WO2023239048A1 - Method for preparing pancreatic cancer organoids mimicking cancer microenvironment and use thereof - Google Patents

Abstract

The present invention relates to a method for preparing pancreatic cancer organoids mimicking a cancer microenvironment. In addition, the present invention relates to a method for evaluating the efficacy of pancreatic cancer organoids prepared by the above preparation method and an anticancer agent using the pancreatic cancer organoids, and a screening method. The preparation method according to the present invention can prepare pancreatic cancer organoids that perfectly mimic a cancer microenvironment. Accordingly, the pancreatic cancer organoids prepared by the above method can directly observe the effects on cancer cell proliferation/metastasis and anticancer agent resistance, and can be used to develop a drug screening platform to confirm patient-specific anticancer agent resistance in advance. In addition, observation of gene expression regulation according to drug treatment can contribute to the study of molecular mechanisms for the purpose of cancer treatment.

Description

Method for manufacturing pancreatic cancer organoids simulating cancer microenvironment and use thereof

The present invention relates to a method for producing pancreatic cancer organoids simulating a cancer microenvironment. In addition, the present invention relates to pancreatic cancer organoids prepared by the above production method and methods for evaluating and screening the efficacy of anticancer drugs using the pancreatic cancer organoids.

Currently, drug evaluation for new drug development is conducted through cell lines or animal models. The method of using cell lines is the easiest to use because it utilizes already established cell lines. For example, pancreatic cancer cell lines such as HPAC, PANC-1, Capan-2, and CFPAC-1 are used. However, because each patient has different genetic characteristics, there is a disadvantage that specific pathogenic mechanisms and drug reactions cannot be observed. As an animal model, a patient-derived cancer tissue transplant model (PDX; Patient-derived mouse xenograft), which is a model made by transplanting tumor tissue into a mouse, is used. This best reflects the patient's characteristics, but has practical disadvantages in that it takes a lot of time to create the model, has a high failure rate, and is labor-intensive.

Accordingly, patient-derived cancer organoid models (PDO) are being proposed as a new alternative. PDO is a model created to closely resemble actual cancer tissue by cultivating organoids derived from the patient's cancer tissue in three dimensions, and has the advantage of maintaining the patient's unique genotype and phenotype. However, because PDO consists only of cancer cells, it has the disadvantage of not reflecting the cancer microenvironment, the interaction between the tumor microenvironment and cancer cells cannot be observed, and it has limitations in not being able to completely reproduce cancer tissue in vivo.

The above-described cancer microenvironment is also called the tumor microenvironment (TME), which is the total environmental environment in which cancer cells proliferate and evolve. In addition to cancer cells, fibroblasts, immune cells, and extracellular matrix ( It is a concept that includes all extracellular matrix. Existing cancer treatment development research was conducted on the principle of killing the cancer cells themselves. However, as research results continue to be published showing that not only the development, growth, and invasion of cancer cells, but also anticancer drug resistance occur through interaction with the cancer microenvironment, attempts are being made to target the cancer microenvironment to overcome the limitations of existing anticancer drugs. is being done.

The present inventors conducted various studies to develop pancreatic cancer organoids that mimic the cancer microenvironment. As a result, when pancreatic cancer organoids and cancer-associated fibroblasts (CAF) are mixed and cultured at a specific ratio or specific cell number, tissue and cytological characteristics very similar to the actual human pancreatic cancer microenvironment can be displayed. By experimentally demonstrating that pancreatic cancer organoids simulating the cancer microenvironment present can be produced, the present invention was completed.

Each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description described below.

Additionally, terms that are not specifically defined in this specification should be understood to have meanings commonly used in the technical field to which the present invention pertains. Additionally, unless specifically defined by context, the singular includes the plural, and the plural includes the singular.

One aspect of the invention provides a method for producing a cancer microenvironment-mimicking pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid) comprising the following steps: (a) in pancreatic ductal adenocarcinoma (PDAC) tissue; Isolating pancreatic cancer cells; (b) culturing the separated cells into pancreatic cancer organoids (PCO) and cancer-associated fibroblasts (CAF), respectively; and (c) mixing and culturing the pancreatic cancer organoids and cancer-related fibroblasts.

