Combined bacterial agent for diagnosis and treatment of colorectal cancer and application thereof

Abstract

The present application relates to the field of microorganisms and biotechnology, and discloses a combined bacterial agent for diagnosis and treatment of colorectal cancer and application thereof. The combined bacterial agent contains separately preserved engineered probiotics and genetically engineered bacteria; the engineered probiotics contain probiotics and a recombinant expression system introduced into the probiotics; the recombinant expression system contains a coding gene of Dickkopf 3 protein; the amino acid sequence of the Dickkopf 3 protein is shown as SEQ ID NO. 1; the probiotics are Escherichia coli Nissle 1917 and / or attenuated Salmonella typhimurium HCS1; the genetically engineered bacteria can co-express speFL protein and luminescent protein; the luminescent protein includes Renilla luciferase and / or superfolder green fluorescent protein. The combined bacterial agent can be used to prepare a drug for early diagnosis and treatment of colorectal cancer.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and biotechnology, specifically to a combined bacterial agent for the diagnosis and treatment of colorectal cancer and its application. Background Technology

[0002] Malignant tumors impact the health and quality of life of many people worldwide. Colorectal cancer (CRC), associated with genetic and environmental risk factors, is one of the most common life-threatening cancers. The 5-year survival rate for advanced-stage CRC is only 20%. This indicates that improving early diagnosis rates is crucial to reducing CRC mortality. Currently, mainstream screening methods for early CRC diagnosis include colonoscopy, fecal occult blood testing, and multi-target fecal DNA testing. However, these methods are invasive, costly, require rigorous bowel preparation, and necessitate specialized medical resources, making them unsuitable for universal screening or lacking sufficient sensitivity. Therefore, there is an urgent need to develop cost-effective, non-invasive alternatives for early CRC diagnosis.

[0003] Currently, treatment options for colorectal cancer mainly include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. While these treatments have made some progress, they still face challenges such as limited efficacy, significant side effects, and drug resistance. In the development of colorectal cancer, dysregulation of the classic Wnt / β-catenin signaling pathway has been well-established as a fundamental driver of colon tumorigenesis, making this pathway one of the most promising molecular targets for colorectal cancer treatment. Current research focuses on developing inhibitors that target and block the binding of ligands to receptors in this pathway or promote β-catenin degradation. However, the narrow therapeutic window and the risk of damaging normal intestinal stem cells significantly limit the effective therapeutic dose of these drugs, hindering their clinical development and resulting in the lack of approved drugs for marketing. Therefore, the key to achieving safe and effective treatment lies in how to specifically deliver inhibitors to tumor tissue while avoiding damage to normal tissues (especially the intestines). Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies, such as high cost, invasiveness, poor accessibility and insufficient sensitivity in the early diagnosis of colorectal cancer, as well as the drug toxicity that occurs in the treatment of colorectal cancer.

[0005] To achieve the above objectives, a first aspect of the present invention provides a combined bacterial agent for the diagnosis and treatment of colorectal cancer, the combined bacterial agent containing separately preserved engineered probiotics and genetically engineered bacteria; the engineered probiotics comprise probiotics and a recombinant expression system introduced into the probiotics; the recombinant expression system comprises a gene encoding the Dickkopf 3 protein; the amino acid sequence of the Dickkopf 3 protein is shown in SEQ ID NO.1; the probiotics are Escherichia coli Nissle1917 and / or attenuated Salmonella Typhimurium HCS1; The genetically engineered bacteria can co-express speFL protein and luminescent protein; the luminescent protein includes Renaissance luciferase and / or superfolded green fluorescent protein. In the combined bacterial agent, the genetically engineered bacteria are used for in vitro culture and for oral administration and / or intravenous injection.

[0006] A second aspect of the present invention provides the use of the combined bacterial agent described in the first aspect in the preparation of a medicament for the early diagnosis and treatment of colorectal cancer.

[0007] Compared with the prior art, the present invention has at least the following advantages through the above technical solution: (1) This invention designs and constructs a genetically engineered bacterium that can respond to the concentration of ornithine metabolite in the feces of individuals with early colorectal cancer and express luminescent protein, providing a new idea for early cancer screening and a non-invasive, convenient and economical detection strategy.

[0008] (2) The present invention uses the method of detecting bioluminescent signals to reduce the interference of other contents in feces on the detection signal and improve the detection sensitivity.

[0009] (3) This invention utilizes the characteristics of bacteria to target and colonize the tumor microenvironment to design and construct engineered probiotics that can target intestinal tumors and continuously express the secretion of the antagonistic protein DKK3 (also known as Dickkopf 3 protein) that inhibits the Wnt / β-catenin signaling pathway in situ. The therapeutic protein is delivered to the tumor tissue, realizing the local precision treatment of intestinal tumors and developing a highly efficient and low-toxicity treatment strategy (the strategy of combining therapeutic protein with intestinal probiotics), which shows great potential in the prevention and treatment of CRC.

[0010] (4) The engineered probiotics provided by the present invention use Escherichia coli Nissle 1917 and / or attenuated Salmonella typhimurium HCS1 as the chassis bacteria. Escherichia coli Nissle 1917 is an intestinal probiotic and HCS1 is attenuated Salmonella typhimurium, thereby making the engineered bacteria of the present invention safer.

[0011] (5) The engineered probiotics provided by the present invention can be used as oral probiotics and / or intravenous injection drugs, which are suitable for multiple administration modes and are beneficial to clinical translation. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the recombinant plasmid constructed in this invention; Figure 2 It is a genetically engineered bacterium E. coli Figure 1. Results of ornithine-induced expression of superfolded green fluorescent protein sfGFP under different culture conditions (Nissle 1917-pJ23119-speF-sfGFP); Figure 3 It is a genetically engineered bacterium E. coli Figure showing the results of Nissle 1917-pJ23119-speF-sfGFP inducing the expression of superfolded green fluorescent protein sfGFP under different concentrations of ornithine; Figure 4 It is a genetically engineered bacterium E. coli Figure 1. Results of Nissle 1917-pJ23119-speF-Rluc8 inducing expression of Renina luciferase Rluc8 under different concentrations of ornithine; Figure 5 It is a genetically engineered bacterium E. coli Nissle 1917-pJ23119-speF-Rluc8 and healthy mice and Apc Min / + Figure showing the results of inducing expression of Renaissance luciferase Rluc8 after co-incubation with fresh mouse feces; Figure 6 It is a genetically engineered bacterium E. coli Nissle 1917-pJ23119-speF-Rluc8 and healthy mice under different storage conditions and Apc Min / + The results of inducing expression of Renaissance luciferase Rluc8 after co-incubation with mouse feces; Figure 7 It is engineered probiotics E. coli Figure showing the protein expression and secretion of Nissle 1917-pJ23119-pelB-DKK3-HA.

[0013] Figure 8 It is engineered probiotics E. coli Image showing the colony formation results after co-incubation of the supernatant of Nissle 1917-pJ23119-pelB-DKK3-HA with HCT116 cells; Figure 9 It is an oral engineered probiotic. E. coliNissle 1917-pJ23119-pelB-DKK3-HA in Apc Min / + The experimental results for mice; Figure 10 It is an oral engineered probiotic. E. coli Figure 1. Experimental results of Nissle 1917-pJ23119-pelB-DKK3-HA in AOM / DSS mouse model; Figure 11 It is an oral engineered probiotic. E. coli Nissle 1917-pJ23119-pelB-DKK3-HA in IL10 - / - Figure of experimental results in the AOM mouse model; Figure 12 It is engineered probiotics E. coli Figure 1. Experimental results of co-culturing the supernatant of Nissle 1917-pJ23119-pelB-DKK3-HA with organoids from colorectal cancer patients; Figure 13 It is the tail vein injection of engineered probiotic HCS1-pJ23119-pelB-DKK3-HA Apc Min / + The experimental results for mice; Figure 14 The figure shows the experimental results of tail vein injection of engineered probiotic HCS1-pJ23119-pelB-DKK3-HA in MC38 subcutaneous tumor model mice. Figure 15 It is an oral administration of engineered probiotics in different doses. E. coli Figure showing the colonization results of Nissle 1917-pJ23119-pelB-DKK3-HA in mouse feces; Figure 16 This is a diagram showing the experimental results of injecting bacterial supernatant containing DKK3 into MC38 subcutaneous tumor model mice; Figure 17 This is a monitoring graph of tumor growth curves in different treatment groups after treatment. Detailed Implementation

[0014] 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 and 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.

