C57BL / 6 transgenic mouse endometrial cancer organ and application thereof

Abstract

The invention belongs to the field of cell engineering, and relates to a C57BL / 6 transgenic mouse endometrial cancer organ and application thereof. The C57BL / 6 transgenic mouse endometrial cancer organ established in the invention has the advantages of strong repeatability in the construction process, good passage stability, realization of tumor formation on the mouse, especially subcutaneous tumor formation and in-situ implantation of tumor in the uterine cavity in the C57BL / 6 mouse with a sound immune system, provides a tool closer to the practical situation for researching the pathogenesis of endometrial cancer, and has a good application prospect. The model is used for screening drugs effective for PTEN and ARID1A combined deletion type endometrial cancer, and an experimental basis is provided for developing a more accurate and more effective endometrial cancer treatment strategy.

Description

Technical Field

[0001] The present invention belongs to the field of cell engineering and relates to a C57BL / 6 transgenic mouse endometrial cancer organoid and its uses. Background Art

[0002] The incidence of endometrial cancer has been continuously rising globally. According to relevant research predictions, the incidence of endometrial cancer will exceed that of colorectal cancer and become the third most common tumor type and the fourth leading cause of cancer-related death in the female population. Basic research and drug development based on endometrial cancer cell lines are important intervention research approaches. Currently, most are human endometrial cancer cell lines transplanted into mice to construct tumor models. Due to species immune rejection, it often affects the accuracy and reliability of experimental results. Murine endometrial cancer cell lines can, to a certain extent, avoid this immune barrier. Murine endometrial cancer cell lines can more precisely understand the natural process of tumorigenesis and development in mice, including the formation of the tumor microenvironment, the interaction between immune cells and tumor cells, etc., and are not interfered by human-mouse immune incompatibility. This not only helps to conduct more realistic and effective exploration of tumor mechanisms in mouse models, such as studying the impact of specific gene mutations on the progression of endometrial cancer and exploring the signal transduction pathways between tumor cells and surrounding stromal cells, but also provides a more practical experimental basis for the development of new treatment methods for endometrial cancer. Based on murine endometrial cancer cell lines for drug screening and efficacy evaluation can more accurately predict the effects of drugs in mice, and then provide more reliable reference data for subsequent translation to human clinical trials, accelerating the R & D process of endometrial cancer treatment drugs. The current shortage of murine endometrial cancer cell lines has indeed caused many obstacles and dilemmas to the basic research and drug development of endometrial cancer.

[0003] Primary endometrial cancer cells usually show low proliferative ability under 2D culture conditions and are difficult to maintain cell viability and passage ability for a long time; in addition, the 2D culture system lacks the interaction between tumor cells and the extracellular matrix (ECM) and immune cells, making it difficult to simulate the complex tumor microenvironment in vivo, which may lead to endometrial cancer cells losing their original biological characteristics easily during passage. Therefore, when constructing an endometrial cancer tumor animal model by subculturing endometrial cancer cells under conventional methods, the success rate is not high, and a large amount of effective cell mass needs to be implanted at the initial stage of the experiment. However, due to the difficulty of subculturing primary endometrial cancer cells in 2D culture, it is very difficult to obtain sufficient effective cancer cells. Summary of the Invention

[0004] The molecular genetic landscape of endometrial cancer has been comprehensively elucidated by The Cancer Genome Atlas (TCGA), and high frequencies of somatic mutations have been detected in known tumor driver genes, including PTEN (65%), PIK3CA (53%), ARID1A (33%), and CTNNB1 (30%). Endometrioid carcinoma accounts for approximately 80% of newly diagnosed endometrial cancer cases, and PTEN (or PIK3CA) and ARID1A mutations highly coincide in endometrioid carcinoma. In endometrial atypical hyperplasia tissue, the precursor lesion of endometrioid carcinoma, the PTEN mutation rate is 16%. Most endometrial atypical hyperplasias are indolent and rarely progress to endometrioid carcinoma unless other molecular genetic events are acquired. ARID1A mutations are very rare in endometrial atypical hyperplasia, but approximately 41.5% of cases of endometrioid carcinoma have concurrent PTEN and ARID1A mutations. Therefore, by constructing a conditional knockout mouse model of Pten and Arid1a genes, the tumorigenesis process in clinical patients can be more accurately simulated, the relationship between PTEN and ARID1A mutations driving the progression of endometrial cancer can be understood, a tool closer to the actual situation can be provided for studying the pathogenesis of endometrial cancer, and drugs effective against PTEN- and ARID1A-deficient endometrial cancer can be screened using this model, providing an experimental basis for developing more precise and effective treatment strategies for endometrial cancer.

[0005] The present invention innovatively constructs a Pten- and Arid1a-knockout endometrial cancer animal model and optimizes conditions to construct murine endometrial cancer PAD organoids for exploring the molecular mechanisms and diagnosis and treatment regimens of endometrial cancer.

[0006] To achieve the above object, in the first aspect of the present invention, C57BL / 6 mouse endometrial cancer PAD organoids are disclosed. The C57BL / 6 mouse endometrial cancer PAD organoids are preserved in the China Center for Type Culture Collection, with the preservation date being December 7, 2023, and the preservation number being CCTCC NO: C2023321.

[0007] In the second aspect of the present invention, the application of the above C57BL / 6 mouse endometrial cancer PAD organoids in constructing a tumor animal model is disclosed.

[0008] In the third aspect of the present invention, a tumor animal model is disclosed. The tumor animal model is constructed by inoculating cells from the above C57BL / 6 mouse endometrial cancer PAD organoids into a mouse.

[0009] Preferably, the tumor animal model is an endometrial cancer mouse model.

[0010] Preferably, the tumor animal model includes a subcutaneous transplanted tumor implantation model, an orthotopic uterine cavity implanted tumor model, or a footpad tumor-lymph node model.

[0011] The fourth aspect of the present invention discloses the application of the above-mentioned C57BL / 6 mouse endometrial cancer PAD organoids or tumor animal models in the preparation of disease diagnosis products.

[0012] Preferably, the application in the preparation of disease diagnosis products includes the development, manufacture, or screening of products related to the immune process of human cells and the application in the manufacture or screening of human antibodies.