As used herein, the term "organoid" refers to a cell mass with a 3D three-dimensional structure, and is manufactured through an artificial culture process rather than collected, acquired, or harvested from animals, etc., and is a reduced and simplified version. It is defined as an organ of Organoids have the advantage of being capable of long-term culture and cryopreservation and are easy to manipulate and observe. At the same time, it is an experimental model that maintains the original characteristics of cells as it does not require immortalization, and can study physiological phenomena at a level higher than that of cells by reproducing the hierarchical and histological structure of cells that can only be seen in vivo. Due to these characteristics, organoids can evaluate drugs with higher accuracy than immortalized cell lines with changed intrinsic cell characteristics or animal models with structures different from the human body. In particular, since they use patient-derived tissues, organoids can be used in human clinical trials. It has the advantage of being able to check not only the safety but also the efficacy of the drug in advance.

As used herein, the term “pancreatic cancer organoid (PCO)” refers to an organoid derived from pancreatic cancer cells. The PCO of the present invention can express the characteristics of pancreatic cancer cells as is, and a marker known to indicate the characteristics of pancreatic cancer cells (e.g., CK19, etc.) through analysis methods such as tissue staining, cytokine analysis, and immunofluorescence analysis. It can be confirmed that it appears. In this specification, the term “pancreatic cancer organoid” may be used interchangeably with “PCO”.

As used herein, the term "cancer-associated fibroblast (CAF)" refers to fibroblasts present inside and/or around a cancer lesion, and refers to fibroblasts whose characteristics have changed to allow cancer cells to grow. do. These CAFs secrete various growth factors such as FGF2, HGF, TGF-beta, SDF-1, VEGF, and IL-6 out of the cells to form a favorable microenvironment for cancer cells. They promote the growth and invasion of cancer cells, It directly causes metastasis and acts as a major cause of anticancer drug resistance. In addition, carcinomas in which CAFs are intensively distributed do not respond to anticancer drugs, and even if the cancer cell killing effect that reduces the size of the tumor appears in response to anticancer drugs, if the cancer microenvironment centered on CAFs is healthy, cancer cells can easily grow. The likelihood of recurrence increases. Because of these characteristics, CAFs are recognized as an essential component among the various elements that make up the cancer microenvironment (Erik Sahai et al. Nat Rev Cancer. 2020 Mar;20(3):174-186.; Leilei Tao et al. Oncol Lett. 2017 Sep; 14(3): 2611-2620.) In this specification, “cancer-related fibroblasts” may be used interchangeably with “CAFs.”

As used herein, the term "cancer microenvironment-mimicking pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid)" refers to an organoid model prepared by mixing pancreatic cancer organoids (PCO) and cancer-related fibroblasts (CAF). This means that the in vivo cancer microenvironment is reproduced in vitro . The in vivo cancer microenvironment is a cellular environment in which blood vessels, immune cells, fibroblasts (CAFs), lymphocytes, signaling molecules, and extracellular matrix (ECM) surround cancer cells. Cancer cells change their microenvironment by interacting with surrounding cells, and the microenvironment can affect the growth or metastasis of cancer cells. Among these, the interaction with CAFs has a particularly large impact. Therefore, CIPCO containing PCO and CAF can express the characteristics of pancreatic cancer that exist in the in vivo cancer microenvironment. In this specification, the term “cancer microenvironment-mimicking pancreatic cancer organoid” may be used interchangeably with “CIPCO”.

The step (a) is a step of obtaining pancreatic cancer cells from pancreatic ductal adenocarcinoma (PDAC) tissue isolated from an individual.

As used herein, the term “individual” includes all individuals who have never developed pancreatic cancer, are likely to develop it, have developed it, or have completely recovered from the disease, and include humans or any non-human animals. It can be included without limitation. The non-human animal may be a vertebrate, such as a primate, dog, cow, horse, pig, rodent, such as mouse, rat, hamster, guinea pig, etc. In this specification, “individual” may be used interchangeably with “subject” or “patient.”

Pancreatic cancer cells obtained from the pancreatic cancer tissue may include stem cells.

