Method for establishing stomach cancer liver metastasis PDX model

Abstract

The invention provides an establishment method of a gastric cancer liver metastasis PDX model, and belongs to the technical field of PDX model establishment. The establishment method of the stomach cancer liver metastasis PDX model comprises the following steps: collecting stomach cancer tissues of a clinical patient, and preprocessing the stomach cancer tissues; cutting the gastric cancer tissue into gastric cancer tissue cubes and storing the gastric cancer tissue cubes; the method comprises the following steps: shaving and preparing abdominal skin of a mouse, disinfecting, transversely cutting the skin and peritoneum of the mouse by using sterile dissecting scissors, and exposing large-lobe tissue of the liver; a sterile syringe needle is used for creating a tiny wound on the liver lobe scratch; suturing the gastric cancer tissue cubes to the tiny wounds; and the peritoneum and skin of the mouse are sutured by absorbable surgical sutures. Compared with a gastric cancer liver metastasis model constructed by a cell line, the model obtained by the method for establishing the gastric cancer liver metastasis PDX model is closer to tumor tissue characteristics, genetic hereditary characteristics and tumor heterogeneity of a patient, and is more beneficial to individualized treatment or mechanism research of the patient.

Description

Technical Field

[0001] The present invention relates to the technical field of PDX model establishment, and in particular to a method for establishing a gastric cancer liver metastasis PDX model. Background Art

[0002] Gastric cancer (GC) is a common malignancy and a leading cause of cancer-related mortality. According to statistics, the global incidence of new GC cases in 2018 was 5.7%, ranking fifth among all cancers, with a mortality rate of 8.2%, second only to lung cancer and colorectal cancer. In addition to these high incidence and mortality rates, most GC patients present with advanced disease, even with distant metastases, making radical treatment difficult.

[0003] Humanized xenograft (PDX) models closely mimic the in vivo growth of tumors, offering excellent clinical predictability. They can be used to screen drugs on behalf of specific patients and guide personalized anti-tumor treatments in clinical settings, known as "Avatar" trials. Some patients have already benefited from these models. Experiments have confirmed that gastric cancer PDX models closely resemble primary tumors in terms of histopathological characteristics, gene expression, and drug response. Their application in gastric cancer research will undoubtedly lead to new breakthroughs in personalized gastric cancer treatment. While significant progress has been made in PDX models for digestive system tumors, particularly colon cancer, research on gastric cancer PDX models is still in its infancy, and studies on gastric cancer metastasis PDX models are even rarer.

[0004] Most existing gastric cancer research uses gastric cancer cell lines to establish subcutaneous or orthotopic mouse models. Gastric cancer metastasis PDX models maximize the preservation of patient tumor tissue characteristics, genetic characteristics, and tumor heterogeneity. This addresses the problem that current tumor cell lines undergo thousands of generations of culture and selection, radically altering their genetic composition and behavior. Consequently, research results from tumor animal models established using these cell lines or cells are less than 5% relevant to clinical practice.

[0005] Therefore, providing a method for establishing a gastric cancer liver metastasis model that is closer to the patient's tumor tissue characteristics, genetic genetic characteristics and tumor heterogeneity has important practical significance for personalized treatment or mechanism research. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for establishing a gastric cancer liver metastasis PDX model. Compared with gastric cancer liver metastasis models constructed using cell lines, the model obtained by the gastric cancer PDX model establishment method of the present invention is closer to the patient's tumor tissue characteristics, genetic characteristics and tumor heterogeneity, and is more conducive to its personalized treatment or mechanism research.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for establishing a gastric cancer liver metastasis PDX model, comprising:

[0009] (1) Collect gastric cancer tissues from clinical patients and pretreat them;

[0010] (2) cutting the gastric cancer tissue into gastric cancer tissue blocks and then preserving them;

[0011] (3) Shave the abdominal skin of the mouse, prepare the skin, disinfect it, and use sterile dissecting scissors to cut the mouse skin and peritoneum horizontally to expose the liver lobe tissue;

[0012] (4) using a sterile syringe needle to create a micro-wound in the liver lobe tissue;

[0013] (5) suturing the gastric cancer tissue block to the micro-wound;

[0014] (6) Use absorbable surgical sutures to suture the mouse peritoneum and skin to complete the model.

[0015] Furthermore, the pretreatment is to remove part of the necrotic tissue in the gastric cancer tissue and wash it with physiological saline.

[0016] Furthermore, the size of the gastric cancer tissue block is 3 mm×3 mm.

[0017] Furthermore, the storage condition is -30 to -5°C.

[0018] Furthermore, the mice are 4 to 6 weeks old.

