A tumor-suppressive Escherichia coli O8 and its uses

Abstract

This application relates to the field of biomedical technology, specifically to Escherichia coli strains and pharmaceutical compositions and their applications, and more specifically to a tumor-suppressive Escherichia coli O8 and its uses. The preservation number of this Escherichia coli strain is CCTCC NO: M20251869. The Escherichia coli strain provided by this invention, after infecting tumor cells, can alter the tumor microenvironment, leading to damage to tumor cells, thereby effectively inhibiting the activity of tumor cells.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to Escherichia coli strains, pharmaceutical compositions, and their applications. Background Technology

[0002] Cervical cancer is a malignant tumor that occurs in the cervix and is the fourth most common malignant tumor among women worldwide. Cervical cancer is mainly divided into squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma. Squamous cell carcinoma is a cancer caused by the malignant transformation of squamous epithelial cells; adenocarcinoma develops from subcolumnar reserve cells of the endometrium that differentiate into both glandular and squamous cells. Adenocarcinoma ranks second in incidence among cervical cancers globally and is increasing year by year, with a trend towards affecting younger populations. Compared to squamous cell carcinoma, adenocarcinoma has multiple pathological types, different cell morphologies, lower sensitivity to radiotherapy and chemotherapy, a higher recurrence rate, a greater tendency to metastasize to distant sites, and significant adverse prognoses. Pathological classification alone cannot adequately guide the treatment of adenocarcinoma patients; currently, treatment plans for cervical adenocarcinoma still refer to those for squamous cell carcinoma, leading to overtreatment of some adenocarcinoma patients and causing various negative effects, complications, and functional impairments. The main clinical manifestations of cervical cancer patients are heavy or abnormal vaginal bleeding, especially after sexual intercourse. Some patients may experience watery, mucous, or foul-smelling vaginal discharge. Advanced patients may experience lower limb edema, flank pain, and pain in the pelvis or lower back.

[0003] Cervical cancer is closely related to persistent infection with high-risk human papillomavirus (HPV), especially HPV types 16 and 18. HPV oncogenic proteins E6 and E7 target p53 and Rb proteins, leading to uncontrolled cell proliferation and genomic instability, and inducing abnormal host DNA methylation or histone modifications. Squamous cell carcinoma is generally considered highly associated with HPV 16, while adenocarcinoma is more closely related to HPV 18. However, studies have shown that HPV infection alone is insufficient to cause cancer; other factors such as immunosuppression (e.g., HIV infection), long-term use of oral contraceptives, multiple pregnancies, and early sexual activity can also promote cervical cancer development.

[0004] The main treatment options for early-stage cervical cancer include hysterectomy, lymph node dissection, radiotherapy, and chemotherapy. Advanced-stage patients receive comprehensive treatment primarily based on radiotherapy, supplemented by chemotherapy drugs such as cisplatin and etoposide. However, these treatment options still face many challenges.

[0005] Recent research indicates that alterations to the tumor microenvironment are also a key factor influencing tumor development. Endogenous microbes, acting as a double-edged sword, truly participate in the tumorigenesis and progression. During their growth, the microbial community secretes various metabolites such as lactic acid, short-chain fatty acids (butyric acid), secondary bile acids, and even bacterial toxins, all of which can alter the tumor microenvironment and exert diverse effects on tumor cells. These effects include promoting cancer cell proliferation and metastasis, suppressing the host immune response, and causing tumor cell immune escape. While the cervix was traditionally considered a sterile environment, recent research using high-throughput sequencing technology has confirmed the existence of a unique microbial community within cervical cancer tissue, including bacteria, fungi, and viruses. It has been reported that the development or metastasis of more than 16% of cancers worldwide (such as lung cancer, cervical cancer, breast cancer, and pancreatic cancer) is related to microorganisms. Researchers can identify a variety of microorganisms in different types of tumors, and the composition of these microorganisms is also affected by a variety of factors, resulting in significant differences between individuals, such as gender, age, physical condition, and daily habits (smoking and drinking). With the development of microbiome and culture omics, researchers can conduct in vitro culture and research on endogenous bacteria or fungi in tumor tissues based on multi-omics, making it possible to explore new targets for cancer treatment and tumor treatment intervention strategies. Summary of the Invention

[0006] In view of this, the present invention provides an Escherichia coli strain, a pharmaceutical composition, and its application. After infecting tumor cells, this Escherichia coli strain can alter the tumor microenvironment, leading to damage to tumor cells, thereby effectively inhibiting tumor cell activity.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides an Escherichia coli strain with the preservation number CCTCCNO: M20251869.