Additionally, the pancreatic cancer cells can be obtained through a process of cutting pancreatic cancer tissue and an enzyme decomposition process. Specifically, the cutting includes both physical cutting and mechanical cutting, and can be performed using general tissue cutting methods known in the art. The enzymatic digestion can be performed under general enzymatic digestion conditions known in the art, for example, using collagenase II enzyme, and more specific examples are collagenase II, HEPES. and GlutaMAX.

Step (b) is a step of culturing the pancreatic cancer cells isolated in step (a) in Matrigel. By culturing the pancreatic cancer cells in three dimensions using Matrigel, pancreatic cancer organoids (PCO) of a tissue-like form are formed. This is the stage of forming a pancreatic cancer organoid. At this time, the culture may be performed by mixing pancreatic cancer cells and Matrigel at a ratio of 0.5 to 1.5:0.5 to 1.5, and specifically, may be performed by mixing them at a ratio of 1:1.

As used herein, the term “matrigel” refers to a protein complex (product name of BD Bioscience) extracted from sarcoma cells of EHS (Engelbreth-Holm-Swarm) mice. The Matrigel contains extracellular matrix (ECM) such as laminin, collagen, and heparan sulfate proteoglycan, and fibroblast growth factor (FGF), epithelial cells. Growth factors (EFG; epiderma growth factor), insulin growth factor (IGF; insulin-like growth factor), transforming growth factor-beta (TGF-β; transforming growth factor-beta), platelet-derived growth factor (PDGF; platelet- It contains growth factors such as derived growth factors.

In addition, the culture of the PCO contains Wnt, R-spondin, B-27, Nicotinamide, HEPES, GlutaMAX, FGF10, N-Acetylcysteine, It may be performed with a culture medium containing one or more selected from the group consisting of EGF, Gastrin 1, and Plasmocin.

At the same time, step (b) is a step of cutting the tissue from step (a) into small pieces, dispensing it on a culture plate, and culturing it while pressing it using a slide glass. ) is the step of forming. Cultivation of the CAF may be performed with a culture medium containing one or more selected from the group consisting of FBS and GlutaMAX.

Step (c) is a step of mixing and culturing the pancreatic cancer organoid (PCO) and cancer-associated fibroblast (CAF) cultured in step (b), which simulates the cancer microenvironment of pancreatic cancer. This is the stage of manufacturing organoids.

Specifically, the mixing may be performed by mixing PCO and CAF at a ratio of 1:1 to 10, and more specifically, may be performed by mixing PCO and CAF at a ratio of 1:3 to 5, preferably 1:3 to 5. It can be performed by mixing at a ratio of 4. Additionally, the mixing may be performed by mixing PCO and CAF with cell numbers of 3.3×10 ⁴ and 1×10 ⁵ .

At this time, the culture of the PCO and CAF mixture is Wnt, R-spondin, B-27, Nicotinamide, HEPES, GlutaMAX, FGF10, N-acetylcysteine (N- It can be performed with a culture medium containing Acetylcysteine, EGF, Gastrin 1, and Plasmocin.

The cancer microenvironment-simulating pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid) prepared through the above-described steps (a) to (c) may express CK19 and Vimentin.

As used herein, the term "CK19 (cytokeratin-19)" refers to keratin or keratin-19 protein expressed by the KRT19 gene, and is mainly a marker of gastroenteropancreatic and hepatobiliary epithelial or pancreatic cancer (PDAC). ; pancreatic ductal adenocarcinoma). In the present invention, CK19 can be used as a marker for PDAC or PCO.

As used herein, the term “Vimentin” refers to a protein expressed by the VIM gene and plays a role in maintaining cell shape and stabilization of the cytoskeleton. In the present invention, CK19 can be used as a marker for CAF.

Therefore, CIPCO according to the present invention, which expresses CK19 and Vimentin, can exhibit both the characteristics of PCO and CAF characteristics, and can perfectly mimic the cancer microenvironment existing in vivo.

In addition, the cancer microenvironment-simulating pancreatic cancer organoid prepared through the above-described steps (a) to (c) has epithelial to mesenchymal transition (EMT), proliferation ability, and cancer organoid compared to pancreatic cancer organoid. This may increase the metastatic ability and anticancer drug resistance.

