Construction method and application of tumor organoid generation model

Abstract

The invention relates to the technical field of biological medicine, in particular to a construction method and application of a tumor organoid generation model, and more particularly relates to an organoid gene operon-based tumor generation model and an evaluation method of a mutation system of the organoid gene operon-based tumor generation model. According to the method, the tumor organoid generation model which can promote normal tissue organoid cancerization and has the in-vivo tumor formation capacity is established through gene manipulation, an evaluation system of the generation model is established, the tumor generation process can be more accurately simulated by utilizing the tumor organoid generation model, the success rate of tumor transplantation is increased, and the method is suitable for large-scale popularization and application. The method is a very valuable tool in research and development of tumor treatment strategies.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a method for constructing a tumor organoid generation model and its uses, and more specifically, to a method for evaluating a tumorigenesis model and its mutation system based on organoid gene manipulation. Background Art

[0002] Endometrial cancer is the fourth most common malignancy among women globally, and its incidence is on the rise worldwide. Most patients are postmenopausal women. Most endometrial cancers occur randomly, and there are also some hereditary cases caused by reproductive system mutations, mainly mismatch repair gene mutations. Endometrial cancer is histologically classified according to the World Health Organization classification system and can be divided into the following subtypes: endometrioid carcinoma (70%-80%), serous carcinoma, not otherwise specified (10%), clear cell carcinoma, not otherwise specified (6%), mixed cell adenocarcinoma, and other relatively rare types, including mucinous adenocarcinoma, neuroendocrine tumors (further divided into small cell, large cell carcinoma, or carcinoid), dedifferentiated carcinoma, and undifferentiated carcinoma. Currently, the use of immunohistochemical markers to characterize endometrial features helps to distinguish subtypes, and common ones include p53, PTEN, estrogen receptor ER, progesterone receptor PR, etc. Reports have analyzed and identified 4 types of endometrial cancer with different clinical, pathological, and molecular characteristics: POLE (ultramutated) (7%), microsatellite instability (MSI) / hypermutated (28%), low-grade intraepithelial neoplasia / microsatellite stable (39%), and serous / high-grade intraepithelial neoplasia (26%). The POLE group contains TP53 mutations; the MSI group contains MLH1, ARID5B, PTEN, PIK3CA, and PIK3R1 mutations; there are also other common mutations such as KRAS, CTNNB1, etc. Studying the pathogenesis of endometrial cancer helps to promote the progress of understanding the molecular biology of endometrial cancer. Developing efficient endometrial cancer or tumor models can improve the success rate of clinical trials and is beneficial in treating advanced and recurrent diseases.

[0003] Currently, the animal models of endometrial cancer commonly used in basic medical research mainly include genetically engineered animal models, tumor cell line xenografts, and tumor tissue xenograft models (PDX, patient-derived xenograft model). However, each of these models has some limitations and drawbacks. Genetically engineered animal models have a better tumor microenvironment and reproducibility, but the cost of preparing transgenic animals is high and the preparation cycle is long. The cell line xenograft model can easily transplant human tumor cell lines into model animals, with simple preparation and high reproducibility. However, the cell line xenograft model requires the use of immunodeficient mice, which may have significant differences from primary tumors in terms of development and pathophysiological conditions. PDX transplants tumor tissues from patients into model animals, with a genotype closer to the actual tumor, but its development is hindered by high costs and low success rates.

[0004] Organoid models provide a new method for studying endometrial tumorigenesis. Organoids are three-dimensional tissue structures cultured in vitro, which can better simulate the real tissue environment and be closer to the actual human situation. In addition, organoid models can be stably passaged for a long time, avoiding the influence of gene mutations or drifts on the model results, and having high reproducibility.

[0005] In the prior art, the samples for culturing endometrial cancer organoids are mostly taken from endometrial tissue samples of clinical endometrial cancer patients or endometrial tissue samples of endometrial cancer animal models. Endometrial cancer organoids cultured based on cancer and adjacent tissue samples of clinical patients are more suitable for the actual situation of patients and are mostly used for drug screening research on specific patients. However, the number of clinical samples is scarce, the sample differences are large, and the organoids directly cultured from endometrial cancer tissue samples cannot simulate the process of endometrial tumorigenesis and cannot conduct more in-depth research on the causes of endometrial cancer in terms of mechanism.

[0006] Therefore, there is an urgent need to develop a tumor organoid generation model and evaluation system that can more accurately simulate the process of endometrial tumorigenesis, improve the success rate of xenografts, and provide a valuable tool for the development of treatment strategies. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems in the related art to some extent.

[0008] To this end, the first aspect of the present invention provides a method for constructing a tumor organoid generation model, including:

[0009] 1) Performing organoid culture on normal tissue samples to obtain organoids of the tissue samples;

[0010] 2) Co-culture the gene editing-related vector with the organoids to obtain gene-edited organoids.

[0011] The gene editing in step 2) includes the insertion, deletion or replacement of proto-oncogenes and / or tumor suppressor genes.

[0012] In the gene-edited organoids, the expression level of proto-oncogenes increases and / or the expression level of tumor suppressor genes decreases.

[0013] The present invention provides a method for constructing a tumor organoid generation model. Using the method of the present invention to construct tumor organoids can more precisely simulate the tumorigenesis process, can predict in advance whether the tumor organoids can successfully form tumors, that is, can estimate the tumorigenic ability of tumor organoids, improve the tumorigenesis rate of organoids in animal models, provide a valuable tool for studying the causes of tumors, and is beneficial to the research and development of treatment strategies for related tumors.

[0014] Using the method for constructing a tumor organoid generation model provided by the present invention, specific genes can be directionally mutated, and by observing the impact of the mutation of specific genes on the formation of organoids, the association between the mutation of specific genes and the causes of tumors or other gene-related diseases can be studied.