[0015] As previously described, a first aspect of the present invention provides a combined bacterial agent for the diagnosis and treatment of colorectal cancer, the combined bacterial agent containing separately preserved engineered probiotics and genetically engineered bacteria; the engineered probiotics comprise probiotics and a recombinant expression system introduced into the probiotics; the recombinant expression system comprises a gene encoding the Dickkopf 3 protein; the amino acid sequence of the Dickkopf 3 protein is shown in SEQ ID NO.1; the probiotics are Escherichia coli Nissle 1917 and / or attenuated Salmonella Typhimurium HCS1; The genetically engineered bacteria can co-express speFL protein and luminescent protein; the luminescent protein includes Renaissance luciferase and / or superfolded green fluorescent protein. In the combined bacterial agent, the genetically engineered bacteria are used for in vitro culture and for oral administration and / or intravenous injection.

[0016] In this invention, the genetically engineered bacteria are able to detect higher bioluminescent signals in the feces of individuals with early-stage cancer.

[0017] SEQ ID NO.1: MQRLGATLLCLLLAAAVPTAPAPAPTATSAPVKPGPALSYPQEEATLNEMFREVEELMEDTQHKLRSAVEEMEAEEAAAKASSEVNLANLPPSYHNETNTDTKVGNNTIHVHREIHKITNNQTGQMVFSETVITSVGDEEGRRSHECIIDEDCGPSMYCQFASFQYTCQPCRGQR MLCTRDSECGDQLCVWGHCTKMATRGSNGTICDNQRDCQPGLCCAFQRGLLFPVCTPLPVEGELCHDPASRLLDLITWELEPDGALDRCPCASGLLCQPHSHSLVYVCKPTFVGSRDQDGEILLPREVPDEYEVGSFMEEVRQELEDLERSLTEEMALREPAAAAAALLGGEEI.

[0018] In some embodiments, the nucleotide sequence of the Dickkopf 3 protein is shown in SEQ ID NO.2.

[0019] SEQ ID NO.2:

[0020] Proteins derived by those skilled in the art from the amino acid sequence disclosed in this invention using techniques known in the art, such as substitution, deletion, or addition of one or more amino acids, whose enzyme activity remains unchanged, should fall within the protection scope of this invention. Unchanged enzyme activity means that, under the same assay conditions, the percentage (relative activity) between the enzyme activity of the protein derived from SEQ ID NO. 1 and the enzyme activity of the amino acid sequence SEQ ID NO. 1 is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%).

[0021] This invention does not impose any special requirements on the "separate preservation" method, as long as it meets the requirements for in vitro culture of the genetically engineered bacteria and for oral administration and / or intravenous injection of the engineered probiotics. For example, they can be separately preserved in two capsules within the same packaging bag, one capsule for preserving the genetically engineered bacteria and the other capsule for preserving the engineered probiotics; alternatively, they can be separately preserved in two packaging bags within the same kit, one packaging bag for preserving the genetically engineered bacteria and the other packaging bag for preserving the engineered probiotics. This invention will not further list the "separate preservation" methods, and those skilled in the art should not interpret this as a limitation of the invention.

[0022] The present invention does not have any special requirements for the oral formulation of the engineered probiotics, such as oral liquid probiotic dispersants or oral lyophilized probiotic powders.

[0023] In some embodiments, the method of applying the combined microbial agent includes: (1) After co-culturing the genetically engineered bacteria with sample I taken from the individual to be diagnosed, the fluorescence signal of the luminescent protein in the genetically engineered bacteria is measured; (2) If the measured value of the fluorescence signal is greater than the threshold, the engineered probiotic is administered orally or intravenously to the corresponding individual; the threshold is the measured value of the fluorescence signal of the genetically engineered bacteria after co-culturing with sample II taken from a healthy individual; Sample I and Sample II are each independently feces and / or the supernatant obtained after grinding the feces; The daily dose of the engineered probiotics administered orally is 1 × 10⁻⁶. 9 -1×10 10 CFU; The daily dose of the engineered probiotics administered intravenously is 1 × 10⁻⁶. 7 -1×10 8 CFU.

[0024] In some embodiments, the daily dose of the engineered probiotic administered orally is 1 × 10⁻⁶. 9 CFU.

[0025] In some embodiments, the engineered probiotics are administered orally 2-3 times per week.

[0026] In some implementations, the engineered probiotics are administered intravenously once or twice every two weeks, or only once.

[0027] In some embodiments, the method of applying the combined microbial agent further includes: storing the sample I in vitro before co-culturing it.

[0028] In some embodiments, the in vitro storage conditions include: a time of 0-20 h and a temperature of 2-30 °C; more preferably, a time of 0-10 h and a temperature of 2-10 °C. Under this preferred condition, the inventors of the present invention have found that after sample I is stored for a long time in a low-temperature environment (2-10 °C) or a room-temperature environment, the response efficiency of the genetically engineered bacteria remains excellent, demonstrating the stability of the genetically engineered bacteria, which is more conducive to clinical development and application.

[0029] In some embodiments, the concentration of the genetically engineered bacteria in the co-culture system is 1 × 10⁻⁶. 7 -1×10 10 CFU / mL.

[0030] In some embodiments, the sample is feces, and the concentration of the feces in the co-culture system is 0.005-0.05 g / mL.

[0031] In some embodiments, the solvent in the co-culture system is M9 medium and / or LB medium.

[0032] In some embodiments, the co-culture conditions include a temperature of 36-37 °C and a time of 4-6 h.

[0033] In some embodiments, the co-cultivation conditions also include a rotation speed of 100-300 rpm.

[0034] In some embodiments, the recombinant expression system includes at least one of a signal peptide encoding gene, an HA tag protein encoding gene, and a promoter.

[0035] In some embodiments, the nucleotide sequence of the signal peptide encoding gene is shown in SEQ ID NO.3.

[0036] SEQ ID NO.3: ATGAAATACCTGCTGCCGACTGCTGCACGGGTCTGTTGCTGCTGGCTGCACAGCCAGCTATGGCG.

[0037] In some embodiments, the nucleotide sequence of the gene encoding the HA tag protein is shown in SEQ ID NO.4.

[0038] SEQ ID NO.4: TATCCATACGACGTTCCGGACTACGCA.

[0039] In some implementations, the promoter is P J23119 Promoter; the P J23119 The nucleotide sequence of the promoter is shown in SEQ ID NO. 5. The P... J23119 The promoter is a constitutive promoter. In this preferred case, the Dickkopf 3 protein-coding gene is expressed through a constitutive promoter, enabling sustained in situ release of the protein and targeted therapy for tumors.

[0040] SEQ ID NO.5: TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC.

[0041] The present invention does not specifically limit the method for introducing the recombinant expression system into the probiotic, nor the method for constructing the recombinant expression system. Any method in the art that can achieve the above objectives is applicable to the present invention.

[0042] In some embodiments, the amino acid sequence of the speFL protein is shown in SEQ ID NO.6.

[0043] SEQ ID NO.6: MENNSRTMPHIRRTTHIMKFAHRNSFDFHFFNAR.

[0044] In some embodiments, the sequence of nucleotides encoding the speFL protein is shown in SEQ ID NO.9.