[0013] The fifth aspect of the present invention discloses the application of the above-mentioned C57BL / 6 mouse endometrial cancer PAD organoids or the above-mentioned tumor animal models in drug screening.

[0014] Preferably, the application in drug screening includes a) screening, efficacy detection, evaluation of efficacy, verification, or evaluation of regulators of endometrial cancer-related pathways in vivo; or,

[0015] b) studying the gene functions of endometrial cancer with combined knockout of Pten and Arid1a, studying the drugs and drug effects targeting the therapeutic target sites of endometrial cancer, and the uses in studying drugs for immune-related diseases and anti-tumor or anti-inflammatory drugs.

[0016] The sixth aspect of the present invention discloses the application of the above-mentioned C57BL / 6 mouse endometrial cancer PAD organoids or tumor animal models in scientific research protocols for non-therapeutic purposes in vitro.

[0017] Preferably, the application in scientific research protocols for non-therapeutic purposes in vitro includes:

[0018] a) Application as a model system in pharmacological, immunological, microbiological, and medical research; or,

[0019] b) Production involving the immune process of human cells and the use of animal experimental disease models for application in etiological research.

[0020] The inventors of the present application constructed a C57BL / 6 mouse endometrial cancer animal model and successfully isolated and cultured mouse endometrial cancer PAD organoids. The C57BL / 6 transgenic mouse endometrial cancer organoids of the present invention have strong repeatability in the construction process and good passage stability, and can form tumors in mice, especially can form tumors subcutaneously and orthotopically implant tumors in the uterine cavity of immune-competent C57BL / 6 mice, providing a tool closer to the actual situation for studying the pathogenesis of endometrial cancer. Using this model to screen drugs effective for PTEN and ARID1A combined deletion endometrial cancer provides an experimental basis for developing more precise and effective endometrial cancer treatment strategies. The scientific description of the C57BL / 6 mouse endometrial cancer animal model claimed in the present invention is "C57BL / 6 mouse endometrial cancer PAD organoids", which is deposited in the China Center for Type Culture Collection, with the deposit number of CCTCC NO: C2023321, the deposit date of December 7, 2023, and the deposit address of Wuhan University, Wuhan, Hubei Province, China. The above-mentioned C57BL / 6 mouse endometrial cancer PAD organoids have been tested in the China Center for Type Culture Collection, and the result is that they are alive. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or in the existing methods and experiments, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0022] Figure 1 For Pten in Example 1 flox / flox Agarose gel electrophoresis result diagram of mice, note: B6 is the negative control, which is the genomic DNAs of C57BL / 6 mice; N is the blank control, the control without template; DL2000 Marker, 2000bp\1000bp\750bp\500bp\250bp\100bp.

[0023] Figure 2 For constructing Arid1a in Example 1 flox / flox Flow chart of obtaining intermediate product AIB1177 - ABC in Arid1a mice.

[0024] Figure 3 For the enzyme digestion verification diagram of taking about 800 ng of plasmid and using FspI: 4273, 2449, 1172 with a 20 μl enzyme digestion system in Example 1 to measure the concentration.

[0025] Figure 4 For Arid1a in Example 1 flox / flox Agarose gel electrophoresis result diagram of mice, note: B6 is the negative control, which is the genomic DNAs of C57BL / 6 mice; N is the blank control, the control without template; 1000Marker, 1000bp\900bp\800bp\700bp\600bp\500bp\400bp\300bp\200bp\100bp; 36, 38, 39 are Arid1a flox / flox homozygous mice, 37 is Arid1a flox / - heterozygous mice.

[0026] Figure 5 Agarose gel electrophoresis results of PR-Cre homozygous mice. B6 is the negative control, which is the genomic DNA of C57BL / 6 mice; N is the blank control, the control without template; 1000Marker, 1000bp\900bp\800bp\700bp\600bp\500bp\400bp\300bp\200bp\100bp; 36 and 39 are PR-Cre mice, and 37 and 38 are wild-type mice.

[0027] Figure 6 WB identification results of PAD organoids in Example 2.

[0028] Figure 7 Results of the subcutaneous xenograft tumor implantation model of PAD organoids in Example 4.

[0029] Figure 8 Results of the in-situ tumor implantation model of PAD organoids in the uterine cavity in Example 5.

[0030] Figure 9 Results of the footpad tumor-lymph node model of PAD organoids in Example 6. Detailed implementation methods

[0031] The present invention will be further elaborated below in conjunction with specific embodiments. For the experimental methods and techniques without specific conditions indicated in the following embodiments, they are generally carried out according to the conventional conditions in the relevant field or according to the conditions recommended by the manufacturer.

[0032] Example 1, Pten flox / flox , Arid1a flox / flox , Construction of PR-Cre endometrial cancer mouse model

[0033] 1. Obtain Pten flox / flox mice

[0034] (1) Pten flox / flox Male mice (MGI ID MGI:3707004) were purchased from The Jackson Laboratory in the United States, and the strain name is B6.129S4-Pten tm1Hwu / J, with loxP sites on both sides of the 5th exon of the Pten gene on chromosome 19. Homozygous mice carrying the "floxed" (inducible gene knockout) allele can survive, be fertile, have a normal body size, and show no obvious physical or behavioral abnormalities. When mated with a strain expressing Cre recombinase, tissue-specific mutants for the "floxed" allele can be generated.

[0035] (2) Pten flox / flox Biological purification of mice

[0036] Three 6- to 8-week-old Pten flox / flox After the male homozygous mice were taken out of isolation, the biological purification process was initiated.

[0037] ① Sperm collection: After sacrificing the mice, the epididymides were removed using sterile ophthalmic scissors and forceps. The cauda epididymis was placed in a Petri dish containing M2 culture medium, and the cauda epididymis was gently squeezed with forceps to allow the sperm to swim out into the buffer. The motility and morphology of the sperm were observed under a microscope.

[0038] ② Fresh sperm transplantation: Three surrogate female mice of the same age with an ICR background were selected. After anesthetizing the surrogate female mice, they were fixed on their backs, an abdominal incision was made to enter the abdominal cavity, the fallopian tubes were located, and a sperm suspension was injected into the position near the fimbria of the fallopian tube using a microsyringe, usually about 5 - 10 μL. The abdominal incision was sutured with absorbable thread.

