Human gastric cancer peritoneal metastasis organoid cell line and culture method and application thereof

Abstract

The application discloses a human gastric cancer peritoneal metastasis organoid cell line and a culture method and application thereof, and belongs to the technical field of digestive system tumor cell line models.The human gastric cancer peritoneal metastasis organoid cell line of the application is a human gastric cancer peritoneal metastasis organoid cell line WGC-247 preserved in the China Center for Type Culture Collection (CCTCC) and having a preservation number of CCTCC NO: C202646, and is obtained by establishing 3D organoid culture in vitro for ascites tumor cells of a gastric cancer and peritoneal metastasis patient with cancerous ascites fluid.The organoid cell line of the application can be stably subcultured in vitro, has good tumorigenicity in nude mice, and can be used for research on pathogenesis, drug resistance mechanism, new drug screening and the like of gastric cancer and peritoneal metastasis.

Description

Technical Field

[0001] This invention relates to the field of digestive system tumor cell line model technology, specifically to a human gastric cancer peritoneal metastasis organoid cell line, its culture method, and its application. Background Technology

[0002] Gastric cancer is the most common malignant tumor worldwide, ranking fifth in incidence and third in mortality globally. In 2025, approximately 1.23 million new cases and 950,000 deaths from gastric cancer were diagnosed globally each year. The vast majority of patients are diagnosed at a locally advanced or late stage. The overall five-year survival rate for gastric cancer is only 33.5%, with significant differences in survival depending on the stage. Early-stage (stage I) patients have a five-year survival rate of 85% to 92%, and can be cured with minimally invasive endoscopic surgery. Stage II and III locally advanced patients have a five-year survival rate between 30% and 60%, while stage IV advanced metastatic gastric cancer patients have a five-year survival rate of less than 13%. Patients with peritoneal metastasis have the worst prognosis, with a median survival of only 3 to 10 months and a five-year survival rate of less than 2%, making them the leading cause of death from advanced gastric cancer. Clinically, approximately 17% to 27% of patients are diagnosed with peritoneal metastasis at initial diagnosis. For patients with gastric cancer reaching T3 or T4 depth, the risk of peritoneal recurrence after radical surgery can reach 28.6% to 50%. Peritoneal metastasis presents multiple treatment challenges, including insidious lesions, a high rate of missed diagnosis of tiny miliary nodules on imaging, the peritoneal plasma barrier hindering the efficacy of intravenous chemotherapy drugs, the strong anti-anodic apoptosis ability of exfoliated tumor cells, the tendency to form malignant ascites and omental cake, and frequent complications such as intestinal obstruction and severe malnutrition.

[0003] Currently, advanced metastatic gastric cancer is entering the precision medicine stage, with targeted therapy, immunotherapy, and chemotherapy regimens selected based on molecular subtyping such as HER2, Claudin18.2, PD-L1, and MSI. However, most advanced patients lack highly matched targeted drugs and are prone to drug resistance after first-line treatment. The poor prognosis, difficulty in early diagnosis, and lack of optimized strategies for intraperitoneal drug administration in clinical practice, particularly in peritoneal and intraperitoneal metastases, have driven the widespread development and application of animal models for gastric cancer peritoneal metastases. Commonly used models include intraperitoneal injection cell line CDX models, spontaneous metastasis models from in situ implantation in the gastric wall, patient-derived PDX models, and genetically engineered GEMM mice, each suited for different research directions such as high-throughput drug screening, research on the mechanisms of natural tumor invasion and metastasis, clinical personalized drug sensitivity testing, and basic research on inflammation and genetic carcinogenesis. However, existing models struggle to fully simulate the gene mutation background and differentiation characteristics of patient tumors, and also fail to replicate the tumor microenvironment of human gastric cancer peritoneal metastases. These limitations restrict the screening of new drugs for intraperitoneal localization, the preclinical evaluation of related second-line treatments for gastric cancer, and the translation of corresponding preclinical research into clinical treatment.

[0004] Therefore, the applicant is considering providing a novel cell line that fully mimics the differentiation characteristics of tumors and replicates the tumor microenvironment of peritoneal metastases from gastric cancer in humans. Compared to PDX models, this tumor organoid model of peritoneal metastasis from gastric cancer patients offers a higher modeling success rate and greater controllability of experimental conditions, facilitating scientific research related to peritoneal metastases of gastric cancer. It also helps in screening effective local therapies, controlling the disease, and prolonging patient survival. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a human gastric cancer peritoneal metastasis organoid cell line, its culture method, and its application. This solves the problem that existing cell lines cannot fully simulate the differentiation characteristics of patient tumors or replicate the microenvironment of human peritoneal metastatic tumors. The cell line provided by the present invention can not only be stably passaged in vitro, but also has strong proliferative activity and the ability to metastasize to tumors in nude mice via subcutaneous and peritoneal metastasis. This provides new in vitro and in vivo research support for further in-depth research on the etiology, pathogenesis, drug resistance mechanisms, and new drug screening of gastric cancer peritoneal metastasis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A human gastric cancer peritoneal metastasis organoid cell line, including the human gastric cancer peritoneal metastasis organoid cell line WGC-247, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202646.

