Method for producing tumor microenvironment implementation and analysis model, and method for evaluating activity of anticancer drug candidate using same

Abstract

The present invention relates to a method for producing a tumor microenvironment implementation and analysis model, and a method for evaluating the activity of an anticancer drug candidate using same. The tumor microenvironment implementation and analysis model of the present invention implements a tumor microenvironment similarly to the actual environment, thereby enabling rapid, highly accurate, and inexpensive analysis, and thus is very suitable for use in analysis such as activity evaluation and screening related to cancer diseases and anticancer drugs.

Description

Method for producing a tumor microenvironment implementation and analysis model and method for evaluating the activity of anticancer drug candidates using the same

[0001] The present invention relates to a method for producing a tumor microenvironment implementation and analysis model and a method for evaluating the activity of an anticancer drug candidate using the same. Specifically, the present invention relates to a method that is highly suitable for use in analyses such as activity evaluations related to cancer and anticancer drugs, as it enables rapid, highly accurate, and inexpensive analysis by implementing a tumor microenvironment similar to the actual one.

[0002] The tumor microenvironment is a complex structure composed not only of cancer cells but also of mesenchymal cells, endothelial cells, the extracellular matrix (ECM), and various types of immune cells. Current immunotherapy activity assessment systems rely on cell activity assessment through 2D culture of cancer cells, in vivo efficacy assessment in syngeneic mice, and in vivo efficacy assessment in humanized mice.

[0003] However, the cell activity evaluation system using 2D cancer cell culture has low reliability of measured efficacy because the culture conditions are significantly different from the actual tumor microenvironment, and efficacy evaluation in a syngeneic mouse model has disadvantages that are different from those of the human body because it is an evaluation system that utilizes the mouse immune system.

[0004] Efficacy evaluation in humanized mice is not only difficult to conduct because it is a system that transplants human-derived blood cells into mice, but also has the disadvantage of being very expensive, with low accuracy in experimental results due to differences in the degree of immune cell engraftment between mice.

[0005] Therefore, in order to effectively search for anticancer drugs that suppress cancer cell growth within the tumor microenvironment, an evaluation technology capable of screening immunotherapy drugs under experimental conditions similar to the tumor microenvironment is needed.

[0006] Accordingly, the problem to be solved by the present invention is to provide a method for producing a tumor microenvironment implementation and analysis model that enables rapid and highly accurate analysis by implementing a tumor microenvironment similar to the actual tumor microenvironment.

[0007] Another problem to be solved by the present invention is to provide a method for evaluating and screening the activity of an immuno-oncology drug candidate using the above model.

[0008] The tasks of the present invention are not limited to the technical tasks mentioned above, and other technical tasks not mentioned will be clearly understood by those skilled in the art from the description below.

[0009] A method for producing a tumor microenvironment implementation and analysis model according to one embodiment of the present invention for solving the above problem comprises: (a) forming a spheroid by co-culturing cancer cells and stromal cells; (b) labeling peripheral blood mononuclear cells (PBMCs); (c) co-culturing the labeled PBMCs with the spheroids; and (d) analyzing the labeled PBMCs that have migrated to the spheroids after co-culturing with the spheroids.

[0010] The cancer cells may be cells of one or more cancers selected from the group consisting of gastric cancer, lung cancer, liver cancer, colon cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, sclerosing adenoma, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer (including leukemia, multiple myeloma, myelodysplastic syndrome), lymphoma (including Hodgkin's disease, non-Hodgkin's lymphoma), psoriasis, and fibroadenoma. Specifically, the cancer cells may be breast cancer cells, and more specifically, triple-negative breast cancer cells (MDA-MB-231).

[0011] The stromal cells may be specifically cancer associated fibroblasts (CAFs), and more specifically MRC5 cells.

[0012] The co-culturing of cancer cells and stromal cells may be a direct co-culturing of the cancer cells and stromal cells. The ratio of cancer cells to stromal cells may be 1:1-10, specifically 1:2-4, and more specifically approximately 1:3. During co-culturing, a phenol red-free medium may be used, but embodiments of the present invention are not limited thereto.