[0039] ③ Obtaining Pten flox / - Heterozygous mice: Ensure that the surrogate female mice are housed in an SPF-class animal room, set appropriate temperature (20 - 26 °C), humidity (40% - 60%), and light cycle (12 h light / 12 h dark), observe the changes in the body weight and abdominal size of the mice, judge the pregnancy status of the mice, and obtain Pten flox / - Heterozygous mice.

[0040] ④ Obtaining Pten flox / flox Homozygous mice: Wait for Pten flox / - After the heterozygous mice reached sexual maturity, the male and female Pten flox / - heterozygous mice were mated at a ratio of 1:2 to obtain Pten flox / flox homozygous mice, and the obtained Pten flox / flox homozygous male and female mice were bred in a breeding cage at a ratio of 1:2.

[0041] (3) Genotype identification

[0042] ① Mouse tail DNA extraction: DNA was extracted according to the DNA extraction kit (Qiagen, Germany).

[0043] ② PCR amplification: The Novizan high-fidelity PCR kit ( Super-Fidelity DNA Polymerase Kit) was used for amplification.

[0044] Primer information:

[0045]

[0046] PCR reaction system:

[0047] Reaction components Volume (μL) Concentration 2×TaqMasterMix (DyePlus) 12.5 / <![CDATA[ddH2O]]> 9.5 / Primer 1 10 μM Primer 1 10 μM Template 1 ≈100 ng / μL

[0048] PCR Amplification Program

[0049] Seg. Temp. Time Cycle 1 95℃ 5 min 2 98℃ 30s 3 65℃ 30s 20× 4 72℃ 45s 5 98℃ 30s 6 55℃ 30s 15× 7 72℃ 45s 8 72℃ 5 min 9 10℃ hold

[0050] Agarose Gel Electrophoresis: Prepare a 2% agarose gel and perform agarose gel electrophoresis on the above PCR products. Take a photo of the agarose gel electrophoresis results as Figure 1 shown.

[0051] 2. Construction of Arid1a flox / flox Mouse and Genotype Identification

[0052] (1) Construction of Arid1a flox / flox Mouse

[0053] Arid1a flox / flox , using the CRISPR / Cas technology, knock in loxP sites on both sides of exons 5-6 of the Arid1a gene on chromosome 4. Mice homozygous for the "floxed" (inducible gene knockout) allele can survive, be fertile, have a normal body size, and show no obvious physical or behavioral abnormalities. When mated with a strain expressing Cre recombinase, tissue-specific mutants for the "floxed" allele can be generated.

[0054] The construction process is as follows:

[0055] ① gRNA Design: Design gRNA at http: / / crispor.tefor.net / , select high-score gRNAs for biosynthesis, and the sequences are as follows:

[0056] gRNA-A1 (matching forward strand of gene): ACATAGACCAGACTACCCCC-TGG (SEQ ID NO: 3);

[0057] gRNA-A2 (matching forward strand of gene): AAAGTTGGTTCGGGCAGTAG-TGG (SEQ ID NO: 4).

[0058] ② Synthesis of gRNA & Homologous Recombination Vector: Fragment amplification (5'arm, cKO, 3'arm) → Ligation (backbone + fragment) / Transformation → Bacterial Detection → Positive Clone Plasmid Extraction → Enzyme Digestion Identification → Submission for Sequencing → Preparation of Plasmid for Injection, specifically as follows:

[0059] a. Fragment Amplification:

[0060]

[0061]

[0062] BAC plasmid, Bacterial artificial chromosome.

[0063] After synthesizing the primers, use Novoprotein P515 high-fidelity enzyme for amplification. The amplification system and procedure are as follows:

[0064] Sample materials Novoprotein P515 enzyme PCR system Template Plasmid: about 5 ng Primer 4 μL each Sterilized water 40ul Enzyme 50ul Total volume 100 μL (mixed well and aliquoted into two tubes)

[0065]

[0066] After the amplification is completed, use the Gel Extraction Kit (28706) from QIAGEN to recover the fragments from the gel. Gel Extraction Kit (28706) to recover the gel-extracted fragments.

[0067] b. Ligation (backbone + fragment) / Transformation

[0068] Use the backbone prepared in advance by VB157. Linearize 4 μg of the backbone with AscI / NotI restriction enzymes, and use Novoprotein C115 ligase to ligate the PCR-amplified fragments ABC to obtain the intermediate product AIB1177-ABC plate (as Figure 2 shown). The ligation reaction and procedure are as follows:

[0069]

[0070] c. Bacterial screening:

[0071] Select 16 colonies from the plate and use Novoprotein P222 PCR Taq enzyme (the system and procedure are as follows) for bacterial screening. Select 4 positive colonies and inoculate them into 4 ml of LB medium and incubate overnight at 37°C. The primers for bacterial screening are as follows:

[0072]

[0073]

[0074] d. Plasmid extraction and restriction enzyme digestion identification of positive clones

[0075] For the small-scale plasmid extraction from the overnight culture, use the Spin Miniprep Kit (27106) from QIAGEN to recover the plasmid. Measure the concentration and take about 800 ng of the plasmid and use FspI: 4273, 2449, 1172 plus 20 μl of restriction enzyme digestion system for verification as Figure 3 shown. The restriction enzyme digestion system is as follows:

[0076] Sample materials Reaction system buffer 2ul Enzyme 0.7ul Plasmid ~800 ng Water Upto 20 μL Total volume 20ul

[0077] e. Submission for sequencing / Preparation of plasmid for injection

[0078] After correct digestion, aspirate 30 μl of the plasmid for sequencing. The sequencing primers are as follows:

[0079]

[0080]

[0081] Plasmid extraction:

[0082] Inoculate the plasmid bacterial solution with correct sequencing into 15 ml of medium and culture it overnight at 37 °C for use. Extract the plasmid using the SpinMiniprep Kit (27106) and identify it by digestion.

[0083] ① Prepare the RNP complex: Mix gRNA (crRNA + tracRNA) and Cas9 protein to prepare the RNP complex for later use.