[0045] SEQ ID NO.9: ATGGAAAATAACAGCCGCACTATGCCCCATATAAGGCGGACAACTCATATTATGAAGTTTGCTCATCGCAATAGCTTCGACTTTCACTTCTTCAATGCCCGTTAG.

[0046] In some embodiments, the amino acid sequence of the Renal luciferase (Rluc8) is shown in SEQ ID NO.7.

[0047] SEQ ID NO.7: MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGAALAFHYAYEHQDRIKAIVHMESVVDVIESWDEW PDIEEDIALIKSEEGEKMVLENNFFVETVLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTSWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFLQEDAPDEMGKYIKSFVERVLKNEQ.

[0048] In some embodiments, the nucleotide sequence encoding the Renal luciferase (Rluc8) is shown in SEQ ID NO. 10.

[0049] SEQ ID NO.10: AtggcttccaaggtgtacgaccccgagcaacgcaaacgcatgatcactgggcctcagtggtgggctcgctgcaagcaaatgaacgtgctggactccttcatcaactactatgattccgagaagcacgccgagaacgccgtgatttttctgcatggtaacgctacctccagctacctgtggaggcacgtcgtgcctcacatcgagcccgtggctagatgcatcatccctgatctgatcggaatgggtaagtccggcaagagcgggaatggctcatatcgcctcctggatcactacaagtacctcaccgcttggttcgagctgctgaaccttccaaagaaaatcatctttgtgggccacgactggggggctgctctggcctttcactacgcctacgagcaccaagacaggatcaaggccatcgtccatatggagagtgtcgtggacgtgatcgagtcctgggacgagtggcctgacatcgaggaggatatcgccctgatcaagagcgaagagggcgagaaaatggtgcttgagaataacttcttcgtcgagaccgtgctcccaagcaagatcatgcggaaactggagcctgaggagttcgctgcctacctggagccattcaaggagaagggcgaggttagacggcctaccctctcctggcctcgcgagatccctctcgttaagggaggcaagcccgacgtcgtccagattgtccgcaactacaacgcctaccttcgggccagcgacgatctgcctaagctgttcatcgagtccgaccctgggttcttttccaacgctattgtcgagggagctaagaagttccctaacaccgagttcgtgaaggtgaagggcctccacttcctccaggaggacgctccagatgaaatgggtaagtacatcaagagcttcgtggagcgcgtgctgaagaacgagcagTAA。

[0050] In some embodiments, the amino acid sequence of the superfolded green fluorescent protein (sfGFP) is shown in SEQ ID NO. 8.

[0051] SEQ ID NO.8: MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.

[0052] In some embodiments, the nucleotide sequence encoding the superfolded green fluorescent protein (sfGFP) is shown in SEQ ID NO.11.

[0053] SEQ ID NO.11: ATGCGTAAAGGTGAAGAACTGTTCACTGGTGTTGTTCCGATTCTGGTTGAACTGGACGGTGACGTTAACGGCCACAAATTCTCTGTACGTGGTGAAGGTGAGGGTGATGCGACCAACGGTAAACTGACTCTGAAGTTCATCTGCACCACCGGCAAACTGCCAGTACCGTGGCCAACTCTGGTGACCACTCTGACCTACGGTGTTCAGTGCTTCGCTCGTTATCCGGATCACATGAAACAGCACGACTTCTTCAAGTCTGCGATGCCAGAAGGTTACGTTCAAGAACGTACCATCTCTTTCAAAGACGACGGCACCTACAAGACTCGTGCGGAAGTTAAATTCGAAGGTGACACTCTGGTTAACCGTATCGAACTGAAAGGTATCGACTTCAAAGAGGACGGTAACATCTTGGGTCACAAACTGGAATACAACTTCAACAGCCACAACGTGTACATCACCGCGGACAAACAGAAGAACGGTATCAAAGCGAACTTCAAGATCCGTCACAACGTTGAGGACGGTTCTGTTCAGCTGGCAGATCACTACCAGCAGAACACTCCGATCGGTGATGGTCCAGTTCTGCTGCCAGACAACCACTACCTGTCTACTCAGTCTGTTCTGTCCAAAGATCCGAACGAGAAACGTGACCACATGGTTCTGCTGGAATTTGTGACCGCAGCAGGTATCACTCACGGTATGGACGAACTGTACAAGTAA。

[0054] Those skilled in the art can reasonably deduce the corresponding nucleotide sequences from the amino acid sequences of the Dickkopf 3 protein, the speFL protein, and the luminescent protein disclosed in this invention, based on publicly known codon tables. Therefore, this invention does not impose a unique limitation on the specific base combination of the nucleotide sequences. The nucleotide sequences include, but are not limited to, any nucleotide sequence that can encode the same amino acid sequence and meets the requirements of conventional genetic engineering operations. Therefore, nucleotide sequences with at least 50% identity to the nucleotide sequences disclosed in this invention should also fall within the scope of protection claimed by this invention. At least 50% identity means that the identity can be 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100%, or a range consisting of any two of the above values, or any intermediate value within that range.

[0055] In some embodiments, the chassis strain of the genetically engineered bacteria is Escherichia coli Nissle 1917.

[0056] In some embodiments, the promoter of the speFL protein-coding gene is the P... J23119 Promoter.

[0057] As previously stated, a second aspect of the present invention provides the use of the combined bacterial agent described in the first aspect in the preparation of a medicament for the early diagnosis and treatment of colorectal cancer.

[0058] In some embodiments, the drug contains an active ingredient; the active ingredient contains the combined bacterial agent.

[0059] In some embodiments, the active ingredient may also contain components and / or excipients that can be used to treat intestinal tumors.

[0060] In some embodiments, the ingredient for treating intestinal tumors is selected from at least one of glucocorticoids, aminosalicylates, immunosuppressants, and second probiotic preparations.

[0061] In this invention, the excipient refers to a component in the pharmaceutical composition that does not itself function as an active ingredient. For example, it may be a component used to assist the active ingredient in maintaining or promoting its activity, or it may be a component used for drug shaping or preservation. The excipient includes at least one of buffers, protectants, stabilizers, coating agents, and flavoring agents.

[0062] In some embodiments, the buffer includes at least one of microcrystalline cellulose, lactose, starch, pregelatinized starch, and dicalcium phosphate.

[0063] In some embodiments, the protective agent includes at least one of sucrose, trehalose, mannitol, and sorbitol.

[0064] In some embodiments, the stabilizer includes at least one of sodium bicarbonate, vitamin C, vitamin E, and polyethylene glycol 1000.

[0065] In some embodiments, the coating agent includes at least one of hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, polyvinyl alcohol, povidone, polyvinyl acetate resin, and polyvinyl acetal diethylaminoacetate.

[0066] In some embodiments, the flavoring agent includes at least one of beef flavoring, chicken flavoring, and compound meat flavoring.

[0067] This invention does not impose any particular limitations on the specific process and method for preparing the engineered probiotics and genetically engineered bacteria in the combined bacterial agent into a drug. Any method in the art that can be used to prepare a drug containing (active) probiotics is applicable to this invention.

[0068] The present invention will be described in detail below through examples. Unless otherwise specified, the methods used in the following examples are conventional methods in the art, and the reagents used are all commercially available. Room temperature refers to 25±2℃.

[0069] Escherichia coli MG1655: purchased from Beijing Bio-Lab Co., Ltd., product number BTN12-211y.

[0070] Escherichia coli Nissle 1917 ( E. coli Nissle 1917): Purchased from Beijing Bio-European Co., Ltd., product number bio-089890.

[0071] Attenuated Salmonella Typhimurium HCS1: Constructed according to the method published in Sun, Y., Guo, Y., Liu, X., Liu, J., Sun, H., Li, Z., Wen, M., Jiang, SN, Tan, W., Zheng, JH (2023). Engineered oncolytic bacteria HCS1 exerts high immune stimulation and safety profiles for cancer therapy. Theranostics, 13(15), 5546-5560.

[0072] The plasmid miniprep kit was purchased from Tiangen Biotech Co., Ltd.