[0013] The temperature for co-culturing cancer cells and stromal cells may be 30-40°C, specifically 35-39°C, more specifically about 37°C, the CO2 may be 1-10%, specifically 2-8%, more specifically about 5%, and the period may be 1-10 days, specifically about 1-5 days, more specifically about 3 days.

[0014] When culturing PBMC, the medium may be a medium containing IL-2 (interleukin-2), and specifically, may be a phenol red-free medium containing IL-2, but the embodiments of the present invention are not limited thereto.

[0015] The temperature for culturing PBMCs may be 30-40°C, specifically 35-39°C, more specifically about 37°C, the CO2 may be 1-10%, specifically 2-8%, more specifically about 5%, and the period may be 1-10 days.

[0016] The step of labeling PBMCs may refer to any method of labeling using a known method for qualitative or quantitative analysis of PBMCs, such as staining PBMCs with fluorescent dyes such as CF dye, FITC, or Cyanine, or binding substances such as Biotin, Digoxygenin, or DNP. In an exemplary embodiment, PBMCs are labeled using pre-warmed CellTracker TM Labeling can be accomplished by staining with a Deep Red dye reagent, but the embodiments of the present invention are not limited thereto.

[0017] The step of co-culturing labeled PBMCs with spheroids may involve placing a vessel containing labeled PBMCs in a medium containing cancer cells and stromal cells, spaced apart from the cancer cells and stromal cells. The vessel containing labeled PBMCs may be formed with pores sized to allow the PBMCs to pass through, such as a transwell. That is, although the cancer cells and stromal cells are spaced apart from the vessel containing labeled PBMCs, they share a medium, allowing the labeled PBMCs to pass through the pores and migrate to the cancer cells, thereby infiltrating the cancer cells. In an exemplary embodiment, the vessel containing labeled PBMCs, such as a transwell, may be placed spaced apart from the cancer cells and stromal cells, and the medium may be filled to a height sufficient to submerge the labeled PBMCs.

[0018] The step of co-culturing labeled PBMCs with spheroids and the step of analyzing the labeled PBMCs may be a step of analyzing labeled PBMCs that have migrated to cancer cells during co-culturing for 1-30 days, specifically 1-20 days, more specifically 1-10 days, and more specifically 1-7 days.

[0019] The step of analyzing the labeled PBMCs that have migrated into the spheroid may be a step of quantitatively analyzing the volume, mass, area, etc. of the labeled PBMCs that have migrated and infiltrated into the spheroid, and specifically, may be a FACS (Fluorescence activated cell sorter) analysis using a staining image.

[0020] According to one embodiment of the present invention for solving the above-described other problems, a method for evaluating and screening the activity of a candidate drug on a tumor microenvironment may further include a step of analyzing or screening characteristics, such as anticancer activity, of the candidate drug by comparing and analyzing a model prepared by the above method with a model treated with the candidate drug and a model not treated. As the candidate drug, a substance having anticancer activity, such as an ENPP1 inhibitor or a STING pathway activator, which can affect cancer, tumors, and the tumor microenvironment, may be used.

[0021] A method for producing a tumor microenvironment implementation and analysis model according to another embodiment of the present invention for solving the above problem comprises: (a) labeling cancer cells; (b) co-culturing the labeled cancer cells and stromal cells to form spheroids; (b) co-culturing peripheral blood mononuclear cells (PBMCs) with the spheroids; and (d) analyzing the spheroids after co-culturing with the spheroids to analyze the cancer cells.

[0022] The step of analyzing cancer cells may be a step of qualitatively or quantitatively analyzing the death of cancer cells due to infiltration of PBMCs, and the specific analysis method may be the same as described above.

[0023] Additionally, a step of measuring cancer-related markers in the medium after co-culture with spheroids may be further included. Cancer-related markers may include, for example, INFβ, CXCL10, TNFα, etc., but the embodiments of the present invention are not limited thereto. By measuring markers in cases where the candidate drug has been treated and not treated, the activity of the candidate drug can be evaluated or screened.

[0024] Specific descriptions of each other step are omitted as they are the same as described above.