[0084] ② Preparation of fertilized eggs: Select C57BL / 6 female mice at 3 - 4 weeks old, inject pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hcg) respectively, with a time interval of 46 - 48 h between the two injections; after injecting hcg, mate the female mice with adult fertile male mice to fertilize the female mice; the next day, after euthanizing the female mice, collect the fertilized eggs from the fallopian tubes and place them in a 37 °C constant temperature 5% CO₂ incubator for later use.

[0085] ③ Pronuclear microinjection: Prepare the injection needle and fixation needle for microinjection; add the prepared injection solution (containing the RNP complex and homologous recombination vector) to the microinjection needle; select fertilized eggs with normal morphology and place them in the injection dish. Under an inverted microscope at 200 - 400 times magnification, inject the injection solution into the nucleus of the fertilized eggs by microinjection; transfer the injected fertilized eggs to M16 medium and place them in a 37 °C constant temperature 5% CO₂ incubator. After culturing for 0.5 - 1 h, perform transplantation; or culture until the 2 - cell stage and perform transplantation the next day;

[0086] ④ Preparation of surrogate mice and embryo transplantation: Select fertile female mice with ICR background at the appropriate age and mate them with male mice sterilized by vasectomy to stimulate a series of pregnancy changes in the female mice to obtain pseudopregnant female mice, which will be used as surrogate mice after the fertilized eggs are transgenic; transplant the fertilized eggs that have been injected with foreign genes into the fallopian tubes of the surrogate female mice on the day of detecting vaginal plugs; after transplantation, place the surrogate female mice in a clean cage and keep them warm until they wake up and then put them back on the cage rack for breeding; after successful tubal transplantation, the female mice generally give birth 19 - 20 days after the operation; one week after the mice are born, the mice can be numbered by cutting their claws, and at the same time, PCR identification can be performed; three weeks after the mice are born, they can be separately caged and raised independently.

[0087] ⑤ Identification of F0 mice born: Collect the tails of young mice at 1-2 weeks of age. After tissue lysis, extract genomic DNA; perform PCR amplification and electrophoresis detection using specific primers for the target gene (see the genotype identification section for details), and screen out the offspring that have successfully integrated the flox sequence; mice with flox are called founder mice and can be passaged and established into lines.

[0088] ⑥ Passage and line establishment of transgenic mice: Mate and passage the mice with the flox sequence with non-transgenic mice; each founder mouse needs to be passaged independently; identify the F1 mice born. For F0 that can be normally transmitted through the germline, 50% of its offspring have the chance to carry the flox sequence; the obtained F1 positive mice can be used for experiments and continued passage.

[0089] (2) Genotype identification

[0090] ① Extract DNAs from mouse tails: Obtain high-purity genomic DNA according to the operation of the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit.

[0091] ② PCR amplification: Use the Novizan high-fidelity PCR kit ( Super-Fidelity DNA Polymerase Kit) for amplification.

[0092] Primer information:

[0093]

[0094] PCR reaction system:

[0095] Reaction components Volume (μL) Concentration 2×TaqMasterMix (DyePlus) 12.5 / <![CDATA[ddH2O]]> 9.5 / Primer 1 10 μM Primer 1 10 μM Template 1 ≈100 ng / μL

[0096] PCR amplification program:

[0097]

[0098] Agarose gel electrophoresis: Prepare a 2% agarose gel and perform agarose gel electrophoresis on the above PCR products. Take a photo of the agarose gel electrophoresis result as Figure 4 shown.

[0099] 3. Obtain PR-Cre homozygous mice and genotype identification

[0100] (1) Obtain PR-Cre homozygous mice

[0101] PR-Cre homozygous mice were purchased from Cyagen Biosciences. The gene encoding progesterone receptor PR is PGR, which is a member of the steroid receptor superfamily. In PR-Cre gene-edited mice, the IRES-Cre element was knocked into the 3'UTR of the PGR gene on chromosome 9 to drive its specific expression. When crossed with mice containing loxP sites, the offspring can generate sequence recombination between loxP sites mediated by Cre recombinase in progesterone-responsive cells / tissues (uterus, ovary, fallopian tube, pituitary gland, and mammary gland). PR-Cre homozygous mice are fertile and have no obvious phenotypic abnormalities.

[0102] (2) Genotype identification

[0103] ① Tail DNA extraction: DNA was extracted according to the DNA extraction kit (Qiagen, Germany).

[0104] ② PCR amplification: Novizan high-fidelity PCR kit ( Super-Fidelity DNA Polymerase Kit) was used for amplification.

[0105] Primer information:

[0106]

[0107] PCR reaction system:

[0108] Reaction components Volume (μL) Concentration 2×TaqMasterMix (DyePlus) 12.5 / <![CDATA[ddH2O]]> 9.5 / Primer 1 10 μM Primer 1 10 μM Template 1 ≈100 ng / μL

[0109] PCR amplification program

[0110]

[0111] Agarose gel electrophoresis: A 2% agarose gel was prepared, and the above PCR products were subjected to agarose gel electrophoresis. The results of agarose gel electrophoresis were photographed as Figure 5 shown.

[0112] 4. Obtain Pten flox / flox , Arid1a flox / flox , PR-Cre endometrial cancer mouse model and genotype identification

[0113] (1) Obtain Pten flox / flox , Arid1a flox / flox mice

[0114] First, sexually mature Pten flox / flox male (female) mice and Arid1a flox / flox female (male) mice were mated to obtain Pten flox / - , Arid1a flox / -After the mice reach sexual maturity, Pten flox / - , Arid1a flox / - Male and female mice are mated to obtain Pten flox / flox , Arid1a flox / flox homozygous mice.

[0115] (2) Obtain Pten flox / - , Arid1a flox / - , PR-Cre mice

[0116] The obtained Pten flox / flox , Arid1a flox / flox homozygous male (female) mice are mated with PR-Cre homozygous female (male) mice to obtain Pten flox / - , Arid1a flox / - , PR-Cre heterozygous mice.

[0117] (3) Obtain Pten flox / flox , Arid1a flox / flox , PR-Cre mice

[0118] The obtained Pten flox / - , Arid1a flox / - , PR-Cre heterozygous male mice are mated with Pten flox / flox , Arid1a flox / flox homozygous female mice in a ratio of 1:2 to obtain Pten flox / flox , Arid1a flox / flox , PR-Cre.

