Pancreatic cancer organoid with tumor microenvironment characteristics as well as construction method and application of pancreatic cancer organoid

Abstract

The invention discloses a pancreatic cancer organoid with tumor microenvironment characteristics and a construction method and application thereof, and belongs to the technical field of organoid. According to the invention, KPC transgenic mouse in-situ pancreatic tumor tissue is adopted as a cell source, and an organ with a pancreatic cancer tissue structure, biological characteristics and a complex tumor microenvironment is successfully constructed in combination with a culture medium formula with optimized culture conditions and characteristics and a multi-component co-culture mode; the obtained organoid is composed of multiple cell types such as tumor cells, cancer-related fibroblasts (CAFs), tumor-related macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), T cells and the like, and a complex pancreatic cancer immune microenvironment can be rebuilt truly. The organ can be widely applied to efficacy evaluation of pancreatic cancer immunotherapy drugs, an efficient and repeatable in-vitro experimental platform is provided for early-stage research and drug development of pancreatic cancer immunotherapy, the research and development process of related drugs is expected to be accelerated, and the clinical conversion efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoids, and in particular relates to a pancreatic cancer organoid with tumor microenvironment characteristics, a construction method thereof, and an application thereof in immunotherapy drug screening. Background Art

[0002] Pancreatic cancer, as a highly malignant digestive system tumor, has clinical characteristics such as insidious onset, rapid progression, and extremely poor prognosis, posing a serious threat to human health. Among them, pancreatic ductal adenocarcinoma (PDAC) is the most common pathological type, accounting for more than 90% of all pancreatic cancers. According to epidemiological data, the 5-year survival rate of pancreatic cancer patients is only about 10%, ranking among the lowest among all solid tumors. The main treatments for pancreatic cancer include surgical resection, chemotherapy, and radiotherapy, but for most patients, especially those in the advanced stage, the treatment effect is still unsatisfactory. Immunotherapy provides a new option for tumor treatment, which activates the body's own immune system to identify and kill tumor cells, but due to the complex tumor microenvironment of pancreatic cancer, such as high fibrosis and immunosuppression, the efficacy of immune cells is not ideal, and there is a lack of effective and accurate immune drug effect evaluation models in clinical practice.

[0003] As an emerging in vitro three-dimensional culture system, organoids have shown great potential in the field of tumor research. Tumor organoids can highly simulate the key biological characteristics of the original tumor in vitro, including cell composition, tissue structure, gene expression profile, and response to drugs. Patient-derived tumor organoids have been widely used in tumor research and have played an important role in evaluating treatment sensitivity and resistance, studying cell-cell interactions, and the interaction between tumor cells and the tumor microenvironment. Tumor organoids have significant advantages in predicting patients' clinical treatment responses. Organoids can retain the heterogeneity of patients' tumors and reflect the sensitivity of different patients to drugs. Multiple drug tests on patients' tumor organoids in vitro can accurately predict patients' responses to different drugs, thereby formulating personalized treatment plans for patients.

[0004] However, the current method for constructing pancreatic cancer organoids only retains tumor epithelial cells, but cannot retain stromal cells and immune cells, making it difficult to fully reproduce the various cellular components and their interactions in the tumor microenvironment. Although some studies have attempted to co-culture primary tumor cells with cancer-associated fibroblasts (CAFs), macrophages, etc. to simulate the interaction between tumor cells and the tumor microenvironment, they cannot fully simulate the complex tumor microenvironment of pancreatic cancer. Therefore, the existing pancreatic cancer organoid models have many limitations when used for immunotherapy drug screening, and cannot accurately reflect the actual situation of the pancreatic cancer immune microenvironment, resulting in the screened drugs often failing to achieve the expected results in clinical trials, wasting a lot of manpower, material resources and time.