[0007] Furthermore, the human gastric cancer peritoneal metastasis organoid cell line WGC-247 was established by in vitro 3D organoid culture of ascites tumor cells from patients with gastric cancer and peritoneal metastasis accompanied by malignant ascites.

[0008] This invention also provides a method for culturing organoid cell lines from peritoneal metastases of gastric cancer, comprising the following steps: 1) Sample collection: Under aseptic conditions, 50 mL of ascites drainage fluid was collected from patients with gastric cancer and peritoneal metastasis accompanied by malignant ascites and sent to the laboratory; 2) Preliminary isolation of tumor cells: Tumor cells were isolated using a multiple centrifugation method. The multiple centrifugation method included: transferring ascites fluid to a 50 mL centrifuge tube, centrifuging at 1000 rpm for 5 min, discarding the supernatant, resuspending the pellet in 5 mL of PBS buffer, centrifuging again at 800 rpm for 5 min, discarding the supernatant, transferring the pellet to a 1.5 mL Eppendorf tube, adding 1.2 mL of PBS buffer for resuspending, centrifuging at 400 rpm for 3 min, discarding the supernatant and upper red blood cells; repeating the resuspension with 1.2 mL of PBS buffer and centrifugation at 400 rpm for 3 min multiple times until no red blood cells were visible to the naked eye, obtaining tumor cell pellets, gently resuspending them with PBS buffer, and counting the number of tumor cell clusters. 3) Seeding cells: Resuspend the pre-cooled tumor cell pellet on ice with matrix gel. Use 30 μL of matrix gel per 3000 cell clusters. Then, seed the tumor cell resuspension into a 24-well plate preheated to 37°C, adding 30 μL to each well. Next, incubate the 24-well plate at 37°C until the matrix gel solidifies into a dome shape. Then, add 650 μL of culture medium to each well for further incubation. 4) 3D organoid culture and routine passage culture: Tumor cell growth in 3D matrix gel was observed daily under a microscope. Some lymphocytes showed transient proliferation but lacked organoid morphology and could not be continuously passaged for expansion. The culture medium was changed every 3-5 days, and passage was performed on days 10-14 based on organoid density. The passage culture procedure was as follows: The culture medium in each well was discarded and the cells were washed once with PBS buffer. 250 μL of TrypLE digestive enzyme was added to each well for digestion. The matrix gel was dispersed by pipetting, and then mixed by pipetting every 5 minutes for 10-15 minutes. Once single cells were dispersed, the cells were transferred to twice the volume of HBSS buffer (containing Ca2+). 2+ Mg 2+ After neutralization, centrifugation, and resuspending, count the cells and continue seeding and passage at 8000 cells per well, repeating steps 3) and 4).

[0009] This invention also provides an application of the above-mentioned human gastric cancer peritoneal metastasis organoid cell line in constructing a nude mouse subcutaneous tumorigenesis animal model or a gastric cancer peritoneal metastasis tumorigenesis animal model. The nude mouse subcutaneous tumorigenesis animal model and the peritoneal injection metastasis tumorigenesis animal model are used to study the pathogenesis of gastric cancer peritoneal metastasis and drug treatment.

[0010] This invention provides a method for constructing a nude mouse subcutaneous tumorigenesis animal model, comprising the following steps: 1) Organoid culture: By passage, seeding and 3D organoid culture of the above-mentioned human gastric cancer peritoneal metastasis organoid cell line, a certain number of WGC-247 organoids were obtained. The culture medium was changed on the 4th, 7th and 9th days after passage. 2) Organoid digestion and seeding: On day 10 post-passage, TrypLE digestive enzyme was added to WGC-247 organoids in the logarithmic growth phase for digestion. 250 μL of TrypLE digestive enzyme was added to each well, and the matrix gel was gently dispersed by pipetting. After 5 min, it was gently mixed again by pipetting. The mixture was then neutralized with twice the volume of HBSS buffer, washed twice with PBS buffer, and centrifuged. Six-well organoids were collected, containing a total of 2 × 10⁶ cells / well. 6 One WGC-247 organoid was injected subcutaneously into nude mice; 3) Tumor formation observation: Observe and record the subcutaneous tumor formation and growth of nude mice once a week.

