Construction and application of two mouse esophageal squamous cell carcinoma organoid models with gender difference

Abstract

The invention discloses construction and application of two mouse esophageal squamous cell carcinoma organoid models with sex difference. The method comprises the steps of 1, tumor sample collection; step 2, carrying out single-cell enzymolysis treatment; step 3, modeling and culturing the tumor organoid; step 4, passage of the tumor organs and construction of a library for cryopreservation; compared with the prior art, the invention has the advantages that two stable and high-purity esophageal squamous carcinoma organoid strains are successfully established and are respectively named as PDO306 (from female mice) and PDO307 (from male mice). The establishment of the two new esophageal squamous cell carcinoma organoid strains makes up for the deficiency of immune microenvironment in the esophageal squamous cell carcinoma in-vitro research process, and valuable experimental materials and tools are provided for subsequent esophageal squamous cell carcinoma research.

Description

Technical Field

[0001] The present invention relates to the technical field of constructing esophageal squamous cell carcinoma organoid models, and specifically to the construction and application of two mouse esophageal squamous cell carcinoma organoid models with gender differences. Background Art

[0002] Esophageal cancer is mainly divided into squamous cell carcinoma (ESCC) and adenocarcinoma (EAC) pathologically. Among them, ESCC mostly originates from the squamous epithelium of the esophagus and is the most common type of esophageal cancer in China. This cancer is characterized by rapid clinical progression and poor prognosis, and its 5-year survival rate is usually less than 30%. At present, the standard treatment methods for ESCC are surgical resection, radiotherapy, and chemotherapy. In recent years, the combined application of immunotherapy and chemotherapy, especially the use of immune checkpoint inhibitors, has become an important strategy for ESCC treatment, especially for advanced treatment. However, the clinical effect of this combined treatment is not ideal, and the proportion of patients with complete remission is less than 40%. As a highly potential treatment method, immunotherapy still needs to deeply study its mechanism of action and clinical effect to further improve the effectiveness of treatment.

[0003] The traditional in vitro research system for tumors is related to cell lines, but it lacks the tumor microenvironment, which leads to limitations in research. However, organoids perfectly solve this problem due to the richness of their tumor microenvironment. Given the high similarity between mice and humans in genetic and pathological characteristics, mouse models play a key role in the research of new drug targets, and their preclinical data are of irreplaceable importance for promoting related research. At present, for the research on the tumor microenvironment of esophageal squamous cell carcinoma, the two commonly used mouse models are the primary mouse model of esophageal squamous cell carcinoma induced by 4-nitroquinoline-1-oxide (4-NQO) and the mouse model of tumor xenotransplantation. However, the 4-NQO-induced model has the limitation of a relatively long time-consuming process, which makes mouse-derived organoids particularly important in the research of the tumor immune microenvironment.

[0004] The information disclosed in this background art section is only intended to enhance the overall understanding of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the above technical defects and provide the construction and application of two organoid models of esophageal squamous cell carcinoma in mice with gender differences. The present invention has successfully established two stable and highly pure esophageal squamous cell carcinoma organoids, named PDO306 (derived from female mice) and PDO307 (derived from male mice) respectively. The establishment of these two new esophageal squamous cell carcinoma organoids makes up for the lack of immune microenvironment in the in vitro study of esophageal squamous cell carcinoma, and provides valuable experimental materials and tools for the subsequent research of esophageal squamous cell carcinoma. By deeply studying the biological characteristics and genetic background of these two organoids, we can more comprehensively understand the pathogenesis of esophageal squamous cell carcinoma and the role of the immune microenvironment therein, so as to provide new ideas and methods for clinical treatment and prevention.