[0073] Human colorectal adenocarcinoma cells HCT116: from the American Type Culture Collection (ATCC), strain number CCL-247.

[0074] MC38 cells: from the American Type Culture Collection (ATCC), strain number CRL-2638.

[0075] Streptomycin / penicillin bispecific antibody solution: purchased from Seville Biopharmaceutical Co., Ltd., product number G4003.

[0076] Organoid growth medium: purchased from Beijing Daxiang Technology Co., Ltd., catalog number OC105131.

[0077] DMEM culture medium: purchased from Seville Biosciences, catalog number G4519.

[0078] Fetal bovine serum: purchased from SeraPure, catalog number SE141-500.

[0079] CCK8 reagent kit: purchased from Icoin (Wuhan) Biotechnology Co., Ltd., catalog number BMU106-CN.

[0080] Azomethane (AOM): Purchased from MP Biomedicals.

[0081] The matrix adhesive was purchased from Bozhen Biotechnology Co., Ltd., product number M315066.

[0082] Preparation method of M9 liquid culture medium: 64g disodium hydrogen phosphate heptahydrate, 15g dipotassium hydrogen phosphate, 5g ammonium chloride, 2.5g sodium chloride, distilled water to a final volume of 1000mL, autoclave at high temperature (121℃) for 20min, cool and take 200mL, add 20mL of 20% glucose solution, 2ml of 1M magnesium sulfate, 0.1mL of 1M calcium chloride, and sterile deionized water to a final volume of 1L, store at 4℃ for later use.

[0083] 20% glucose solution: Dissolve 20g of glucose powder in 80mL of sterile water and bring the volume up to 100mL. Filter the solution through a 0.22μm filter membrane for sterilization.

[0084] 1M Magnesium Sulfate: Dissolve 1.2g of magnesium sulfate powder in 8mL of distilled water and bring the volume up to 10mL. Sterilize at high temperature (121℃) under high pressure for 20min.

[0085] 1M Calcium Chloride: Dissolve 1.47g of calcium chloride powder in 8mL of distilled water and bring the volume up to 10mL. Sterilize at high temperature (121℃) under high pressure for 20min.

[0086] Preparation method of LB liquid culture medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, double distilled water to a final volume of 1000mL, autoclave at high temperature (121℃) for 20min, and store at 4℃ for later use.

[0087] 2% sodium dextran sulfate (DSS) solution: purchased from MP Biomedicals.

[0088] Apc Min / + Mouse model: purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.

[0089] C57BL / 6 mice: purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.

[0090] male IL10 - / - Mice: Purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.

[0091] Hematoxylin-eosin (HE) staining: Immerse 5 μm thick paraffin sections twice in xylene for 10 minutes each time to fully dewax. Then, hydrate sequentially in 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each, and finally rinse with distilled water for 2 minutes. Immerse the sections in hematoxylin staining solution for 6 minutes, rinse with running water for 1 minute to return to blue. Differentiation and blueing: Differentiate using 1% hydrochloric acid-ethanol differentiation solution for 2 seconds, rinse rapidly with running water for 10 minutes to ensure clear nuclear staining and cytoplasmic decolorization. Counterstain the sections in 0.5% eosin (water-soluble) staining solution for 3 minutes, and rinse briefly with distilled water. Dehydrate rapidly sequentially in 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol for 2 minutes each, then immerse twice in xylene for 3 minutes each time. After slightly drying the sections, add neutral resin and seal with a coverslip. Images were observed and acquired using a Motic Panthera S upright optical microscope.

[0092] The morphological assessment method was performed in accordance with A Practical Guide to the Histology of the Mouse [J]. 2014. DOI:10.1002 / 9781118789568.

[0093] Preparation Example 1 This embodiment illustrates the construction of engineered probiotics and genetically engineered bacteria provided by the present invention.

[0094] 1. Construction of recombinant plasmids Amplification and synthesis of recombinant gene sequence: The gene sequence of speFL (nucleotide sequence is SEQ ID NO.9) was obtained by consulting literature and GenBank. The speFL gene fragment was amplified from Escherichia coli MG1655 using primers 1 and 2.

[0095] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the sfGFP gene fragment (nucleotide sequence as SEQ ID NO.11) and the Rluc8 gene fragment (nucleotide sequence as SEQ ID NO.10).

[0096] Beijing Qingke Biotechnology Co., Ltd. was commissioned to splice the signal peptide (i.e., the secretory peptide pelB gene with nucleotide sequence SEQ ID NO.3), the DKK3 protein-coding gene (nucleotide sequence SEQ ID NO.2), and the HA tag protein-coding gene (nucleotide sequence SEQ ID NO.4) into the plasmid recipient bacterium DH5α using the cloning plasmid PUC57, and then transform them into the plasmid recipient bacterium DH5α, to obtain the recombinant bacterium DH5α-PUC57-pelB-DKK3-HA containing the recombinant cloning plasmid.

[0097] SEQ ID NO.12: 5'-GTCGACATGGAAAATAACAGCCGCACTATGC-3' (Primer name is primer 1); SEQ ID NO.13: 5'-TTCACCTTTACGCATTTTTGACATTTTTCATCTCTTTATTTTAGG-3' (Primer name is primer 2); Synthesis of recombinant plasmid pJ23119-speFL-sfGFP: The speFL gene fragment and the sfGFP gene fragment were amplified by overlap extension PCR, and then spliced ​​with the expression plasmid pJ23119 by homologous recombination to obtain the recombinant plasmid pJ23119-speFL-sfGFP.

[0098] Synthesis of recombinant plasmid pJ23119-speFL-Rluc8: The speFL gene fragment and the Rluc8 gene fragment were amplified by overlap extension PCR, and then spliced ​​with the expression plasmid pJ23119 by homologous recombination to obtain the recombinant plasmid pJ23119-speFL-Rluc8.

[0099] Synthesis of recombinant plasmid pJ23119-pelB-DKK3-HA: The plasmid extracted from the recombinant cloning bacterium DH5α-PUC57-pelB-DKK3-HA was used as a template to obtain the target sequence pelB-DKK3-HA through a full-length PCR synthesis reaction. The target sequence was then ligated into the expression plasmid pJ23119 to obtain pJ23119-pelB-DKK3-HA.

[0100] The recombinant plasmids were transformed into the plasmid recipient bacteria DH5α to obtain recombinant bacteria.

[0101] Figure 1 This is a map showing the construction of plasmids. Figure 1 In the image, A represents the spectrum of pJ23119-speFL-sfGFP. Figure 1 B in the image represents the spectrum of pJ23119-speFL-Rluc8. Figure 1 C in the diagram represents the spectrum of pJ23119-pelB-DKK3-HA.

[0102] 2. Construction of engineered bacteria 2.1 Construction of genetically engineered bacteria and engineered probiotics using Escherichia coli Nissle 1917 as a chassis Recombinant plasmids pJ23119-speFL-sfGFP, pJ23119-speFL-Rluc8, and pJ23119-pelB-DKK3-HA were extracted from recombinant bacteria using a plasmid miniprep kit. These plasmids were then introduced into competent *Escherichia coli* Nissle 1917 via heat shock. The transformed bacterial culture was plated on LB agar plates containing 10 mg / L chloramphenicol and incubated overnight at 37°C. Single colonies from these plates were picked, and plasmids were extracted and amplified by PCR to verify the gene fragments, yielding positive clones.

[0103] Positive clones obtained by transforming pJ23119-speFL-sfGFP, pJ23119-speFL-Rluc8, and pJ23119-pelB-DKK3-HA into Escherichia coli Nissle 1917 were named EcO-sfGFP (i.e., E. coli Nissle1917-pJ23119-speF-sfGFP), EcO-Rluc8 (i.e., E. coli Nissle 1917-pJ23119-speF-Rluc8) and EcD (i.e., E. coli Nissle 1917-pJ23119-pelB-DKK3-HA).

