A method for constructing an orthotopic tumor model of malignant tumors and its application
By co-culturing pancreatic cancer tissue cells with a 3D tumor scaffold and transfecting them with a reporter gene before injecting them into the pancreas of animals, a highly efficient orthotopic pancreatic cancer tumor model was constructed. This addresses the shortcomings of existing technologies in simulating the pancreatic cancer microenvironment and achieves efficient construction and clinical relevance of the tumor model.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGZHOU INSTITUTES OF BIOMEDICINE AND HEALTH CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2026-04-24
- Publication Date
- 2026-07-17
AI Technical Summary
Existing 2D and 3D tumor cell culture methods cannot fully simulate the natural microenvironment of pancreatic cancer, resulting in significant differences from in vivo tumors. Animal model construction is complex and difficult, and there is a lack of efficient methods for constructing in situ pancreatic cancer tumor models.
Pancreatic cancer organoids were prepared by co-culturing pancreatic cancer tissue cells with 3D tumor scaffolds, and then injected into the pancreas of experimental animals after transfecting them with reporter genes to construct an in situ pancreatic cancer tumor model.
A highly efficient and clinically relevant pancreatic cancer tumor model was successfully established, which can realistically simulate the growth and development of tumors in vivo. It is suitable for tumor biology research, drug screening and immunotherapy research. The tumor histological characteristics of the model are consistent with those of the original patient's pancreatic cancer tissue.
Smart Images
Figure CN122397684A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology and relates to a method for constructing an in situ tumor model of malignant tumors and its application. Background Technology
[0002] Pancreatic cancer (PC) is a highly malignant digestive system tumor. Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, originating from pancreatic duct epithelial cells. PDAC has unique biological behavior and histological characteristics. The tumor microenvironment is complex, containing a large amount of fibrous tissue and immune cell infiltration. These fibrous tissues not only provide physical support for tumor cells, but also secrete a variety of cytokines and growth factors, promoting the proliferation, invasion and metastasis of tumor cells.
[0003] Constructing and studying preclinical models that realistically simulate tumor biological behavior is crucial for deeply elucidating the complex molecular mechanisms driving the occurrence, development, and invasion of pancreatic cancer. Simultaneously, these models constitute an indispensable validation and prediction platform, enabling systematic evaluation and screening of the efficacy and safety of candidate compounds in the early stages of drug development, thereby accelerating the development of new anti-tumor drugs. Currently, there are three main methods for constructing tumor models: 2D tumor cell culture, 3D tumor cell culture, and in vivo tumor culture in animals.
[0004] 2D culture systems can provide information for tumor pathology research; however, tumor cells proliferate in a monolayer within 2D models, lacking cell-cell and cell-extracellular matrix (ECM) interactions, leading to significant differences in gene expression and protein synthesis compared to the in vivo environment. In contrast to 2D culture, 3D culture can reconstruct the 3D communication network of cell-cell and cell-ECM interactions, reproducing the in vivo tumor microenvironment and tumor cell biological behavior, thereby reducing the differences between in vitro cell models and in vivo tumors. However, it still lags behind animal tumor models. Animal models carrying tumors are important tools for monitoring drug utilization, therapeutic efficacy, and dose-toxicity, but constructing animal tumor models is complex and challenging.
[0005] In conclusion, developing efficient methods for constructing in situ tumor models of pancreatic cancer is of great significance for the treatment of pancreatic cancer. Summary of the Invention
[0006] In view of the shortcomings of existing technologies and practical needs, this invention provides a method for constructing an in situ tumor model of malignant tumors and its application, with the aim of efficiently constructing an in situ tumor model that can more realistically simulate the growth and development of tumors in their natural microenvironment.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for constructing an orthotopic malignant tumor model, the method comprising: Pancreatic cancer tissue cell suspension was mixed with 3D tumor scaffold and cultured to obtain pancreatic cancer organoids; A reporter gene was transfected into the pancreatic cancer organoids, and the transfected pancreatic cancer organoids were injected into the pancreas of experimental animals for feeding, thus obtaining an in situ pancreatic cancer tumor model.
