A method for constructing a gastric MiNENs model and application thereof

Abstract

The application provides a gastric MiNENs model and application, and a novel gastric MiNENs conditional gene knockout mouse model (Pou2f3-CreERT;Rosa26-tdTomato;Rb1 fl / fl ;Trp53 fl / fl ;LSL-Myc T58A , referred to as PTD-RPM mouse) is established, the model can rapidly induce the formation of gastric MiNENs within one month, the histopathological characteristics and molecular phenotype of human gastric MiNENs are reproduced, and three different morphological tumor cells are observed: atypical hyperplasia (M1 subtype), adenocarcinoma (M2 subtype) and neuroendocrine carcinoma (M3 subtype). The establishment of the model provides an important experimental tool for further research of the gastric MiNENs, and is crucial for in-depth research of the origin and pathogenesis, and finding of new treatment targets and strategies.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the construction method and application of gastric MiNENs models. Background Technology

[0002] Mixed neuroendocrine-nonneuroendocrine tumors (MiNENs) are defined as tumors containing two or more different histological components, each of which occupies at least 30% of the tumor volume. The neuroendocrine component is usually a neuroendocrine carcinoma, while the nonneuroendocrine component is mainly adenocarcinoma, accounting for up to 90%, and may also include other epithelial tumors such as squamous cell carcinoma. Gastric MiNENs, due to their complex composition, high invasiveness, and high malignancy, often have distant metastases at initial diagnosis and are less sensitive to chemotherapy drugs than gastric adenocarcinoma, leading to poor patient prognosis. Although recent studies have attempted to reveal the key genetic and epigenetic abnormalities behind MiNENs and explore the biological similarities and potential common therapeutic targets among their mixed components, key factors remain incompletely elucidated, and research is largely limited to sites such as the lung and pancreas. Research on the molecular drivers and pathogenesis of gastric MiNENs is scarce, largely due to the lack of corresponding animal models. Therefore, developing a stable animal model of gastric MiNENs is crucial for in-depth research into its origin and pathogenesis, and for identifying new therapeutic targets and strategies. Summary of the Invention

[0003] This invention provides a model for establishing and applying a gastric MiNENs model. This model can rapidly induce the formation of gastric MiNENs within one month, reproducing the histopathological features and molecular phenotype of human gastric MiNENs, and observing three different morphologies of tumor cells: dysplasia (M1 subtype), adenocarcinoma (M2 subtype), and neuroendocrine carcinoma (M3 subtype). The establishment of this model provides an important experimental tool for further research on gastric MiNENs, and is crucial for in-depth research into their origin and pathogenesis, and for finding new therapeutic targets and strategies.

[0004] This invention provides a method for constructing a gastric MiNENs model, comprising the following steps:

[0005] Step (1): Mice expressing the Pou2f3 promoter-driven Cre recombinase were crossed with mice carrying conditional alleles of Rb1 deletion, Trp53 deletion, and Myc overexpression to obtain mice with the genotype Pou2f3-CreERT; Rosa26-tdTomato; Rb1 fl / fl Trp53 fl / fl LSL-Myc T58A Transgenic mice;

[0006] Step (2): The transgenic mice obtained in step (1) were given an inducer to activate Cre recombinase, thereby specifically knocking out Rb1 and Trp53 genes and activating Myc expression in Pou2f3 positive cells in the stomach, thus inducing the formation of gastric MiNENs.

[0007] Furthermore, the Rosa26-tdTomato reporter gene was introduced in step (1) above for lineage tracing.

[0008] Furthermore, the aforementioned inducer is tamoxifen.

[0009] Furthermore, the induction method described above is intraperitoneal injection, with a dosage of 40-60 mg kg / day, administered continuously for 1-2 days.

[0010] Furthermore, the above model formed tumors with histopathological and functional phenotypes consistent with human primary gastric MiNENs within one month after induction.

[0011] Furthermore, the aforementioned tumors exhibit a mixed morphology of glandular neuroendocrine carcinoma, including dysplastic morphology, adenocarcinoma morphology, and neuroendocrine carcinoma.