[0104] 2.2 Construction of engineered probiotics using attenuated Salmonella Typhimurium HCS1 as a chassis pJ23119-pelB-DKK3-HA was transformed into attenuated Salmonella Typhimurium HCS1 competent cells to obtain a recombinant strain, named HCS1 DKK3. 2.3 Construction of control strains The empty pJ23119 plasmid was transformed into attenuated Salmonella Typhimurium HCS1 competent cells to obtain a recombinant strain, named HCS1 empty.

[0105] The empty vector pJ23119 was transformed into competent Escherichia coli Nissle 1917 to obtain a recombinant strain, which was named EcN.

[0106] The empty vector pJ23119 was transformed into Escherichia coli MG1655 to obtain a recombinant strain, named MG1655-empty.

[0107] Preparation Example 2 This preparation example uses a similar method to Preparation Example 1 to construct engineered probiotics. The difference is that Escherichia coli MG1655 is used instead of attenuated Salmonella Typhimurium HCS1 in Preparation Example 1. The remaining methods were the same as in Preparation Example 1, and a positive clone strain was obtained by transforming pJ23119-pelB-DKK3-HA into Escherichia coli MG1655, which was named MG1655-DKK3.

[0108] Example 1: Performance Testing of Genetically Engineered Bacteria 1. Detection of the performance of genetically engineered bacteria in responding to ornithine expression of sfGFP in different culture media EcO-sfGFP was inoculated into LB liquid medium containing chloramphenicol and cultured at 37°C for 12 h. A certain amount of bacteria was then inoculated into 10 mL of fresh M9 liquid medium and LB liquid medium containing chloramphenicol, respectively, to achieve an initial concentration of 1 × 10⁻⁶. 7 CFU / mL was added, and ornithine was added to final concentrations of 0 mM, 2 mM, and 10 mM, respectively. The mixture was incubated at 37 ℃ and 180 rpm for 6 h. 100 μL of the bacterial culture was then used to determine the OD. 600 Values ​​and OD 488 / 510 Value (sfGFP), calculated in different culture media, OD 488 / 510 Value and OD 600 The ratio of values ​​(defined as sfGFP / OD) 600 ), the result is as follows Figure 2 As shown. Figure 2 The results showed that the genetically engineered bacteria EcO-sfGFP responded better to sfGFP expression in M9 medium than in LB medium.

[0109] 2. Detection of the performance of genetically engineered bacteria in responding to ornithine expression sfGFP EcO-sfGFP was inoculated into LB liquid medium containing chloramphenicol and cultured overnight at 37°C; a certain amount of bacteria was then inoculated into 10 mL of fresh M9 liquid medium containing chloramphenicol to achieve an initial concentration of 1×10⁻⁶. 7 CFU / mL, and ornithine was added to final concentrations of 0 mM, 2 mM, and 10 mM, respectively. After incubation at 37 ℃ and 180 rpm for 6 h, 12 h, and 24 h, 100 μL of bacterial culture was taken to determine OD. 600 Values ​​and OD 488 / 510 Value (sfGFP), calculate OD 488 / 510 Value and OD 600 The ratio of values ​​(defined as sfGFP / OD) 600 ), to obtain sfGFP / OD 600 The value changes over time, and the result is as follows: Figure 3 As shown, when the ornithine concentration is 10 mM, the engineered bacteria EcO-sfGFP can respond sensitively to the presence of ornithine and express sfGFP after 6 h.

[0110] 3. Performance Detection of Genetically Engineered Bacteria Responding to Rluc8 Ornithine Expression EcO-Rluc8 was inoculated into LB liquid medium containing chloramphenicol and cultured overnight at 37°C; a certain amount of bacteria was then inoculated into 10 mL of fresh M9 liquid medium containing chloramphenicol to achieve an initial concentration of 1×10⁻⁶. 7 CFU / mL, and ornithine was added to final concentrations of 0 mM, 2 mM, 5 mM, and 10 mM, respectively. After co-culturing at 37 ℃ and 180 rpm for 6 hours, 100 μL of bacterial culture was transferred to a 96-well black ELISA plate, and 2 μg of coelenterate was added. The bioluminescent signal was measured using a small animal imaging system, and the results are as follows. Figure 4 As shown, when the ornithine concentration is 10 mM, the genetically engineered bacterium EcO-Rluc8 can sensitively respond to the presence of ornithine and express Rluc8 after 6 hours.

[0111] Example 2 This embodiment illustrates the response effect of the genetically engineered bacterium EcO-Rluc8 provided by the present invention in the feces of tumor-bearing mice.

[0112] (I) Response effects of genetically engineered bacteria in different mouse models In this embodiment, 10-week-old male healthy C57BL / 6 mice (WT) and Apc Min / +Mouse feces were weighed and added to M9 medium to a final concentration of 1 g / mL. After thorough vortexing, the solution was diluted with M9 medium, and then EcO-Rluc8 was added to achieve a final concentration of 1×10⁻⁶. 10 The final concentration of feces was 0.01 g / mL (CFU / mL). The mixture was co-cultured at 37 ℃ and 180 rpm for 6 hours. 100 μL of the co-cultured solution was then transferred to a 96-well black microplate, and 2 μg of coelenterate was added. The bioluminescent signal was measured using a small animal imaging system. The results are as follows: Figure 5 As shown. Figure 5 The display shows that EcO-Rluc8 and Apc Min / + The bioluminescent signal obtained after co-culturing mouse feces was significantly higher than that of healthy C57BL / 6 mice.

[0113] (ii) Stability of the response of genetically engineered bacteria in feces In this embodiment, 10-week-old male healthy C57BL / 6 mice and Apc Min / + Mouse feces were weighed and added to M9 medium to a final concentration of 1 g / mL. After thorough vortexing, the solution was diluted with M9 medium and stored overnight at 4°C or for 12 h at room temperature. EcO-Rluc8 was then added to achieve a final concentration of 1 × 10⁻⁶ g / mL. 10 The final concentration of feces was 0.01 g / mL, and the mixture was co-cultured at 37 ℃ and 180 rpm for 6 hours. 100 μL of the co-cultured mixture was then placed in a 96-well black microplate, and 2 μg of coenzyme was added. The bioluminescent signal was measured using a small animal imaging system.

[0114] Figure 6 It is engineered probiotics E. coli Nissle 1917-pJ23119-speF-Rluc8 was used in healthy mice under different storage conditions and Apc Min / + The results of inducing expression of Renaissance luciferase Rluc8 after co-incubation with mouse feces; Figure 6 A in the figure represents the result of storing Renal luciferase Rluc8 at 4℃; Figure 6 B in the figure represents the result of storing Renaissance luciferase Rluc8 at room temperature.

[0115] Figure 6 The results show that, under the above conditions, EcO-Rluc8 and Apc Min / + The bioluminescent signal obtained after co-culturing the fecal supernatant of mice was still significantly higher than that of healthy C57BL / 6 mice.

[0116] Example 3 This embodiment illustrates the inhibitory effect of the engineered probiotic EcD provided by the present invention on the proliferation of HCT116 cells.

[0117] (I) Detection of EcD expression and secreted protein in engineered probiotics Bacterial EcN and EcD were inoculated separately into LB liquid medium containing chloramphenicol and cultured overnight at 37°C. Then, they were inoculated into 5 mL of fresh LB liquid medium containing chloramphenicol at a volume ratio of 1:100. After 6 hours, 1×10⁻⁶ cells were collected from each culture medium. 8 The expression of DKK3 protein was detected by Western blotting of the bacterial culture and supernatant corresponding to CFU. The results are as follows: Figure 7 As shown, T refers to the total bacterial cells and supernatant; S refers to the bacterial culture supernatant. Figure 7 The results showed that strain EcD was able to express and secrete the target protein DKK3 under in vitro culture conditions.