[0119] (4) Genotype identification of Pten flox / flox , Arid1a flox / flox , PR-Cre mice

[0120] The identification method is the same as above.

[0121] Example 2, Establishment of PAD endometrial cancer model and PAD endometrial cancer organoids

[0122] Raise Pten flox / flox , Arid1a flox / flox , PR-Cre female mice. When the mice reach 12 weeks of age, the mice are sacrificed. Under sterile conditions, the uterine tissues of the mice are taken out and placed in a 10 cm sterile culture dish. Part of the tissue is used for HE staining to clarify the pathological morphology, and the other part of the tissue is used to isolate and digest the tumor tissue for organoid establishment. The specific operation steps are as follows:

[0123] 1. HE staining of tissues: 1) Tissue fixation: Mouse uterine tissues were fixed in 10% neutral formalin overnight; 2) Tissue dehydration: Starting from 70% ethanol, the tissues were successively soaked in 80%, 90%, 95% and 100% ethanol for 30 - 60 minutes respectively to ensure that the water in the tissues was fully removed; 3) Tissue clearing: The dehydrated tissues were placed in xylene and soaked for 10 - 30 minutes; 4) Paraffin infiltration and embedding: The cleared tissues were placed in pre-melted paraffin and soaked in an incubator at 60 - 62°C for 1 - 2 hours. The operation was repeated to ensure that the paraffin fully infiltrated the tissues, and the tissues were paraffin-embedded into plastic molds; 5) Making continuous paraffin sections with an average thickness of 4 μm; 6) Dewaxing, hematoxylin staining, differentiation with hydrochloric acid alcohol, blue return, eosin staining, dehydration, and sealing with neutral resin were carried out according to the HE staining operation steps for preservation and observation.

[0124] 2. Digestion and isolation of PAD mouse endometrial tissues: 1) Prepare 1× tissue digestion solution in advance: In DMEM / F12 serum-free medium, add molecular compound Y-27632 with a final concentration of 5 - 10 μM; add type IV or V collagenase with a final concentration of 0.4 - 2 mg / mL; add neutral protease II with a final concentration of 1.25 U / mL; 2) Preheat the prepared 1× tissue digestion solution in a 37°C incubator; 3) After washing twice with phosphate buffer solution PBS in a biosafety cabinet, use sterile tissue scissors to cut off necrotic tissues and suck out the phosphate buffer solution in the culture dish; 4) Add 2 ml of the preheated tissue digestion solution to the culture dish or centrifuge tube, and use sterile tissue scissors to cut the tissues into pieces about 0.25 - 0.5 mm in the culture dish or centrifuge tube. 3Tissue blocks; 5) Set the constant temperature shaking incubator at 37°C and 220 rpm, shake and digest for 60 minutes. During digestion, pipette the tissue digestion suspension every 15 minutes to reduce tissue block aggregation and ensure complete digestion; 6) Add 10% fetal bovine serum to the digested tissue suspension to slow down the digestion, and gently pipette 5 - 10 times, then let it stand at room temperature for 3 minutes; 7) First, filter the above tissue supernatant through a 100-μm cell strainer, discard the larger tissue fragments and cells, then use a 40-μm cell strainer to isolate endometrial cancer epithelial cells. Invert the 40-μm cell filter on a 50-ml centrifuge tube, and use PBS to rinse the endometrial cancer epithelial cells in the inverted filter into the 50-ml centrifuge tube. Centrifuge at 300 g for 5 minutes, aspirate the supernatant, and retain the cell pellet; 8) Add an appropriate amount of sterile 1× red blood cell lysate to lyse red blood cells, centrifuge at 300 g for 5 minutes, aspirate the supernatant, and retain the cell pellet; 9) Wash the pellet twice with a basal medium of Advanced DMEM F12 containing 1% double antibiotics (centrifuge at 300 g for 5 minutes after mixing) to remove FBS and residual red blood cell lysate; 10) Take a small amount of the suspension for live cell detection and counting.

[0125] 3. Culture of PAD organoids: 1) Add the obtained endometrial epithelial cells to the extracellular matrix gel at a density of 100,000 - 500,000 cells / mL (calculate the amount of extracellular matrix gel used as 40 μL per well for a 24-well cell culture plate), gently pipette and mix on ice, and then place it on ice; 2) Use a 200-μl pipette to aspirate the mixture of extracellular matrix gel and cells and transfer it to the cell culture well plate. Add 40 μL of the mixed suspension to each well of the 24-well cell culture plate. The mixed suspension must be pipetted to the center of the bottom of the culture well, and then spread it slightly with a pipette tip. After spreading, it should not touch the side wall of the culture well; 3) Place the culture plate in an incubator at 37°C and 5% CO2 for 30 minutes. After the extracellular matrix gel solidifies and stops flowing, slowly add the complete medium along the well wall (for a 24-well plate, add 500 μL of the complete medium), and then place it in an incubator at 37°C and 5% CO2; 4) Observe the growth status of the organoids under an inverted microscope every day. When the organoids grow to a size of 100 - 500 μm in diameter, they can be passaged or cryopreserved (about 5 - 7 days per generation).

[0126] 4. Passaging of PAD organoids: 1) Aspirate the culture medium of the well where the organoids are located, wash twice with PBS, add 500 μL of Advance DMEM F12 basal culture medium containing 1% double antibody, gently blow away the matrix gel mixed with organoids and transfer to a 1.5 ml EP tube, blow 10-30 times to separate the organoids and extracellular matrix, centrifuge at 300g for 3 minutes, and repeat the above steps until there is no organoid adherence to the wall of the EP tube after centrifugation; 2) Remove the supernatant, add 0.05% trypsin and gently blow the precipitate thoroughly, place it in a 37℃ 5% CO2 cell culture incubator and digest it for 3-5 minutes until it becomes a single cell; 3) Add 3 times the volume of digestion suspension and Advance DMEM F12 basal culture medium containing 1% double antibody to dilute the digested organoid suspension to terminate digestion, and blow thoroughly to mix, centrifuge at 300g for 4 minutes, and after centrifugation, use Advance DMEM containing 1% double antibody Wash twice with F12 basal medium to remove the residual digestion solution; 4) After washing, the cells in the centrifuged precipitate are subcultured (same as above).