[0011] This invention provides a method for constructing an animal model of peritoneal metastasis of gastric cancer, comprising the following steps: 1) Organoid culture: By passage, seeding and 3D organoid culture of the above-mentioned human gastric cancer peritoneal metastasis organoid cell line, a certain number of WGC-247 organoids were obtained, and the medium was changed on the 4th, 7th and 9th days after passage. 2) Organoid digestion and seeding: On day 10 post-passage, TrypLE digestive enzyme was added to WGC-247 organoids in the logarithmic growth phase for digestion. 250 μL of TrypLE digestive enzyme was added to each well, and the matrix gel was gently dispersed by pipetting. After 5 min, it was gently mixed again by pipetting. The mixture was then neutralized with twice the volume of HBSS buffer, washed twice with PBS buffer, and centrifuged. Organoids from 24 wells were collected, containing a total of 8 × 10⁶ cells / well. 6 One WGC-247 organoid was injected into the abdominal cavity of nude mice; 3) Tumor formation observation: Observe and record the tumor formation and weight changes of nude mice weekly, and observe the activity, feeding and abdominal condition of nude mice.

[0012] The present invention also provides the application of the above-mentioned human gastric cancer peritoneal metastasis organoid cell line in overexpressing luciferase or in preparing cell products overexpressing luciferase.

[0013] This invention also provides the application of the above-mentioned human gastric cancer peritoneal metastasis organoid cell line in screening drugs for the treatment of gastric cancer and gastric cancer peritoneal metastasis tumors.

[0014] As a preferred embodiment, the application includes: applying a therapeutic drug to an in vitro cell model, wherein the drug that inhibits cell proliferation or induces apoptosis after application is a therapeutically sensitive drug; or applying a test compound to an in vitro animal model, wherein the drug that inhibits disease progression, reduces ascites formation, improves the survival status of nude mice, and prolongs survival time after application is a therapeutically effective drug; wherein the in vitro cell model or in vitro animal model is obtained by constructing the human gastric cancer peritoneal metastasis organoid cell line according to claim 1 or 2.

[0015] The present invention also provides the application of the above-mentioned human gastric cancer peritoneal metastasis organoid cell line in the preparation of products for screening drugs for the treatment of gastric cancer and gastric cancer peritoneal metastasis tumors.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The WGC-247 cell line, a human gastric cancer peritoneal metastasis organoid cell line cultured in this invention, was derived from a newly diagnosed gastric cancer patient with peritoneal metastasis and malignant ascites. This cell line exhibits stable in vitro passage capacity and strong peritoneal metastatic tumorigenicity, providing a new cell line tool for research on metastatic gastric cancer. Compared to traditionally passaged cell lines, its tumorigenic tissues are similar in histopathological characteristics to those of human gastric cancer patients, facilitating the translation of preclinical research into clinical treatment for tumor therapy-related research. This provides new tools and models for in vivo and in vitro studies of metastatic gastric cancer.

[0017] In this invention, animal models are constructed using organoid implantation. In the subcutaneous tumorigenesis model in nude mice, local injection of 2 million cells results in tumor formation in approximately 2-3 weeks, indicating a short tumorigenesis cycle. In the intraperitoneal injection metastasis tumorigenesis model, due to the high cell count requirement for intraperitoneal metastasis, injections of 1 million, 2 million, and 4 million cells did not result in tumor formation. Injection of 8 million cells resulted in noticeable tumor formation after 4 weeks, a small amount of ascites after 8 weeks, and significant ascites and intraperitoneal metastasis after 14 weeks.

[0018] The human gastric cancer peritoneal metastasis organoid cell line WGC-247 obtained by this invention exhibits good 3D organoid morphology in vitro, and is stable in characteristics, can be continuously and stably passaged, and can well tolerate viral infection. Currently, a WGC-247 cell line overexpressing luciferase has been constructed, which further facilitates in vivo animal research. Attached Figure Description