[0118] (II) Inhibitory effect of engineered probiotic EcD on cell proliferation 1. Cell culture: HCT116 cells were cultured in DMEM medium (containing 10% fetal bovine serum and 1% streptomycin / penicillin antibiotic solution) at 37°C and 5% CO2.

[0119] 2. Colony formation assay: The supernatants of bacterial EcN and EcD were concentrated 10-fold using ultrafiltration. HCT116 cells were seeded at 1000 cells / well in six-well cell culture plates. After 48 hours of culture, 30 μL of the concentrated 10-fold ultrafiltration supernatant of bacterial EcN and EcD was added to each well. Fresh bacterial supernatant was added directly to the original culture supernatant every 48 hours. After 6 days of co-culture, the culture medium was aspirated, the cells were washed twice with PBS, fixed with 4% PFA, and stained with 1 mL of crystal violet solution. After incubation for 10 minutes, the stain was washed with PBS, and the cells were photographed and the formed cell colonies were counted. Wells without bacterial supernatant were set up as controls (DMEM group). The colony formation rate (%) was calculated as: (number of colonies formed in each group / number of colonies formed in the DMEM group) × 100%.

[0120] 3. CCK8 Assay: The supernatants of bacterial EcN and EcD were concentrated 10-fold using ultrafiltration. HCT116 cells were seeded at 1000 cells / well in 96-well cell culture plates. After 12 hours of culture, 10 μL of the concentrated 10-fold ultrafiltration supernatant of bacterial EcN and EcD was added to each well. Fresh bacterial supernatant was added directly to the original culture supernatant every 48 hours. Wells without bacterial supernatant were set up as controls (control wells), and wells without HCT116 cells and bacterial supernatant, with only culture medium added, were set up as blank controls (blank wells). After 6 days of co-culture, cell proliferation was detected using the CCK8 assay kit. Cell viability (%) was calculated using the formula: [(experimental wells - blank wells) / (control wells - blank wells)] × 100%.

[0121] Figure 8 In this context, A represents engineered probiotics. E. coli Image showing colony formation results after co-incubation of Nissle 1917-pJ23119-pelB-DKK3-HA supernatant with HCT116 cells; from Figure 8 As shown in A, the formation of HCT116 cells was significantly reduced after treatment with EcD supernatant.

[0122] Figure 8 B in the text refers to engineered bacteria. E. coli Image showing cell proliferation results after co-incubation of the supernatant of Nissle 1917-pJ23119-pelB-DKK3-HA with HCT116 cells; from Figure 8 As shown in B, the proliferation of HCT116 cells was significantly inhibited after co-culturing with EcD supernatant.

[0123] from Figure 8 It can be seen that the culture supernatant of EcD strain can significantly inhibit the proliferation of human colorectal adenocarcinoma cells HCT116.

[0124] Example 4 This embodiment follows Figure 9 The experimental procedure shown in A evaluates the engineered probiotic EcD provided by this invention in [the context of the invention]. Apc Min / + Antitumor effects in mouse models: In this embodiment, 9-week-old males were selected for the animal experiment. Apc Min / + Mice were randomly divided into two groups as described below: EcN group: Mice were administered EcN strain orally via gavage, 200 μL per mouse, containing 1×10⁻⁶ bacteria. 9 CFU, administered via gavage twice a week.

[0125] EcD group: Mice were administered EcD strain via oral gavage, 200 μL per mouse, containing 1×10⁻⁶ bacteria. 9 CFU, administered via gavage twice a week.

[0126] At 20 weeks of age, mice from each group were sacrificed and dissected. The small intestine of the mice was separated, and the adenomas were counted, photographed and sampled. Hematoxylin-eosin (HE) sections of the small intestinal tissue were prepared and stained for pathological and morphological evaluation.

[0127] Figure 9 For oral engineered probiotics E. coli Nissle 1917-pJ23119-pelB-DKK3-HA in Apc Min / + The experimental results for mice are shown in the figure.

[0128] Figure 9 Image B in the figure shows representative images of tumor development in the small intestine of mice in different treatment groups; Figure 9 C in the figure shows the statistical results of the number of tumors in the small intestine of mice in different treatment groups; Figure 9 D in the figure shows the statistical results of the total area of ​​tumors in the small intestine of mice in different treatment groups; from Figure 9 B in Figure 9 C in Figure 9 As can be seen from D, EcD treatment significantly reduced the number of adenomas and the total area of ​​the tumor region in the small intestine of mice, thus inhibiting the progression of the tumor.

[0129] Figure 9 E in the figure shows the H&E staining results of small intestinal tissues from mice in different treatment groups; Figure 9 The E staining results showed that the reduction in the number of tumors after EcD treatment occurred in all regions of the small intestine.

[0130] In addition, the same experimental mouse model was used, and the same engineered bacteria were administered via gavage. The survival period of the mice was evaluated, and the survival rate (%) was calculated as: number of mice surviving on day n after probiotic injection / number of mice initially injected × 100%. Figure 9 F in the figure represents the survival time monitoring results of mice after different treatments, showing the survival rate (%) of mice in different treatment groups.

[0131] Figure 9 The F-value in the study showed that EcD treatment could significantly prolong [the duration of life]. Apc Min / + The survival time of the tumor-bearing mice showed a good anti-cancer effect.

[0132] Figure 9 This indicates that EcD is in ApcMin / + The treatment showed good therapeutic effects in mouse models.

[0133] Example 5 This embodiment follows Figure 10 The experimental procedure shown in A evaluates the antitumor effect of the engineered probiotic EcD provided by this invention in the AOM / DSS mouse model: In this embodiment, healthy male C57BL / 6 mice aged 9 weeks were selected for the animal experiment. Each mouse was intraperitoneally injected with AOM at a final concentration of 10 mg / kg. One week later, they were given free access to 2% DSS solution for 7 days, followed by normal drinking water for 2 weeks. Each cycle consisted of 1 week of DSS and 2 weeks of normal drinking water, and this cycle was repeated for 3 cycles. At the beginning of the first cycle of free access to water (i.e., after the first week of intraperitoneal injection of AOM), the mice were randomly divided into the following two groups: EcN group: Mice were administered EcN strain orally via gavage, 200 μL per mouse, containing 1×10⁻⁶ bacteria. 9 CFU, administered via gavage twice a week.

[0134] EcD group: Mice were administered EcD strain via oral gavage, 200 μL per mouse, containing 1×10⁻⁶ bacteria. 9 CFU, administered via gavage twice a week.

[0135] When the mice reached 22 weeks of age, the mice in each group were sacrificed and dissected. The colons of the mice were separated, and the tumors were counted, photographed and sampled. Hematoxylin-eosin (HE) sections of colon tissue were prepared for staining and pathological and morphological evaluation.

[0136] Figure 10 It is an oral engineered probiotic. E. coli Figure showing experimental results of Nissle 1917-pJ23119-pelB-DKK3-HA in the AOM / DSS mouse model.

[0137] Figure 10 Image B in the figure shows representative images of tumor development in the colon of mice in different treatment groups; Figure 10 C in the figure shows the statistical results of the number of tumors in the colon of mice in different treatment groups; Figure 10 D in the figure shows the statistical results of the total area of ​​tumors in the colon of mice in different treatment groups; Figure 10 E in the figure shows the HE staining results of colon tissue.

[0138] from Figure 10 B in Figure 10 C in Figure 10 As can be seen from D, EcD treatment significantly reduced the number and total area of ​​tumors in the colon of mice, inhibiting tumor development. Furthermore, Figure 10 The "E" indicates that the number and size of tumors were significantly suppressed after EcD treatment.

[0139] Figure 10 This indicates that EcD shows good therapeutic effects in the AOM / DSS mouse model.