[0127] 5. Cryopreservation of PAD organoids: 1) Prepare a cell cryopreservation cooling box and place it at room temperature to balance the temperature, ensuring that the isopropanol in the box is completely melted when used; 2) Select organoids in good growth condition for digestion to obtain cell pellets, add 500-1000μL pre-cooled cryopreservation solution, mix well by pipetting, and quickly transfer to low-temperature cryopreservation tubes; 3) Place the low-temperature cryopreservation tubes in the cell cryopreservation cooling box, and then quickly place the cell cryopreservation cooling box in a -80℃ ultra-low temperature refrigerator. After 24 hours, transfer the cryopreservation tubes to liquid nitrogen (-196℃) for long-term storage.

[0128] 6. Recovery of PAD organoids: 1) Prepare basal culture medium and 37℃ water bath, preheat basal culture medium in 37℃ water bath; 2) Take out the cryotube from the liquid nitrogen tank, immediately put it into 37℃ water bath, shake it quickly to melt it as soon as possible, stop the water bath before the ice is completely melted, transfer the organoid cryo suspension to a 15mL centrifuge tube, slowly add 5-10 times the volume of basal culture medium preheated at 37℃ to dilute the cryo suspension, centrifuge at 200-300g for 5 minutes, remove the supernatant, add 1mL basal culture medium to resuspend, transfer to a 1.5mL centrifuge tube, centrifuge at 200-300g for 5 minutes, repeat washing 1-2 times to remove residual cryo solution; 3) After washing, add extracellular matrix gel to the cell pellet according to the amount of organoid pellet, and culture the organoid according to the above steps.

[0129] 7. Preparation of complete medium for PAD organoids: DMEM / F12 serum-reduced medium (1×), neural / stem cell culture additive or similar additive (1×), HEPES buffer (10 mM), L-glutamine or similar additive (2 - 4 mM), streptomycin and penicillin mixed antibiotics (1×), N-acetylcysteine (1.25 mM), N2 (1×), lipid concentrate (1×), estradiol (10 nM), nicotinamide (5 mM), small molecule compound SB202190 (0.1 μM), small molecule compound Y-27632 (10 μM), small molecule compound A83-01 (0.25 μM), recombinant human R-spondin protein (500 ng / mL), recombinant human Noggin protein (100 ng / mL), recombinant human EGF protein (50 ng / mL), recombinant human HGF protein (50 ng / mL), recombinant human FGF10 protein (100 ng / mL), recombinant human IGF-1 (40 ng / mL); Note: All recombinant proteins must have biological activity, purity higher than 90% (SDS-PAGE silver staining analysis method), and endotoxin level controlled below 0.10 EU per 1 μg protein (LAL test method).

[0130] 8. WB identification of PAD organoids: 1) Sacrifice 12-week-old Pten flox / flox , Arid1a flox / flox wild-type female mice, dissect out the uterus and place it in a Petri dish containing PBS. Use ophthalmic scissors to cut the uterus along the longitudinal axis, and use the back of a scalpel to bluntly scrape out the endometrial tissue cells in the uterine cavity. Centrifuge at 300 g to collect cell pellets, and culture mouse endometrial cells with 1640 complete medium. Collect and extract proteins when the cell confluence reaches more than 80%; 2) Collect proteins from well-conditioned PAD organoids; 3) After protein quantification, perform WB experiments to identify the expression of PTEN and ARID1A proteins.

[0131] As Figure 6 shown in A - E, during the breeding of Pten flox / flox , Arid1a flox / flox , PR-Cre female mice, a small amount of vaginal secretion was found in female mice at 4 weeks of age. During the breeding process, the vaginal secretion increased significantly. At 8 weeks of age, the vaginal orifice was significantly red and swollen, and the abdomen was distended ( Figure 6 shown in A). Sacrifice the mice at 12 weeks of age, take out the thickened and edematous uterine tissue under sterile conditions, and place it in a 10 cm sterile Petri dish ( Figure 6 shown in B). Perform HE staining and observe that the glandular structure of the endometrial tissue is disordered, accompanied by nuclear atypia, tumor necrosis in some areas, and massive infiltration of inflammatory cells ( Figure 6In C). Mouse endometrial epithelial cells were extracted for PAD organoid culture. Under the microscope, it was observed that the PAD organoids grew in a solid sphere-like shape and grew vigorously, as shown in Figure 6 Figure D in the manuscript. WB verification showed that both PTEN and ARID1A were absent in the PAD organoids, as shown in Figure 6 Figure E in the manuscript.

[0132] Example 3. Identification of short tandem repeats in PAD endometrial cancer tissues and organoids

[0133] DNA of PAD endometrial cancer tissues and PAD organoids was extracted using Axygen's genomic extraction kit, amplified using a 10-STR amplification protocol, and the STR loci and the gender gene Amelogenin were detected on an ABI 3730XL genetic analyzer. The test results are as follows:

[0134]

[0135] No matching cell lines were found in the cell line search for the DNA typing of PAD endometrial cancer tissues and PAD organoids. No multi-alleles were found in this cell line. The matching degree between the PAD organoid sample and the PAD endometrial cancer tissue sample was 86%, indicating the same origin.

[0136] Note: The STR data of the cell line to be tested is matched with the STR data included in ATCC, DSMZ (DSMZ includes STR data of 2,490 cell lines from ATCC, DSMZ, JCRB, and RIKEN, etc.), and ExPASy cell bank (ExPASy includes STR data of approximately 8,000 human cell lines from databases such as ATCC, DSMZ, JCRB, ECACC, and Riken). Cells not included in the above cell banks will not be matched. According to the identification standard of the ATCC Standards Committee (ANSI / ATCC ASN-0002-2022), a matching degree of EV≥80% is considered to have a correlation and may be derived from a common ancestral cell; when the matching degree is between 55% and 80%, other methods need to be combined for further identification and verification of their correlation. Among the following loci, D4S2408 is a human locus, which is used to detect whether the cell has human contamination.