[0009] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned Escherichia coli strain.

[0010] In embodiments of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0011] In embodiments of the present invention, the pharmaceutical composition may further include at least one of a chemotherapy drug, an immune checkpoint inhibitor, an immune cell therapy drug, a ferroptosis inducer, and a KRAS inhibitor.

[0012] In a specific embodiment of the present invention, the chemotherapy drugs include at least one of cisplatin, etoposide, paclitaxel, camptothecin, 5-fluorouracil, doxorubicin, mitomycin, and epirubicin.

[0013] In a specific embodiment of the present invention, immune checkpoint inhibitors include inhibitors 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, and KLRA.

[0014] In a specific embodiment of the present invention, the immune cell therapy drug includes at least one of T cell therapy drugs, tumor-infiltrating lymphocyte therapy drugs, and NK cell therapy drugs.

[0015] In embodiments of the present invention, the dosage forms of the pharmaceutical composition include non-gastrointestinal dosage forms and / or gastrointestinal dosage forms.

[0016] In specific embodiments of the present invention, non-gastrointestinal drug delivery dosage forms include at least one of injection dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0017] In specific embodiments of the present invention, the gastrointestinal dosage form includes at least one of tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups, and drops.

[0018] Thirdly, the present invention provides the use of the above-mentioned Escherichia coli strains and / or pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of cancer.

[0019] In embodiments of the present invention, cancer includes at least one of cervical cancer, liver cancer, or ovarian cancer.

[0020] In a specific embodiment of the present invention, cervical cancer includes at least one of cervical adenocarcinoma, cervical squamous cell carcinoma, and cervical adenosquamous carcinoma.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The *E. coli* strain obtained by screening in this invention has the preservation number CCTCC NO: M20251869. Escherichia coli HZ_Ec_8, which will also be referred to as "HZ_Ec_8" in this article. E. coli 8) exhibits a significant inhibitory effect on tumor cell activity. Simultaneously, during interaction with tumor cells, the strain can either secrete its own lactic acid or promote the secretion of lactic acid by tumor cells. It can also significantly increase the gene expression of factors related to tumor cell proliferation and migration, thereby altering the tumor microenvironment through autocrine or paracrine systems by secreting metabolites or modifying tumor cell metabolic processes, thus influencing its further development. Compared to 17 other Escherichia coli strains, E. coli 8 exhibits stronger tumor cell inhibitory activity and interaction with host cells, suggesting... E. coli The gene in strain 8 may undergo large-scale recombination or mutation at key gene sites, resulting in significant differences in activity compared to other strains.

[0023] The interaction between intratumoral microbiota and tumor provides a novel perspective on tumor treatment "from microbes to the host." The complex interaction network between microbiota and tumor is driving the development of personalized and combination therapies, revealing the key role of microbiota in tumor occurrence, metastasis, and treatment. Therefore, the antitumor active bacteria isolated from clinical tumor samples in this invention provide important theoretical and material support for subsequent bacterial intervention therapies.

[0024] Biological Preservation Instructions

[0025] Escherichia coli HZ_Ec_8, Escherichia coli HZ_Ec_8, deposited on August 21, 2025, with accession number CCTCC NO: M20251869, classified and named as: Escherichia coli HZ_Ec_8. Escherichia coli HZ_Ec_8, the name of the depository is: China Center for Type Culture Collection, the address of the depository is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0026] Figure 1 Analysis of vaginal flora composition and functional annotation in cervical cancer patients and healthy women; where A is LefSeq analysis and genus-level intratumoral flora composition analysis; B is KEGG database flora functional annotation.

[0027] Figure 1 The Latin translation is as follows:

[0028] Figure 2 The results of species annotation in the vagina of cervical cancer patients and healthy women are shown.

[0029] Figure 2 The Chinese Latin or English translations are as follows:

[0030]

[0031] Figure 3This study presents the correlation analysis results between vaginal flora and C-reactive protein (CRP) in cervical cancer patients. Specifically, A represents a comparison of serum CRP concentrations between healthy women and cervical cancer patients (Wilcoxon rank-sum test, ***P=0.0003); B represents the correlation analysis between CRP and Enterobacteriaceae; C represents the correlation analysis between CRP and Enterobacteriaceae; D represents the correlation analysis between CRP and Escherichia coli-Shigella spp.; and E represents the correlation analysis between CRP and Staphylococcus spp.