[0012] The present invention also provides a transgenic mouse model of MiNENs constructed using the above-described construction method.

[0013] Furthermore, this invention also provides the application of the above-mentioned model in studying the origin and pathogenesis of gastric mixed neuroendocrine tumors and in finding new therapeutic targets.

[0014] In this invention, Rb1 is a key tumor suppressor gene responsible for regulating the cell cycle; fl / fl indicates that both alleles of this gene are marked by LoxP sites flanking their key exons. Trp53 is considered the most well-known tumor suppressor gene; fl / fl similarly indicates that both alleles are marked by LoxP sites. Myc is a potent proto-oncogene that promotes cell proliferation and metabolism. LSL (Lox-Stop-Lox): This is a "transcriptional arrest" tape. LSL: A termination sequence surrounded by LoxP sites is inserted before the Myc gene sequence. As long as this Stop sequence is present, the Myc gene cannot be expressed. MycT58A is a mutant form of the Myc protein, where the threonine (T) at position 58 is replaced with alanine (A). This mutation prevents the phosphorylation and degradation of the Myc protein, making it more stable, more active, and more carcinogenic.

[0015] This invention provides a method for constructing a transgenic mouse model of inducing gastric MiNENs and the application of the model, establishing a novel conditional gene knockout mouse model of gastric MiNENs (Pou2f3-CreERT; Rosa26-tdTomato; Rb1fl / fl; Trp53fl / fl; LSL-MycT58A, abbreviated as PTD-RPM mouse). The establishment of this model provides an important experimental tool for further research on gastric MiNENs, and can be used to screen drugs and study scientific challenges such as how tumors transform from adenocarcinoma to neuroendocrine carcinoma. The model demonstrated that it rapidly formed tumors within just one month of induction, significantly shortening the experimental cycle and improving research efficiency. It perfectly reproduced the characteristics of human mixed gastric adeno-neuroendocrine carcinoma (MiNENs), a special type of mixed tumor containing both adenocarcinoma and neuroendocrine carcinoma components, making it clinically difficult to treat. The model observed three cell morphologies, corresponding to different stages or types of tumor development: M1 subtype (dysplasia): a precancerous lesion where cells are just beginning to deteriorate; M2 subtype (adenocarcinoma): a typical gastric cancer morphology; and M3 subtype (neuroendocrine carcinoma): a morphology typically with a higher degree of malignancy. This proves that the model can simulate the "mixed" nature of this cancer. Attached Figure Description

[0016] Figure 1 (A) Pou2f3-CreERT; Rosa26-tdTomato; Rb1 fl / fl Trp53 fl / fl LSL-Myc T58A (A) Schematic diagram of gene structure of mouse model (PTD-RPM); (B) Gastric tumor formation in PTD-RPM mice after 5 weeks of Tmx induction, scale bar: 5 mm; (C) HE staining showing the pathological histology and functional phenotype of human primary gastric MiNENs reproduced by the model, scale bar: 50 μm; (DE) Staining map of perigastric lymph nodes visible in the model, scale bar: 100 μm; (F) Overall survival curve of the model; (G) HE staining map of tumor, scale bar: 100 μm; (HJ) Immunofluorescence staining map of tumor, scale bar: 100 μm.

[0017] Figure 2 (A) Map of a mouse neuroendocrine carcinoma (NEC); (B) Map of a human neuroendocrine carcinoma (NEC); (C) Map of vascular invasion of gastric neuroendocrine carcinoma in a mouse model; (D) Lung metastases in a mouse model; (E) Liver metastases in a mouse model. Detailed Implementation

[0018] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained in the art without creative effort should fall within the scope of protection of the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0020] Example 1: Breeding of transgenic mice

[0021] Rb1loxp / loxp; Trp53loxp / loxp; H11lsl-MycT58A (RPM; RRID: IMSR_JAX:029971), Rosa26-tdTomato (RRID: IMSR_JAX: 007914), and Pou2f3CreERT2 (RRID:IMSR_JAX: 037511) mice were purchased from Jackson Laboratory.