[0025] The tumor microenvironment implementation and analysis model manufactured according to the method of the present invention as described above can simulate the actual tumor microenvironment more closely than conventional 2D models. Furthermore, when treated with a specific drug whose activity has been verified through in vivo experiments (e.g., an ENPP1 inhibitor), the change in the expression amount and / or expression rate of the related indicator may be greater than that of the control group, and may exhibit a more concentration-dependent trend. In other words, it has the advantage of being able to show results closer to the in vivo environment than the control group.

[0026] This enables reliable, rapid, and cost-effective analysis. Therefore, it is highly suitable for use in assays such as activity assessment and screening for cancer and anticancer drugs.

[0027] Specific details of other embodiments are included in the detailed description.

[0028] The tumor microenvironment implementation and analysis model according to embodiments of the present invention implements a tumor microenvironment similar to the actual one, enabling rapid, highly accurate, and cost-saving analysis, and thus has the advantage of being highly suitable for use in analysis such as activity evaluation and screening related to cancer and anticancer drugs.

[0029] The effects according to the embodiments of the present invention are not limited to the contents exemplified above, and more diverse effects are included in the present specification.

[0030] Figure 1 is a schematic diagram illustrating a lymphocyte infiltration analysis process according to an experimental example of the present invention.

[0031] Figure 2 is an image of FACS analysis of immune cells stained according to lymphocyte infiltration analysis according to an experimental example of the present invention.

[0032] Figure 3 is a graph quantitatively showing the degree of immune cell infiltration according to a lymphocyte infiltration analysis according to an experimental example of the present invention.

[0033] Figure 4 is a graph quantitatively showing the degree of immune cell infiltration according to a lymphocyte infiltration analysis according to an experimental example of the present invention.

[0034] Figure 5 is a schematic diagram illustrating the process of analyzing a tumor microenvironment in a dish according to an experimental example of the present invention.

[0035] Figure 6 is an image of FACS analysis of stained cancer cells according to the tumor microenvironment analysis in a dish according to an experimental example of the present invention.

[0036] Figure 7 is an image of FACS analysis of stained cancer cells according to the tumor microenvironment analysis in a dish according to an experimental example of the present invention.

[0037] Figure 8 is a graph quantitatively showing the size and degree of death of cancer cell spheroids according to the tumor microenvironment analysis in a dish according to an experimental example of the present invention.

[0038] Figure 9 is a graph quantitatively showing the size and degree of death of cancer cell spheroids according to the tumor microenvironment analysis in a dish according to an experimental example of the present invention.

[0039] Figure 10 is a graph showing the ENPP1 inhibitory activity of the compounds according to the experimental example of the present invention.

[0040] Figure 11 is a graph measuring the degree of IRF3 transcriptional activity according to the concentration of compound A according to an experimental example of the present invention.

[0041] Figure 12 is a graph showing the results of qPCR analysis of THP-1 cells treated with each compound according to an experimental example of the present invention.

[0042] Figure 13 is a graph showing the results of measuring the amount of each protein using ELISA after treating each compound according to an experimental example of the present invention.

[0043] Figure 14 is a graph showing the results of analyzing the number of immune cells (immune cell population) after 2D co-culture according to an embodiment of the present invention.

[0044] Figure 15 is a graph showing the results of measuring the tumor inhibition rate-related values ​​of compound A in vivo according to an experimental example of the present invention.

[0045] Figure 16 is a graph showing the results of measuring the tumor inhibition rate-related values ​​of compound A in vivo according to an experimental example of the present invention.

[0046] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention. The present invention is defined solely by the scope of the claims.

[0047] The terminology used herein is for the purpose of describing embodiments only and is not intended to limit the present invention. As used herein, "and / or" includes each and any combination of one or more of the mentioned items. In addition, the singular also includes the plural unless specifically stated otherwise in the phrase. As used herein, "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components in addition to the mentioned components. A numerical range indicated by using "-" or "to" indicates a numerical range that includes the values ​​stated before and after it as the lower and upper limits, respectively, unless otherwise stated. The terms "about" or "approximately" mean a value or numerical range that is within 20% of the value or numerical range stated thereafter.