[0140] Example 6 This embodiment follows Figure 11 The experimental procedure shown in A evaluates the engineered probiotic EcD provided by this invention in [the context of the invention]. IL10 - / - Antitumor effects in the AOM mouse model: In this embodiment, 10-week-old males were selected for the animal experiment. IL10 - / - Mice were intraperitoneally injected with AOM at 10 and 15 weeks of age, with a final concentration of 10 mg / kg per mouse. The mice were randomly divided into two groups as follows: EcN group: Mice were administered EcN strain orally via gavage, 200 μL per mouse, containing 1×10⁻⁶ bacteria. 9 CFU, administered via gavage twice a week.

[0141] EcD group: Mice were administered EcD strain via oral gavage, 200 μL per mouse, containing 1×10⁻⁶ bacteria. 9 CFU, administered via gavage twice a week.

[0142] When the mice reached 20 weeks of age, the mice in each group were sacrificed and dissected. The colons of the mice were separated, and the tumors were counted, photographed and sampled. Hematoxylin-eosin (HE) sections of colon tissue were prepared for staining and pathological and morphological evaluation.

[0143] Figure 11 For oral engineered probiotics E. coli Nissle 1917-pJ23119-pelB-DKK3-HA in IL10 - / - Figure of experimental results in the AOM mouse model.

[0144] Figure 11 Image B in the figure shows representative images of tumor development in the colon of mice in different treatment groups; Figure 11 C in the figure shows the statistical results of the number of tumors in the colon of mice in different treatment groups; Figure 11 D in the figure shows the statistical results of the total area of ​​tumors in the colon of mice in different treatment groups; Figure 11 E in the figure shows the HE staining results of colon tissue.

[0145] from Figure 11 B in Figure 11 C in Figure 11 As can be seen from D, EcD treatment significantly reduced the number and total area of ​​tumors in the colon of mice, thus delaying disease progression. Furthermore, Figure 11 The presence of E indicates that oral EcD can significantly inhibit the number and size of tumors.

[0146] Figure 11 This indicates that EcD is in IL10 - / - It showed good therapeutic effects in the / AOM mouse model.

[0147] Example 7 This embodiment follows Figure 12 The experimental procedure shown in A evaluates the inhibitory effect of the engineered probiotic EcD provided by this invention on organoids derived from colorectal cancer patients: This embodiment included colorectal cancer patients from the Second Xiangya Hospital of Central South University. Informed consent has been obtained from the relevant personnel regarding the research content of this embodiment.

[0148] This embodiment refers to the paper by Mahe, MM, Sundaram, N., Watson, CL, Shroyer, NF, Helmrath, MA, "Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy," J. Vis. Exp. (97), e52483, which evaluates the effects of EcN and EcD bacterial supernatants on organoid proliferation. Specifically: (1) Tumor isolation: A 1 cm long human colon biopsy sample was excised and thoroughly washed with ice-cold phosphate-buffered saline (PBS) to remove residual feces. The tissue was then longitudinally cut into small pieces and washed with ice-cold PBS to remove cell debris. Next, the sample was placed in 10 mM EDTA (prepared in PBS) and incubated at 4 °C with shaking for 30 minutes to allow dissociation. Afterward, the sample was shaken vigorously to loosen the crypt structures, and the resulting supernatant was filtered through a 70 μm cell sieve to remove larger tissue fragments. The filtrate containing the crypts was then centrifuged at 300 g for 2 minutes to precipitate the crypts, and finally, the crypts were resuspended in PBS for quantification.

[0149] (2) Organoid culture and assessment of organoid proliferation: A certain number of crypts were centrifuged at 300 g for 2 minutes and resuspended in organoid growth medium at a concentration of 40 crypts per 15 μL. This suspension was mixed with Matrigel at a 1:1 volume ratio, and 25 μL of the mixture was inoculated into each well of a 48-well plate. After polymerization at 37°C and 5% CO2 for 30 minutes, 200 μL of organoid growth medium containing 1× penicillin-streptomycin (purchased from Invitrogen) was added to each well. 20 μL of bacterial EcN and EcD supernatants, concentrated 10-fold by ultrafiltration, were added respectively. Every 48 hours, 20 μL of bacterial EcN and EcD supernatants, concentrated 10-fold by ultrafiltration, were added again. After 6 days of co-culture, the proliferation of organoids was assessed. A control group (DMEM group) was set up without the addition of bacterial supernatant.

[0150] The formula for calculating organoid spheroidization rate (i.e., organoid spheroidization ratio) % is: (Total number of inoculated cells / Number of organoids observed on day n of culture) × 100%.

[0151] Figure 12 Engineered probiotics E. coli The experimental results of co-culturing the supernatant of Nissle 1917-pJ23119-pelB-DKK3-HA with human organs from CRC disease.

[0152] Figure 12 Image B in the figure shows representative images of organoid growth at different time points in different treatment groups; Figure 12 C in the figure shows a quantitative analysis of the changes in organoid spheroid formation over time during co-culture; Figure 12 D in the figure shows a quantitative analysis of the average area of ​​organoid spheres formed in different treatment groups on day 6.

[0153] Figure 12 B and Figure 12 The C-value in the figure shows that the organoid formation rate was significantly inhibited during the culture process when co-cultured with EcD bacterial supernatant. Figure 12 The D-values ​​in the study showed that the organoid area formed after co-culturing with EcD bacterial supernatant was also significantly reduced.

[0154] Figure 12 This indicates that the engineered bacteria EcD provided by the present invention has a significant inhibitory effect on the growth of organoids derived from colorectal cancer patients.

[0155] Example 8 This embodiment follows Figure 13 The process shown in A evaluates the engineered probiotic HCS1 DKK3 provided by this invention in [the following context is missing from the original text] ApcMin / + Antitumor effects in mouse models: In this embodiment, 9-week-old males were selected for the animal experiment. Apc Min / + Mice were randomly divided into two groups as described below: HCS1 empty group: Mice were injected with HCS1 empty strain via tail vein, 100 μL per mouse, containing 1×10⁶ HCS1 strains. 8 CFU, administered once every two weeks.

[0156] HCS1 DKK3 group: Mice were injected with HCS1 DKK3 strain via tail vein, 100 μL per mouse, containing 1×10⁶ bacteria. 8 CFU, administered once every two weeks.

[0157] At 20 weeks of age, mice from each group were sacrificed and dissected. The small intestine of the mice was separated, and the adenomas were counted.

[0158] Figure 13 It is the tail vein injection of engineered probiotic HCS1-pJ23119-pelB-DKK3-HA Apc Min / + The experimental results for mice are shown in the figure. Figure 13 B in the figure shows the statistical results of the number of tumors in the small intestine of mice in different treatment groups. Figure 13 B in the study showed that HCS1 DKK3 treatment significantly reduced the number of adenomas in the small intestine of mice and inhibited tumor progression.

[0159] Figure 13 This indicates that HCS1 DKK3 in Apc Min / + It showed good anti-cancer effects in mouse models.

[0160] Example 9 This embodiment follows Figure 14 The flowchart shown in section A illustrates the evaluation of the anti-tumor effect of the engineered probiotic HCS1DKK3 provided by this invention in the MC38 xenograft mouse model: In this embodiment, 9-week-old male healthy C57BL / 6 mice were selected for the animal experiment. Each mouse was injected subcutaneously with 50 μL of MC38 cell suspension (containing 1×10⁻⁶ cells) on its right side using a 0.5 mL syringe. 6 (1 cell, solvent: DPBS), until the tumor size reaches 100 mm. 3 At that time, they were randomly divided into the following two groups: HCS1 empty group: Mice were injected with HCS1 empty strain via tail vein, 100 μL per mouse, containing 1×10⁶ HCS1 strains. 7CFU is injected once during the entire process, and the day of injection is defined as day 0.

[0161] HCS1 DKK3 group: Mice were injected with HCS1 DKK3 strain via tail vein, 100 μL per mouse, containing 1×10⁶ bacteria. 7 CFU is injected once during the entire process, and the day of injection is defined as day 0.

[0162] Tumor size and mouse weight were measured every 3 days, and mouse survival time was recorded.