[0008] This invention presents a method for constructing an orthotopic pancreatic cancer tumor model. After preparing in vitro organoids containing reporter genes, orthotopic transplantation is performed, which more realistically simulates the orthotopic growth of pancreatic cancer. This provides an efficient pancreatic cancer tumor model with good clinical relevance, suitable for tumor biology research, drug screening, and immunotherapy research. Verification using H&E staining and MUC1-specific IHC staining confirmed that the tumor histological characteristics were consistent with the original patient's pancreatic cancer tissue, further validating the reliability of this tumor model.
[0009] Optionally, the 3D tumor scaffold comprises at least one of the following: matrix adhesive, gelatin, sodium alginate, gelatin methacrylate, collagen, silk fibroin, polylactic acid-glycolic acid copolymer, polyvinyl alcohol, or porous material.
[0010] Optionally, the matrix adhesive is selected from Matrigel.
[0011] Optionally, the culture medium contains basal medium, hydroxyethylpiperazine ethanesulfonic acid, glutamine substitute, B27 supplement, N-acetylcysteine, nicotinamide, epidermal growth factor, fibroblast growth factor 10, Noggin protein, R-vertebral protein 1, Wnt-3A cell conditioned medium, gastrin I, transforming growth factor-β receptor I inhibitor, Rho-related protein kinase inhibitor, heparin, and antibiotics.
[0012] This invention designs a specific organoid culture medium that can effectively improve the success rate of establishing pancreatic cancer organoids and maintain their long-term expansion capacity and tumor biological characteristics.
[0013] Optionally, the basal culture medium is selected from Advanced DMEM / F12 medium.
[0014] Optionally, the culture medium contains 5-25 mM hydroxyethylpiperazine ethanesulfonic acid, 1-3 mM glutamine substitute, 1-2 mg / mL B27 supplement, 1-3 mM N-acetylcysteine, 1-3 mM nicotinamide, 45-55 ng / mL epidermal growth factor, 95-105 ng / mL fibroblast growth factor 10, 95-105 ng / mL Noggin protein, 490-510 ng / mL R-vertebral protein 1, 30%-50% Wnt-3A cell conditioned medium, 5-15 nM gastrin I, 490-510 nM transforming growth factor-β receptor I inhibitor, 4-6 μM Rho-associated protein kinase inhibitor, 45-55 μg / mL heparin, and 0.3-0.6 mg / mL antibiotics.
[0015] Optionally, the ratio of pancreatic cancer tissue cells to 3D tumor scaffolds is 5 × 10⁶ cells per 30 μL of 3D tumor scaffold. 3 ~2×10 4 Each cell.
[0016] Optionally, the reporter gene includes a luciferase gene and / or a green fluorescent protein gene.
[0017] Optionally, the experimental animals include any one of mice, rats, nude mice, rabbits, humanized mice, dogs, pigs, monkeys, or gerbils.
[0018] Optionally, the injection includes injecting the transfected pancreatic cancer organoids mixed with a 3D tumor scaffold.
[0019] Optionally, the number of transfected pancreatic cancer organoids injected is 450 to 550.
[0020] Secondly, the present invention provides the application of the method for constructing the orthotopic tumor model of malignant tumor described in the first aspect in screening drugs for pancreatic cancer treatment.
[0021] Optionally, the screening includes: constructing a pancreatic cancer in situ tumor model using the method for constructing a pancreatic cancer in situ tumor model as described in the first aspect, administering candidate drugs to the pancreatic cancer in situ tumor model, and screening candidate drugs with therapeutic effects based on disease indicators of the pancreatic cancer in situ tumor model.