[0032] Figure 4 The results of Escherichia coli isolation and identification are shown; where A is the bacterial morphology observed under SEM (scale bar is 2 µm); B is the phylogenetic tree constructed based on the 16S rRNA gene sequence.

[0033] Figure 5 This study demonstrates the effects of different strains of Escherichia coli on the growth of cervical cancer cells.

[0034] Figure 6 This diagram illustrates the effect of *E. coli* on tumor cell activity; where A represents *E. coli*. E. coli 8. Effects on HeLa cells, B represents Escherichia coli E. coli 8. Effects on SiHa cells.

[0035] Figure 7 The results show the lactate concentration detection results after co-incubation of Escherichia coli and tumor cells; where A is the L-lactic acid concentration detection result in the co-culture system of Escherichia coli with HeLa and SiHa, and B is the D-lactic acid concentration detection result in the co-culture system of Escherichia coli with HeLa and SiHa.

[0036] Figure 8 Transmission electron microscopy observation of cell damage (scale bar 2 µm); where A is... E. coli 5 and E. coli 8. HeLa cell damage after infection, B is E. coli 5 and E. coli 8. Damage to SiHa cells after infection.

[0037] Figure 9 This study shows the relative gene expression in cervical cancer cells after infection with Escherichia coli.

[0038] Figure 10 The study investigated the effects of inactivated Escherichia coli and the supernatant from bacterial culture on the growth of cervical cancer cells. Specifically, A represents the effect of the inactivated bacterial culture on the HeLa activity of cervical cancer cells, B represents the effect of the inactivated bacterial culture on the SiHa activity of cervical cancer cells, C represents the effect of the supernatant from bacterial culture on the HeLa activity of cervical cancer cells, and D represents the effect of the supernatant from bacterial culture on the SiHa activity of cervical cancer cells.

[0039] Figure 11This study demonstrates the effects of *Escherichia coli* on the activity of different tumor cells; where A represents... E. coli Effects of 8 on human liver cancer cells (HepG2), B being... E. coli Effects of 8 on human ovarian cancer cells (A2780). Detailed Implementation

[0040] This invention discloses an Escherichia coli strain, a pharmaceutical composition, and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0041] In the description of this invention, the list of items connected by the term "at least one of" or other similar terms may mean any combination of the listed items. For example, if items A and B are listed, then the phrase "at least one of A and B" means only A; only B; or A and B. In another example, if items A, B, and C are listed, then the phrase "at least one of A, B, and 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 may contain a single element or multiple elements. Item B may contain a single element or multiple elements. Item C may contain a single element or multiple elements.

[0042] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0043] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges or individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] The term "strain" refers to a member of a bacterial species that possesses genetic characteristics that distinguish it from closely related members of the same bacterial species. These genetic characteristics may include the complete or partial absence of at least one gene, the complete or partial absence 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 mutant gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one non-natural plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. 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 cases where a strain (compared to another strain of the same species) acquires or loses antibiotic resistance or acquires or loses biosynthetic capacity (e.g., auxotrophic strains), the strain or nutrient / metabolite can be distinguished by selection or anti-selection using antibiotics. It is known in the art that bacterial species can be classified and identified using conventional taxonomic methods and molecular biological methods. Conventional taxonomic methods include, for example, cell morphology observation, Gram staining, flagellar staining, various metabolic experiments, etc. Molecular biology methods include ribosomal RNA sequencing and whole-genome sequencing-based methods.

[0045] The term "immune checkpoint inhibitor" is an antagonist that targets an immune checkpoint protein. This immune checkpoint inhibitor enhances proteins that stimulate the immune response or blocks proteins that inhibit the immune response, thereby exhibiting an anti-cancer effect through the immune response. In some embodiments, the immune checkpoint inhibitor can be a protein or peptide, such as a soluble fusion protein; it can be an antibody or its antigen-binding fragment that binds to the immune checkpoint protein to be inhibited; or it can be an inhibitory nucleic acid (e.g., siRNA molecules, shRNA molecules, antisense RNA) that specifically binds to mRNA encoding the immune checkpoint protein.

[0046] Specifically, the present invention adopts the following technical solution:

[0047] In a first aspect, the present invention provides an Escherichia coli strain with the preservation number CCTCCNO: M20251869.