[0005] Therefore, developing an organoid that can truly simulate the immune microenvironment of pancreatic cancer and applying it to immunotherapy drug screening has urgent practical needs and important scientific significance for improving the treatment effect of pancreatic cancer and promoting the development of precision immunotherapy for pancreatic cancer. Summary of the Invention

[0006] In order to address the deficiencies in the prior art, the purpose of the present invention is to provide a mouse pancreatic cancer organoid with tumor microenvironment characteristics, a construction method thereof, and its application in immunotherapy drug screening. In view of the problem that the existing organoid models are generally single in cell composition and only contain epithelial cells, which is difficult to reflect the complex immune microenvironment of real tumor tissue, the present invention significantly improves the structural and functional simulation of the organoid by constructing a multi-cell co-culture system. The present invention uses the in situ pancreatic tumor tissue of KPC transgenic mice as the tissue source, and establishes a three-dimensional pancreatic cancer organoid model that simulates the real tumor immunosuppressive microenvironment and hypoxia state by optimizing the culture method and conditions of the organoid. The organoid not only contains tumor epithelial cells, but also integrates a variety of important immune-related cell populations such as cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) and T cells, which can truly reproduce the characteristics of the pancreatic cancer microenvironment at the spatial structure and cell interaction levels. The organoid model generated using this construction method can be widely used in immunotherapy drug efficacy screening and mechanism research, providing reliable data support for drug functional evaluation on tumor cell proliferation, apoptosis, and immune cell activation and killing. It is expected to serve as an in vitro experimental platform for pancreatic cancer immunotherapy research, significantly improving the relevance and efficiency of drug screening and providing a solid foundation for the development of novel immunotherapy strategies.

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

[0008] In a first aspect, the present invention provides a method for constructing pancreatic cancer organoids with tumor microenvironment characteristics, comprising the following steps:

[0009] (1) The tumor tissue is washed with a buffer solution, minced, and digested with an enzyme in a digestion solution. The resulting digestion solution is passed through a sieve, and the resulting cell suspension is centrifuged to obtain a pancreatic cancer single cell pellet;

[0010] (2) adding red blood cell lysis buffer to the pancreatic cancer single cell pellet obtained in step (1), incubating at room temperature for 3 to 10 minutes, stopping the lysis, centrifuging, discarding the supernatant, adding pancreatic cancer organoid culture medium to the obtained cell pellet, and dispersing to obtain a pancreatic cancer single cell suspension;

[0011] (3) The pancreatic cancer single cell suspension obtained in step (2) is evenly mixed with the matrix material, and the resulting mixture is inoculated dropwise into the culture plate. After the gel droplets solidify, pancreatic cancer organoid culture medium is added, and pancreatic cancer organoids are obtained after culture.

[0012] Based on the above technical solution, further, the pancreatic cancer tumor in step (1) includes spontaneous pancreatic cancer tumor tissue extracted from KPC transgenic mice.

[0013] Based on the above technical solution, further, in step (1), 1×PBS buffer containing 30-100 μg / ml gentamicin is used to wash the tumor tissue to remove surface contaminants.

[0014] Based on the above technical solution, further, the size of the tissue after cutting in step (1) is 1 to 10 mm 3 .

[0015] Based on the above technical solution, further, the digestion solution in step (1) is RPMI-1640 basal culture medium containing 1-5 mg / ml collagenase I and 1-5 mg / ml collagenase IV.

[0016] Based on the above technical solution, further, the conditions for enzymatic digestion in step (1) are digestion at 36-38° C. for 20-60 min, with shaking once every 10 min.

[0017] Based on the above technical solution, further, the pore size of the sieve in step (1) is 50 to 150 μm.

[0018] Based on the above technical solution, further, the centrifugal condition in step (1) is 1500-2000 rpm for 2-10 min.

[0019] Based on the above technical solution, further, the centrifugal condition in step (2) is 1500-2000 rpm for 2-10 min.

[0020] Based on the above technical solution, further, the pancreatic cancer organoid culture medium described in step (2) and step (3) is composed of: DMEM / F12 basal medium, 10-20% FBS, 10-60 μg / ml Gentamicin, 5-20 ng / ml FGF-10, 1-2 mM N-Acetylcysteine, 5-15 μM Y-27632, 5-50 ng / ml mEGF, and 10-50 ng / ml M-CSF.

[0021] Based on the above technical solution, further, the matrix material in step (3) is Matrigel matrix glue.

[0022] Based on the above technical solution, further, in step (3), the volume ratio of the matrix material to the pancreatic cancer single cell suspension is 1:5 to 1:1.

[0023] Based on the above technical solution, further, the glue droplet described in step (3) contains 3000 to 10000 cells per droplet.