[0015] According to an embodiment of the present invention, the gene editing includes knocking out tumor suppressor genes and / or increasing the copy number of proto-oncogenes.

[0016] According to an embodiment of the present invention, the proto-oncogenes include at least one selected from the group consisting of ras, myc, sis, myb, and src.

[0017] According to an embodiment of the present invention, the tumor suppressor genes include at least one selected from the group consisting of Rb, P53, APC, nm23, CDKN2A, WT, DCC, Axin, VHL, WTI, MSH, and MLH.

[0018] According to an embodiment of the present invention, the gene editing includes at least one of the following:

[0019] i. Knock out the MLH1 gene;

[0020] ii. Knock out the LKB1 gene;

[0021] iii. Knock out the p53 gene;

[0022] iv. Knock out the PTEN gene;

[0023] v. Overexpress the KRAS mutant gene;

[0024] vi. Overexpress the MYC gene;

[0025] vii. Overexpress the Ctnnb1 mutant gene;

[0026] viii. Overexpress the PIK3CA mutant gene,

[0027] wherein,

[0028] the protein expressed by the KRAS mutant gene has p.Gly12Val compared with the protein expressed by the wild-type KRAS gene,

[0029] the protein expressed by the Ctnnb1 mutant gene has p.Pro3del compared with the protein expressed by the wild-type Ctnnb1 gene,

[0030] the protein expressed by the PIK3CA mutant gene has p.Glu545Lys compared with the protein expressed by the wild-type PIK3CA gene.

[0031] According to an embodiment of the present invention, compared with the protein expressed by the wild-type KRAS gene, the 12th amino acid of the protein expressed by the KRAS mutant gene is mutated from G to V; compared with the protein expressed by the wild-type Ctnnb1 gene, the 545th amino acid of the protein expressed by the Ctnnb1 mutant gene is mutated from E to K; compared with the protein expressed by the wild-type PIK3CA gene, the 3rd amino acid (proline) of the protein expressed by the PIK3CA mutant gene is lost.

[0032] In the present invention, the nucleotide of the wild-type KRAS gene is a human sequence, and the gene accession number is NCBI ID: 3845; the nucleotide sequence of the wild-type Ctnnb1 gene is a human sequence, and the gene accession number is NCBI ID: 1499; the nucleotide sequence of the wild-type PIK3CA gene is a human sequence, and the gene accession number is NCBI ID: 5290.

[0033] In the present invention, the nucleic acid sequence of the MLH1 gene is a human sequence, and the gene accession number is NCBI ID: 4292; the nucleic acid sequence of the LKB1 gene is a human sequence, and the gene accession number is NCBI ID: 6794; the nucleic acid sequence of the p53 gene is a human sequence, and the gene accession number is NCBI ID: 7157; the nucleotide sequence of the PTEN gene is a human sequence, and the gene accession number is NCBI ID: 5728; the nucleotide sequence of the MYC gene is a human sequence, and the gene accession number is NCBI ID: 4609.

[0034] According to an embodiment of the present invention, the tissue sample includes at least one of an endometrial tissue sample, a liver tissue sample, a heart tissue sample, and an intestinal tissue sample. It should be noted that other types of tumor organoid generation models known in the art can be constructed by the method described in the present invention.

[0035] According to a specific embodiment of the present invention, the culture medium for organoid culture includes any one of a complete endometrial organoid medium, a complete heart organoid medium, a complete liver organoid medium, and a complete intestinal organoid medium. For different organoid cultures, the culture media used are different.

[0036] According to an embodiment of the present invention, the gene editing technology includes at least one of shRNA-based, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1, and zinc finger nuclease technology. It should be noted that other gene editing technologies known in the art are within the scope of application of the present invention.

[0037] According to an embodiment of the present invention, the gene editing-related vector includes any one of a viral vector, a plasmid, and a phage.

[0038] According to an embodiment of the present invention, the viral vector includes at least one of a retroviral vector, a lentiviral vector, and an adenoviral vector.

[0039] According to an embodiment of the present invention, the gene-edited viral vector includes a lentivirus with knockdown of tumor suppressor genes and / or a retrovirus with overexpression of proto-oncogenes.

[0040] According to a specific embodiment of the present invention, the organoid culture in step 1) includes primary culture and passage culture.

[0041] According to a specific embodiment of the present invention, the organoid culture in step 1) includes:

[0042] a) Digest the normal endometrial tissue sample to obtain single cells and / or cell clusters of the endometrial tissue sample;

[0043] b) Co-culture Matrigel with the endometrial single cells and / or cell clusters after removing red blood cells to obtain Matrigel with attached organoids;

[0044] c) Perform passage culture on the Matrigel with attached organoids.

[0045] According to a specific embodiment of the present invention, the organoid culture medium is an endometrial organoid culture medium. It should be noted that any culture medium known in the art that can culture endometrial organoids is applicable to the present invention. Preferred medium components include: DMEM / F12 serum-reduced medium, neural / stem cell culture additives or similar additives, HEPES buffer, L-glutamine or similar additives, a mixture of streptomycin and penicillin antibiotics, N-Acetylcysteine, N2, lipid concentrate, estradiol, Nicotinamide, SB202190, Y-27632, A83-01, R-spondin 1, Noggin, EGF, HGF50, FGF10, IGF.

[0046] According to a specific embodiment of the present invention, when the concentration of the HEPES buffer is 10 mM, the concentration of the L-glutamine or similar additives is 2 - 4 mM, the concentration of the N-Acetylcysteine is 1 - 3 mM, the dilution ratio of the N2 is 100 ± 5 times, the concentration of the Nicotinamide is 3 - 10 mM, the concentration of the SB202190 is 0.01 - 1 μM, the concentration of the Y-27632 is 5 - 20 μM, the concentration of the A83-01 is 0.1 - 0.8 μM, the concentration of the R-spondin 1 is 200 - 800 ng / mL, the concentration of the Noggin is 50 - 300 ng / mL, the concentration of the EGF is 10 - 90 ng / mL, the concentration of the HGF is 10 - 100 ng / mL, the concentration of the FGF10 is 50 - 400 ng / mL, and the concentration of the IGF is 20 - 60 ng / mL, the effect of culturing endometrial organoids is better.