The term “epithelial to mesenchymal transition (EMT)” used in this specification refers to the transition from epithelial cell properties to mesenchymal cell properties, and is an important process in the movement and metastasis of cancer cells. When EMT increases, the expression of mesenchymal cell marker genes Vimentin, Fibronectin, and ZEB-1 increases. CIPCO according to the present invention has increased EMT compared to general PCO that does not contain CAF, and accordingly, mobility and metastatic ability can be increased compared to PCO. In addition, in subjects transplanted with CIPCO, compared to subjects transplanted with PCO, characteristics such as reduced death of cancer organoids, increased volume of cancer organoids, decreased body weight of transplanted subjects, and/or decreased survival rate of transplanted subjects were observed. You can.

As used herein, the term “proliferative ability of cancer organoids” refers to the cell division ability of cancer organoids, and means that cancer organoids divide themselves to increase their absolute number and quantity. As used herein, the term “metastatic ability of cancer organoids” is also called “migratory ability of cancer organoids,” where cancer organoids infiltrate the extracellular matrix surrounding them and migrate to other parts or other organs. It means transitioning to . The proliferative or metastatic ability of cancer organoids is controlled by the cancer microenvironment surrounding the cancer organoid, and in particular, the proliferative or metastatic ability is increased by CAF. Therefore, CIPCO according to the present invention can increase the metastatic and migration ability of cancer organoids compared to general PCO that does not contain CAF. In this case, in individuals transplanted with CIPCO, compared to individuals transplanted with PCO, features such as reduced death of cancer organoids, increased volume of cancer organoids, decreased body weight of transplanted individuals, and/or decreased survival rate of transplanted individuals. can indicate.

The term "anticancer drug resistance" used in this specification is also called "anticancer drug resistance" or "anticancer drug refractoriness", and when treating cancer patients with anticancer drugs, there is no effect from the beginning of treatment, or it is effective initially but continued treatment. This means that the effect is reduced or lost over the course of the treatment, or that the response to the treatment does not last for a long period of time. Anticancer drug resistance of cancer cells is controlled by the cancer microenvironment surrounding the cancer cells, and in particular, CAF increases anticancer drug resistance. Therefore, CIPCO according to the present invention can increase the anticancer drug resistance of cancer organoids compared to general PCO that does not contain CAF. In this case, in subjects transplanted with CIPCO, when treated with anticancer drugs, the death of cancer organoids is reduced, the volume of cancer organoids increases, the body weight of the transplanted subjects decreases, and/or the survival rate of the transplanted subjects decreases compared to subjects implanted with PCO. It can show characteristics such as:

Another aspect of the present invention provides a cancer microenvironment simulating pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid) prepared by the above manufacturing method.

In CIPCO according to the present invention, each term has the same meaning as described in the manufacturing method of CIPCO above, unless otherwise specified.

CIPCO according to the present invention is an organoid model containing pancreatic cancer organoid (PCO) and cancer-associated fibroblast (CAF), and is a tissue/tissue very similar to the microenvironment of pancreatic cancer existing in vivo. It has cytological characteristics and functions. In addition, the CIPCO exhibits the characteristics of increased EMT, proliferation ability of cancer organoids, metastatic ability of cancer organoids, and anticancer drug resistance compared to PCO that does not contain CAF.

Therefore, the CIPCO has the advantage of being able to embody the in vivo cancer microenvironment very similarly in vitro , and can be used as a treatment for malignant tumors or a treatment that can overcome anticancer drug resistance. It can be useful for screening, efficacy evaluation, etc. In particular, when the CIPCO is manufactured using cells or tissues derived from a patient, it can be used to confirm patient-specific anticancer drug resistance in advance.

Another aspect of the present invention provides a method for evaluating the efficacy of an anticancer agent, comprising the following steps: (a) injecting an anticancer agent candidate into the cancer microenvironment simulating pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid); processing steps; (b) Measurement of one or more levels selected from the group consisting of epithelial to mesenchymal transition (EMT), proliferative ability of cancer organoids, metastatic ability of cancer organoids, and anticancer drug resistance in CIPCO treated with the anticancer drug candidate. steps; and (c) determining the anticancer drug candidate as an anticancer drug when the level measured by CIPCO according to step (b) is lower than the level measured by the control group.