[0024] Based on the above technical solution, further, the curing conditions in step (3) are: incubation in an incubator at 36-38°C for 10-40 minutes.

[0025] Based on the above technical solution, further, the culture in step (3) is continued in a 37° C., 5% CO 2 incubator for 5 to 10 days, and the culture medium is replaced every 2 to 3 days.

[0026] Based on the above technical solution, the pancreatic cancer organoids described in step (3) and the activated T cells are further mixed in a co-culture system with 8-12% matrigel and 5-15 ng / mL IL-2 at a ratio of 1:1 to 20:1 between the number of T cells and the number of organoids, and co-cultured at 37°C and 5% CO2 for 36-72 hours.

[0027] Based on the above technical solution, the ratio of T cells to organoids is 1:5.

[0028] In a second aspect, the present invention provides pancreatic cancer organoids having tumor microenvironment characteristics obtained by the above preparation method.

[0029] In a third aspect, the present invention provides the use of the above-mentioned pancreatic cancer organoids with tumor microenvironment characteristics in anticancer drug screening.

[0030] The present invention has the following beneficial effects compared to the prior art:

[0031] The present invention successfully constructs an organoid model with pancreatic cancer histological characteristics and tumor microenvironment characteristics in vitro by collecting tumor tissue from pancreatic cancer mice and combining it with an optimized three-dimensional tissue culture system. The constructed organoid not only retains the cellular heterogeneity and tissue structure of the primary tumor, but also truly reproduces the hypoxic state and immunosuppressive microenvironment unique to pancreatic cancer, providing a highly relevant in vitro model platform for tumor immune mechanism research and drug evaluation. In terms of immunotherapy drug screening, the organoid model has the following significant advantages: (1) The high consistency of biological characteristics between the organoid and the primary tumor enables it to more accurately simulate the growth and metastasis behavior of pancreatic cancer in vivo, thereby improving the accuracy and reliability of drug prediction. (2) Compared with traditional animal models or two-dimensional culture systems, the organoid model can perform high-throughput drug evaluation in a short period of time, significantly shortening drug development time and reducing R&D costs. (3) The candidate immune drugs screened by the organoid are closer to clinical practice in terms of mechanism and efficacy, and can provide more accurate and personalized treatment plans for pancreatic cancer patients, which is expected to improve treatment effects and patient quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the construction route of pancreatic cancer organoids in Example 1 of the present invention;

[0033] Figure 2 This is a bright field image of the growth process of pancreatic cancer organoids in Example 1 of the present invention;

[0034] Figure 3 HE staining and IHC staining of pancreatic cancer organoids and pancreatic cancer tissues in Example 1 of the present invention;

[0035] Figure 4 Schematic diagram of co-culture of pancreatic cancer organoids and T cells in Example 2 of the present invention;

[0036] Figure 5 This is a graph showing the proportion of various cell types in the pancreatic cancer organoids in Example 2 of the present invention;

[0037] Figure 6 This is a diagram showing the flow cytometry analysis results of the immune microenvironment regulated by immune drugs in Example 3 of the present invention. DETAILED DESCRIPTION

[0038] Example 1

[0039] Four-week-old female KPC-positive mice (genotype Kras) were selected. tm4Tyj Trp53 tm1BrnTg (Pdx1-cre / Esr1*)#Dam / J, purchased from Jackson Laboratory) were induced with tamoxifen: tamoxifen was dissolved in corn oil and prepared into a 100 mg / ml solution for storage. The solution was administered intraperitoneally at a dose of 100 mg / kg body weight every 24 hours for 7 consecutive days.

[0040] KPC mice with terminal pancreatic cancer were euthanized and orthotopic pancreatic tumor tissue was removed under sterile conditions. After removing necrotic and adipose tissue, the tumor tissue was placed in a sterile PBS solution for later use. The tumor was then divided into two parts: one part was fixed with 4% paraformaldehyde for subsequent histological analysis, and the other part was used to generate mouse pancreatic cancer organoids.