[0022] Compared with the prior art, the present invention has at least the following beneficial effects: This invention, by constructing an in vitro organoid system carrying a specific reporter gene and performing orthotopic transplantation, can more accurately simulate the actual growth environment and evolution process of pancreatic cancer in vivo, thus successfully establishing a highly efficient, stable, and clinically relevant pancreatic cancer tumor research model. This model system is suitable for exploring the basic biological mechanisms of tumor development and progression, high-throughput drug screening and evaluation, and the research and validation of novel immunotherapy strategies. Detailed H&E staining and MUC1-specific immunohistochemical staining analysis confirmed that the tumor tissue in the constructed model maintains a high degree of consistency with the original patient's pancreatic cancer tissue in terms of morphology, cell arrangement, and molecular marker expression, further validating the reliability and biomimetic realism of the tumor model at the histological level. This platform provides a powerful new research tool and technical support for deeply elucidating the early metastasis mechanism of pancreatic cancer, objectively evaluating the efficacy of different treatment regimens, and accelerating the research and development of new anti-pancreatic cancer drugs. Attached Figure Description
[0023] Figure 1 Bright-field image of a three-dimensional culture of a clinical pancreatic cancer sample after digestion into a mixture of single cells and matrix gel and seeding. Scale bar: 70 μm. Figure 2 The images are taken under bright field and green fluorescence after pancreatic cancer organoids were transfected with reporter genes (luciferase gene and GFP gene). The scale bar is 70 μm. Figure 3A In vivo bioluminescence images one week after organoids were injected into the pancreas of immunodeficient mice (approximately 500 organoids were injected into each mouse); Figure 3B In vivo bioluminescence images two weeks after organoids were injected into the pancreas of immunodeficient mice (approximately 500 organoids were injected into each mouse); Figure 3C In vivo bioluminescence images three weeks after organoids were injected into the pancreas of immunodeficient mice (approximately 500 organoids were injected into each mouse); Figure 4 The image shows the results of hematoxylin-eosin staining and MUC1 antibody immunohistochemical staining. The scale bar is 100 μm. Detailed Implementation
[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.
[0025] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.
[0026] Example 1 This embodiment provides a method for constructing and histologically identifying pancreatic ductal adenocarcinoma organoids.
[0027] 1. Construction of organoids from pancreatic ductal adenocarcinoma After obtaining pancreatic ductal adenocarcinoma tumor samples, they were cut into small pieces, placed in 6-well plates, and ground with pre-chilled PBS and a filter. The ground samples were then filtered and collected into two separate EP tubes. The tumor cells were collected after digestion with primary tissue digestion solution at 300 g for 5 min. The supernatant was discarded by centrifugation, and red blood cell lysis buffer (RBC) was added to lyse the RBCs for 5 min. The digestion was then terminated by adding 5 times the volume of RBCs in phosphate buffer, centrifuged again to discard the supernatant, and mixed with matrix gel at a 1:1 volume ratio to form a three-dimensional culture system. The mixture was then dropwise seeded into culture plates (bright field image as shown). Figure 1 As shown in the figure, after the matrix gel solidifies, organoid culture medium is added, and the culture is carried out at 37°C and 5% CO2. During the culture process, the culture medium is changed every 2-3 days. Within 7-14 days, pancreatic cancer organoids with clear structures and typical glandular structures can be formed. After continuous passage culture, the organoids still maintain stable proliferative capacity and tumor histological characteristics.
[0028] The mass fractions of each component in the organoid culture medium are as follows: The basal medium consisted of Advanced DMEM / F12 (1:1 mixture), 10 mM HEPES (hydroxyethylpiperazine ethanesulfonic acid), 2 mM glutamine substitute (GlutaMAX), 1×B27 supplement (final concentration approximately 1.5 mg / mL, catalog number: 17504-044, Thermo Fisher Scientific), 1 mM N-acetylcysteine, 10 mM nicotinamide, 50 ng / mL epidermal growth factor (EGF), 100 ng / mL fibroblast growth factor 10 (FGF10), 100 ng / mL Noggin, 500 ng / mL R-spondin 1, Wnt-3A cell conditioned medium (catalog number: S2734, Selleckchem), comprising 30%–50% of the total medium volume, 10 nM gastrin I, 500 nM transforming growth factor-β receptor I inhibitor A83-01, and 5 μM Rho The relevant protein kinase inhibitor Y-27632, 50 μg / mL heparin, and 0.5 mg / mL antibiotic (penicillin-streptomycin solution).