[0048] In embodiments of the present invention, the *E. coli* strain can also be a variant of the *E. coli* strain with accession number CCTCC NO: M20251869. All of the above are... Escherichia coli Any variant, mutant, recombinant, or derived strain obtained from HZ_Ec_8 as the starting material through natural mutation, mutagenesis, screening, genetic engineering, gene editing, recombination, or other conventional molecular biology methods in the field, as long as it substantially retains or can achieve the same characteristics as HZ_Ec_8. Escherichia coliTumor-suppressive effects and applicable uses of HZ_Ec_8 that are identical or similar to those of HZ_Ec_8 are considered to fall within the scope of protection claimed in this invention. To eliminate any ambiguity, protection of this invention can be determined based on structural features or functional equivalence (i.e., maintaining tumor-suppressive activity), and includes, but is not limited to, the specific alternative embodiments shown in this specification and claims. Any manufacture, use, sale, or offer for sale of the strains or uses falling within the scope of protection without the written permission of the inventor may constitute an infringement of the rights of this invention. The inventor will uphold its legal rights and pursue corresponding legal liabilities in accordance with the law.

[0049] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned Escherichia coli strain.

[0050] In embodiments of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0051] In specific embodiments of the present invention, pharmaceutically acceptable excipients include at least one of excipients, diluents, carriers, lubricants, wetting agents, binders, emulsifiers, suspension stabilizers, solubilizers, preservatives, sweeteners, and flavorings.

[0052] In embodiments of the present invention, the pharmaceutical composition may further include at least one of a chemotherapy drug, an immune checkpoint inhibitor, an immune cell therapy drug, a ferroptosis inducer, and a KRAS inhibitor.

[0053] In a specific embodiment of the present invention, the chemotherapy drugs include at least one of cisplatin, etoposide, paclitaxel, camptothecin, 5-fluorouracil, doxorubicin, mitomycin, and epirubicin.

[0054] In a specific embodiment of the present invention, immune checkpoint inhibitors include inhibitors 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, and KLRA.

[0055] In a specific embodiment of the present invention, the immune cell therapy drug includes at least one of T cell therapy drugs (such as CAR-T, TCR-T), tumor-infiltrating lymphocyte therapy drugs, and NK cell therapy drugs.

[0056] In a specific embodiment of the present invention, the ferroptosis inducer includes RSL3 and / or Errastin.

[0057] In a specific embodiment of the present invention, the KRAS inhibitor includes at least one of D3S-001, AMG-510, and MRTX849.

[0058] In embodiments of the present invention, the dosage forms of the pharmaceutical composition include non-gastrointestinal dosage forms and / or gastrointestinal dosage forms.

[0059] In specific embodiments of the present invention, non-gastrointestinal drug delivery dosage forms include at least one of injection dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0060] In specific embodiments of the present invention, the gastrointestinal dosage form includes at least one of tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups, and drops.

[0061] In a specific embodiment of the present invention, the content of Escherichia coli strain in the pharmaceutical composition is 1×10⁻⁶. 2 CFU / g ~ 1×10 12 CFU / g. For example, the content of *E. coli* strain in the pharmaceutical composition is 1 × 10⁻⁶. 2 CFU / g, 11×10 3 CFU / g, 1×10 4 CFU / g, 1×10 5 CFU / g, 1×10 6 CFU / g, 1×10 7 CFU / g, 1×10 8 CFU / g, 1×10 9 CFU / g, ×10 10 CFU / g, 1×10 11 CFU / g, 1×10 12 Any value in CFU / g or any value within the range formed by any two of the above values.

[0062] Thirdly, the present invention provides the use of the above-mentioned Escherichia coli strains and / or pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of cancer.

[0063] In one embodiment of the present invention, the cancer includes at least one of cervical cancer, liver cancer, or ovarian cancer.

[0064] In a specific embodiment of the present invention, cervical cancer includes at least one of cervical adenocarcinoma, cervical squamous cell carcinoma, and cervical adenosquamous carcinoma.

[0065] In another embodiment of the present invention, cancer may also include oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cavity cancer, sinus cancer, laryngeal cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, small intestinal cancer, appendix cancer, colon cancer, rectal cancer, anal canal cancer, intrahepatic bile duct cancer, gallbladder cancer, hilar bile duct cancer, distal bile duct cancer, hepatopancreatic ampulla cancer, pancreatic cancer, gastric neuroendocrine tumor, duodenal and ampulla neuroendocrine tumor, jejunal-ileal neuroendocrine tumor, appendiceal neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, angiosarcoma, desmoidoma, Ewing sarcoma, fibrosarcoma, gastrointestinal stromal tumor, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, and malignant peripheral nerve sheath tumor. Rhabdomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, cutaneous fibrosarcoma protuberans, Merkel cell carcinoma, cutaneous malignant melanoma, vulvar cancer, vaginal cancer, endometrial cancer, uterine carcinosarcoma, endometrial sarcoma, fallopian tube cancer, primary peritoneal cancer, gestational trophoblastoma, penile cancer, prostate cancer, testicular cancer, renal cancer, renal pelvis cancer, ureteral cancer, bladder cancer, urethral cancer, eyelid cancer, conjunctival cancer, conjunctival melanoma, uveal melanoma, retinoblastoma, lacrimal gland cancer, orbital sarcoma, lymphoma of the ocular adnexa, brain cancer, spinal cord tumor, differentiated thyroid carcinoma, undifferentiated thyroid carcinoma, medullary thyroid carcinoma, parathyroid carcinoma, adrenocortical carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous lymphoma, plasma cell myeloma, and leukemia.