[0006] To solve the above problems, the technical solution of the present invention is the construction of two organoid models of esophageal squamous cell carcinoma in mice with gender differences: the male organoids in the two esophageal squamous cell carcinoma organoids are named PDO307, and the female organoids are named PDO306. The method for constructing the organoid model includes the following steps:

[0007] Step 1: Tumor sample collection. Select 8-week-old C57BL / 6J male and female mice and feed them with 4-NQO solution for 16 weeks, and then feed them with normal water for 12 weeks. The mice are euthanized, and the multiple lesion areas existing in the esophagus are selected. After dissection, they are put into tissue specimen preservation solution, the sample information is marked, the tube cap is sealed tightly, and they are transported to the cell culture room at high activity in a 4°C transport box;

[0008] Step 2: Single-cell enzymatic digestion treatment. Malignant pleural and peritoneal effusions are centrifuged and filtered to enrich tumor cells. The tumor cells are washed twice with phosphate buffer solution (PBS) containing compound antibiotics, and then an enzymatic digestion complex solution is added. The mixture is placed on a shaker and digested at 200 rpm for 30 minutes. The digested cells pass through a cell sieve to obtain a single-cell suspension; the single-cell suspension is centrifuged twice at 200 g, resuspended with PBS, and centrifuged to separate the tumor cell layer;

[0009] Step 3: Tumor organoid modeling culture. The isolated tumor cells are added to a three-dimensional culture matrix and planted in a special cell culture device at low temperature. After incubating at 37°C for 30 minutes, after the solid tumor layer is formed, a culture base layer is added; the expanded organoids are enzymatically digested by protease, reverted to single cells, centrifuged and separated, resuspended with a culture medium, the resuspended cells are counted, and after re-centrifugation, Step 3 is repeated for subculture at a passage ratio of 1:2 to 1:4;

[0010] Step 4: Subculture and cryopreservation of tumor organoids. Collect the cultured tumor organoids with a Pasteur pipette, add enzymatic digestion solution to digest the tumor organoids to form a single-cell suspension. After cell counting, adjust the cell concentration with organoid cryopreservation buffer, add organoid cryopreservation solution, and then perform programmed cooling, and finally transfer it to liquid nitrogen for ultra-low temperature long-term preservation.

[0011] Further, in Step 1, the concentration of 4-NQO is 100 μg / mL.

[0012] Further, in Step 2, the temperature of the enzymatic digestion process is 37°C.

[0013] Further, in Step 3, the culture substrate contains optimized growth factors and nutrients, which can provide a microenvironment for the rapid growth of tumor organoids. Observe the growth state of the organoids under a microscope at any time.

[0014] Further, in Step 4, adjust the cell concentration to 1×10 6 ce l l s / ml, and add organoid cryopreservation solution according to 5×10 5 ce l l s / tube.

[0015] Further, the application of the two organoid models of esophageal squamous cell carcinoma with gender differences is characterized in that: fix and section the tumor organoids and perform HE staining. After observing under the microscope that the rapidly growing tumor organoid cells grow to a certain size and quantity, use DMEM medium to dilute and centrifuge to remove the Matrigel in the culture well plate. After the organoid cells are centrifuged and precipitated at low speed, add paraformaldehyde for fixation, and then perform sectioning and HE staining.

[0016] The advantages of the present invention compared with the existing technology are as follows:

[0017] 1. The present invention successfully established two stable and highly pure esophageal squamous cell carcinoma organoids, named PDO306 (derived from female mice) and PDO307 (derived from male mice) respectively. The establishment of these two new esophageal squamous cell carcinoma organoids makes up for the lack of immune microenvironment in the in vitro study of esophageal squamous cell carcinoma, and provides valuable experimental materials and tools for the subsequent research of esophageal squamous cell carcinoma. By deeply studying the biological characteristics and genetic background of these two organoids, we can more comprehensively understand the pathogenesis of esophageal squamous cell carcinoma and the role of the immune microenvironment therein, so as to provide new ideas and methods for clinical treatment and prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the sample collection and preservation diagram of the present invention.

[0019] Figure 2 is the single-cell enzymatic digestion diagram of the present invention.