[0022] Tamoxifen (Sigma, product number T5648) and sunflower oil (Sigma, product number S5007) were purchased from Sigma.

[0023] Hybridization process: Pou2f3-CreERT × Rosa26-tdTomato -> F1; F1 × Rb1 / Trp53 / Myc -> ... -> finally obtaining homozygous PTD-RPM mice.

[0024] Example 2: Model Induction and Establishment

[0025] Experimental methods:

[0026] The expression of Tdtomato in Pou2f3-positive cells in the stomach of mice was induced by tamoxifen, and the tumor formation of Pou2f3-positive cells was tracked by lineage. Stomach tissues were collected from mice on days 1, 2, 3, 7, 14, 28, 35, 42, 49, and 56 after induction, and paraffin and frozen sections were prepared. Immunofluorescence was used to analyze the tumor formation of Pou2f3-positive cells.

[0027] 1. Reagents and consumables:

[0028] Tamoxifen (Sigma, T5648)

[0029] Sunflower seed oil (Sigma, S5007)

[0030] 1.5 mL / 15 mL centrifuge tubes (protected from light)

[0031] Disposable syringe (1 mL)

[0032] Electronic balance, vortex oscillator, aluminum foil (to protect from light)

[0033] 2. Weighing and Dissolving

[0034] Weigh the Tamoxifen powder using a light-protected centrifuge tube.

[0035] Add the solvent at a ratio of 10 mg tamoxifen + 1 mL S5007 sunflower seed oil (10 mg / ml).

[0036] Vortex thoroughly for 1–2 minutes, then shake overnight until completely clear and free of precipitate.

[0037] After complete dissolution, wrap the centrifuge tubes with aluminum foil for later use.

[0038] 3. Injection method

[0039] The mice used in the experiment were adult mice with no sex difference, with the optimal age being 6-8 weeks. The Pou2f3CreERT2 mouse strain was injected intraperitoneally with tamoxifen (50 mg kg / day) to induce Cre recombinase activity, and the injection was repeated for 1 day.

[0040] Experimental results:

[0041] Dissection of the stomachs of mice 5 weeks after tamoxifen injection revealed multiple gastric tumors, often accompanied by large ulcerative lesions (such as...). Figure 1 (B) The tumor exhibits a mixed histological morphology, containing both neuroendocrine carcinoma and adenocarcinoma components, and highly mimics human gastric mixed neuroendocrine-non-neuroendocrine tumors (MiNENs) in morphological and phenotypic characteristics (Figures 1C-F, 2A-E). The neuroendocrine carcinoma (NEC) region is morphologically similar to human gastric neuroendocrine carcinoma, with dense cell arrangement and reduced intercellular spaces, consistent with the phenotypic characteristics of small cell neuroendocrine carcinoma (Figures 2A-B).

[0042] By 35 days post-modeling (35 dpi), the tumor exhibited multifocal growth and varying degrees of infiltration: there were micronodules and macronodular lesions confined to the mucosal layer, as well as diffuse infiltration of the mucosal and submucosal layers (Figure 1C). As the disease progressed, the tumor could metastasize extensively, with vascular metastasis (18 / 24) and lymphatic metastasis (16 / 24) being the most common, while lung metastasis (3 / 24) and liver metastasis (5 / 24) could also occur (Figure 1D-E, Figure 2C-E).

[0043] The survival time of tumor-bearing mice was significantly shortened, with a median survival of only 48.5 days (Figure 1F).

[0044] Example 3: Immunohistochemical analysis of the model

[0045] Experimental methods:

[0046] Immunohistochemistry and scoring: Gastric tumor tissue sections (4 μm) embedded in paraffin were dried, dewaxed with turpentine oil and graded alcohols, and antigens were repaired using high-pressure chromatography. After incubation at non-binding sites, the sections were washed with PBS, primary antibody was added, and incubation was performed overnight at 4°C. Secondary antibody was added, and incubation was performed at room temperature for 20 min. After washing, DAB staining was performed, and the sections were counterstained with hematoxylin. The sections were dehydrated and mounted with neutral resin. A blank control was constructed using PBS instead of primary antibody. Cell staining intensity and the proportion of positive tumor cells were independently scored. The percentage and intensity of cells positive for the target protein in randomly selected fields of view were assessed to indicate protein expression levels.