[0066] The reagents and biological materials used in this invention are all commercially available.

[0067] The present invention will be further illustrated below with reference to the embodiments:

[0068] Example 1: Isolation, identification, and in vitro antitumor activity detection of Escherichia coli

[0069] 1. Identification, composition, and functional analysis of vaginal flora in cervical cancer patients and healthy women.

[0070] 16S rRNA sequencing and diversity analysis were performed on bacteria from vaginal secretions of 30 healthy women and 63 patients with cervical cancer. The specific methods were as follows: bacterial genomic DNA was extracted from the samples using a kit method, and purity and concentration were detected using Nanodrop 2000. The DNA was fragmented using Covaris M 220, and DNA fragments of approximately 400 bp were screened and enriched. Subsequently, a PE library was constructed using a kit 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. The filtered sequences were spliced ​​and assembled using MEGA HIT, and contigs longer than 300 bp were selected 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 sample was calculated. The non-redundant gene sequences were compared with the NR database using the DIAMOND online analysis tool to obtain annotation information of species at different taxonomic levels.

[0071] LefSeq analysis showed that Firmicutes, Clostridium, and Bacteroidetes were highly abundant flora in the vagina of cancer patients. At the class level, Staphylococcus, anaerobic cocci, and Corynebacterium were significantly enriched, and the bacterial composition differed significantly between the two cohorts. The tumor group showed a decreased relative abundance of beneficial bacteria, such as Lactobacillus and Bifidobacterium; while the relative abundance of some opportunistic pathogens, such as Proteus, Staphylococcus, and Streptococcus, increased, suggesting that tumor lesions caused changes in the vaginal environment. Figure 1 (A in the text). This also indicates that the dysbiosis of the tumor microbiota caused changes in dissolved oxygen and pH in the tumor microenvironment. KEGG annotation results show that the functions of the tumor microbiota are mainly enriched in the biodegradation and metabolism of exogenous substances such as amino acids, carbohydrates, energy metabolism, and membrane transport functions, which is speculated to be related to the energy supply from excessive proliferation, invasion, or migration of tumor cells. Figure 1 (B in the original text). 16S rRNA sequencing results can detect strains of varying abundance, such as Escherichia coli-Shigella, Fusobacterium, and Corynebacterium, etc. Figure 2 ).

[0072] 2. Correlation analysis between intratumoral flora and C-reactive protein (CRP) in cervical cancer

[0073] Analysis of serum biochemical factors in 63 cervical cancer patients and 30 healthy women showed that C-reactive protein was significantly elevated in cervical cancer patients. Figure 3 (A in the middle).

[0074] Pearson analysis was used to analyze the correlation between the relatively abundant bacterial communities and CRP concentration in the two cohorts. The results showed that high CRP concentration was positively correlated with the relative abundance of Enterobacteriaceae in the vagina of cervical cancer patients (R=0.2, P<0.1), and negatively correlated with Staphylococcus spp. (R=-0.099, P<0.1). Figure 3 (Figures B-E) suggest that Enterobacteriaceae (Escherichia coli-Shigella) may activate the host immune response or regulate the tumor microenvironment, affecting cell activity.

[0075] 3. Isolation and Identification of Escherichia coli

[0076] (1) Isolation of Escherichia coli

[0077] Vaginal secretions and cervical tissues collected from 32 cervical cancer patients were used for in vitro bacterial culture to isolate bacterial strains. The specific method for bacterial isolation and culture was as follows: Cervical tissues and vaginal secretions from cervical cancer patients were collected and pretreated under sterile laboratory conditions. The tissues were washed five times with sterile physiological saline, homogenized, and spread onto Brain Heart Extract (BHI) agar. Vaginal swabs were directly spread onto the same medium and incubated at 37°C for 24 hours. Samples under anaerobic conditions were treated in the same manner. The strains were purified by 3-4 consecutive cultures. Single bacterial clones were picked and cultured on BHI liquid medium until the logarithmic growth phase for preservation, and then frozen at -80°C. A total of 18 strains of *Escherichia coli* were isolated and cultured, and designated as follows: E. coli 1 to E. coli 18.