[0029] 2. Pathological characteristics of pancreatic ductal adenocarcinoma organoids (1) Sample fixation and embedding The constructed pancreatic ductal adenocarcinoma organoids were obtained and fixed at 4°C for 24 h with pre-cooled 4% paraformaldehyde fixative (PFA, pH 7.4). After graded dehydration with ethanol (70%→85%→95%→100%, 1 h per grade), and clearing with xylene, they were embedded in molten paraffin (60°C) to prepare tissue blocks.
[0030] (2) Slice preparation The wax block was placed in a microtome (Leica RM2235) and sections with a thickness of 4-5 μm were continuously cut. The sections were then attached to poly-L-lysine-treated glass slides and baked at 60°C for 2 h to enhance adhesion.
[0031] (3) Hematoxylin-eosin (H&E) staining After dewaxing and rehydration, the sections were immersed in the following solutions in sequence: hematoxylin stain (Harris modified) for 5 min → differentiation with 1% hydrochloric acid ethanol → blueing with saturated lithium carbonate solution, staining with eosin (0.5% aqueous solution) for 3 min → dehydration with graded ethanol → clearing with xylene, mounting with neutral resin, and acquiring tissue morphology images under a bright field optical microscope (Nikon Eclipse E100).
[0032] (4) Immunohistochemical (IHC) analysis Step 1: Target staining After antigen retrieval, endogenous peroxidase was blocked with 3% H2O2 for 30 min. Specific primary antibody (anti-MUC1, catalog number AB109185) was added, and the sections were incubated overnight at 4°C.
[0033] Step 2: Signal Amplification HRP-labeled goat anti-rabbit secondary antibody (EnVision™ system) was incubated at room temperature for 30 min, followed by DAB staining for 5 min, and hematoxylin counterstaining of cell nuclei. Results were interpreted to assess positive expression rate and localization (cell membrane / cytoplasm / nucleus). A positive criterion was ≥10% staining of tumor cells.
[0034] Immunohistochemical images: confirmed that the expression of MUC1 in the organoids was consistent with that in the original clinical samples.
[0035] Bright-field image: Showing typical acinar / solid structures of three-dimensional organoids.
[0036] H&E staining: reproduces the pathological features of the primary tumor adenocarcinoma.
[0037] Immunohistochemistry: to verify the consistency of expression of the key target (MUC1) with that of the primary tumor (Kappa value > 0.85).
[0038] Example 2 This embodiment provides a method and its application for transfecting target genes into organoids constructed from clinical pancreatic ductal adenocarcinoma (PDAC) patient tissue. The method uses lentivirus-mediated transfection to stably introduce a reporter gene into PDAC organoids, thereby obtaining a gene-modified pancreatic cancer organoid model suitable for in vivo imaging and in vitro fluorescence observation. In this embodiment, the reporter gene preferably includes the luciferase gene and the green fluorescent protein (GFP) gene, but is not limited thereto.
[0039] (1) Pretreatment and single-cell preparation of PDAC organoids Specifically, PDAC organoids (refer to Example 1) constructed from pancreatic cancer tissue samples obtained through clinical surgical resection or puncture were mechanically sheared and enzymatically digested to obtain single cells or small cell clusters. The resulting cells were resuspended in organoid complete culture medium for later use.
[0040] (2) Lentiviral transfection and stability screening of the luciferase gene Prepare cell culture plates in advance, setting up replicates with different lentivirus transfection conditions, including but not limited to MOI = 5, 10, 20, 30, and 50. Add an appropriate amount of organoid complete culture medium to each replicate and keep at room temperature.
[0041] Add the PDAC organoid single cells obtained in step (1) to each replicate well, shake gently, and then culture in a cell culture incubator at 37°C, 5% CO2, and 20% O2.