[0019] Figure 1 This is a diagram showing the relevant examination results of a patient with gastric cancer and peritoneal metastasis with malignant ascites, derived from the organoid cell line WGC-247 in Example 1; among them, Figure 1 Image A shows the results of a gastroscopy performed during the initial consultation in early July 2024. Figure 1B shows the patient's abdominal CT scan results during follow-up examinations in August and October 2024, indicating poor ascites control. Figure 2 for Figure 1 Image of HE and immunohistochemical staining results of tissue obtained from a gastroscopy biopsy; based on morphology and immunohistochemistry, the diagnosis is poorly differentiated carcinoma (gastric body), leaning towards adenocarcinoma. Figure 2 Image A shows the HE staining results of gastroscopy biopsy tissue; Figure 2 B shows the results of immunohistochemical protein expression: PCK (+), CK7 (-), Her-2 (2+, with a few tumor cells showing ectopic nuclei +), Claudin18.2 (approximately 95% strongly +), CEA (+), P53 (scattered +, suggesting wild type), Ki-67 (LI hotspot area approximately 60%), MLH1 (+), PMS2 (+), MSH2 (+), MSH6 (+); Figure 2 C is the FISH fluorescence in situ hybridization diagram of the Her-2 gene, indicating that the Her-2 gene was not amplified; Figure 3 This is a diagram showing the ascites tumor cells and culture results from Example 1. Figure 3 The upper part of A is... Figure 1 Corresponding to the patient's ascites image and the bright field image of tumor cells in the ascites, it can be seen that in addition to scattered lymphocytes and large single tumor cells, there are many scattered tumor cell clusters in the field of view. Figure 3 The lower image in A shows the cytological staining results of ascites sediment, which contains a large number of lymphocytes and clusters of tumor cells. Figure 3 B shows bright-field images of tumor organoids in culture taken under a microscope. The left image is a bright-field morphological image of organoids in the middle region of a 3D matrix gel, while the right image is a bright-field morphological image of organoids at the edge of the matrix gel and tumor cells released from the organoids in the surrounding culture medium. Figure 3 C is Figure 3 Paraffin-embedded HE-stained images of the corresponding organoids in section B; Figure 4 Images of subcutaneous tumor formation in nude mice and corresponding HE staining morphological results are shown in Example 2. Figure 4 A shows the results of subcutaneous tumor formation in nude mice; Figure 4 Image B shows the HE staining results of tumor tissue from nude mice, revealing the poorly differentiated state of the tumor-forming tissue. Figure 5 This is a diagram showing the results of constructing a nude mouse peritoneal metastasis tumor model in Example 3; Figure 5 Image A shows the mesenteric metastatic lesions and abdominal wall nodules. Figure 5 B is Figure 5 Figure A shows the HE staining results of the intestine and mesentery tissues and the abdominal wall nodules; Figure 5C shows the HE staining results of the lungs, liver, and spleen of the nude mouse. Nodular foci were found around the splenic hilum, but no metastatic foci were found in the spleen, liver, and lungs. Figure 6 This is a diagram showing the staining results of mesenteric nodules in the nude mouse peritoneal metastasis tumor model of Example 3; Figure 6 Image A shows the HE staining results of mesenteric nodules; Figure 6 B shows the corresponding immunohistochemical staining results. Figure 2 B. The differentiation and immunohistochemical staining of the gastroscopy biopsy tissue were consistent. Figure 7 This is a statistical graph of fluorescence intensity in the WGC-247 organoid and the organoid after luciferase overexpression in Example 4. Detailed Implementation

[0020] The invention is further illustrated below with reference to specific examples and accompanying drawings. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0021] This invention relates to the preservation of biological materials, homo sapiens, CCTCC NO: C202646, wherein the depository is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, postcode: 430072, and the deposit date is June 16, 2026.

[0022] Unless otherwise specified, all instruments, raw materials, and reagents used in the following examples are commercially available products well-known in the art and can be obtained through commercial means. The specific materials and their sources listed in the following examples are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or function as the tissues, cells, reagents, and instruments described below can be used to implement the invention. The culture media used in the following examples for ascites tumor cell culture and line establishment, passage culture of the human gastric cancer peritoneal metastasis organoid cell line WGC-247, and 3D organoid culture are organoid culture media, the components, concentrations, and functions of which are shown in Table 1. Information on some reagents and their manufacturers in the examples is shown in Table 2.

[0023] Table 1: Components, concentrations, and effects of organoid culture media In Table 1, X represents the concentration factor unit. For example, if the concentration of the mother liquor is 50X and the concentration used is 1X, it needs to be diluted 50 times. Then, 50mL of the final solution only needs to contain 1mL of the mother liquor.

[0024] Table 2: Information on some reagents and their manufacturers Example 1 The method for establishing the human gastric cancer peritoneal metastasis organoid cell line WGC-247 includes the following steps: 1. Source of the specimen: The specimen used in this embodiment is ascites from a patient with gastric cancer and peritoneal metastasis accompanied by malignant ascites. The ascites specimen came from a 53-year-old patient newly diagnosed with gastric cancer and peritoneal metastasis.

[0025] The patient presented in July 2024 with persistent abdominal distension and pain for more than 10 days and underwent a CT scan. The enhanced CT scan of the upper and lower abdomen revealed: 1. Uneven thickening of the gastric wall in the gastric body, with localized bulging into the intestinal lumen, and enlarged perigastric lymph nodes; 2. Blurred abdominal fat spaces with multiple small nodules, suggestive of a possible omental neoplasia; 3. Abdominal and pelvic effusion. Tumor marker tests showed: CEA 15.69 ng / mL (≤5); CA72-4 > 600 U / mL (< 6.9). Follow-up CT scans were performed in August and October, with the following results... Figure 1 As shown in B, the ascites is poorly controlled.