[0078] (2) Observation of bacterial morphology using scanning electron microscopy (SEM)

[0079] Will E. coli 8. Incubate in BHI liquid medium for 3-4 hours (37℃, 180 rpm) until the logarithmic growth phase (OD). 600 The bacterial cells were recovered (5000 g, 3 min) with a concentration of 0.5. The supernatant culture medium was then discarded, and the samples were washed three times with phosphate-buffered saline (PBS). The samples were then fixed overnight at 4°C with 2.5% glutaraldehyde solution. Following alcohol gradient dehydration, critical point drying, and gold sputtering, bacterial morphology was observed under SEM. All samples were imaged using a field emission scanning electron microscope (JOEL). The results showed... E. coli 8 are Enterobacteriaceae 1-2 µm in length, with flagella present in the bacterial cells ( Figure 4 (A in the middle).

[0080] (3) 16S rRNA sequencing and evolutionary analysis

[0081] After reviving and culturing the bacteria, their genomic DNA was extracted, and their 16S rRNA gene was amplified using 27F and 1492R primers and sequenced. Phylogenetic analysis (nearest neighbor method, bootstrap value = 2000) was performed using NCBI tools and MEGA software, and the strain was preliminarily identified.

[0082] The results showed that multiple copies of the gene encoding 16S rRNA were present in the E. coli genome. Figure 4 The 18 Escherichia coli strains (B) were mainly clustered into 4 groups, and their genes showed a high degree of consistency with the Escherichia coli reference strains in the NCBI database. Based on the above methods, the isolation, purification and identification of Escherichia coli were completed.

[0083] 4. Detection of in vitro antitumor activity of Escherichia coli

[0084] Escherichia coli (18 strains and DH5α control strain) frozen at -80 ℃ were streaked onto BHI agar medium and incubated at 37 ℃ for 16-18 h. Single colonies were picked and inoculated into BHI broth medium and cultured to the logarithmic growth phase (37 ℃, 180 rpm). The bacterial cells were then recovered by centrifugation (7000 g, 5 min), washed three times with sterile PBS solution, and resuspended in sterile PBS solution for subsequent co-culture with cells.

[0085] One day before the experiment, administer 1 × 10 4 HeLa (cervical adenocarcinoma cell line) and SiHa (cervical squamous cell carcinoma cell line) were seeded into 96-well plates at cell / mL ratios, respectively. The cell supernatant was discarded, and 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 PBS control group (Ctrol). The cells were incubated for 1 h, then the bacterial supernatant was discarded, and the cells were washed three times with PBS buffer. 100 μL of DMEM medium containing 2% FBS and 2% PS was added to each well, and the cells were incubated for 24 h. The medium was then aspirated, 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 assess the effect of the bacteria on tumor cell viability. Cell viability was calculated and analyzed using the CCK-8 method.

[0086] The results show that, E. coli 8. E. coli 11. E. coli 15 and E. coli 18 Escherichia coli can inhibit the activity of SiHa. E. coli 8 and E. coli 18 showed the strongest inhibitory effect on tumor cell activity, with an inhibition rate of 90%-100%. E. coli 11 and E. coli 15 Escherichia coli can inhibit the survival rate of SiHa by approximately 50% ( Figure 5 Then select E. coli_ 8 and E. coli Eighteen bacterial strains were further compared for antitumor activity, with different bacterial infection doses of 10 CFU, 20 CFU, 40 CFU, and 60 CFU. The results showed that *Escherichia coli*... E. coli 8. Antitumor activity and bacterial infection are dose-dependent. Figure 6 E. coli E. coli 8. Biological preservation was carried out, with accession number CCTCC NO: M20251869, and named... Escherichia coli HZ_Ec_8.

[0087] Example 2: Detection of lactic acid content in a co-culture system of Escherichia coli and tumor cells

[0088] Eighteen strains of *Escherichia coli* and DH5α *Escherichia coli* were selected and infected with HeLa and SiHa cells at an MOI of 50, respectively. A mock control group and a PBS control group were also included. After 5 h, the supernatant was collected, and the lactate content in the supernatant was detected using an L-lactate assay kit (AATBioquest) and a D-lactate assay kit (AAT Bioquest). Standard curves were prepared according to the reagent manufacturers' instructions, and the samples were processed and analyzed using a Varioskan LUX microplate reader (Thermo).