[0137] Example 4. Subcutaneous transplantation tumor implantation model of PAD organoids

[0138] To evaluate the tumorigenicity rate of mouse endometrial cancer PAD organoids subcutaneously in C57BL / 6 mice with normal immune function, we successfully established a subcutaneous transplantation tumor implantation model of PAD organoids. The specific operation steps are as follows:

[0139] 1. Preparation of PAD organoids: 1) Culture and expand PAD organoids; 2) Collect and process PAD organoids when they grow to a sufficient quantity and appropriate size; 3) Wash twice with PBS and then perform cell counting; 4) Prepare a cell suspension with a mixed solution of a medium containing 5 mg / mL Matrigel, place it on ice, and prepare for subcutaneous transplantation.

[0140] 2. Preparation of C57BL / 6 female mice: 1) Purchase 6-8-week-old immunocompetent C57BL / 6 female mice from Cyagen Biosciences, and raise them in an SPF-level animal room for 7-10 days, setting appropriate temperature (20-26 °C), humidity (40%-60%), and light cycle (12 h light / 12 h dark); 2) After the mice adapt, prepare for subcutaneous transplantation of PAD organoids into mice.

[0141] 3. Subcutaneous transplantation: 1) Grasp the mouse with one hand and select the lateral abdomen of the mouse as the subcutaneous injection site; 2) Use a 1 mL syringe to aspirate the corresponding organoid cells, slowly insert the needle in a "Z" shape to avoid leakage of the organoid suspension, and perform cell injection according to different cell amounts. Set 3 cell amounts for subcutaneous injection of mice, which are 7.0×10 5 cells, 1.05×10 6 cells, 1.4×10 6 cells, and the injection volume is 100-200 μL.

[0142] 4. Observation and evaluation: 1) Observe once a week, measure the growth of subcutaneous tumors in mice, evaluate the tumor formation rate, and plot the tumor growth curve; 2) Euthanize the mice when the maximum tumor diameter grows to 2 cm, and dissect the subcutaneous transplanted tumor tissue of the mice; 3) Perform HE staining on the subcutaneous transplanted tumor tissue of PAD organoids.

[0143] As Figure 7 shown in A-D below, after 6-8-week-old immunocompetent C57BL / 6 female mice are raised in an SPF-level animal room for 7-10 days, subcutaneous transplantation of PAD organoids is performed according to different cell amounts. After 12 weeks of implantation, the in-vivo pictures of the subcutaneous transplanted tumors in mice are shown in Figure 7 A below. Measure the tumor volume weekly and plot the growth curve as shown in Figure 7 B below. Euthanize the mice after 12 weeks of implantation and dissect the subcutaneous transplanted tumors, as shown in Figure 7 C below. Perform HE staining on the subcutaneous transplanted tumors, and it can be seen that the glandular structure of the endometrial tissue is disordered, showing a sieve-like shape, accompanied by changes in nuclear atypia, as shown in Figure 7 D below.

[0144] Example 5: Tumor model of in-situ implantation of PAD organoids in the uterine cavity

[0145] To evaluate the tumorigenesis rate of PAD organoids of mouse endometrial cancer implanted in situ in the uterine cavity of immunocompetent C57BL / 6 mice, we successfully established a tumor model of PAD organoids implanted in situ in the uterine cavity. The specific operation steps are as follows:

[0146] 1. Preparation of PAD organoids: 1) Culture and expand PAD organoids; 2) Collect and process PAD organoids when they grow to sufficient quantity and appropriate size; 3) Wash twice with PBS and then perform cell counting; 4) Prepare a cell suspension with a mixed solution of medium containing 5 mg / mL Matrigel, with a concentration of 2×10 6 cells / 100 μL, and place it on ice for uterine cavity transplantation.

[0147] 2. Preparation of C57BL / 6 female mice: 1) Purchase 6-8-week-old immunocompetent C57BL / 6 female mice from Cyagen Biosciences, and raise them in an SPF-level animal room for 7-10 days, setting appropriate temperature (20-26 °C), humidity (40%-60%), and light cycle (12 h light / 12 h dark); 2) After the mice are adapted, perform uterine cavity injection of PAD organoids on the mice.

[0148] 3. Uterine cavity transplantation in mice: 1) Perform vaginal smear on C57BL / 6 female mice. Since the uterus of mice in estrus is edematous and thickened, which is convenient for uterine cavity injection, select mice in estrus for in situ implantation in the uterine cavity; 2) Anesthesia and fixation: Inject sodium pentobarbital intraperitoneally at a dose of 40-50 mg / kg body weight. After the animal enters the anesthetic state, fix it on the operating table with the back facing up. 3) Disinfect the skin about 0.5-1 cm beside the spinal column on the back of the mouse with iodophor, and make a longitudinal incision about 1-2 cm long with sterile scissors to enter the abdominal cavity and expose one side of the ovary and uterus. 3) Curettage: To facilitate the retention of PAD organoid cells in the uterine cavity, make a curette-like tool and insert it into the uterine cavity through the incision of the uterine horn, carefully scrape and damage the endometrium of the uterine cavity, avoiding penetrating the uterine wall; 4) Slowly inject the prepared tumor cell suspension into the uterine cavity using a microinjector. The injection volume depends on the size of the uterus and the experimental design, and 10-50 μL is injected into each uterine cavity. Pay attention to avoid leakage of the cell suspension into other parts of the abdominal cavity during injection, so as not to affect the accuracy of the model; 5) After injection, carefully place the uterus back into the abdominal cavity, and suture the peritoneum and skin layer by layer with silk thread. After the operation, place the animal in a warm and quiet environment for anesthetic recovery, and drip a small amount of penicillin & gentamicin at the back incision to prevent infection; 6) Apply a small amount of picric acid to the incision site of the mouse on the second day after the operation to prevent the mouse from biting the incision and affecting the incision healing.

[0149] 4. Observation and evaluation: 1) Observe once a week to check for increased vaginal discharge, abdominal distension, etc. in the mice; 2) When symptoms such as increased vaginal discharge and abdominal distension are observed around 2 months, sacrifice the mice and dissect the uterine tissues.

[0150] 3) Perform HE staining on the uterine tissues transplanted in situ into the uterine cavity of PAD organoids.