[0047] The second aspect of the present invention provides a tumor organoid generation model constructed by the method described in the first aspect.

[0048] According to a specific embodiment of the present invention, the tumor organoids include at least one of endometrial organoids, liver organoids, heart organoids, and intestinal organoids.

[0049] The third aspect of the present invention provides an animal model by injecting the tumor organoid generation model described in the second aspect subcutaneously into an animal.

[0050] According to a specific embodiment of the present invention, the animal is a mammalian animal, and the mammalian animal includes any one of mouse, pig, cow, and sheep.

[0051] The fourth aspect of the present invention provides the use of the tumor organoid generation model described in the second aspect and the animal model described in the third aspect in studying tumor causes and drug screening.

[0052] The organoid formation model constructed by the method described in the first aspect of the present invention can study the association between the mutation of a specific gene and the cause of tumorigenesis or other gene-related diseases by performing directed mutation on the specific gene and observing the effect of the mutation of the specific gene on organoid formation.

[0053] By applying different drugs to tumor organoid formation models or animal models constructed based on different gene mutations, drugs suitable for specific gene mutations can be screened out, providing value for the research and development of treatment strategies.

[0054] The fifth aspect of the present invention provides a method for evaluating a tumor organoid formation model mutation system, which performs single-cell clone formation analysis on the tumor organoid formation model described in the second aspect.

[0055] According to the specific embodiments of the present invention, the tumorigenic ability of the tumor organoid formation model is proportional to the number of clones of the single cells per unit time. Within a unit time, the higher the number of clones of a single cell, the higher the probability that the organoid formed by the single cell will form a tumor.

[0056] The object of the present invention is to provide a method for constructing an organoid tumorigenesis model by gene manipulation, and a method for an evaluation system that can predict in advance whether an organoid can successfully form a tumor. A method for evaluating a tumorigenesis model mutation system based on organoid gene manipulation can predict in advance the tumorigenic ability of tumor organoids and improve the tumorigenesis rate of organoids in an animal model in vivo.

[0057] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0059] Figure 1 is a schematic diagram of tumor model construction in an embodiment of the present invention;

[0060] Figure 2 is the map of PLKO.1 empty plasmid in Example 3 of the present invention;

[0061] Figure 3 is the map of PWPI empty plasmid in Example 3 of the present invention;

[0062] Figure 4 is the bright field picture of the growth of gene-edited organoids in Example 3 of the present invention;

[0063] Figure 5It is a bar chart showing the number of clones after one month of single - cell organoid cloning culture of gene - edited successful organoids in Example 4 of the present invention;

[0064] Figure 6 It is a subcutaneous tumor formation diagram of a mouse tumor in Example 4 of the present invention. Detailed implementation manners

[0065] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0066] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "a plurality" is two or more.

[0067] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0068] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0069] In this document, the term "comprising" or "including" is an open - ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0070] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequent event or condition may or may not occur, and this description includes the case where the event or condition occurs and the case where the event or condition does not occur.

[0071] In this article, "gene editing" refers to the editing (directed modification) of target genes and their transcripts, achieving the addition, deletion of specific DNA fragments, the deletion, substitution of specific DNA bases, etc., so as to change the sequence, expression level or function of the target gene or regulatory element. The basic principle is that sequence-specific endonucleases composed of a sequence-specific DNA-binding domain and a non-specific DNA-modifying domain recognize DNA target sites on chromosomes, cut them to generate DNA double-strand breaks, and induce DNA damage repair, thereby achieving the directed editing of the specified genome.

[0072] In this article, the term "proto-oncogene (cellular oncogene)" refers to an oncogene existing in the genome of normal cells of an organism. Under normal circumstances, the proto-oncogene existing in the genome is in a low-expression or non-expression state and plays an important physiological function. However, under certain conditions, such as viral infection, chemical carcinogens or radiation, the proto-oncogene can be abnormally activated, transformed into an oncogene, and induce cell carcinogenesis.

[0073] In this article, the term "tumor suppressor gene" or anti-oncogene is synonymous with tumor suppressor gene, which refers to a certain gene whose inhibition, inactivation, loss, or the loss of function of its expression product can lead to malignant transformation of cells; conversely, under experimental conditions, if it is introduced or activated, it can inhibit the malignant phenotype of cells.

[0074] In this article, the term "viral vector" can introduce genetic material into cells. The principle is to utilize the molecular mechanism by which a virus transmits its genome into other cells for infection. This can occur in intact living organisms (in vivo) or in cell culture (in vitro). It can be applied to basic research, gene therapy or vaccines.

[0075] In this article, the term "lentiviral vector" can effectively integrate foreign genes or foreign shRNAs into the host chromosome, thereby achieving the effect of persistent expression of the target sequence. In terms of infection ability, it can effectively infect various types of cells such as neuronal cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells, etc., thereby achieving good gene therapy effects. For some cells that are difficult to transfect, such as primary cells, stem cells, undifferentiated cells, etc., using lentiviral vectors can greatly improve the transduction efficiency of the target gene or target shRNA, and the probability of the target gene or target shRNA integrating into the host cell genome is greatly increased, enabling the long-term and stable expression of the target gene or target shRNA to be achieved more conveniently and quickly.

[0076] In this article, the term "Organoid" refers to a three-dimensional structure and physiological function that can simulate an in-vivo organ (or tissue) under normal (or diseased) conditions through 3D culture in vitro. Generally speaking, an organoid is a three-dimensional cell culture obtained by culturing stem cells in Matrigel and under the action of chemical small molecule inhibitors / activators, cytokines, culture medium additives, etc., resulting in a tissue structure similar to the corresponding organ, with characteristics such as cell proliferation and differentiation, self-renewal, self-assembly, long-term culturing, and genetic stability.