[0020] Figure 3 It is a figure of esophageal squamous cell carcinoma organoids of the present invention.

[0021] Figure 4 It is a HE staining figure of the organoids of the present invention.

[0022] Figure 5 It is a figure of the sample collection process of the present invention.

[0023] Figure 6 It is a figure of the organoid construction process of the present invention. Detailed implementation manners

[0024] In order to make the content of the present invention be more clearly understood, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention.

[0025] The construction and application of two gender-differentiated mouse esophageal squamous cell carcinoma organoid models of the present invention. The name of the male organoid is PDO307, and the name of the female organoid is PDO306. The method for constructing the organoids includes the following steps:

[0026] Step 1: Tumor sample collection. Select 8-week-old male and female C57BL / 6J mice and feed them with 4-NQO solution for 16 weeks. The concentration of 4-NQO is 100 μg / mL, and then feed them with normal water for 12 weeks. The mice are euthanized, and the multiple lesion areas present in the esophagus are selected. After dissection, they are put into the tissue specimen preservation solution, the sample information is marked, the tube cap is sealed tightly, and they are transported to the cell culture room at high activity in a 4°C transport box;

[0027] Step 2: Single-cell enzymatic digestion treatment. Centrifuge and filter the malignant pleural and peritoneal effusions to enrich the tumor cells. After the tumor cells are washed twice with phosphate buffer solution (PBS) containing compound antibiotics, add the enzymatic digestion complex solution, place it on a shaker, and digest at 200 rpm for 30 minutes. The temperature during the enzymatic digestion process is 37°C. The enzymatically digested cells pass through a cell sieve to obtain a single-cell suspension; after the cell suspension is centrifuged twice at 200 g, it is resuspended with PBS, and the tumor cell layer is obtained by centrifugal separation;

[0028] Step 3: Tumor organoid modeling culture. Add the isolated tumor cells into the three-dimensional culture matrix, plant them in a special cell culture device at low temperature, incubate at 37°C for 30 minutes, and after the solid tumor layer is formed, add the culture base layer; the culture base layer contains optimized growth factors and nutrients, which can provide a microenvironment for the rapid growth of tumor organoids. Observe the growth state of the organoids through a microscope at any time; the expanded organoids are enzymatically digested by protease, become single cells again, are centrifuged and separated, resuspended with the culture medium, the resuspended cells are counted, and after centrifugation again, repeat Step 3 to amplify the culture at a passage ratio of 1:2 to 1:4;

[0029] Step 4: Subculture and cryopreservation of tumor organoids. Use a Pasteur pipette to collect the cultured tumor organoids, add enzymatic digestion solution to digest the tumor organoids to form a single-cell suspension. After cell counting, use organoid cryopreservation buffer to adjust the cell concentration to 1×10 6 cells / ml, add organoid cryopreservation solution at 5×10 6 cells / tube, and then cool the cells through a programmed cooling process and finally transfer them to liquid nitrogen for ultra-low temperature long-term preservation.

[0030] Application of two gender-different esophageal squamous cell carcinoma organoid models. It is characterized in that: for the fixed sections and HE staining of the tumor organoids, after observing under the microscope that the rapidly growing tumor organoid cells grow to a certain size and quantity, use DMEM medium to dilute and centrifuge to remove the Matrigel in the culture well plate. After the organoid cells are centrifuged and precipitated at low speed, add paraformaldehyde for fixation, and then perform sectioning and HE staining.

[0031] The establishment of these two new esophageal squamous cell carcinoma organoids makes up for the lack of immune microenvironment in the in vitro study of esophageal squamous cell carcinoma, and provides valuable experimental materials and tools for subsequent research on esophageal squamous cell carcinoma. By deeply studying the biological characteristics and genetic background of these two organoids, we can more comprehensively understand the pathogenesis of esophageal squamous cell carcinoma and the role of the immune microenvironment therein, so as to provide new ideas and methods for clinical treatment and prevention.