[0048] Additionally, in describing components of embodiments of the present invention, terms such as first, second, A, B, (a), (b), etc. may be used. These terms are only intended to distinguish the components from other components, and the nature, order, or sequence of the components are not limited by the terms.

[0049] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in their common sense to those of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0050] And when describing an embodiment of the present invention, if it is determined that a specific description of a related known configuration or function hinders understanding of the embodiment of the present invention, the detailed description is omitted.

[0051] In this specification, 'mono-culture' may mean culturing a single type of cell in an incubator or the like so that no interaction with other cells occurs.

[0052] In this specification, 'co-culture' may mean culturing two or more types of cells together in a culture medium or the like, thereby inducing interaction between heterologous cells.

[0053] In this specification, 'direct co-culture' may mean co-culture that allows for physical contact as well as interaction between heterologous cells through secreted substances such as cytokines.

[0054] In this specification, 'indirect co-culture' may mean co-culture in which heterologous cells can interact with each other through secreted substances such as cytokines, for example, by sharing the same medium, but are physically separated and do not come into contact.

[0055] In this specification, the term 'control group' may refer to a model in which all other conditions are the same as the culture method of the present invention, but only the arrangement form of the heterologous cells is different, such as a single culture model, a 2D culture model, etc.

[0056] Hereinafter, embodiments of the present invention will be described in detail through manufacturing examples and experimental examples, but it is obvious that the effects of the present invention are not limited by the following experimental examples.

[0057] Experimental Example 1: Lymphocyte infiltration analysis

[0058] Experimental Example 1-1: Cell Culture and Analysis Method

[0059] Cancer cells (human triple-negative breast cancer cell line, MDA-MB-231) 1x10 4 cells / well and MRC5 (CAF) 3x10 4Cells / well were prepared and seeded at a cancer cell:CAF ratio of 1:3 (in a 96-well U-bottom plate, ultra-low attachment, Sterile), and then cultured in a 37°C, 5% CO2 incubator for 3 days to form spheroids. Phenol red-free medium was used at this time.

[0060] Peripheral blood mononuclear cells (PBMC) were thawed, suspended in phenol red-free medium (containing 0.5 ng / ml of IL-2), and pre-warmed CellTracker TM Staining with Deep Red dye (incubation for 30 minutes in a 37°C, 5% CO2 incubator) was performed. After staining, washing was performed and cells were prepared.

[0061] After installing the HTS transwell® 96-well plate on a 96-well plate, stained PBMCs were seeded (1.6x10 5 After 100 cells / well, they were cultured in a 37°C, 5% CO2 incubator.

[0062] For lymphocyte infiltration analysis, dye-labeled PBMCs that migrated toward cancer cells through transwells after 1-7 days of culture were captured using a fluorescence microscope, and images of migrated immune cells were obtained, and quantitative analysis was performed using a fluorescence activated cell sorter (FACS) after immunofluorescence staining with immune antigen antibodies.

[0063] Figure 1 is a schematic diagram illustrating a lymphocyte infiltration analysis process according to an experimental example of the present invention.

[0064] Experimental Example 1-2: Analysis Results

[0065] Figure 2 is an image of FACS analysis of immune cells stained according to lymphocyte infiltration analysis according to an experimental example of the present invention.

[0066] Figures 3 and 4 are graphs quantitatively showing the degree of immune cell infiltration according to lymphocyte infiltration analysis according to an experimental example of the present invention.

[0067] In the lymphocyte infiltration analysis, the effectiveness of the tumor microenvironment implementation and analysis model of the present invention was evaluated by treating compounds MV-658 and Compound A, which are innate immune anticancer agents. Compound MV-658 is a known substance having the following chemical formula known to have ENPP1 (Ectonucleotide pyrophosphatase-phosphodiesterase 1) inhibitory activity, and Compound A is a benzotriazole-based compound (TXN10128) with ENPP1 inhibitory and STING (stimulator of interferon genes) pathway stimulatory activity.