[0041] The tissue was washed 2-3 times in 1×PBS solution containing 50 μg / mL gentamicin to remove surface contaminants. The tumor tissue was minced into approximately 5 mm pieces using sterile surgical scissors. 3 Size and transfer to a 5mL sterile centrifuge tube. Add 3mL of digestion solution (RPMI-1640 basal medium with 2mg / ml collagenase I and 2mg / ml collagenase IV) to the centrifuge tube, mix thoroughly and place in a 37°C incubator for 45 minutes, gently shaking every 10 minutes to promote tissue dissociation. After digestion, place a 100μm cell sieve at the mouth of a 50mL centrifuge tube, transfer the digestion solution to the cell sieve, and gently grind with a syringe plunger, while rinsing the cell sieve with DMEM / F12 medium. The resulting cell suspension was centrifuged at 1800rpm for 5min, and the supernatant was discarded. Add 2mL of red blood cell lysis buffer, incubate at room temperature for 5min, add 5mL of 1×PBS to terminate lysis, centrifuge again at 1800rpm for 5min, and discard the supernatant. Add 1 mL of pancreatic cancer organoid culture medium (basal medium is DMEM / F12, which contains 20% FBS, 50 μg / ml Gentamicin, 10 ng / ml FGF-10, 1.5 mM N-Acetylcysteine, 10 μM Y-27632, 10 ng / ml mEGF, 20 ng / ml M-CSF) and gently pipette to resuspend the cells. Count the cells using a hemocytometer. After counting, add the cell suspension (3.3 × 10 5cells / ml) with pre-cooled Matrigel in a volume ratio of 3:2, and gently blow evenly with a pipette to avoid bubbles. Use a pipette to inoculate 25μL of the cell-Matrigel mixture dropwise into a 24-well culture plate, with 3 drops in each well. Incubate the culture plate upside down in a 37°C incubator for 30 minutes. After the Matrigel solidifies and forms a stable gel droplet structure, restore the culture plate to its upright position. Slowly add 1mL of preheated organoid culture medium along the wall of the well to avoid disturbing the gel droplet. Place the culture plate in a 37°C, 5% CO2 incubator for continuous culture. Change the culture medium every 2 to 3 days and observe the growth morphology and structural stability of the organoids.

[0042] The results are as follows Figure 2 As shown in the figure, on Day 0 (initial inoculation), the tumor cells or cell clusters were dispersed, with a small overall volume and sparse structure, and no obvious three-dimensional structure had yet formed. Compared with Day 0, on Day 2, it can be observed that some cell clusters gradually aggregated and began to form clear spherical or round organoid structures. Cell proliferation was initially active, indicating that the organoids had started the growth and morphological remodeling process. On Day 4, the number of organoids increased significantly, and the size expanded significantly, forming multiple round vesicle-like organoid structures of different sizes. Compared with Day 2, the growth and differentiation of organoids were clear, indicating that the organoids were in a stage of rapid proliferation and tissue reconstruction. On Day 6, the morphology of the organoids tended to be more complex and diversified, showing a larger diameter (average of about 200μm). Some organoids showed layered or cavity structures, and the number further increased, indicating that the culture system supports the continuous growth and spatial structure formation of organoids. During the 6-day culture period, the organoids underwent a dynamic evolution process from initial inoculation state, aggregation and proliferation, structural construction, and diversified maturation, showing good biological activity and growth trend, further verifying the effectiveness of this construction system in maintaining the physiological characteristics of pancreatic cancer organoids.

[0043] In addition, to verify the fidelity of organoids in morphology and cellular composition, pancreatic cancer tissues and pancreatic cancer organoids were comparatively analyzed using H&E staining and IHC staining.

[0044] The results are as follows Figure 3 As shown, the results showed that the organoids had a certain degree of similarity with pancreatic cancer tissue in histological structure; the epithelial cell marker CK19 and the proliferation-related marker Ki67 were both positively expressed, indicating that the organoids had the characteristic cell composition and high proliferation activity of the source tissue.

[0045] Example 2

[0046] The pancreatic tumor tissue from the KPC mice in Example 1 was placed in a sterile culture dish and rinsed 2-3 times with pre-cooled sterile PBS buffer to remove blood, impurities and necrotic tissue on the surface. The tumor tissue was cut into 1-3 mm pieces using sterile surgical instruments. 3 Transfer the tumor mass to a sterile centrifuge tube, add 3 mL of digestion solution (RPMI-1640 basal medium with 2 mg / ml collagenase I and 2 mg / ml collagenase IV), incubate at 37°C, and shake every 10 minutes to ensure thorough digestion. When most of the cells are observed to be individually dispersed under a microscope, add an equal volume of complete medium containing serum to terminate digestion. Centrifuge at 1800 rpm for 5 minutes and discard the supernatant. Add 3 mL of red blood cell lysis buffer, incubate at room temperature for 5 minutes, add an equal volume of PBS to terminate lysis, and centrifuge at 1800 rpm for 5 minutes. Resuspend the resulting single cell pellet in PBS for organoid culture and tumor antigen preparation.