[0077] In this article, the term "Matrigel" is a soluble basement membrane preparation extracted from EHS mouse tumors rich in extracellular matrix proteins, whose main components consist of laminin, type IV collagen, heparan sulfate proteoglycan (HSPG), and nestin, etc., and also include various growth factors such as TGF-β, EGF, IGF, and FGF. At room temperature, Matrigel polymerizes to form a biologically active three-dimensional matrix that can simulate the structure, composition, physical properties, and functions of the in-vivo cell basement membrane, facilitating the culturing and differentiation of cells in vitro.

[0078] The present invention provides a method for constructing a tumor organoidogenesis model, including:

[0079] 1) Performing organoid culture on a normal tissue sample to obtain the organoid of the tissue sample;

[0080] 2) Co-culturing a gene editing-related vector with the organoid to obtain a gene-edited organoid,

[0081] The gene editing in step 2) includes the insertion, deletion, or replacement of proto-oncogenes and / or tumor suppressor genes,

[0082] The expression level of proto-oncogenes in the gene-edited organoid increases and / or the expression level of tumor suppressor genes decreases.

[0083] The present invention provides a method for constructing a tumor organoidogenesis model. Using the method of the present invention to construct tumor organoids can more precisely simulate the tumorigenesis process, can predict in advance whether a tumor organoid can successfully form a tumor, that is, can estimate the tumorigenic ability of the tumor organoid, improve the tumorigenesis rate of the organoid in an animal model in vivo, provide a valuable tool for studying the causes of tumors, and is more conducive to the research and development of treatment strategies for related tumors.

[0084] Using the method for constructing a tumor organoidogenesis model provided by the present invention, specific genes can be directionally mutated, and by observing the impact of the mutation of specific genes on the formation of organoids, the association between the mutation of specific genes and the causes of tumors or other gene-related diseases can be studied.

[0085] According to an embodiment of the present invention, the gene editing includes knocking out tumor suppressor genes and / or increasing the copy number of proto-oncogenes.

[0086] According to an embodiment of the present invention, the proto-oncogenes include at least one selected from the group consisting of ras, myc, sis, myb, and src.

[0087] According to an embodiment of the present invention, the tumor suppressor genes include at least one selected from the group consisting of Rb, P53, APC, nm23, CDKN2A, WT, DCC, Axin, VHL, WTI, MSH, and MLH.

[0088] According to an embodiment of the present invention, the gene editing includes at least one of the following:

[0089] i. Knocking out the MLH1 gene;

[0090] ii. Knocking out the LKB1 gene;

[0091] iii. Knocking out the p53 gene;

[0092] iv. Knocking out the PTEN gene;

[0093] v. Overexpressing the KRAS mutant gene;

[0094] vi. Overexpressing the MYC gene;

[0095] vii. Overexpressing the Ctnnb1 mutant gene;

[0096] viii. Overexpressing the PIK3CA mutant gene,

[0097] wherein,

[0098] compared with the wild-type KRAS gene, the KRAS mutant gene has p.12G>V,

[0099] compared with the wild-type Ctnnb1 gene, the Ctnnb1 mutant gene has p.3del P,

[0100] compared with the wild-type PIK3CA gene, the PIK3CA mutant gene has p.545E>K.

[0101] According to an embodiment of the present invention, the tissue sample includes at least one of an endometrial tissue sample, a liver tissue sample, a heart tissue sample, and an intestinal tissue sample. It should be noted that other types of tumor organoid generation models known in the art can be constructed by the method described in the present invention.

[0102] According to a specific embodiment of the present invention, the organoid culture in step 1) includes primary culture and passage culture.

[0103] According to a specific embodiment of the present invention, the organoid culture in step 1) includes:

[0104] a) digesting the normal endometrial tissue sample to obtain single cells and / or cell clusters of the endometrial tissue sample;

[0105] b) co-culturing the Matrigel with the single cells and / or cell clusters after removing red blood cells to obtain Matrigel with attached organoids;

[0106] c) subculturing the Matrigel with attached organoids.

[0107] According to a specific embodiment of the present invention, the culture medium for organoid culture includes any one of endometrial organoid complete medium, cardiac organoid complete medium, hepatic organoid complete medium, and intestinal organoid complete medium. Different organoid cultures use different culture media.

[0108] According to a specific embodiment of the present invention, the organoid culture medium is endometrial organoid medium. It should be noted that any culture medium known in the art that can culture endometrial organoids is applicable to the present invention. Preferred culture medium components include: DMEM / F12 serum-free medium, neural / stem cell culture additives or similar additives, HEPES buffer, L-glutamine or similar additives, a mixture of streptomycin and penicillin antibiotics, N-Acetylcysteine, N2, lipid concentrate, estradiol, Nicotinamide, SB202190, Y-27632, A83-01, R-spondin 1, Noggin, EGF, HGF50, FGF10, IGF.

[0109] According to the specific embodiments of the present invention, when the concentration of the HEPES buffer is 10 mM, the concentration of L-glutamine or the same type of additive is 2 - 4 mM, the concentration of N-Acetylcysteine is 1 - 3 mM, the dilution ratio of N2 is 100 ± 5 times, the concentration of Nicotinamide is 3 - 10 mM, the concentration of SB202190 is 0.01 - 1 μM, the concentration of Y-27632 is 5 - 20 μM, the concentration of A83-01 is 0.1 - 0.8 μM, the concentration of R-spondin 1 is 200 - 800 ng / mL, the concentration of Noggin is 50 - 300 ng / mL, the concentration of EGF is 10 - 90 ng / mL, the concentration of HGF is 10 - 100 ng / mL, and the concentration of FGF10 is 50 - 400 ng / mL, the effect of culturing endometrial organoids is better.