[0026] The patient underwent gastroscopy and biopsy on July 10, 2024. The gastroscopy results are shown below. Figure 1 A, Figure 1 Results A showed a large gastric ulcer (Ca? pending pathological examination). The biopsy tissue underwent HE and immunohistochemical staining. Immunohistochemical staining was used to detect the expression of pan-cytokeratin (PCK), pan-cytokeratin (CK7), human epidermal growth factor receptor 2 (Her-2), tight junction protein 18.2 (Claudin18.2), carcinoembryonic antigen (CEA), tumor protein 53 (p53), MutL homolog 1 (MLH1), postmeiotic segregation enhancement protein 2 (PMS2), MutS homolog 2 (MSH2), and MutS homolog 6 (MSH6), as well as the Ki-67 proliferation index. Results are shown below. Figure 2 Combining Figure 2 As a result, the gastroscopic biopsy tissue was diagnosed as poorly differentiated carcinoma (of the gastric body) based on morphology and immunohistochemistry, with a tendency towards adenocarcinoma. Figure 2 Image A shows the HE staining results of gastroscopy biopsy tissue; Figure 2 B shows the results of immunohistochemical protein expression: PCK (+), CK7 (-), Her-2 (2+, with a few tumor cells showing ectopic nuclei +), Claudin18.2 (approximately 95% strongly +), CEA (+), P53 (scattered +, suggesting wild type), Ki-67 (LI hotspot area approximately 60%), MLH1 (+), PMS2 (+), MSH2 (+), MSH6 (+); Figure 2C represents FISH fluorescence in situ hybridization, indicating no Her-2 gene amplification. The patient had a large amount of ascites; paracentesis was performed to drain the ascites. Ascites endoscopic images and cytological staining results are shown below. Figure 3 A. In addition to scattered lymphocytes and large single tumor cells, scattered clusters of tumor cells are frequently seen in the field of view.

[0027] The above results indicate that the patient has gastric cancer with peritoneal metastasis and malignant ascites.

[0028] 2. Specimen processing: 2.1) Sample collection: Under aseptic conditions, collect 50 mL of the patient's abdominal drainage fluid, place it in a 4°C ice box, and quickly send it to the laboratory; 2.2) Tumor cell isolation: Tumor cells were isolated using a repeated centrifugation method (without using red blood cell lysis buffer). Ascites fluid was transferred to a 50 mL sterile centrifuge tube in a sterile laminar flow hood and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in 5 mL of PBS buffer and centrifuged at 800 rpm for 5 min. The supernatant was then discarded. The cell pellet was transferred to a 1.5 mL Eppendorf tube, resuspended in 1.2 mL of PBS buffer, and centrifuged at 400 rpm for 3 min. The supernatant and upper red blood cells were discarded. This resuspension and centrifugation at 400 rpm were repeated multiple times until no red blood cell pellet was visible to the naked eye, thus obtaining the tumor cell pellet.

[0029] 2.3) Seeding cells: Take the above tumor cell pellet, resuspend it with PBS buffer, and count the viable cells and cell clusters to obtain the total number of viable cells and cell clusters. After centrifugation and discarding the supernatant, resuspend the pre-cooled cells (clusters) in matrix gel on crushed ice at low temperature to 3000 cells / 30μL to obtain tumor cell resuspension. Then, seed the tumor cell resuspension at 30μL / well into 24-well plates that have been preheated at 37℃ and incubated at 37℃ for 30 min. After the dome-shaped matrix gel solidifies, add 650μL of organoid culture medium to each well for further culture. 2.4) Routine Culture and Passaging: The growth status of tumor cells in 3D matrix gel was observed under a microscope daily. Some lymphocytes could proliferate briefly, but they did not exhibit organoid morphology and could not be continuously passaged for expansion. The culture medium was changed every 3-5 days. Passaging was performed on days 10-14 after the organoids showed good growth and the number of cells had significantly increased. The passage culture procedure was as follows: First, the culture medium in each well was discarded and the cells were washed once with PBS buffer. Then, 250 μL of TrypLE digestive enzyme was added to each well for digestion. The cells were mixed by pipetting every 5 minutes. After 10-15 minutes, the cells were digested into a single cell suspension. The cells were then neutralized with HBSS buffer, centrifuged, resuspended, and counted. Passaging was continued at 8000 cells per well. Steps 3) and 4) were repeated thereafter.

[0030] The above culture method can be used to culture organoid cell lines with good 3D organoid morphology, stable characteristics, sustainable passage, and good tolerance to viral infections. Cells from different passages from the 2nd to the 10th generation were deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on June 16, 2026, with accession number CCTCC NO: C202646.

[0031] Human gastric cancer peritoneal metastasis organoid cell line WGC-247, after passaged culture, was used for in vitro 3D organoid culture. After cell expansion and differentiation to form a three-dimensional structure (e.g., ... Figure 3 By culturing B and C, WGC-247 organoids can be obtained. The results of WGC-247 organoid culture are shown in [link to culture results]. Figure 3 B, of which Figure 3 The left image shows that the WGC-247 organoid has both solid and cystic properties, while the right image shows that the WGC-247 organoid at the edge of the 3D stroma gel releases a large number of single tumor cells into the nearby culture medium, which in vitro simulates the characteristic of gastric cancer that is prone to peritoneal dissemination and metastasis. Figure 3 Image C shows an HE staining image of the corresponding WGC-247 organoid, further demonstrating that the WGC-247 organoid possesses the cell arrangement morphology and differentiation state of the corresponding tumor tissue. This indicates that the WGC-247 organoid embodies the heterogeneity of tumors and their tendency to readily disseminate and release tumor cells.