[0032] The above description of the present invention and its implementation manners is not restrictive. Generally speaking, if those of ordinary skill in the art are inspired by it and without departing from the gist of the present invention, they design similar structural modes and embodiments to this technical solution without creative efforts, which shall fall within the protection scope of the present invention.

Claims

1. Construction of two sex-differentiated mouse esophageal squamous cell carcinoma organoid models, characterized by: The male organoid of the two esophageal squamous cell carcinoma organoids is named PDO307, and the female organoid is named PDO306. The organoid model construction method comprises the following steps: Step 1: Tumor sample collection: 8-week-old C57BL / 6J male and female mice were selected and fed with 4-NQO solution for 16 weeks and then with normal water for 12 weeks. The mice were euthanized, and multiple lesion areas in the esophagus were selected. After dissection, the samples were placed in tissue specimen preservation solution, labeled with sample information, and tightly sealed. The samples were then transported to the cell culture room in a 4°C transport box with high activity. Step 2: single cell enzymatic hydrolysis treatment: malignant pleural and abdominal effusions are centrifuged and filtered to enrich tumor cells. The tumor cells are washed twice with phosphate buffered saline (PBS) containing compound antibiotics, and then the enzymatic complex solution is added and placed in a shaker for enzymatic hydrolysis at 200 rpm for 30 minutes. The enzymatically hydrolyzed cells are passed through a cell sieve to obtain a single cell suspension; the cell suspension is centrifuged twice at 200 g, resuspended with PBS, and centrifuged to obtain a tumor cell layer; Step 3: Modeling and culturing tumor organoids: adding the isolated tumor cells to a three-dimensional culture matrix, planting them in a special cell culture device at low temperature, incubating them at 37°C for 30 minutes, and adding a culture medium layer after a solid tumor layer is formed; the expanded cultured organoids are hydrolyzed by protease to become single cells again, centrifuged and resuspended in culture medium, the resuspended cells are counted, and after re-centrifugation, step 3 is repeated to expand the culture at a subculture ratio of 1:2 to 1:4; Step 4: Tumor organoids are passaged and cryopreserved. Use a Pasteur pipette to collect the cultured tumor organoids, add enzymatic solution to hydrolyze the tumor organoids to form a single-cell suspension. After cell counting, use organoid freezing buffer to adjust the cell concentration, add organoid freezing solution, and then cool through a program, and finally transfer to liquid nitrogen for ultra-low temperature long-term storage.

2. The construction of the two sex-differentiated mouse esophageal squamous cell carcinoma organoid model according to claim 1, characterized in that: In step 1, the concentration of 4-NQO is 100 μg / mL.

3. The construction of the two sex-differentiated mouse esophageal squamous cell carcinoma organoid model according to claim 1, characterized in that: In step 2, the temperature of the enzymatic hydrolysis process is 37°C.

4. The construction of the two sex-differentiated mouse esophageal squamous cell carcinoma organoid model according to claim 1, characterized in that: In step three, the culture medium layer contains optimized growth factors and nutrients, which can provide a microenvironment for the rapid growth of tumor organoids, and the growth status of the organoids can be observed under a microscope.

5. The construction of the two sex-differentiated mouse esophageal squamous cell carcinoma organoid model according to claim 1, characterized in that: In step 4, adjust the cell concentration to 1×10 6 cells / ml, based on 5×10 5 cells / tube and add organoid freezing solution.

6. The use of the two sex-differentiated mouse esophageal squamous cell carcinoma organoid model according to claims 1-5, characterized in that: The tumor organoids were fixed, sectioned and stained with HE. After the rapidly growing tumor organoid cells grew to a certain size and number under a microscope, DMEM culture medium was used to dilute and centrifuge to remove the matrix gel in the culture well plate. After the organoid cells were precipitated by low-speed centrifugation, paraformaldehyde was added for fixation, and then sectioned and stained with HE.

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