[0110] According to the embodiments of the present invention, the preferred primary culture method of the organoids is as follows: Wash the endometrial tissue with 1×PBS, and use a medical scissor to cut the tissue into fragments of 0.25 mm 3 fragments. Treat the tissue with normal tissue digestive fluid at 37°C and 220 rpm for 60 min to obtain endometrial cells. Take an appropriate amount of Matrigel resuspension, seed it in a 24-well plate at 30 μL / well, place it in a 37°C incubator, wait for 30 minutes, and then add the complete medium for endometrial organoids for culture.

[0111] According to the embodiments of the present invention, the preferred subculture method of the organoids is as follows: Remove the original organoid medium, add cold Advanced DMEM / F12, pipette to recover the organoids from the Matrigel, centrifuge to remove the supernatant and Matrigel, add 0.05% trypsin for digestion, treat at 37°C for 3 min, pipette evenly, wash the cells with Advanced DMEM / F12, remove the supernatant, mix the endometrial cells with Matrigel and seed them in a 24-well plate. After the Matrigel solidifies, add the complete medium for endometrial organoids for culture.

[0112] According to the specific embodiments of the present invention, the present invention provides an animal model, which includes injecting the tumor organoid generation model constructed by the above method subcutaneously into an animal.

[0113] According to the specific embodiments of the present invention, the animal is a mammal, including any one of mouse, pig, cow, and sheep.

[0114] According to the specific embodiments of the present invention, the present invention provides an animal model, and the method for constructing the animal model is as Figure 1 described, specifically including:

[0115] ①Perform organoid culture on normal tissue samples to obtain organoids of the tissue;

[0116] ②Co - culture the virus infection solution of the virus vector capable of gene editing with the organoids to obtain gene - edited organoids;

[0117] ③Inject the gene - edited endometrial organoids subcutaneously into an animal,

[0118] Wherein, the gene editing in step ② includes insertion, deletion or substitution of proto - oncogenes and / or tumor suppressor genes, and the expression level of proto - oncogenes is enhanced and / or the expression level of tumor suppressor genes is reduced in the gene - edited organoids.

[0119] According to the embodiments of the present invention, the techniques for gene editing include at least one of shRNA, antisense nucleic acid, ribozyme, dominant - negative mutation, CRISPR - Cas9, CRISPR - Cpf1 and zinc - finger nuclease. It should be noted that other techniques known in the art that can be used for gene editing are within the scope of the present invention.

[0120] According to the embodiments of the present invention, the virus vector includes at least one of retroviral vectors, lentiviral vectors, and adenoviral vectors.

[0121] According to the embodiments of the present invention, the gene - edited virus vector includes a lentivirus with knocked - down tumor suppressor genes and / or a retrovirus with overexpressed proto - oncogenes.

[0122] On the other hand, the present invention provides a method for evaluating a tumor organoid formation model mutation system. Perform single - cell clone formation analysis on the tumor organoid formation model constructed by the aforementioned method. Within a unit time, the higher the number of clones, the higher the probability that the single - cell belonging to the organoid forms a tumor.

[0123] According to the examples of the present invention, the preferred single - cell organoid cloning culture method is as follows: Remove the original organoid culture medium, add cold Advanced DMEM / F12, pipette the Matrigel to recover the organoids, centrifuge to remove the supernatant and Matrigel, add organoid digestive solution, treat at 37 °C for 5 min, pipette evenly and filter through a 40 - μm filter, wash the cells with 1×PBS, remove the supernatant, mix the endometrial cells with Matrigel and seed them in a 96 - well plate (3 μL of cell - Matrigel resuspension per well). After the Matrigel solidifies, add the complete endometrial organoid culture medium for culture.

[0124] According to the specific implementation scheme of the present invention, the present invention separates normal human endometrial cells and establishes normal human endometrial organoids with a specific culture medium, and then performs gene editing on the high-frequency mutant genes of endometrial cancer on the normal endometrial organoids, thereby establishing an endometrial tumor organoid occurrence model that can promote the carcinogenesis of normal endometrial organoids and has the ability to form tumors in vivo. The gene manipulation is to simulate the commonly abnormally expressed genes of endometrial cancer by means of lentivirus and / or retrovirus infection, and evaluate the efficacy of different genes and their common combinations in driving the occurrence of normal endometrium to endometrial cancer. The gene manipulation includes: knocking out the MLH1 gene; knocking out the LKB1 gene; knocking out the p53 gene; knocking out the PTEN gene; overexpressing the KRAS mutant gene; overexpressing the MYC gene; overexpressing the Ctnnb1 mutant gene; overexpressing the PIK3CA mutant gene; knocking out the MLH1 gene and overexpressing the KRAS mutant gene; knocking out the p53 gene and overexpressing the MYC gene.

[0125] According to the specific implementation scheme of the present invention, the present invention conducts a morphological evaluation on the endometrial epithelial organoids obtained after different gene interventions. During the continuous maintenance culture of the constructed organoids, most of the organoids show a cavity-like structure, while some in the MYC overexpression group show a solid-like structure. The organoids after the superposition of p53 and MYC also show a multi-layer solid-like structure similar to that of the MYC overexpressed organoids.

[0126] According to the specific implementation scheme of the present invention, the present invention conducts an evaluation on the colony formation ability of the endometrial epithelial organoids obtained after different gene interventions. After 1 month of continuous maintenance culture of the constructed organoids, the colony formation ability of PTEN in the knockdown group increases, and the overexpression groups of MYC, CTNNB1, and PIK3CA show an increase in colony formation ability. The double-gene superposition groups all show a slight upregulation of cloning ability. In the early stage, the colony formation ability of the PTEN knockdown group and the MYC overexpression group is stronger than that of other intervention groups, indicating that the endometrial cancer occurrence model constructed by single-gene manipulation has a faster proliferation rate and the potential to form tumors.