[0042] After approximately 12 hours of culture and cell adhesion, the culture medium was replaced with fresh medium, and luciferase virus mix was added according to the pre-set transfection conditions for lentivirus transfection. Fresh medium was then added again approximately 12 hours after transfection.
[0043] 48–72 h after transfection, the appropriate antibiotics were added to the culture system for screening to obtain PDAC organoid cell lines with stable expression of the luciferase gene (luc-PDAC).
[0044] Preferably, in the above transfection step, the lentivirus and the complete organoid culture medium can be added simultaneously before the organoid cells adhere to the culture medium to improve the lentivirus infection efficiency.
[0045] (3) Secondary transfection of the GFP gene After obtaining a stable luc-PDAC cell line, the GFP gene was introduced using the same lentiviral transfection strategy as in step (2). Specifically, different gradient transfection conditions were set according to experimental needs, and GFP lentiviral transfection mixture was added to the luc-PDAC cell line.
[0046] After transfection and culture for the predetermined time, the appropriate antibiotic was added for screening, preferably at 48-72 h. Through two consecutive rounds of lentivirus transfection and resistance screening, a PDAC organoid cell line (luc-GFP-PDAC) that stably expresses both the luciferase gene and the GFP gene was finally obtained.
[0047] It should be understood that, depending on the actual experimental needs, one may choose to transfect only the luciferase gene (for in vivo imaging), or only the GFP gene (for in vitro fluorescence observation), or further screen pancreatic cancer organoid cell lines that stably express, knock down, or knock out other target genes.
[0048] Images of the reporter genes (luciferase gene, GFP gene) stained under bright field and green fluorescence are shown below. Figure 2 As shown.
[0049] (4) Three-dimensional reconstruction of gene-modified PDAC organoids Prepare preheated culture plates, overnight thawed and chilled matrix gel, and organoid complete culture medium brought to room temperature.
[0050] After digesting and counting the obtained luc-GFP-PDAC cell lines or untransfected PDAC organoids, take a certain number of cells as needed for the experiment, centrifuge and discard the supernatant, and place on ice. Add an appropriate volume of matrix gel to the cell pellet, gently mix to ensure uniform cell dispersion without the formation of air bubbles.
[0051] The cell-containing matrix gel was evenly dotted into the culture plate. After the matrix gel had completely solidified, organoid culture medium (refer to Example 1) was slowly added along the side wall of the plate. The plate was then cultured for several days at 37°C, 5% CO2, and 20% O2 to allow the cells to reform pancreatic cancer organoids with a three-dimensional structure.
[0052] (5) Matrix gel and inoculation conditions The preferred matrix gel is Matrigel. The volume of matrix gel added is adjusted according to the number of seeded cells, preferably 5 × 10⁶ cells per 30 μL of matrix gel. 3 ~2×10 4 The procedure is performed at a ratio of 1.5 × 10⁶ cells / mL, preferably seeded at a ratio of 1.5 × 10⁶ cells / mL for every 30 μL of matrix gel. 4 Each cell.
[0053] Example 3 This embodiment provides a method and its application for establishing an in situ pancreatic cancer tumor model by injecting organoids constructed from samples of pancreatic ductal adenocarcinoma (PDAC) patients into the pancreas of immunodeficient mice. The method can stably reconstruct the in situ growth environment of pancreatic cancer in vivo and is suitable for tumor development and progression studies, drug efficacy evaluation, and metastasis mechanism research.
[0054] The specific method is as follows: (1) Preparation of organoid suspension for injection The PDAC organoids constructed in Example 2, which stably express both the luciferase and GFP genes, were subjected to enzymatic digestion to obtain single cells or small cell clusters. After counting the obtained cells, they were thoroughly mixed with matrix gel to prepare an organoid suspension for pancreatic injection.
[0055] Preferably, the matrix gel is Matrigel, and air bubbles are avoided during mixing to ensure the uniformity and stability of the injection suspension.