[0047] Preparation of tumor antigens: Freeze a portion of the single-cell suspension at -80°C, then thaw in a 37°C water bath. Repeat the freeze-thaw cycle six times to promote tumor cell lysis and release tumor antigens. The resulting cell lysate is the supernatant containing tumor antigens.

[0048] Induction and Culture of Mouse Bone Marrow-Derived DCs (BMDCs): 7-8 week-old C57BL / 6J mice were euthanized and disinfected with ethanol. The femurs and tibias were dissected, and the attached muscle was removed. The femurs and tibias were cut open to expose the bone marrow cavity. Bone marrow cells were flushed out using a sterile syringe (10 mL) filled with 1640 medium. The resulting bone marrow cells were centrifuged at 1800 rpm for 5 minutes, and the cell pellet was collected. 2 mL of erythrocyte lysis buffer was added and incubated for 5 minutes. Lysis was terminated by adding 8 mL of PBS, and the cells were centrifuged again at 1800 rpm for 5 minutes, and the erythrocyte lysis buffer was removed. The cells were resuspended in 1640 complete medium supplemented with 20 ng / mL GM-CSF and 20 ng / mL IL-4 and plated in a 10 cm culture dish. Fresh 1640 complete medium supplemented with 20 ng / mL GM-CSF and 20 ng / mL IL-4 was added on day 3. On day 6, suspended and semi-adherent cells were collected to obtain immature BMDCs.

[0049] BMDCs were loaded with tumor antigens: On the sixth day of BMDC culture, the cells were centrifuged at 1800 rpm for 5 min, the supernatant was discarded, 200 μL of tumor cell lysate was added, and the cells were incubated in a 37°C, 5% CO2 incubator for 24 h to promote BMDCs to take up tumor antigens.

[0050] Co-culture of organoids with mouse splenic T cells: 7-8 week-old C57BL / 6J mice were euthanized and disinfected with ethanol. The spleens were removed and placed in sterile culture dishes. The cells were washed three times with 1× sterile PBS. A 70 μm cell strainer was placed in a 50 mL centrifuge tube. The spleen tissue was triturated with a syringe plunger and the cells were rinsed with 1× sterile PBS to prepare a single-cell suspension. The suspension was centrifuged at 1800 rpm for 5 minutes, and the supernatant was discarded. 3 mL of red blood cell lysis buffer was added, and the cells were incubated at room temperature for 5 minutes. After lysis, an equal volume of sterile PBS was added to terminate the reaction. The cells were centrifuged again at 1800 rpm for 5 minutes, and the supernatant was discarded to obtain a cell pellet. BMDCs loaded with tumor antigens were mixed with isolated mouse splenic T cells at a cell ratio of 1:5, seeded in 6-well plates, and co-cultured at 37°C, 5% CO2 for 24 hours. The mature mouse pancreatic cancer organoids cultured on day 6 in Example 1 were mixed evenly with the above-mentioned activated T cells at different cell number ratios (T cell: organoid = 1:1, 5:1, 10:1, 20:1). 10% Matrigel and 10 ng / mL IL-2 were added to the co-culture system, and co-cultured at 37°C, 5% CO2 for 48 h.

[0051] By comparing the T cell infiltration at different ratios, we finally determined that 1:5 was the optimal co-culture ratio. At this ratio, the T cell infiltration level was close to that of pancreatic in situ tumor tissue in mice ( Figure 5 ).