[0127] The following will explain the solution of the present disclosure in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. For those not specified in the examples regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0128] Example 1 A primary culture method for normal human endometrial tissue organoids

[0129] In this embodiment, a primary culture method for human endometrial normal tissue organoids is provided, which comprises the following steps:

[0130] (1) Tissue sample collection: After the operation, take 0.5*0.5*1 cm 3 of endometrial tissue, soak the endometrial tissue in tissue protective solution, and transfer it from the hospital to the laboratory. This study has passed the review of the ethics committee;

[0131] (2) Tissue sample washing: Clamp the tissue with forceps on ice and put it into PBS washing solution, gently rinse it 4 - 5 times, discard the PBS, and cut off the excess smooth muscle layer, etc.;

[0132] (3) Add 1 mL of digestive solution (composition of digestive solution: 0.4 mg / mL collagenase, dispaseⅡ1.25 U / mL; 1% FBS; 1X double antibody), and transfer the tissue to an untreated 6 cm dish at the bottom, fully cut the tissue into pieces to ensure that the tissue can be easily aspirated with a 10 mL pipette;

[0133] (4) Incubate on a shaker at 37°C at 220 rpm for 1 hour, and shake vigorously every 20 minutes;

[0134] (5) After the tissue is digested, take out the centrifuge tube, add 1 mL of FBS to 10 mL of digestive solution, mix well and let it stand for 3 minutes;

[0135] (6) Filter the supernatant through a 100 μm filter mesh respectively, and immediately filter the filtrate through a 40 μm filter mesh;

[0136] (7) Invert the 40 μm filter mesh and place it in a new 50 ML centrifuge tube;

[0137] (8) Use advanced DMEM / F12 to back - flush the 40 μm filter mesh to wash down the undigested endometrial epithelial cells sufficiently;

[0138] (9) Centrifuge at 300 g for 3 minutes, discard the supernatant (it can be seen that there is some red precipitate mixed in the upper layer of the white precipitate, which is the red blood cell precipitate);

[0139] (10) Add an appropriate amount of red blood cell lysate to resuspend the precipitate and let it stand at room temperature for 3 minutes;

[0140] (11) Centrifuge at 300 g for 3 minutes, discard the supernatant, and then resuspend the cell precipitate with 1 mL of advanced DMEM / F12;

[0141] (12) Centrifuge at 300 g for 3 minutes, discard the supernatant, and resuspend the cell precipitate with 1 mL of advanced DMEM / F12 again;

[0142] (13) Discard the supernatant and place the cells on ice;

[0143] (14) For the photoreceptor cell precipitate, add an appropriate amount of Matrigel and resuspend the precipitate.

[0144] (15) Take an appropriate amount of the Matrigel resuspension and seed it in a 24-well plate at 30 μL / well. Place it in an incubator at 37 °C and wait for 30 minutes. Then add 510 μL of endometrial organoid complete medium organoid culture medium. The primary culture should contain Y-27632 and fungicidal antibiotics. Change the medium after 3-4 days of P0 generation culture. At this time, the medium does not contain Y-27632 and fungicidal antibiotics.

[0145] (16) Culture in a cell incubator at 37 °C and 5% CO2, and change the medium every 2-3 days.

[0146] (17) Composition of endometrial complete medium:

[0147] DMEM / F12 serum-reduced medium, neural / stem cell culture supplement or similar supplement, 10 mM HEPES buffer, 2-4 mM L-glutamine or similar supplement, a mixture of streptomycin and penicillin antibiotics, 1.25 mM N-Acetylcysteine, N2 diluted 100 ± 5 times, lipid concentrate, 10 nM estradiol, 5 mM Nicotinamide, 0.1 μM SB202190, 10 μM Y-27632, 0.25 μM A83-01, 500 ng / mL R-spondin 1, 100 ng / mL Noggin, 50 ng / mL EGF, 50 ng / mL HGF, 100 ng / mL FGF10, 40 ng / mL IGF.

[0148] Example 2 A method for subculture of human endometrial normal tissue organoids

[0149] In this example, a method for subculture of human endometrial normal tissue organoids is provided, including the following steps:

[0150] (1) Remove the original medium of the organoids, add cold Advance DMEM / F12, and pipette the Matrigel with the organoids growing on it.

[0151] (2) Transfer the organoid Matrigel suspension to a 15 mL centrifuge tube and let it stand on ice for 15 min.

[0152] (3) Discard the supernatant and Matrigel by centrifuging at 300 g for 3 min, add 0.05% trypsin for digestion, and incubate at 37 °C for 3 min.

[0153] (4) Pipette the organoid suspension with a 200 μL pipette tip and wash the obtained cells with Advance DMEM / F12.

[0154] (5) After discarding the supernatant of 300g for 3 minutes, resuspend the precipitate with Matrigel and seed the cells in a 24-well plate;

[0155] (6) Incubate the culture plate in an incubator at 37°C for 30 minutes to solidify the Matrigel;

[0156] (7) Slowly add 500 μL of complete growth medium for human endometrial organoids along the well wall;

[0157] (8) Culture in a cell incubator at 37°C and 5% CO2, and change the medium every 2 - 3 days to culture a sufficient number of normal human endometrial organoids;

[0158] (9) The formula of the complete growth medium is as in Example 1.