[0019] Furthermore, during the modeling process, sterile cotton swabs or cotton balls are used to absorb blood.

[0020] The present invention also provides a gastric cancer liver metastasis PDX model obtained by the method for establishing a gastric cancer liver metastasis PDX model described in the above technical solution.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0022] Compared with the gastric cancer liver metastasis model constructed by cell line, the method for establishing the gastric cancer liver metastasis PDX model described in the present invention is closer to the patient's tumor tissue characteristics, genetic characteristics and tumor heterogeneity, and is more conducive to its individualized treatment or mechanism research. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a surface image of the mouse gastric cancer liver metastasis model obtained in Example 1;

[0024] Figure 2 This is a schematic diagram of the liver of a mouse with gastric cancer liver metastasis obtained in Example 1;

[0025] Figure 3 Schematic diagram of pathological HE staining of the liver of gastric cancer liver metastasis in mice obtained in Example 1. DETAILED DESCRIPTION

[0026] Humanized xenograft (PDX) models closely mimic the in vivo growth of tumors, offering excellent clinical predictability. They can be used to screen drugs on behalf of specific patients and guide personalized anti-tumor treatments in clinical settings, known as "Avatar" trials. Some patients have already benefited from these models. Experiments have confirmed that gastric cancer PDX models closely resemble primary tumors in terms of histopathological characteristics, gene expression, and drug response. Their application in gastric cancer research will undoubtedly lead to new breakthroughs in personalized gastric cancer treatment. While significant progress has been made in PDX models for digestive system tumors, particularly colon cancer, research on gastric cancer PDX models is still in its infancy, and studies on gastric cancer metastasis PDX models are even rarer.

[0027] Most existing gastric cancer research uses gastric cancer cell lines to establish subcutaneous or orthotopic mouse models. Gastric cancer metastasis PDX models maximize the preservation of patient tumor tissue characteristics, genetic characteristics, and tumor heterogeneity. This addresses the problem that current tumor cell lines undergo thousands of generations of culture and selection, radically altering their genetic composition and behavior. Consequently, research results from tumor animal models established using these cell lines or cells are less than 5% relevant to clinical practice.

[0028] Therefore, providing a method for establishing a gastric cancer liver metastasis model that is closer to the patient's tumor tissue characteristics, genetic genetic characteristics and tumor heterogeneity has important practical significance for personalized treatment or mechanism research.

[0029] The present invention provides a method for establishing a gastric cancer liver metastasis PDX model, comprising:

[0030] (1) Collect gastric cancer tissues from clinical patients and pretreat them;

[0031] (2) cutting the gastric cancer tissue into gastric cancer tissue blocks and then preserving them;

[0032] (3) Shave the abdominal skin of the mouse, prepare the skin, disinfect it, and use sterile dissecting scissors to cut the mouse skin and peritoneum horizontally to expose the liver lobe tissue;

[0033] (4) using a sterile syringe needle to create a micro-wound in the liver lobe tissue;

[0034] (5) suturing the gastric cancer tissue block to the micro-wound;

[0035] (6) Use absorbable surgical sutures to suture the mouse peritoneum and skin to complete the model.

[0036] Gastric cancer tissues were collected from clinical patients and pretreated;

[0037] In the present invention, the pretreatment is preferably to remove part of the necrotic tissue in the gastric cancer tissue and wash it with physiological saline;

[0038] The present invention pre-treats gastric cancer tissue collected from clinical patients to purify it, reduce the interference of other substances in the experiment, and improve the accuracy and reliability of subsequent analysis; in addition, it can also maintain the activity of gastric cancer tissue, facilitate the normal growth of mice in the subsequent modeling process, and ensure the repeatability of the experiment.

[0039] cutting the gastric cancer tissue into gastric cancer tissue blocks and then storing them;

[0040] The gastric cancer tissue sample collected by the present invention is not suitable for direct transplantation into mice. It is convenient to transplant it into mice after cutting it into cubes of appropriate size.

[0041] In the present invention, the size of the gastric cancer tissue block is preferably 3 mm×3 mm.

[0042] In the present invention, the storage condition is preferably -30 to -5°C.

[0043] After the gastric cancer tissue is cut into pieces, the pieces are stored for future use. The gastric cancer tissue pieces can maintain activity under the storage conditions of the present invention, making it easy to grow normally after being subsequently transplanted into mice.

[0044] The abdominal skin of the mouse was shaved and disinfected, and the skin and peritoneum were cut transversely using sterile dissecting scissors to expose the liver lobe tissue.