[0032] Example 2 A method for establishing a nude mouse subcutaneous tumorigenesis model includes the following steps: 1) Organoid culture: The human gastric cancer peritoneal metastasis organoid cell line WGC-247 obtained in Example 1 was passaged, seeded, and cultured as 3D organoids to obtain a certain number of WGC-247 organoids. The medium was changed on the 4th, 7th, and 9th days after passage. 2) Organoid digestion and seeding: On day 10 post-passage, TrypLE digestive enzyme was added to WGC-247 organoids in the logarithmic growth phase for digestion. 250 μL of TrypLE digestive enzyme was added to each well, and the organoids were gently agitated. After 5 minutes, the agitation was repeated. The mixture was then neutralized with HBSS buffer, washed twice with PBS buffer, and centrifuged. Six-well organoids were collected, containing a total of 2 × 10⁶ organoids. 6 One cell was injected subcutaneously into nude mice; 3) Tumor formation observation: Observe and record the subcutaneous tumor formation and growth of nude mice once a week.

[0033] Tumors began to form in nude mice two weeks after implantation, small nodules appeared at three weeks, and the tumors grew to the size of [missing information] at fourteen weeks. Figure 4 A shows a 1.5cm area. The tumor tissue was stained with hematoxylin and eosin (HE), and the staining results are shown in Figure A. Figure 4 B, the figure shows that the tumorigenic tissue maintained the poorly differentiated characteristics of the patient's gastric cancer tissue, and... Figure 2 The HE staining results of the gastroscopy biopsy tissue from Patient A were consistent.

[0034] Example 3 A method for constructing an animal tumorigenesis model of gastric cancer by intraperitoneal injection metastasis includes the following steps: 1) Organoid culture: The human gastric cancer peritoneal metastasis organoid cell line WGC-247 obtained in Example 1 was passaged, seeded, and cultured as 3D organoids to obtain a certain number of WGC-247 organoids. The medium was changed on the 4th, 7th, and 9th days after passage. 2) Organoid digestion and seeding: On day 10 after passage, TrypLE digestive enzyme was added to WGC-247 organoids in the logarithmic growth phase for digestion. 250 μL of TrypLE digestive enzyme was added to each well, and the organoids were gently agitated. After 5 minutes, the agitation was repeated. The mixture was then neutralized with HBSS buffer, washed twice with PBS buffer, and centrifuged. Organoids from 24 wells were collected, containing a total of 8 × 10⁶ organoids. 6 One cell was injected into the peritoneal cavity of nude mice; 3) Tumor formation observation: Observe and record the tumor formation and weight changes of nude mice weekly, and observe the activity, feeding and abdominal condition of nude mice.

[0035] In preliminary experiments, approximately 1 million, 2 million, and 4 million cells were injected intraperitoneally into 6-week-old nude mice, and no peritoneal metastasis or tumor formation was observed. In this embodiment, 8 million cells were injected.

[0036] Tumorigenesis observation showed that tumor formation was perceptible in nude mice 4 weeks after tumor cell injection, a small amount of ascites was observed at 8 weeks, and significant ascites and peritoneal metastasis were observed at 14 weeks. In the first 4 weeks, the mice's body weight increased slightly according to natural growth patterns. From weeks 4 to 8, no significant increase or decrease in body weight was observed. At week 14, the mice's body weight increased by approximately 0.5g compared to week 8. Their activity level remained unchanged, but a noticeable fluctuation was perceptible in the abdomen, and bowel movements were normal. Following the experimental protocol, cervical dislocation was performed, and mesenteric metastases and abdominal wall nodules were observed. The results were as follows... Figure 5 As shown in Figure A, mesenteric metastatic lesions and abdominal wall metastatic nodules are visible to the naked eye in mice; HE staining of mouse intestinal mesenteric tissue and abdominal wall nodules yields the following results: Figure 5 As shown in Figure B, the mesenteric and abdominal wall nodules are all peritoneal metastatic tumor lesions, maintaining the poorly differentiated characteristics of the corresponding patients. Furthermore, the mesenteric nodules are all located outside the intestinal wall and between the mesentery. HE staining of mouse lung, liver, and spleen tissues yielded the following results: Figure 5 As shown in Figure C, the splenic hilar nodule is a tumor nodule, and it maintains the poor differentiation characteristics of the corresponding patient's tumor. In addition, no metastatic lesions were found in the spleen, liver, lungs, etc.