The method for evaluating the efficacy of the anticancer agent may be a screening method for the anticancer agent.

In the method for evaluating the efficacy of an anticancer agent or the screening method for an anticancer agent according to the present invention, each term has the same meaning as described in the CIPCO manufacturing method unless otherwise specified.

The step (a) is a step of treating the anticancer drug candidate to the CIPCO manufactured according to the above manufacturing method.

As used herein, the term "candidate" refers to a substance expected to be able to treat cancer. Specifically, any substance that is expected to inhibit or improve the growth, movement, and metastasis of cancer cells, or increase the death of cancer cells can be used without limitation, and all substances expected to be treatable, such as compounds, genes, or proteins, can be used. Includes.

Treatment of the anticancer drug candidate may be performed using methods known in the art. As a specific example, the CIPCO can be treated with an anti-cancer drug candidate and cultured together, or the anti-cancer drug candidate can be treated by administering the anti-cancer drug candidate into a living body containing the CIPCO, but the method is not limited thereto, and a method suitable for the purpose of the present invention is not limited thereto. can be used.

In addition, step (b) is a step of measuring one or more levels selected from the group consisting of EMT of CIPCO treated with the candidate material, proliferation ability of cancer organoids, metastatic ability of cancer organoids, and anticancer drug resistance.

Specifically, the level of EMT, proliferation ability of cancer organoids, metastatic ability of cancer organoids, or anticancer drug resistance can be measured using methods known to those skilled in the art. For example, the EMT can be analyzed by measuring the expression levels of Vimentin, Fibronectin, and/or ZEB-1. In addition, the proliferation ability, metastatic ability, and/or anticancer drug resistance of the cancer organoid can be analyzed by measuring the increase or decrease in death of the cancer organoid, increase or decrease in volume, increase or decrease in body weight of the transplanted individual, and/or increase or decrease in survival rate of the transplanted individual, etc. .

Additionally, as means for measurement, Western blot, Co-Immunoprecipitation assay, ELISA (Enzyme Linked Immunosorbent Assay), PCR (Polymerase chain reaction), reverse transcription PCR (RT-PCR; Reverse transcription PCR), quantitative reverse transcription PCR (RT-qPCR; Quantitative reverse transcription PCR), tissue staining such as H&E, Masson's trichrome, tissue immunostaining, Tissue immunocytochemistry, flow cytometry analysis, fluorescence-based assays, electron microscopic analysis, etc. may be used, but are not limited thereto, and those skilled in the art will be able to use the present invention. You can use any method that suits your purpose.

In addition, step (c) is when the level of EMT, proliferation ability of cancer organoids, metastatic ability of cancer organoids, and/or anticancer drug resistance measured by CIPCO according to step (b) above decreases compared to the level measured in the control group. , This is the step of determining the anticancer drug candidate as an anticancer drug.

As used herein, the term "control group" refers to CIPCO that has not been treated with the anticancer drug candidate or CIPCO that has been treated with the anticancer drug. It is used to compare the level of metastatic potential or anticancer drug resistance.

As a specific example, if the EMT measured by CIPCO decreases from the level measured in the control group, the expression of Vimentin, Fibronectin, and/or ZEB-1 measured by CIPCO decreases compared to the level measured in the control group. This may be the case.

As another specific example, if the proliferative capacity of cancer organoids measured by CIPCO decreases from the level measured in the control group, 1) the volume of cancer organoids measured in the CIPCO-implanted subject decreases compared to the level measured in the control group, or 2) the body weight of the individual implanted with CIPCO increases compared to the level measured in the control group, and/or 3) the survival rate of the individual measured in the CIPCO-implanted individual increases compared to the level measured in the control group. You can.

As another specific example, if the metastatic ability of cancer organoids measured by CIPCO decreases from the level measured in the control group, 1) the volume of the cancer organoid measured in the CIPCO-implanted subject decreases compared to the level measured in the control group, or 2) the body weight of the individual implanted with CIPCO increases compared to the level measured in the control group, and/or 3) the survival rate of the individual measured in the CIPCO-implanted individual increases compared to the level measured in the control group. You can.