[0068] [Compound MV-658]

[0069]

[0070] As shown in Figures 2 to 4, the model treated with compound A was observed to have 1.8 times more blood cells migrated than the control group (vehicle), and when this was analyzed by FACS, it was analyzed that activated T cells among the immune cells that infiltrated tumor cells increased by 1.4 times. In other words, since it was observed that the innate immune response was activated by an ENPP1 inhibitor such as compound A, it can be seen that the model of the present invention functions like an actual tumor microenvironment.

[0071] Experimental Example 2: Analysis of the Tumor Microenvironment in a Dish

[0072] Experimental Example 2-1: Cell Culture and Analysis Method

[0073] Cell culture and analysis were performed in the same manner as in Experimental Example 1 above, but before co-culturing cancer cells with MRC5, they were stained in the same manner as the staining method for PBMC.

[0074] To analyze the tumor microenvironment in dishes, spheroid images were acquired, size and morphology were analyzed, and cell suspensions were stained with Calcein AM. The apoptosis of RFP-labeled cancer cells was quantitatively analyzed using FACS.

[0075] Figure 5 is a schematic diagram illustrating the process of analyzing a tumor microenvironment in a dish according to an experimental example of the present invention.

[0076] Experimental Example 2-2: Analysis Results

[0077] Figures 6 and 7 are images of FACS analysis of stained cancer cells according to the tumor microenvironment analysis in a dish according to an experimental example of the present invention.

[0078] Figures 8 and 9 are graphs quantitatively showing the size and degree of death of cancer cell spheroids according to the analysis of the tumor microenvironment in a dish according to an experimental example of the present invention.

[0079] In the tumor microenvironment analysis, the validity of the tumor microenvironment implementation and analysis model of the present invention was evaluated by treating with compounds MV-658 and A. The area of ​​the spheroids was calculated as a ratio using imgae J, with the area on DMSO Day 1 being 100%.

[0080] As shown in Figures 6 to 9, in the model treated with Compound A, scattering of cancer cells, MDA-MB-231 cells, was observed on the 5th day, and death of fluorescently labeled cancer cells was observed on the 7th day. In addition, Compound A and Compound MV-658 showed a 40% and 25% increase in the death of spheroid cancer cells, respectively, indicating that the model of the present invention functioned like an actual tumor microenvironment.

[0081] Experimental Example 3: Measurement of ENPP1 inhibitory activity of compounds MV-658 and A using AMP-Glo ​​enzyme assay using 2'3'-cGAMP as a substrate.

[0082] The ENPP1 inhibitory activity of compounds MV-658 and A used in the validation of the model of the present invention was measured. Since ENPP1 hydrolyzes 2' 3'-cGAMP to produce 5'-adenosine monophosphate (AMP) and 5'-guanosine monophosphate (GMP), the ENPP1 inhibitory activity was measured by measuring the produced AMP.

[0083] AMP generated by the reaction of ENPP1 protein, compound MV-658, and compound A with cGAMP was measured. The analysis was performed using the AMP-Glo® kit (Promega). The recombinantly generated AMP was converted to ADP and ATP, and the light generated through the luciferase reaction was measured.

[0084] IC for compound concentration 50 The values ​​were determined by fitting inhibition curves using the three-parameter method in GraphPad Prism® software. Serially diluted samples of a compound were tested in duplicate or more, and the average IC for each compound was calculated. 50 The values ​​were calculated. IC of compound MV-658 50 The value was determined to be 0.0043, and the IC of compound A 50 The value was determined to be 0.0030.

[0085] Figure 10 is a graph showing the ENPP1 inhibitory activity of the compounds according to the experimental examples of the present invention. As such, it can be seen that compounds MV-658 and Compound A, used in the efficacy evaluation of the model of the present invention, have ENPP1 inhibitory activity that can act as innate immune anticancer agents, and are therefore suitable for use in the efficacy evaluation of the model of the present invention that embodies a tumor microenvironment.

[0086] Experimental Example 4: Measurement of IRF3 (Interferon regulatory factor 3) stimulating activity of compound A

[0087] STING exists in the endoplasmic reticulum, and when cGAMP binds, it activates the IRF3 (Interferon regulatory factor 3) and NF-KB signaling pathways, promoting the expression of type 1 interferon and inflammatory cytokines.

