH5α was detected using the kit, and the results are shown in Figure 9 A and B in the accompanying drawings.

[0087] It can be seen that the D- and L-lactic acid concentrations in the HeLa and SiHa systems co-cultured with different E. coli isolates have obvious differences compared with the control group, and the change trend of acidic substances in the two systems is consistent, among which, E. coli _8, HZ_Ec_18 (P<0.0001), E. coli _11 and E. coli _15 (P<0.01) have significantly higher L-lactic acid content in the supernatant co-cultured with HeLa cells than other samples; E. coli _11 and E. coli _15 (P<0.0001) have significantly higher D-lactic acid content in the supernatant co-cultured with SiHa cells than other samples; E. coli _8, E. coli _11,E. coli The D-lactic acid content in the supernatant after co-incubation with HeLa cells of HZ_Ec_15 and HZ_Ec_18 (P<0.0001) is significantly higher than that of other samples; E. coli _11、 E. coli The D-lactic acid content in the supernatant after co-incubation with SiHa cells of HZ_Ec_15 and HZ_Ec_18 (P<0.0001) is significantly higher than that of other samples, and the interaction effect of HeLa and Escherichia coli is the most significant, and the change of lactic acid concentration is the most obvious, indicating that after the interaction of intratumoral flora and tumor cells, both bacteria and tumor cells can affect the tumor microenvironment through metabolic reprogramming, ultimately affecting tumor development.

[0088] In summary, through 16S rRNA sequencing diversity analysis of vaginal flora of 30 healthy women and 63 cervical cancer patients, it is found that the composition of bacterial community in the vagina of patients is disordered, the relative abundance of beneficial bacteria is reduced, and the relative abundance of conditional pathogenic bacteria such as Staphylococcus, Clostridium and Enterobacteriaceae is increased. Further statistical analysis of biochemical indicators of healthy women and patients and Pearson correlation analysis of the relative abundance of vaginal flora show that the relative abundance of Enterobacteriaceae in the vagina of patients is positively correlated with CRP (P<0.1). The results of in vitro anti-tumor cell activity of bacterial strains show that the selected Escherichia coli can produce different degrees of inhibitory activity on tumor cells after co-culturing with cervical cancer tumor cells for 1 hour, while inactivated bacteria or supernatant after bacterial culture do not have inhibitory activity on tumor cells. Subsequent lactic acid detection and RT-qPCR experiments show that during the interaction of Escherichia coli and tumor cells, the strain can secrete or promote tumor cells to secrete lactic acid, and can also promote the significant increase of gene expression of tumor cells related to proliferation or migration, indicating that the Escherichia coli isolated in the application can not only directly inhibit tumor cell activity, but also can change tumor cell metabolism by secreting metabolites or changing tumor cell metabolism, change tumor microenvironment through autocrine or paracrine system, and affect further development. The application further observes the morphological changes of Escherichia coli and cervical cancer tumor cells after co-incubation by transmission electron microscopy, showing that the mitochondria are swollen, the lysosomes are destroyed, the intracellular substances are degraded, and the cell membrane is damaged.

[0089] The above has carried out the detailed introduction to the embodiment of the application, the principle and implementation mode of the application have been described by applying specific examples in this paper, and the above embodiment description is only used to help understand the method and core idea of the application; at the same time, for those skilled in the art, according to the idea of the application, the specific implementation mode and application range will be changed, and the above description should not be understood as the limitation of the application.

Claims

1. A tumor-inhibiting E. coli 18, with strain number HZ_Ec_18, classified as Escherichia coli HZ_Ec_18, which has been deposited with the China Center for Type Culture Collection, with the accession number CCTCC No: M20251870.

2. Use of the tumor-inhibiting Escherichia coli 18 according to claim 1 for the preparation of a medicament having at least one of the following effects (1) - (3): (1) a medicament for the prevention and / or treatment of a tumor; (2) a medicament having anticancer cell activity; (3) a medicament having anti-pan-cancer cell activity.

3. Use according to claim 2, characterized in that, The tumor includes cervical cancer, liver cancer, or ovarian cancer.

4. Use according to claim 2, characterized in that, The cancer cell includes cervical cancer cell HeLa, cervical cancer cell SiHa, human liver cancer cell HepG2, or human ovarian cancer cell A 2780.

5. Use according to any one of claims 2 to 4, characterized in that, The tumor-inhibiting Escherichia coli 18 exists in the form of a living cell.

6. Use according to claim 5, characterized in that, The number of living cells of the tumor-inhibiting Escherichia coli 18 is MOI = 10 - 60.

7. Use according to any one of claims 2 to 4, characterized in that, The tumor-inhibiting Escherichia coli 18 is used as the only active ingredient.

8. A pharmaceutical preparation, characterized by, The pharmaceutical preparation includes a therapeutically effective amount of the tumor-inhibiting Escherichia coli 18 according to claim 1.

9. The pharmaceutical preparation according to claim 8, characterized in that, The pharmaceutical preparation further includes other pharmaceutically acceptable carriers and / or adjuvants compatible with the tumor-inhibiting Escherichia coli 18.

10. The pharmaceutical preparation according to claim 8 or 9, characterized in that, The pharmaceutical preparation includes a parenteral administration dosage form and / or a gastrointestinal administration dosage form. The parenteral administration dosage form includes at least one of an injection administration dosage form, a cavity administration dosage form, a mucosa administration dosage form, and a skin administration dosage form. The gastrointestinal administration dosage form includes at least one of a tablet, a granule, a capsule, a solution, a powder, a sustained-release preparation, an emulsion, a suspension, a syrup, and a drop.

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