[0056] (2) Oral injection of pancreas into immunodeficient mice Immunodeficient mice were selected as experimental animals. Under sterile conditions, the organoid suspension prepared in step (1) was slowly injected into the pancreatic parenchyma of the mice using a microinjector.
[0057] Preferably, each mouse is injected with approximately 500 organoids (or an equivalent number of organoid cells).
[0058] (3) Post-treatment feeding and live imaging monitoring After treatment, the mice were fed and monitored for health as usual. At different time points after organoid injection, in vivo bioluminescence imaging was performed on the mice to monitor the survival, proliferation, and tumor formation of organoids within the pancreas in real time.
[0059] (4) Verification of in situ tumor formation effect like Figures 3A-3C As shown, in the first, second and third weeks after injection, gradually increasing bioluminescent signals could be detected in the pancreas of mice, and the signal intensity increased significantly over time, indicating that the PDAC organoids successfully survived and continued to grow in the mouse pancreas.
[0060] Furthermore, bioluminescent signals were observed in the peritoneum or liver in some mice, suggesting distant spread or metastasis of the tumor. These results demonstrate the successful construction of an orthotopic pancreatic tumor model derived from pancreatic organoids.
[0061] (5) Pathological characterization of tumors formed by injecting pancreatic cancer organoids into mice 1. Sample fixation and embedding One month after injecting organoids into the mouse pancreas, the mice were sacrificed, and the pancreatic tumors were harvested and fixed in 4% paraformaldehyde fixative (PFA, pH 7.4) for 24 h. The tumors were then dehydrated in a gradient of ethanol (70%→85%→95%→100%, 1 h per step), cleared with xylene, and embedded in molten paraffin (60°C) to prepare tissue blocks. Clinical tumor samples and organoids were used as control groups.
[0062] 2. Slice preparation The wax block was placed in a microtome (Leica RM2235) and sections with a thickness of 4-5 μm were continuously cut. The sections were then attached to poly-L-lysine-treated glass slides and baked at 60°C for 2 hours to enhance adhesion.
[0063] 3. Hematoxylin-eosin (H&E) staining After dewaxing and rehydration, the sections were sequentially immersed in: hematoxylin stain (Harris modified) for 5 min → differentiation with 1% hydrochloric acid ethanol → blueing with saturated lithium carbonate solution. Eosin stain (0.5% aqueous solution) was applied for 3 min → dehydration with graded ethanol → clearing with xylene. The sections were mounted with neutral resin, and tissue morphology images were acquired under a bright field optical microscope (Nikon Eclipse E100).
[0064] 4. Immunohistochemical (IHC) analysis Step 1: Target staining After antigen retrieval, endogenous peroxidase was blocked with 3% H2O2 for 30 min. Specific primary antibody (anti-MUC1) was added, and the mixture was incubated overnight at 4°C.
[0065] Step 2: Signal Amplification HRP-labeled goat anti-rabbit secondary antibody (EnVision™ system) was incubated at room temperature for 30 min, followed by DAB staining for 5 min, and hematoxylin counterstaining of cell nuclei. Results were interpreted to assess positive expression rate and localization (cell membrane / cytoplasm / nucleus). A positive criterion was ≥10% staining of tumor cells.
[0066] H&E staining: reproduces the adenocarcinoma pathological features of primary pancreatic cancer tumors.
[0067] Immunohistochemistry: to verify the consistency of expression of the key target (MUC1) with that of the primary tumor (Kappa value > 0.85).
[0068] The results of hematoxylin-eosin staining and immunohistochemical staining with MUC1 antibody are as follows: Figure 4 As shown, the mouse orthotopic tumor model constructed in this experiment is consistent with the histological characteristics of the patient clinical samples.