[0088] Since inhibition of ENPP1 activity by compound A reduces cGAMP degradation and increases cGAMP-mediated STING pathway activation, the STING signaling pathway activation by compound A was evaluated by measuring the level of transcriptional activity of IRF3, a downstream signaling protein. After seeding 20,000 cells / well in 96 wells, cGAMP and compound A were diluted and added 24 hours later, and cultured at 37°C for 2 days. After adding 20 μl of supernatant and 50 μl of Quanti-Luc, luminescence detection was performed immediately.

[0089] Figure 11 is a graph measuring the degree of IRF3 transcriptional activity according to the concentration of compound A according to an experimental example of the present invention. As shown in Figure 11, the IRF3 transcriptional activity dependent on compound A concentration is consistent with the increase in cytokine expression measured in the tumor microenvironment analysis in the dish of Experimental Example 2, thereby supporting that the model of the present invention closely mimics the actual tumor microenvironment.

[0090] Experimental Example 5: qPCR Analysis and ELISA Analysis

[0091] THP-1 cell line (1x10 6After treating cells in a 6-well plate with each of the above compounds for 48 hours, the cells were harvested and RNA was isolated. After synthesizing cDNA with the isolated RNA, qPCR was performed using each primer. Figure 12 is a graph showing the results of qPCR analysis of THP-1 cells treated with each compound according to an experimental example of the present invention.

[0092] In addition, after analyzing the tumor microenvironment in the dish, the protein amounts of INFβ, CXCL10, and TNFα in 100 μl of the medium were measured by ELISA. Figure 13 is a graph showing the results of measuring the amount of each protein by ELISA after treating each compound according to an experimental example of the present invention.

[0093] The results of FIGS. 12 and 13 also support that the model of the present invention closely mimics the actual tumor microenvironment, as described above.

[0094] Experimental Example 6: 2D Co-culture Analysis

[0095] In a 2D culture environment, MDA-MB-231 was cultured on day -1, and hPBMC treated with Compound A on day 0 were co-cultured, and FACS analysis was performed on day 7. Figure 14 is a graph showing the results of analyzing the immune cell population after 2D co-culture according to an embodiment of the present invention. As shown in Figure 14, Compound A exhibits concentration-dependent innate immune activity even in a 2D culture environment, and thus can be seen to be suitable for use in evaluating the effectiveness of the model of the present invention.

[0096] Experimental Example 7: In vivo analysis

[0097] MDA-MB-231 cells (5x10 6Cells in 50 μl PBS / each mouse) and Matrigel (50% volume, 50 μl) were mixed and 100 μl of the mixture was subcutaneously inoculated into the mammary fat pad of the mouse twice. Seven days after cell line inoculation, the tumor volume was 100-250 mm 3 Individuals reaching 1x10 were selected and the groups were separated by zigzag distribution so that the average size of the tumors in each group was distributed as evenly as possible. Human PBMC (1x10 7 (cells in 200 μl PBS / each mouse) were inoculated into the tail vein, and compound A was administered orally once a day. The short and long axes of the tumors were measured three times a week using a caliper. Tumor growth inhibition rate (TGI%) was calculated as a percentage using the tumor volumes of the control and test substance administration groups as follows.

[0098] Tumor growth inhibition; TGI% = {1-(Ti-T0) / (Vi-V0)} x 100

[0099] Figures 15 and 16 are graphs showing the results of measuring the tumor inhibition rate of compound A in vivo according to an experimental example of the present invention. According to the measurement results, the tumor inhibition rate of 100 mg / kg of compound A was 19.4% on the 3rd day, 29.4% on the 5th day, 48.5% on the 8th day, 37.0% on the 9th day, 19.6% on the 11th day, 41.7% on the 16th day, and 34.9% on the 18th day.