[0159] Example 3 Construction of a novel endometrial cancer model

[0160] In this example, a method for constructing a novel endometrial cancer model is provided, including the following steps:

[0161] (1) Pre-culture, the same as in Example 1;

[0162] (2) Expansion culture, the same as in Example 2;

[0163] (3) Gene editing, the types of gene editing are shown in Table 1:

[0164] a) Package the ShRNA lentivirus for knocking down tumor suppressor genes and the retrovirus for overexpressing proto-oncogenes. The inserted sequences of the relevant tumor suppressor genes and proto-oncogenes in the plasmid are shown in Table 2. Among them, all shRNA plasmids are carried by the PLKO.1 vector ( Figure 2 ); The overexpression sequences of MYC, PIK3CA, CTNNB1, and KRAS are carried by the PWPI vector ( Figure 3 );

[0165] b) Prepare the virus infection solution: Add 230 μL of complete medium for endometrial organoids (the growth factor concentration is doubled, and 10 nM of Y27632 and 25 μg of poloxamer 407 are added) to the cell amount in each 24-well plate. At the same time, add 20 μL of the concentrated virus solution with the relevant genes, and mix well;

[0166] c) After uniformly mixing the virus infection solution with the organoids, place them in a 48-well plate. Wrap the edge of the plate with sealing film, put it in a tabletop centrifuge, set the temperature to 32°C, centrifuge at 600g for 60 minutes, and culture in a cell incubator for 6 hours. The gene combinations are shown in Table 1;

[0167] d) Resuspend the cell precipitate in the plate and centrifuge to collect the cells;

[0168] e) Discard the supernatant. A small amount of liquid can be retained. Add 80 μL of Matrigel to resuspend the cell pellet, and then seed the cells into two 24-well plates. Place them in the cell culture incubator for 30 minutes, and then add 510 μL of endometrial organoid complete medium (containing 10 μM Y27632).

[0169] f) After culturing for 2 - 3 days, add puromycin (1 μg / mL) or G418 (400 mg / mL) to screen the organoids and continue to maintain for 2 weeks. Observe and photograph the organoids. Figure 4 This is the bright-field image of the successfully gene-edited organoids. During the continuous maintenance culture, most of the organoids showed a hollow structure, while some in the MYC overexpression group showed a solid-like organoid. The organoids after the superposition of p53 and MYC also showed a multi-layer solid-like structure similar to that of the MYC overexpressed organoids.

[0170] Table 1 Gene combinations of the tumor model

[0171] Gene combination Knocked-out gene Overexpressed gene Control - - 1 MLH1 - 2 LKB1 - 3 P53 - 4 PTEN - 5 - KRAS(G12V) 6 - MYC 7 - Ctnnb1(Δ3) 8 - PIK3CA(E545K) 9 MLH1 KRAS(G12V) 10 P53 MYC

[0172] Note: G12V means that the 12th amino acid of the protein is mutated from G to V, E545K means that the 545th amino acid of the protein is mutated from E to K, and Δ3 means that the 3rd amino acid of the protein is lost.

[0173] Table 2

[0174]

[0175]

[0176]

[0177] Among them, all shRNA plasmids were carried by the PLKO.1 vector; the overexpression sequences of MYC, PIK3CA, CTNNB1, and KRAS were carried by the PWPI vector.

[0178] Example 4 Evaluation method for the construction of a novel endometrial cancer model

[0179] 1. Take the successfully gene-edited organoids in Example 3 for single-cell organoid cloning culture. The specific steps are as follows:

[0180] a) Take two 24-well plates with well-grown organoids and discard the medium.

[0181] b) Take an appropriate amount of 0.05% trypsin and preheat it in the cell culture incubator for standby.

[0182] c) Resuspend the organoids by pipetting with 1 mL of advanced DMEM / F12. Pipette the organoids thoroughly, centrifuge at 300 g for 3 minutes, and discard the supernatant.

[0183] d) Add 500 μL of pre-warmed 0.05% trypsin into an EP tube, and pipette the organoids thoroughly. Place it in the cell culture incubator for digestion for 3 minutes;

[0184] e) Take out the EP tube, and pipette the organoids forcefully again using a 200 μL pipette tip. After there are no large cell clumps visible to the naked eye, add 500 μL of advanced DMEM / F12 and pipette to resuspend the organoids, then centrifuge at 300 g for 3 minutes and discard the supernatant;

[0185] f) Resuspend the organoids by pipetting with 1 mL of advanced DMEM / F12, centrifuge at 300 g for 3 minutes, and discard the supernatant;

[0186] g) Wash again, resuspend the organoids by pipetting with 1 mL of advanced DMEM / F12, filter the suspension through a 40 μm nylon filter, centrifuge at 300 g for 3 minutes, and discard the supernatant;

[0187] h) Resuspend the cell pellet by pipetting with 300 μL of advanced DMEM / F12. Then take 10 μL of the cell suspension and mix it with 10 μL of trypan blue solution, add it into a cell counting chamber, let it stand for 15 seconds, insert it into a thermofisher cell counter, record the cell number, and repeat the measurement 3 times;

[0188] i) Calculate the cell quantity. Take 1×10 4 cells and add 55 μL of Matrigel. Plate 10 96-well plates, with 5 μL of Matrigel in each well. Place it in the cell culture incubator for 10 minutes, and then add 120 μL of endometrial organoid complete medium;

[0189] j) Take a photo to record the growth status of the organoids on the 30th day.

[0190] Statistical analysis of the clone number of the organoids cultured for 30 days was performed. Specifically, after plating single cells, clones with a diameter greater than 50 μm were counted for cloning formation, and the t-test was used to evaluate the differences among groups to compare the single-cell organoid cloning formation numbers under different gene editing conditions. Figure 5 It is a graph showing the analysis of the clone number of single-cell clones cultured for one month in the organoids with successful gene editing. Among them, the Control group is the single-cell clone group of normal organoids without gene editing. The results show that the endometrial cancer occurrence model constructed by the method of the present invention has a faster reproduction rate and tumorigenic potential.