[0069] In summary, the pancreatic cancer organoid model derived from clinical patient samples constructed using the method of this invention can achieve in vitro transfection with the target gene. After infection, it can be amplified and cultured in vitro, and then injected orally into the pancreatic tumors of immunodeficient mice. This orthotopic model can more realistically simulate the growth and development of tumors in their natural microenvironment, particularly in terms of the tumor's immune microenvironment, angiogenesis, and metastatic potential. Compared to traditional xenograft models, the orthotopic model can better reproduce the interaction between tumor cells and host tissues, thus providing more clinically relevant experimental data.
[0070] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.
Claims
1. A method for constructing an orthotopic malignant tumor model, characterized in that, The method includes: Pancreatic cancer tissue cell suspension was mixed with 3D tumor scaffold and cultured to obtain pancreatic cancer organoids; A reporter gene was transfected into the pancreatic cancer organoids, and the transfected pancreatic cancer organoids were injected into the pancreas of experimental animals for feeding, thus obtaining an in situ pancreatic cancer tumor model.
2. The method for constructing an orthotopic malignant tumor model according to claim 1, characterized in that, The 3D tumor scaffold comprises at least one of the following: matrix gelatin, gelatin, sodium alginate, gelatin methacrylate, collagen, silk fibroin, polylactic acid-glycolic acid copolymer, polyvinyl alcohol, or porous material.
3. The method for constructing an orthotopic malignant tumor model according to claim 1 or 2, characterized in that, The culture medium contained basal medium, hydroxyethylpiperazine ethanesulfonic acid, glutamine substitute, B27 supplement, N-acetylcysteine, nicotinamide, epidermal growth factor, fibroblast growth factor 10, Noggin protein, R-vertebral protein 1, Wnt-3A cell conditioned medium, gastrin I, transforming growth factor-β receptor I inhibitor, Rho-related protein kinase inhibitor, heparin, and antibiotics.
4. The method for constructing an orthotopic malignant tumor model according to claim 3, characterized in that, The culture medium contained 5-25 mM hydroxyethylpiperazine ethanesulfonic acid, 1-3 mM glutamine substitute, 1-2 mg / mL B27 supplement, 1-3 mM N-acetylcysteine, 1-3 mM nicotinamide, 45-55 ng / mL epidermal growth factor, 95-105 ng / mL fibroblast growth factor 10, 95-105 ng / mL Noggin protein, 490-510 ng / mL R-vertebral protein 1, 30%-50% Wnt-3A cell conditioned medium, 5-15 nM gastrin I, 490-510 nM transforming growth factor-β receptor I inhibitor, 4-6 μM Rho-associated protein kinase inhibitor, 45-55 μg / mL heparin, and 0.3-0.6 mg / mL antibiotics.
5. The method for constructing an orthotopic malignant tumor model according to any one of claims 1-4, characterized in that, The ratio of pancreatic cancer tissue cells to 3D tumor scaffolds is 5 × 10⁶ cells per 30 μL of 3D tumor scaffold. 3 ~2×10 4 Each cell.
6. The method for constructing an orthotopic malignant tumor model according to any one of claims 1-5, characterized in that, The reporter genes include the luciferase gene and / or the green fluorescent protein gene.
7. The method for constructing an orthotopic malignant tumor model according to any one of claims 1-6, characterized in that, The experimental animals include any one of mice, rats, nude mice, rabbits, dogs, pigs, monkeys, or gerbils.
8. The method for constructing an orthotopic malignant tumor model according to any one of claims 1-7, characterized in that, The injection includes mixing the transfected pancreatic cancer organoids with a 3D tumor scaffold and then injecting them; optionally, the injection volume of the transfected pancreatic cancer organoids is 450 to 550.
9. The application of the method for constructing an orthotopic malignant tumor model according to any one of claims 1-8 in screening drugs for pancreatic cancer treatment.
10. The application according to claim 9, characterized in that, The screening includes: constructing a pancreatic cancer in situ tumor model using the method for constructing a malignant tumor in situ tumor model according to any one of claims 1-8; administering candidate drugs to the pancreatic cancer in situ tumor model; and screening candidate drugs with therapeutic effects based on the disease indicators of the pancreatic cancer in situ tumor model.