[0089] The results showed that after co-culturing with different E. coli isolates, the concentrations of D-lactic acid and L-lactic acid in the HeLa and SiHa systems were significantly different from those in the control group, and the trends of acidic substances in both systems were consistent. E. coli 8. E. coli_ 18 ( P <0.0001), E. coli 11 and E. coli 15 ( P <0.01), the L-lactic acid content in the supernatant after co-incubation with HeLa cells was significantly higher than that in other samples; E. coli 11 and E. coli 15 ( P The D-lactic acid content in the supernatant after co-incubation with SiHa cells (<0.0001) was significantly higher than that in other samples; E. coli 8. E. coli 11. E. coli 15 and E. coli_ 18 ( P The D-lactic acid content in the supernatant after co-incubation with HeLa cells (<0.0001) was significantly higher than that in other samples; E. coli_ 11. E. coli 15 and E. coli 18 ( P The D-lactic acid content in the supernatant after co-incubation with SiHa cells (<0.0001) was significantly higher than that in other samples. The interaction between HeLa and E. coli was the most significant, resulting in the most pronounced change in lactic acid concentration. Figure 7 This indicates that after the interaction between the intratumoral microbiota and tumor cells, both bacteria and tumor cells can influence the tumor microenvironment through metabolic reprogramming. D-lactic acid and L-lactic acid play an important role in tumor development and ultimately affect tumor progression.

[0090] Example 3: Observation of bacterial-induced tumor cell damage using transmission electron microscopy (TEM)

[0091] Escherichia coli ( E. coli 5 and E. coli 8) HeLa and SiHa cells were infected with MOI = 50 for 1 h, respectively. The supernatant was discarded, and the cells were washed three times with PBS, then added to DMEM medium containing 2% FBS and 2% PS and cultured in a cell culture incubator. Cell samples were collected after 3 h. Cell samples were washed twice with PBS and fixed with 2.5% glutaraldehyde solution. The fixed samples were washed three times with PBS, fixed with 1% osmotic acid for 2 h, washed three times with PBS, and dehydrated sequentially in gradient concentrations (50%, 70%, 90%) of ethanol for 5 min each time. Then, they were washed twice with 100% ethanol for 7 min each time, followed by dehydration. The cells were then embedded in resin at 25℃ for 4 h, polymerized at 65℃, and sectioned after 48 h. The sections were stained with uranyl acetate for 20 min and with basic lead citrate for 10 min, and bacterial morphology was observed under a transmission electron microscope. All samples were imaged using a biological transmission electron microscope (JOEL).

[0092] Observation using transmission electron microscopy E. coli 8. Damage to HeLa and SiHa cells after infection: massive necrosis of tumor cells, mitochondrial vacuolation and disappearance of cristae structure, eventually swelling and necrosis, with a large number of autolysosomes and lipofuscin accumulation in the cells. E. coli 5. The infected tumor cells were intact, with slight swelling of the mitochondria. Figure 8 ).

[0093] Example 4: Detection of immune-related gene expression in tumor cells by real-time quantitative PCR

[0094] Escherichia coli ( E. coli5 and E. coli 8) Five hours after infecting HeLa and SiHa cells, the relative expression levels of tumor cells and genes related to immunity or invasion (CCL1, GM-CSF, IL-8, ICAM-1) were detected by RT-qPCR. The specific steps were as follows: Escherichia coli ( E. coli 5. E. coli 8) HeLa and SiHa cells were infected with MOI = 50, respectively, and a PBS control group (Ctrol) was set up. Cell samples were collected after 5 h. Total RNA was extracted from the cells using the Novizan Rapid RNA Extraction Kit (RC112-01), and then the reaction was performed according to the instructions using the Novizan HiScript II One Step RT-qPCR SYBR Green Kit (Q221) (primers are shown in Table 1). The QuantStudio real-time fluorescence quantitative PCR instrument was used to detect the data.

[0095] Table 1 Primers used for RT-qPCR detection

[0096]

[0097] The results are as follows Figure 9 As shown, via E. coli 5 and E. coli Following infection with IL-8, the relative expression levels of four genes—CCL1, GM-CSF, IL-8, and ICAM-1—were significantly increased in both HeLa and SiHa cells. P <0.001), and after E. coli 8. Tumor cells after treatment were compared with those after... E. coli The tumor cells in treatment group 5 showed higher relative expression levels of the four genes, suggesting that co-culturing tumor cells with bacteria can secrete factors that promote tumor proliferation or migration through autocrine or paracrine systems, thereby altering the tumor microenvironment and influencing tumor development.