[0045] The disinfection described in the present invention is preferably carried out by wiping with 75% alcohol. It can also be disinfected by a disinfection method well known to those skilled in the art. The present invention does not specifically limit the disinfection method.

[0046] In the present invention, the experimental mice are mice with an age of 4 to 6 weeks.

[0047] Then, a sterile syringe needle was used to create a micro-wound in the liver lobe tissue of the mouse.

[0048] Then, the gastric cancer tissue block is sutured to the micro-wound;

[0049] The suturing described in the present invention can be performed by suturing means well known to those skilled in the art and is not particularly limited thereto.

[0050] Finally, absorbable surgical sutures were used to suture the mouse peritoneum and skin to complete the model.

[0051] In the present invention, a sterile cotton swab or cotton ball is used to absorb blood during the modeling process.

[0052] The present invention continuously uses sterile cotton swabs or cotton balls to absorb blood during the modeling process to avoid blurring and affecting the experiment. The surface diagram of the mouse gastric cancer liver metastasis model completed by the present invention is as follows: Figure 1 shown.

[0053] The present invention also provides a gastric cancer liver metastasis PDX model obtained by the above technical solution.

[0054] In the present invention, unless otherwise specified, the raw materials required for preparation are all commercially available products well known to those skilled in the art.

[0055] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] (1) Gastric cancer tissue was collected from a clinical patient with gastric cancer. After the gastric cancer tissue was isolated from the body, it was rinsed with appropriate saline and then transferred into a 50 mL centrifuge tube containing PMI-1640 / DMEM for transplantation. Afterwards, some necrotic tissue in the gastric cancer tissue was removed and washed with physiological saline to complete the pretreatment.

[0058] (2) The pretreated gastric cancer tissue was cut into 3 mm × 3 mm gastric cancer tissue cubes and stored at -30°C;

[0059] (3) The abdominal skin of 10 mice aged 4 to 6 weeks was shaved and prepared, and disinfected with medical alcohol. Then, sterile dissecting scissors were used to cut the abdominal skin and peritoneum of the mice transversely to expose the liver lobes.

[0060] (4) Using a sterile syringe needle, scratch the liver lobes of all mice to create micro-wounds;

[0061] (5) The gastric cancer tissue blocks were sequentially sutured to the tiny wounds on the liver lobes of all mice;

[0062] (6) Finally, absorbable surgical sutures were used to suture the peritoneum and skin of all mice;

[0063] (7) All mice were cultured under natural conditions, and the various indicators of the mice were recorded regularly. The tumor formation was observed after 8 weeks.

[0064] test

[0065] (1) The mice successfully modeled in Example 1 were dissected to obtain the liver of the gastric cancer liver metastasis. Figure 2 As shown, based on Figure 2 It can be seen that the modeling experiment described in Example 1 of the present invention successfully obtained the tumor volume.

[0066] (2) The liver of the gastric cancer liver metastasis mouse model successfully established in Example 1 was subjected to pathological HE staining. Figure 3 As shown, based on Figure 3 It can be seen that in Figure 2 The success of the model was further verified on this basis.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for establishing a gastric cancer liver metastasis PDX model, characterized in that: include: (1) Collect gastric cancer tissues from clinical patients and pretreat them; (2) cutting the gastric cancer tissue into gastric cancer tissue blocks and then storing them; (3) The abdominal skin of the mouse was shaved and disinfected, and the mouse skin and peritoneum were cut transversely using sterile dissecting scissors to expose the liver lobe tissue; (4) using a sterile syringe needle to create a micro-incision in the liver lobe tissue; (5) suturing the gastric cancer tissue block to the micro-incision; (6) The mouse peritoneum and skin are sutured using absorbable surgical sutures to complete the model.

2. The method for establishing a gastric cancer liver metastasis PDX model according to claim 1, characterized in that: The pretreatment is to remove part of the necrotic tissue in the gastric cancer tissue and wash it with physiological saline.

3. The method for establishing a gastric cancer liver metastasis PDX model according to claim 1, characterized in that: The size of the gastric cancer tissue block is 3 mm×3 mm.

4. The method for establishing a gastric cancer liver metastasis PDX model according to claim 1, characterized in that: The storage condition is -30 to -5°C.

5. The method for establishing a gastric cancer liver metastasis PDX model according to claim 1, characterized in that: The mice are 4 to 6 weeks old.

6. The method for establishing a gastric cancer liver metastasis PDX model according to claim 1, characterized in that: During the modeling process, a sterile cotton swab or cotton ball is used to absorb blood.

7. A gastric cancer liver metastasis PDX model obtained by the method for establishing a gastric cancer liver metastasis PDX model according to any one of claims 1 to 6.

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