As another specific example, if the anticancer drug resistance of the cancer organoid measured by CIPCO decreases compared to the level measured in the control group, when the subject is treated with an anticancer drug or anticancer drug candidate, 1) the cancer organoid measured in the subject transplanted with CIPCO It may be the case that the death of increases compared to the level measured in the control group, and/or 2) the survival rate of the individual measured in the CIPCO-implanted individual increases compared to the level measured in the control group.

The level of the above-described EMT, proliferation ability of cancer organoids, metastatic ability of cancer organoids, or anticancer drug resistance can be measured using methods known to those skilled in the art. For example, the EMT can be analyzed by measuring the expression levels of Vimentin, Fibronectin, and/or ZEB-1. In addition, the proliferation ability, metastatic ability, and/or anticancer drug resistance of the cancer organoid can be analyzed by measuring the increase or decrease in death of the cancer organoid, increase or decrease in volume, increase or decrease in body weight of the transplanted individual, and/or increase or decrease in survival rate of the transplanted individual, etc. there is.

The production method according to the present invention can produce pancreatic cancer organoids that mimic the cancer microenvironment. Therefore, pancreatic cancer organoids prepared by the above method can be directly observed for their effects on cancer cell proliferation/metastasis and anticancer drug resistance, and can be used in the development of a drug screening platform to confirm patient-specific anticancer drug resistance in advance. In addition, it can contribute to research on molecular mechanisms for cancer treatment by observing the regulation of gene expression according to drug treatment.

Figure 1 relates to the preparation of a cancer microenvironment-mimicking pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid). A is a schematic diagram of the CIPCO manufacturing method, B is an image showing the degree of CIPCO formation according to the mixing ratio of pancreatic cancer organoid (PCO) and cancer-associated fibroblast (CAF), and C is an image showing the degree of CIPCO formation. This image shows the degree of fusion of CAF in PCO, CAF, and CIPCO. BF (bright field) shows pictures taken with an optical microscope.

Figure 2 relates to the production of CIPCO and is an image showing the degree of formation of CIPCO according to the mixing ratio of PCO and CAF. This is the result of mixing and culturing PCO and CAF at a cell number ratio of 1:1, 1:2, 1:3, 1:4, or 1:5, respectively.

Figure 3 relates to the growth of CIPCO according to the culture period. A is an image showing the culture period of PCO, CAF, and CIPCO, and B is an image showing CK19 and Vimentin expressed in PCO and CIPCO.

Figure 4 relates to the organizational similarity of CIPCO. A is an image showing the results of H&E staining and expressed vimentin for PCO, CIPCO, and human pancreatic cancer tissue, and B is an image showing the results of Masson's trichrome staining for PCO, CIPCO, and human pancreatic cancer tissue. C is an image showing the results of immunohistochemical staining for Nuclei, Vimentin, and Collagen I for PCO and CIPCO, and D is an image showing collagen I expressed in PCO and CIPCO. This is a graph analyzing the amount of.

Figure 5 relates to CIPCO's CAF subtype. A is an image showing the expression level of vimentin, IL-6, CK19, α-SMA, and MHC II expressed in human pancreatic cancer tissue (Tissue), CAF, PCO, and CIPCO, and B is an image showing the expression level of human pancreatic cancer tissue (hPCT), CIPCO. This is a graph showing the ratio of CAF subtypes present in .

Figure 6 relates to CIPCO's epithelial to mesenchymal transition (EMT). A is an image showing the results of H&E staining and the expression level of CK19, vimentin, and CK19 for PCO, CIPCO, and human pancreatic cancer tissue, and B is CIPCO without ATRA (Control) or CIPCO with ATRA. This is a graph showing the expression levels of ZEB-1, Fibronectin, and Vimentin in (ATRA).

Figure 7 relates to the EMT and metastatic ability of CIPCO using the Xenograft mouse model. A is a graph showing the body weight of mice implanted with PCO or CIPCO, and B is the results of H&E and Masson's trichrome staining for PCO or CIPCO implanted in mice, as well as α-SMA, vimentin, nucleus, CK19, and Twist-1. This image shows the results of immunohistochemical staining.

Figure 8 relates to anticancer drug resistance of CIPCO. A is an image showing the results of immunohistochemical staining with PI (Propidium iodide) on PCO or CIPCO treated with anticancer drug (gemcitabine) at different concentrations, and B is a graph showing the proportion of PI area in the image of A. . Vehicle refers to the control group that was not treated with anything, and collagenase refers to the experimental group treated with collagenase.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples.