[0037] Mouse mesenteric nodules were analyzed by HE staining and immunohistochemical staining. Immunohistochemical staining was used to detect the expression of PCK, CK7, Her-2, Claudin18.2, CEA, p53, MLH1, PMS2, MSH2, and MSH6 proteins, as well as the Ki-67 proliferation index. The results are shown in [the table below]. Figure 6 As shown in the figure, intraperitoneal injection of tumor-forming tissues from WGC-247 organoid nude mice maintained the low-differentiation characteristics of the corresponding patient's tumor tissue and the consistent expression of corresponding proteins, such as high expression of Claudin18.2 and CEA positivity.

[0038] Example 4 A method for overexpressing luciferase in the human gastric cancer peritoneal metastasis organoid cell line WGC-247 includes the following steps: 1) Plasmid extraction: The packaging plasmid psPAX2, the envelope plasmid pMD2G, and the target plasmid PLVX-Luc-IRES-Neo were extracted using the Omega endotoxin-free plasmid extraction kit, and their concentrations were measured. All three plasmid-containing *E. coli* strains were purchased from Dianjun Biotechnology.

[0039] 2) Virus Packaging: Resuscitate 293T cells and passage them into 10cm culture dishes. Culture the 293T cells in high-glucose DMEM medium with 10% fetal bovine serum. When the confluence of cells reaches 80%, add 4μg, 3μg, and 1μg of the target plasmids PLVX-Luc-IRES-Neo, psPAX2, and pMD2G to the same 1.5mL Eppendorf tube. Then add 500µL of Opti-MEM medium. At the same time, add 32μL of polyethyleneimine (PEI) and 500μL of Opti-MEM medium to another 1.5mL Eppendorf tube. Mix well and let stand at room temperature for 5 minutes. Then mix the two Eppendorf tubes and let stand for 15 minutes. Add the mixture dropwise to the 293T cell culture dish. Change the medium after 12 hours. After 48 hours, collect the supernatant and filter it through a 0.48μm pore size filter. The collected filtrate is the virus supernatant containing the luciferase gene.

[0040] 3) Preparation of organoid culture plates and cell seeding: Take a standard 96-well culture plate, seal the perimeter with water, and then spread diluted matrix gel (Corning, 356234) at the bottom of 10 wells. Before use, the matrix gel is diluted with Advanced DMEM medium at a ratio of 1:2. Add 30µL of diluted matrix gel to each well, covering the bottom, and then incubate at 37℃ for 30 minutes to allow the gel to form. At the same time, digest and resuspend the WGC-247 organoids to be infected into a single-cell suspension with a cell count of 3×10⁶ cells. 4 / mL, seeded into the above 10 wells at a rate of 100µL / well.

[0041] 4) Adding viral supernatant: When WGC-247 cells divide into 4-8 cell clusters, add the viral supernatant containing the luciferase gene prepared in step 2) above to 9 wells instead of the culture medium. Add 100µL of viral supernatant to each well and add polybrene at a concentration of 5μg / mL. The other well serves as a blank control. After 12 hours of incubation, switch back to the organoid culture medium listed in Table 1 and continue culturing for 24 hours. Then, use 500μg / mL of genistein G418 for selection for about 5-7 days. After all cells in the control wells have died, continue adding G418 until passage and removal.

[0042] 5) Passaging and Assay: 10-14 days after cell selection in step 4), perform digestion and passaging as before, transferring to 24-well plates for further culture, selection, and cryopreservation. Simultaneously, pass 3 wells to 96-well plates for luciferase expression activity assay. After 1 week of culture, collect WGC-247 organoids overexpressing luciferase from 3 wells infected with lentivirus into 1.5 mL Eppendorf tubes. After centrifugation and discarding the supernatant, add 10 µL and 10 mg / mL VivoGlo™ luciferase reagent to each tube, mix well, and measure the luminescence intensity on a Gloma 20 / 20 luminometer. Simultaneously, use corresponding uninfected WGC-247 organoids as controls to clarify luciferase expression. Results are shown below. Figure 7 .Depend on Figure 7 Analysis of the luciferase intensity results showed that, compared with the control group, the WGC-247 organoids infected with lentivirus that overexpressed luciferase significantly expressed luciferase, promoting substrate luminescence, indicating successful infection and expression. This demonstrates that the WGC-247 organoid cell line can well tolerate lentivirus infection for gene expression intervention.

[0043] The human gastric cancer peritoneal metastasis organoid cell line WGC-247 in this invention is an organoid cell line obtained by culturing tumor cells from malignant ascites of gastric cancer peritoneal metastasis. Compared with traditional gastric cancer cell lines AZP7a and MKN-45P that have peritoneal metastasis characteristics, it lacks the process of artificially inducing and screening cells with high metastatic characteristics from the original cell lines AZ-521 and MKN45 in vitro, making WGC-247 closer to the original metastatic characteristics of the patient's tumor.