[0047] HE staining: After dewaxing and hydration of the target paraffin sections, stain the cell nuclei with hematoxylin for 1 minute, rinse with running water, differentiate with 1% hydrochloric acid for a few seconds, rinse with running water, return to blue with 0.6% ammonia, rinse with running water, stain the cytoplasm with eosin for 1-3 minutes, dehydrate and mount, examine under a microscope, and acquire and analyze the images.

[0048] Experimental results:

[0049] HE staining showed a mixed glandular neuroendocrine carcinoma morphology. Figure 1 G), the tumor shows a mixed adeno-neuronal carcinoma morphology. The yellow circle indicates atypical hyperplasia (M1 subtype), the blue circle indicates adenocarcinoma (M2 subtype), and the remainder shows poorly differentiated small cell neuroendocrine carcinoma (M3 subtype). Immunofluorescence staining indicates that the tumor is heterogeneous and highly proliferative (KI67+), and can be divided into three different morphologies and molecular characteristics. Figure 2The M1 subgroup (M2) comprises atypical hyperplasia (M1), adenocarcinoma (M2), and neuroendocrine carcinoma (M3). The M1 subgroup presents as well-differentiated adenocarcinoma or atypical hyperplasia, expressing CD44V9 and TROP2 (a marker of atypical hyperplasia) (I), and is negative for SYP, CHGA, and INSM1. The M2 subgroup presents as moderately to poorly differentiated adenocarcinoma, without expressing neuroendocrine markers such as SYP, CHGA, and INSM1 (H, J). The M3 subgroup presents as poorly differentiated small cell neuroendocrine carcinoma, with high SYP expression (H), partial expression of CHGA (H) and INSM1 (J), and absent CLDN18 expression (J).

[0050] Based on the above HE staining and immunohistochemical results, and compared with human sample data, it is demonstrated that the phenotype of human primary gastric MiNENs was reproduced.

Claims

1. A method for constructing a gastric MiNENs model, characterized in that, Includes the following steps: Step (1): Mice expressing the Pou2f3 promoter-driven Cre recombinase were crossed with mice carrying conditional alleles of Rb1 deletion, Trp53 deletion, and Myc overexpression to obtain mice with the genotype Pou2f3-CreERT;Rosa26-tdTomato;Rb1 fl / fl Trp53 fl / fl LSL-Myc T58A Transgenic mice; Step (2): The transgenic mice obtained in step (1) were given an inducer to activate Cre recombinase, thereby specifically knocking out Rb1 and Trp53 genes and activating Myc expression in Pou2f3 positive cells in the stomach, inducing the formation of gastric MiNENs.

2. The method according to claim 1, characterized in that, In step (1), the Rosa26-tdTomato reporter gene is also introduced for lineage tracing.

3. The method according to claim 1, characterized in that, The inducer is tamoxifen.

4. The method according to claim 3, characterized in that, The induction method is intraperitoneal injection, with a dose of 40-60 mg kg / day, administered continuously for 1-2 days.

5. The method according to claim 1, characterized in that, The model developed tumors with histopathological and functional phenotypes consistent with human primary gastric MiNENs within one month after induction.

6. The method according to claim 5, characterized in that: The tumor exhibits a mixed glandular neuroendocrine carcinoma morphology. It includes the following three forms: atypical hyperplasia, adenocarcinoma, and neuroendocrine carcinoma.

7. A transgenic mouse model of gastric MiNENs constructed according to any one of claims 1-6.

8. The application of the transgenic mouse model of claim 7 in studying the origin and pathogenesis of gastric mixed neuroendocrine tumors and in finding new therapeutic targets.

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