[0100] In this way, since compound A, which exhibits actual tumor inhibition activity in an in vivo environment, also exhibits the same tumor inhibition activity in the model of the present invention, it can be seen that the tumor microenvironment implementation and analysis model of the present invention sufficiently closely simulates the actual tumor microenvironment and is therefore very suitable for use in analysis such as activity evaluation and screening related to cancer and anticancer drugs.

[0101] While the present invention has been described above with reference to embodiments thereof, these are merely examples and are not intended to limit the scope of the present invention. Those skilled in the art will appreciate that various modifications and applications not exemplified above are possible without departing from the essential characteristics of the embodiments of the present invention. For example, each component specifically shown in the embodiments of the present invention can be modified and implemented. Furthermore, any differences related to such modifications and applications should be construed as being included within the scope of the present invention as defined in the appended claims.

Claims

1. (a) A step of forming a spheroid by co-culturing cancer cells and stromal cells; (b) a step of labeling peripheral blood mononuclear cells (PBMC); (c) a step of co-culturing the labeled PBMC with the spheroid; and (d) a step of analyzing the labeled PBMCs that have migrated to the spheroids after co-culture with the spheroids; Method for producing a tumor microenvironment implementation and analysis model.

2. In claim 1, The above cancer cells are breast cancer cells. Method for producing a tumor microenvironment implementation and analysis model.

3. In claim 1, The above stromal cells are cancer associated fibroblasts (CAF). Method for producing a tumor microenvironment implementation and analysis model.

4. In claim 1, In the step of co-culturing the cancer cells and the stromal cells, the cell number ratio of the cancer cells and the stromal cells is 1:1-10. Method for producing a tumor microenvironment implementation and analysis model.

5. In claim 1, The step of labeling the above PBMC is a step of staining the above PBMC. Method for producing a tumor microenvironment implementation and analysis model.

6. In claim 1, The step of co-culturing the above-mentioned labeled PBMC with the above-mentioned spheroids is: A step of culturing the container containing the labeled PBMC by placing it in a medium of the cancer cells and stromal cells and separating it from the cancer cells and stromal cells. Method for producing a tumor microenvironment implementation and analysis model.

7. In claim 6, The container containing the above-mentioned labeled PBMCs has pores formed in a size through which the PBMCs can pass. Method for producing a tumor microenvironment implementation and analysis model.

8. In claim 1, The step of analyzing the above labeled PBMC is a step of analyzing the amount of the above labeled PBMC that has infiltrated the cancer cells. Method for producing a tumor microenvironment implementation and analysis model.

9. Compare and analyze the analysis results of the model treated with the candidate drug and the model not treated with the candidate drug. The above model is manufactured by any one of the methods of claims 1 to 8. Methods for evaluating the activity of candidate drugs on the tumor microenvironment. 10.(a) Step of labeling cancer cells; (b) a step of co-culturing the dyed cancer cells and stromal cells to form a spheroid; (b) a step of co-culturing peripheral blood mononuclear cells (PBMCs) with the spheroids; and (d) a step of analyzing the cancer cells by analyzing the spheroids after co-culturing with the spheroids; Method for producing a tumor microenvironment implementation and analysis model.

11. In claim 10, The above cancer cells are breast cancer cells. Method for producing a tumor microenvironment implementation and analysis model.

12. In claim 10, The above stromal cells are cancer associated fibroblasts (CAF). Method for producing a tumor microenvironment implementation and analysis model.

13. In claim 10, In the step of co-culturing the cancer cells and the stromal cells, the cell number ratio of the cancer cells and the stromal cells is 1:1-10. Method for producing a tumor microenvironment implementation and analysis model.

14. In claim 10, The step of co-culturing the above PBMC with the above spheroid is: A step of culturing the container containing the PBMC by placing it in a medium of the cancer cells and stromal cells and separating it from the cancer cells and stromal cells. Method for producing a tumor microenvironment implementation and analysis model.

15. In claim 14, The container containing the above PBMC has pores formed in a size through which the PBMC can pass. Method for producing a tumor microenvironment implementation and analysis model.

16. In claim 10, The step of analyzing the cancer cells above is a step of analyzing the death of cancer cells due to the infiltration of the PBMC. Method for producing a tumor microenvironment implementation and analysis model.

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