[0191] 2. Take the organoids with successful gene editing in Example 3 for animal experiments (organoid mouse tumor seeding). The specific steps are as follows:

[0192] a) Take a well - grown 24 - well organoid dosage, discard the culture medium, and add 1 mL of advanced DMEM / F12 to resuspend the organoids by pipetting;

[0193] b) Centrifuge at 300 g for 3 minutes, discard the supernatant, and add 1 mL of advanced DMEM / F12 again to resuspend the organoids by pipetting;

[0194] c) After resuspension and centrifugation until there is no obvious residual Matrigel in the organoids, discard the supernatant;

[0195] d) Add 100 μL of endometrial organoid complete medium and 100 μL of Matrigel, resuspend the organoid pellet and keep it on ice for later use;

[0196] e) Take female nude mice at 4 - 6 weeks old, wipe and disinfect the back skin with 75% ethanol;

[0197] f) Inject 200 μL of the organoid mixture into the subcutaneous tissue of the mouse back by the labyrinth method;

[0198] g) Observe the tumor formation after 2 months.

[0199] After 2 months, sacrifice the mice, dissect and remove the tumor masses respectively. Figure 6 This is the subcutaneous tumor formation diagram of the mice. Among them, the normal group is the normally developed mice without treatment. The results show that the organoids based on successful editing in Example 3 can form tumors in mice.

[0200] The present invention has established an endometrial tumor organoidogenesis model with the ability to promote the carcinogenesis of normal endometrial organoids and tumor formation in vivo through gene manipulation, and has established a model evaluation system, which can more accurately simulate the occurrence process of endometrial tumors, improve the success rate of transplanted tumors, and provide a valuable tool for the research and development of treatment strategies.

[0201] It should be noted that other known types of tumor organoidogenesis models in the art can be constructed by the method described in the present invention, such as liver tissue samples, heart tissue samples, intestinal tissue samples, etc. Other known gene - editing technologies in the art are also within the scope of application of the present invention. Using the method for constructing a tumor organoidogenesis model provided by the present invention, specific genes can be directionally mutated, and by observing the influence of the mutation of specific genes on organoid formation, the association between the mutation of specific genes and the causes of tumorigenesis or other gene - related diseases can be studied. Constructing tumor organoids by the method of the present invention can more accurately simulate the occurrence process of tumors, can predict in advance whether the tumor organoids can successfully form tumors, that is, can estimate the tumor - forming ability of tumor organoids, improve the tumor - forming rate of organoids in animal models, provide a valuable tool for studying the causes of tumors, and is more conducive to the research and development of treatment strategies for related tumors.

[0202] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", "some implementation manners" or "some examples", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0203] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a tumor organoid generation model, characterized in that, Comprising: 1) Performing organoid culture on a normal tissue sample to obtain organoids of the tissue sample; 2) Co-culturing a gene editing-related vector with the organoids to obtain gene-edited organoids, wherein the gene editing in step 2) includes insertion, deletion or replacement of a proto-oncogene and / or a tumor suppressor gene, and the expression level of the proto-oncogene increases and / or the expression level of the tumor suppressor gene decreases in the gene-edited organoids.

2. The method according to claim 1, wherein The gene editing includes knocking out a tumor suppressor gene and / or increasing the copy number of a proto-oncogene; Optionally, the proto-oncogene includes at least one selected from ras, myc, sis, myb, src; Optionally, the tumor suppressor gene includes at least one selected from Rb, P53, APC, nm23, CDKN2A, WT, DCC, Axin, VHL, WTI, MSH, MLH.

3. The method according to claim 1, wherein The gene editing includes at least one of the following: i. Knocking out the MLH1 gene; ii. Knocking out the LKB1 gene; iii. Knocking out the p53 gene; iv. Knocking out the PTEN gene; v. Overexpressing a KRAS mutant gene; vi. Overexpressing the MYC gene; vii. Overexpressing a Ctnnb1 mutant gene; viii. Overexpressing a PIK3CA mutant gene, wherein, the protein expressed by the KRAS mutant gene has p.Gly12Val compared with the protein expressed by the wild-type KRAS gene, the protein expressed by the Ctnnb1 mutant gene has p.Pro3del compared with the protein expressed by the wild-type Ctnnb1 gene, the protein expressed by the PIK3CA mutant gene has p.Glu545Lys compared with the protein expressed by the wild-type PIK3CA gene.

4. The method according to claim 1, wherein The tissue sample includes at least one of an endometrial tissue sample, a liver tissue sample, a heart tissue sample, an intestinal tissue sample; Optionally, the culture medium for performing organoid culture includes any one of an endometrial organoid complete medium, a heart organoid complete medium, a liver organoid complete medium, an intestinal organoid complete medium.

5. The method according to claim 1, wherein The gene editing technology includes at least one of the technologies based on shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1 or zinc finger nuclease technology; Optionally, the gene editing-related vector includes any one of a viral vector, a plasmid, a phage; Optionally, the viral vector includes at least one of a retroviral vector, a lentiviral vector, an adenoviral vector; Optionally, the gene-edited viral vector includes a lentivirus with a knocked-down tumor suppressor gene and / or a retrovirus with an overexpressed proto-oncogene.

6. A tumor organoid generation model, characterized in that, Constructed by the method according to any one of claims 1-5.

7. The tumor organoid generation model according to claim 6, wherein The tumor organoids include at least one of endometrial organoids, liver organoids, heart organoids, intestinal organoids.

8. An animal model, characterized in that, Injecting the tumor organoid generation model according to claim 6 or 7 subcutaneously into an animal.

9. The animal model according to claim 8, characterized in that, The animal is a mammalian animal, and the mammalian animal includes any one of a mouse, a pig, a cow, a sheep.

10. Use of the tumor organoid generation model according to claim 6 or 7 and the animal model according to claim 8 or 9 in studying the causes of tumors and drug screening.

11. An evaluation method for a tumor organoid generation model mutation system, characterized in that, Performing single-cell clone formation analysis on the tumor organoid generation model according to claim 6 or 7; Optionally, the tumorigenic ability of the tumor organoid generation model is proportional to the number of clones of the single cells per unit time.

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