[0163] The survival rate (%) is calculated as follows: (Number of mice surviving on day n after probiotic injection / Number of mice on the day of injection) × 100%.

[0164] Figure 14 This is a diagram showing the experimental results of tail vein injection of engineered probiotic HCS1-pJ23119-pelB-DKK3-HA in MC38 subcutaneous tumor model mice.

[0165] Figure 14 B in the figure shows the tumor growth curve monitoring graphs for different treatment groups after treatment; Figure 14 B in the figure indicates that HCS1 DKK3 treatment significantly inhibits the growth of MC38 tumors.

[0166] Figure 14 C in the figure shows a graph of mouse weight monitoring in different treatment groups after treatment; Figure 14 The C value indicates that HCS1DKK3 treatment did not significantly reduce the weight of mice.

[0167] Figure 14 D in the figure shows a survival time monitoring graph of mice in different treatment groups after treatment. Figure 14 The D in the figure indicates that treatment with HCS1 DKK3 can prolong the survival of tumor-bearing mice.

[0168] Figure 14 This indicates that HCS1 DKK3 still exhibits good anti-cancer effects in MC38 xenograft mouse models via tail vein treatment.

[0169] Example 10 This example illustrates the colonization of different doses of engineered probiotic EcD in the intestines of wild-type mice.

[0170] In this embodiment, 8-week-old male C57BL / 6J mice were randomly divided into the following two groups: 1×10 9 Group: Mice were administered EcD strain orally via gavage, 200 μL per mouse, containing 1×10⁻⁶ bacteria. 9 CFU, administered via gavage only once.

[0171] 1×10 8 Group: Mice were administered EcD strain orally via gavage, 200 μL per mouse, containing 1×10⁻⁶ bacteria. 8 CFU, administered via gavage only once.

[0172] Fresh feces were collected from mice at 6, 24, 72 and 168 hours after gavage, weighed, and added to sterile PBS (the volume of sterile PBS (mL) was 9 times the mass of feces (g)). The mixture was vortexed thoroughly, and 100 μL of the suspension was serially diluted with 900 μL of sterile PBS. 100 μL of the diluted suspension was then spread on LB agar plates containing chloramphenicol resistance. The colonization of EcD in the intestines of wild-type mice was obtained by counting the number of single colonies on the plates.

[0173] Figure 15 It is an oral administration of engineered probiotics in different doses. E. coli Figure showing the colonization results of Nissle 1917-pJ23119-pelB-DKK3-HA in mouse feces.

[0174] Figure 15 This indicates that a single dose of 1×10 9 CFU bacteria showed better colonization in the mouse gut.

[0175] Comparative Example 1 In this comparative study, protein supernatant containing DKK3 was used to treat MC38 mouse models with anti-tumor therapy.

[0176] (1) Inoculate bacteria EcN and EcD into LB liquid medium containing chloramphenicol, incubate overnight at 37°C, and then inoculate into 15 mL of fresh LB liquid medium containing chloramphenicol at a volume ratio of 1:100. (2) After culturing for 12 hours, 10 mL of EcD bacterial supernatant and EcN bacterial supernatant were filtered through a 0.22 µm filter membrane, concentrated 10 times to 1 mL using an ultrafiltration tube, and then replaced with sterile PBS 3 times to obtain 1 mL of protein solution. (3) After measuring the protein concentration in the protein solution using a Nanodrop 2000 spectrophotometer, the protein solution was injected into the tumor site of the MC38 mouse model. Each mouse was injected with 50 µL of protein solution (containing 100 µg of protein). (4) Monitor the tumor volume of different treatment groups and plot the tumor growth curves of different treatment groups. The results are shown in […]. Figure 16 .

[0177] Figure 16 This indicates that using only protein supernatant containing DKK3 does not significantly inhibit the growth of MC38 tumors.

[0178] Comparative Example 2 The method is similar to that in Example 9, except that MG1655-DKK3 is used instead of the engineered probiotic HCS1 DKK3 in Example 9, and MG1655-empty is used instead of the HCS1 empty strain in Example 9. The remaining steps were the same as in Example 9. The tumor volume of different treatment groups was monitored, and the tumor growth curves of different treatment groups were plotted to obtain the anti-tumor effect of MG1655-DKK3 in the MC38 mouse model.

[0179] Figure 17 The following graphs show tumor growth curves monitoring different treatment groups after treatment. Figure 17 This indicates that MG1655-DKK3 has no significant anti-cancer effect in the MC38 xenograft mouse model.

[0180] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A combined bacterial agent for the diagnosis and treatment of colorectal cancer, characterized in that, This combined bacterial agent contains separately preserved engineered probiotics and genetically engineered bacteria; the engineered probiotics include probiotics and a recombinant expression system introduced into the probiotics; the recombinant expression system contains the coding gene for the Dickkopf 3 protein; the amino acid sequence of the Dickkopf 3 protein is shown in SEQ ID NO.1; the probiotics are Escherichia coli Nissle 1917 and / or attenuated Salmonella Typhimurium HCS1; The genetically engineered bacteria can co-express speFL protein and luminescent protein; the luminescent protein includes Renaissance luciferase and / or superfolded green fluorescent protein. In the combined bacterial agent, the genetically engineered bacteria are used for in vitro culture and for oral administration and / or intravenous injection.

2. The combined microbial agent according to claim 1, characterized in that, The method of applying the combined microbial agent includes: (1) After co-culturing the genetically engineered bacteria with sample I taken from the individual to be diagnosed, the fluorescence signal of the luminescent protein in the genetically engineered bacteria is measured; (2) If the measured value of the fluorescence signal is greater than the threshold, the engineered probiotic is administered orally or intravenously to the corresponding individual; the threshold is the measured value of the fluorescence signal of the genetically engineered bacteria after co-culturing with sample II taken from a healthy individual; Sample I and Sample II are each independently feces and / or the supernatant obtained after grinding the feces; The daily dose of the engineered probiotics administered orally is 1 × 10⁻⁶. 9 -1×10 10 CFU; The daily dose of the engineered probiotics administered intravenously is 1 × 10⁻⁶. 7 -1×10 8 CFU.

3. The combined microbial agent according to claim 2, characterized in that, In the co-culture system, the concentration of the genetically engineered bacteria is 1 × 10⁻⁶. 7 -1×10 10 CFU / mL; And / or, the co-culture conditions include: a temperature of 36-37°C and a time of 4-6 h.

4. The combined microbial agent according to any one of claims 1-3, characterized in that, The nucleotide sequence of the Dickkopf 3 protein is shown in SEQ ID NO.2; And / or, the recombinant expression system comprises at least one of a signal peptide encoding gene, an HA tag protein encoding gene, and a promoter.

5. The combined microbial agent according to claim 4, characterized in that, The nucleotide sequence of the signal peptide encoding gene is shown in SEQ ID NO.3; And / or, the nucleotide sequence of the gene encoding the HA tag protein is shown in SEQ ID NO.4; And / or, the promoter is P J23119 Promoter; the P J23119 The nucleotide sequence of the promoter is shown in SEQ ID NO.

5.

6. The combined microbial agent according to any one of claims 1-3, characterized in that, The amino acid sequence of the speFL protein is shown in SEQ ID NO.

6.

7. The combined microbial agent according to any one of claims 1-3, characterized in that, The amino acid sequence of the Renaissance luciferase is shown in SEQ ID NO.7; And / or, the amino acid sequence of the superfolded green fluorescent protein is shown in SEQ ID NO.

8.

8. The combined microbial agent according to any one of claims 1-3, characterized in that, The chassis strain of the genetically engineered bacteria is Escherichia coli Nissle 1917.

9. The combined microbial agent according to any one of claims 1-3, characterized in that, The promoter of the speFL protein-encoding gene is the P J23119 Promoter.

10. The use of the combined bacterial agent according to any one of claims 1-9 in the preparation of a medicament for the early diagnosis and treatment of colorectal cancer.

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