[0044] In summary, the WGC-247 cell line, derived from ascites fluid in gastric cancer peritoneal metastases, established in this invention exhibits good 3D organoid morphology and stable characteristics. It can be stably passaged in vitro and tolerates viral infections well. Studies have shown that it possesses very strong subcutaneous tumorigenicity and good peritoneal tumorigenicity, successfully establishing a gastric cancer peritoneal metastasis model. This provides new in vitro and in vivo research support for further in-depth studies on the etiology, pathogenesis, drug resistance mechanisms, and new drug screening of gastric cancer peritoneal metastases.

Claims

1. A human gastric cancer peritoneal metastasis organoid cell line, characterized in that, This includes the human gastric cancer peritoneal metastasis organoid cell line WGC-247, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202646.

2. The human gastric cancer peritoneal metastasis organoid cell line according to claim 1, characterized in that, The human gastric cancer peritoneal metastasis organoid cell line WGC-247 was established by in vitro 3D organoid culture of ascites tumor cells from patients with gastric cancer and peritoneal metastasis accompanied by malignant ascites.

3. A method for culturing a human gastric cancer peritoneal metastasis organoid cell line, characterized in that, Includes the following steps: 1) Sample collection: Ascites fluid was collected from patients with gastric cancer and peritoneal metastasis accompanied by malignant ascites under aseptic conditions; 2) Preliminary isolation of tumor cells: Tumor cells were isolated using a multiple centrifugation method. The multiple centrifugation method included: centrifuging the ascites fluid sequentially at 1000 r / min for 5 min, 800 r / min for 5 min, and 400 r / min for 3 min, and finally repeating the centrifugation at 400 r / min for 3 min. The process was stopped when no visible red blood cells were precipitated, and the tumor cell pellet was obtained. After each centrifugation, the supernatant was discarded and the pellet was resuspended in PBS buffer. 3) Seeding cells: After resuspending the tumor cell pellet in matrix gel, seed the cells into 24-well plates and incubate at 37°C until the matrix gel solidifies into a dome shape. Then, add culture medium for further incubation. 4) 3D organoid culture and routine passage culture: Change the culture medium every 3-5 days, and perform cell seeding and passage operations on days 10-14, repeating steps 3) and 4).

4. The application of the human gastric cancer peritoneal metastasis organoid cell line according to claim 1 or 2 in constructing a nude mouse subcutaneous tumorigenesis animal model or a gastric cancer peritoneal metastasis tumorigenesis animal model.

5. A method for constructing a nude mouse subcutaneous tumor-forming animal model, characterized in that, Includes the following steps: 1) Organoid culture: By culturing, passaged, and reculturing the human gastric cancer peritoneal metastasis organoid cell line described in claim 1, a certain number of WGC-247 organoids are obtained; 2) Organoid Collection and Planting: On day 10 after subculturing, TrypLE digestive enzyme was added to WGC-247 organoids for digestion, followed by neutralization with HBSS buffer, washing with PBS buffer, centrifugation, and collection of 1.5 × 10⁻⁶ organoids. 6 ~2.5×10 6 One WGC-247 organoid was injected subcutaneously into nude mice; 3) Tumor formation observation: Regularly observe and record the subcutaneous tumor formation in nude mice.

6. A method for constructing an animal model of gastric cancer metastasis in the peritoneum, characterized in that, Includes the following steps: 1) Organoid culture: By culturing, passaged, and reculturing the human gastric cancer peritoneal metastasis organoid cell line described in claim 1, a certain number of WGC-247 organoids are obtained; 2) Organoid Collection and Planting: On day 10 after subculturing, TrypLE digestive enzyme was added to WGC-247 organoids for digestion, followed by neutralization with HBSS buffer, washing with PBS buffer, centrifugation, and collection of 7×10⁶ organoids. 6 ~9×10 6 One WGC-247 organoid was injected into the abdominal cavity of nude mice; 3) Tumor formation observation: Regularly observe and record the tumor formation and weight changes of nude mice, and observe the activity, diet and abdominal condition of nude mice.

7. The use of the human gastric cancer peritoneal metastasis organoid cell line of claim 1 or 2 in overexpressing luciferase or in preparing cell products overexpressing luciferase.

8. The use of the human gastric cancer peritoneal metastasis organoid cell line as described in claim 1 or 2 in screening drugs for the treatment of gastric cancer and gastric cancer peritoneal metastasis.

9. The application according to claim 8, characterized in that, include: The therapeutic drug is applied to an in vitro cell model, and if it inhibits cell proliferation or induces apoptosis after application, it is a therapeutically sensitive drug; or the test compound is applied to an in vitro animal model, and if it inhibits disease progression, reduces ascites formation, improves the survival status of nude mice, and prolongs survival time after application, it is a therapeutically effective drug; the in vitro cell model or in vitro animal model is obtained by constructing the human gastric cancer peritoneal metastasis organoid cell line as described in claim 1 or 2.

10. The use of the human gastric cancer peritoneal metastasis organoid cell line of claim 1 or 2 in the preparation of products for screening drugs for the treatment of gastric cancer and gastric cancer peritoneal metastasis.

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