[0052] Example 3

[0053] On day 6, mature mouse pancreatic cancer organoids were co-cultured with the activated T cells described in Example 2 (at a ratio of 1:5) for 24 hours. The immunostimulatory drug R848 (1 μg / ml) was then added, and the cells were incubated at 37°C, 5% CO2 for another 24 hours. The co-culture system was digested into a single-cell suspension, and a pre-prepared flow cytometry antibody cocktail (CD45-APC / Cy7, CD206-PE / Cy7, F4 / 80-FITC, CD86-BV421, CD11b-PE, Gr1-APC) was added. The cells were incubated at room temperature in the dark for 1 hour. The suspension was then washed once with 1 mL of FACS buffer, centrifuged at 1800 rpm for 5 minutes, and the supernatant discarded. The cells were fixed with 800 μL of 1% paraformaldehyde (PFA) and incubated at 4°C for 45 minutes. After further centrifugation to remove the PFA, the cells were resuspended in 300 μL of FACS buffer, passed through a 300-mesh sieve, and transferred to a flow cytometry tube. Flow cytometry was used for detection, and the obtained data were subsequently analyzed using FlowJo software.

[0054] The results are as follows Figure 6As shown in the results, after treatment with R848, the proportion of M1 macrophages in the organoids increased significantly, while the proportion of M2 immunosuppressive macrophages decreased significantly. This result is consistent with the known immune mechanism of action of R848, indicating that the organoid platform can effectively simulate the real immune response environment and has reliable drug efficacy evaluation capabilities.

Claims

1. A method for constructing pancreatic cancer organoids with tumor microenvironment characteristics, characterized in that: The following steps are involved: (1) The tumor tissue is washed with a buffer solution, minced, and digested with an enzyme in a digestion solution. The resulting digestion solution is passed through a sieve, and the resulting cell suspension is centrifuged to obtain a pancreatic cancer single cell pellet; (2) adding red blood cell lysis buffer to the pancreatic cancer single cell pellet obtained in step (1), incubating at room temperature for 3 to 10 minutes, stopping the lysis, centrifuging, discarding the supernatant, adding pancreatic cancer organoid culture medium to the obtained cell pellet, and dispersing to obtain a pancreatic cancer single cell suspension; (3) The pancreatic cancer single cell suspension obtained in step (2) is evenly mixed with the matrix material, and the resulting mixture is inoculated dropwise into the culture plate. After the gel droplets solidify, pancreatic cancer organoid culture medium is added, and pancreatic cancer organoids are obtained after culture.

2. The construction method according to claim 1, characterized in that The pancreatic cancer tumor in step (1) includes spontaneous pancreatic cancer tumor tissue extracted from KPC transgenic mice; the tumor tissue is washed with 1×PBS buffer containing 30-100 μg / ml gentamicin to remove surface contaminants; the size of the minced tissue is 1-10 mm 3 .

3. The construction method according to claim 1, characterized in that The digestion solution described in step (1) is RPMI-1640 basal culture medium containing 1-5 mg / ml collagenase I and 1-5 mg / ml collagenase IV; the enzymatic digestion conditions are digestion at 36-38°C for 20-60 min, with oscillation once every 10 min; the pore size of the sieve is 50-150 μm; and the centrifugation conditions are centrifugation at 1500-2000 rpm for 2-10 min.

4. The construction method according to claim 1, characterized in that The centrifugal conditions in step (2) are 1500-2000 rpm for 2-10 min.

5. The construction method according to claim 1, characterized in that The pancreatic cancer organoid culture medium described in step (2) and step (3) is composed of: DMEM / F12 basal medium, 10-20% FBS, 10-60 μg / ml Gentamicin, 5-20 ng / ml FGF-10, 1-2 mM N-Acetylcysteine, 5-15 μM Y-27632, 5-50 ng / ml mEGF, and 10-50 ng / ml M-CSF.

6. The construction method according to claim 1, characterized in that In step (3), the matrix material is Matrigel; the volume ratio of the matrix material to the pancreatic cancer single cell suspension is 1:5 to 1:1; and the gel droplets contain 3,000 to 10,000 cells per drop.

7. The construction method according to claim 1, characterized in that The curing conditions in step (3) are: incubation in a 36-38°C incubator for 10-40 minutes; the culture is continued in a 37°C, 5% CO2 incubator for 5-10 days, and the culture medium is replaced every 2-3 days.

8. Pancreatic cancer organoids with tumor microenvironment characteristics obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the pancreatic cancer organoid with tumor microenvironment characteristics according to claim 8 in anticancer drug screening.

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