[0151] As Figure 8 shown in A - C below, 6 - 8 - week - old immunocompetent C57BL / 6 female mice, after being housed in an SPF - level animal room for 7 - 10 days, had their vaginal secretions examined to confirm the estrus phase. Two months after in situ implantation into the uterine cavity, it was observed that the mice had increased vaginal secretions and abdominal distension. After sacrificing the mice and dissecting the abdominal cavity, the uterus was found to be significantly thickened and there were adhesions with masses. As Figure 8 shown in A below, the dissected uterine tissues are as Figure 8 shown in B below. HE staining was performed on the in situ implanted uterine tissues, and it was found that the glandular structure of the endometrial tissue was disordered, showing a sieve - like pattern, mostly solid areas, accompanied by changes in nuclear atypia, as Figure 8 shown in C below.

[0152] Example 6, Tumorigenesis in the Footpad - Lymph Node Model of PAD Organoids

[0153] Since the main way of endometrial cancer metastasis is lymph node metastasis, but no pelvic or inguinal lymph node metastasis was observed in the subcutaneous transplantation tumor model and in situ uterine cavity implantation model of PAD organoids. The mouse footpad has a clear and relatively direct lymphatic drainage system, and tumor cells will first enter the local lymph nodes from the inoculation site in the footpad, which is conducive to studying the metastasis process of tumor cells in the lymphatic system. Therefore, a tumorigenesis model of PAD organoids in the footpad was established to evaluate the lymph node metastasis situation. The specific operation steps are as follows:

[0154] 1. Preparation of PAD organoids: 1) Culture and expand PAD organoids; 2) Collect and process PAD organoids when they grow to a sufficient quantity and appropriate size; 3) Wash twice with PBS and then perform cell counting; 4) Prepare a cell suspension with a mixed solution of a medium containing 5 mg / mL Matrigel, with a concentration of 2×10 6 cells / 100 μL, and place it on ice for implantation under the footpad.

[0155] 2. Preparation of C57BL / 6 female mice: 1) Purchase 6 - 8 - week - old immunocompetent C57BL / 6 female mice from Cyagen Biosciences, house them in an SPF - level animal room for 7 - 10 days, and set appropriate temperature (20 - 26 °C), humidity (40% - 60%), and light cycle (12 h light / 12 h dark); 2) After the mice are adapted, a PAD organoid mouse footpad implantation model is to be performed.

[0156] 3. Subcutaneous implantation under the mouse footpad: 1) Mouse fixation: The assistant holds the mouse with one hand and fixes one hind foot of the mouse with the other hand, preparing for subcutaneous implantation under the mouse footpad; 2) Slowly inject the prepared tumor cell suspension into the mouse footpad with a 1 ml syringe, and the injection volume is 40 - 50 μL.

[0157] 4. Observation and evaluation: 1) Observe once a week to observe the swelling of the hind footpad of the mouse; 2) Observe obvious swelling of the footpad after about 2 months, sacrifice the mouse and dissect the tumor tissue of the mouse footpad, popliteal fossa and inguinal lymph nodes; 3) Perform HE staining on the tumor tissue of the mouse footpad, popliteal fossa and inguinal lymph nodes of PAD - like organ mice.

[0158] As Figure 9 shown in A - C below, C57BL / 6 female mice with normal immune function at 6 - 8 weeks of age are raised in an SPF - level animal house for 7 - 10 days and then subcutaneous implantation is performed under the mouse footpad. Obvious swelling of the footpad is observed after about 2 months ( Figure 9 shown in A below), sacrifice the mouse and dissect the tumor tissue of the mouse footpad, popliteal fossa and inguinal lymph nodes. Perform HE staining on the tumor tissue of the mouse footpad, popliteal fossa and inguinal lymph nodes, and it is seen that the glandular structure of the endometrial tissue is disordered, showing a sieve - like pattern, accompanied by changes in nuclear atypia ( Figure 9 shown in B below), and tumor cell infiltration and metastasis are seen in the popliteal lymph node tissue ( Figure 9 shown in C below).

Claims

1. C57BL / 6 mouse endometrial cancer PAD organoids, characterized in that, The preservation number of the C57BL / 6 mouse endometrial cancer PAD organoids is CCTCC NO: C2023321.

2. The application of the C57BL / 6 mouse endometrial cancer PAD organoids according to claim 1 in constructing a tumor animal model.

3. A tumor animal model, characterized in that, The tumor animal model is constructed by inoculating the cells in the C57BL / 6 mouse endometrial cancer PAD organoids according to claim 1 into a mouse.

4. The tumor animal model according to claim 3, wherein, The tumor animal model includes a subcutaneous transplanted tumor implantation model, an in-situ uterine cavity implanted tumor model, or a footpad tumorigenesis-lymph node model.

5. The application of the C57BL / 6 mouse endometrial cancer PAD organoids according to claim 1 or the tumor animal model according to any one of claims 3-4 in the preparation of a disease diagnosis product.

6. The application according to claim 5, characterized in that The application in the preparation of a disease diagnosis product includes the development, manufacture, or screening of products involving the immune process of human cells and the application in the production of human antibodies.

7. The application of the C57BL / 6 mouse endometrial cancer PAD organoids according to claim 1 or the tumor animal model according to any one of claims 3-4 in drug screening.

8. The application according to claim 7, wherein The application includes a) screening, pharmacodynamic detection, efficacy evaluation, verification, or assessment of regulators of endometrial cancer-related pathways in vivo; or, b) studying the gene functions of endometrial cancer with combined knockout of Pten and Arid1a, studying the drugs and pharmacodynamics of therapeutic target sites for endometrial cancer, studying drugs for immune-related diseases, and anti-tumor or anti-inflammatory drugs.

9. The application of the C57BL / 6 mouse endometrial cancer PAD organoids according to claim 1 or the tumor animal model according to any one of claims 3-4 in a scientific research protocol for non-therapeutic purposes in vitro.

10. The application according to claim 9, characterized in that, The application in the scientific research protocol for non-therapeutic purposes in vitro includes: a) application as a model system in pharmacological, immunological, microbiological, and medical research; or, b) application in the production and utilization of animal experimental disease models involving the immune process of human cells for etiological research.

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