[0098] Example 5: Effects of inactivated Escherichia coli and bacterial culture supernatant on the activity of cervical cancer tumor cells

[0099] Inactivated Escherichia coli was determined using the CCK8 assay. E. coli 1. E. coli 5. E. coli 8. E. coli 11. E. coli 18. The effects of DH-5α and the supernatant after bacterial culture on the growth of cervical cancer cells (HeLa and SiHa) were investigated, and a PBS control group (Ctrol) was established. Results are as follows: Figure 10 As shown, this indicates that the inactivated strain ( Figure 10 (A and B) and bacterial culture supernatant ( Figure 10 C and D) do not affect tumor cell activity.

[0100] Example 6: Effects of Escherichia coli on the activity of different tumor cells

[0101] Determination of Escherichia coli using the CCK8 assay E. coli The effects of 8 on the growth of human hepatocellular carcinoma cells (HepG2) and human ovarian cancer cells (A2780) were investigated. The specific experimental procedure was as follows: 1 × 10⁸ mg / L was administered the day before the experiment. 4 HepG2 (human hepatocellular carcinoma cell line) and A2780 (human ovarian carcinoma cell line) were seeded into 96-well plates at cell / mL ratios, respectively. The cell supernatant was discarded, and 100 μL of DMEM medium was added to each well. To verify the effect of bacterial infection dose on tumor cell viability (dose-dependent), cells were cultured at MOIs of 10, 20, 40, and 60, with a PBS control group (Ctrol). The cells were incubated for 1 h, then the bacterial supernatant was discarded, and the cells were washed three times with PBS buffer. 100 μL of DMEM medium containing 2% FBS and 2% PS was added to each well, and the cells were incubated for 24 h. The medium was then aspirated, and 100 µL of fresh cell culture medium containing 10% CCK-8 was added. After incubation at 37°C for 1–2 h, the absorbance at 450 nm was measured to detect the effect of bacteria on tumor cell viability. Cell viability was calculated and analyzed using the CCK-8 assay.

[0102] The results are as follows Figure 11 As shown, E. coli 8 significantly inhibited the activity of HepG2 cells, with the inhibition rate reaching a maximum of 35% at an MOI of 60. P <0.0001); E. coli 8 also significantly inhibited the activity of A2780 cells in a dose-dependent manner, with the inhibition rate reaching its maximum of 80% at an MOI of 60. P <0.0001), the above results indicate that the isolated and purified Escherichia coli has potential anti-pan-cancer activity and has a wider range of applications.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An Escherichia coli strain (a) characterized in that, Escherichia coli The preservation number of the Escherichia coli strain is CCTCC NO: M20251869. ​ 2. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the Escherichia coli strain of claim 1, wherein the strain is a live bacterium.

3. The pharmaceutical composition of claim 2, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

4. The pharmaceutical composition according to claim 2 or 3, characterized in that, The pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immune checkpoint inhibitor, an immune cell therapy drug, an iron death inducer, and a KRAS inhibitor.

5. The pharmaceutical composition of claim 4, wherein, The chemotherapeutic drug comprises at least one of cisplatin, etoposide, paclitaxel, camptothecin, 5-fluorouracil, doxorubicin, mitomycin, and epirubicin. The immune checkpoint inhibitor comprises 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, and KLRA. The immune cell therapy drug comprises at least one of a T cell therapy drug, a tumor infiltrating lymphocyte therapy drug, and an NK cell therapy drug.

6. The pharmaceutical composition of claim 2, wherein, The dosage form of the pharmaceutical composition comprises a parenteral administration dosage form and / or a gastrointestinal administration dosage form.

7. The pharmaceutical composition of claim 6, wherein, The parenteral administration dosage form comprises 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 comprises at least one of a tablet, a granule, a capsule, a solution, a powder, an emulsion, and a suspension.

8. The pharmaceutical composition of claim 6, wherein, The gastrointestinal administration dosage form comprises a sustained release preparation.

9. Use of the Escherichia coli strain of claim 1 and / or the pharmaceutical composition of any one of claims 2-8 in the preparation of a medicament for preventing and / or treating cancer, wherein the strain is a live bacterium, and the cancer comprises at least one of cervical cancer, liver cancer, and ovarian cancer.

10. Use according to claim 9, characterized in that, The cervical cancer comprises at least one of cervical adenocarcinoma, cervical squamous carcinoma, and cervical adenosquamous carcinoma.

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