Example 1. Method for manufacturing a cancer microenvironment-simulating pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid)

By similarly reproducing the environment in which cancer exists in vivo, we sought to establish an in vitro drug screening and efficacy evaluation system that can accurately predict the efficacy of drugs when administered in vivo. For this purpose, a cancer microenvironment simulating pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid) was prepared using cancer organoids and cancer-related fibroblasts derived from pancreatic cancer tissue.

Specifically, cancer tissue (PDAC; pancreatic ductal adenocarcinoma) from a pancreatic cancer patient was cut into small pieces and cultured using separation buffer at 37°C for a total of 1 hour. Afterwards, the clump-shaped pancreatic cancer cells isolated from the pancreatic cancer tissue were mixed with Matrigel at a ratio of 1:1, and cultured using PCO culture medium to prepare a pancreatic cancer organoid (PCO). did. Additionally, cancer-associated fibroblasts (CAF) were prepared by placing the pancreatic cancer tissue in a 12-well plate, pressing the tissue with a glass slide, and culturing it using CAF culture medium. The composition of the buffer and medium used at this time is shown below.

- Separation buffer composition: DMEM/F12 medium containing 5 mg/ml collagenase II, 10 mM HEPES, 1X GlutaMAX and 1X P/S (penicillin-streptomycin)

- PCO culture medium composition: 50% conditioned-Wnt medium, 10% conditioned-R-spondin medium, 1X B-27, 10 mM nicotinamide, 10 mM HEPES, 1 Advanced DMEM/F12 medium containing 25 μg/ml Plasmocin

- CAF culture medium composition: high glucose DMEM medium containing 10% FBS, 1X GlutaMAX and 1X P/S

Then, in order to prepare a cancer microenvironment-mimicking pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid), PCO and CAF prepared by the above method were mixed and cultured at various ratios of 1:1 to 5, where PCO Culture medium was used. In addition, the optimal ratio of PCO and CAF was identified by analyzing the degree of CIPCO formation and the degree of fusion of PCO and CAF according to the mixing ratio. A schematic diagram of this manufacturing method is shown in Figure 1A.

As a result, as shown in Figure 1B, it was confirmed that when the ratio of CAF during co-culture was lower or similar to that of PCO, the formation of CAF-induced stroma did not occur. Specifically, when mixing CAF with 1 × 10 ⁴ cells for PCO with 3.3 × 10 ⁴ cells (i.e., when the mixing ratio of PCO and CAF is 3.3: 1) or with PCO with 3.3 × 10 ⁴ cells. For example, when mixing 5 × 10 ⁴ cells of CAF (i.e., when the mixing ratio of PCO and CAF is 1:1.5), it was confirmed that CIPCO did not maintain its proper shape because the matrix induced by CAF was not formed. (B in Figure 1).

On the other hand, when the ratio of CAF to PCO was high, it was confirmed that the shape of CIPCO was well maintained by the substrate induced by CAF (Figure 1B). Specifically, when mixing PCO with 3.3 × 10 ⁴ cells and CAF with 1 × 10 ⁵ cells (i.e., when the mixing ratio of PCO and CAF is 1: 3), tissue formation by the CAF matrix is well maintained. , even if the culture continues, its shape remains constant (Figure 1B), and it was confirmed that the fusion of PCO and CAF gradually increases as the culture period increases (Figure 1C).

In particular, as shown in Figure 2, when the mixing ratio of PCO is 1:1 or 1:2, the matrix by CAF is not formed well and the shape of the dome is not maintained or the organoids are shifted to one side within the dome. It was confirmed that PCO and CAF do not fuse well, including bias. On the other hand, when the mixing ratio of PCO and CAF was 1:3 to 1:5, the shape of CIPCO was well maintained, and it was confirmed that the density of organoids and CAF within the dome was uniformly distributed without being biased to one side. . In particular, it was confirmed that as the mixing ratio with CAF increases, the cystic organoid is surrounded by the surrounding CAF-derived matrix and changes into a small-sized organoid with a dense lumen, which is a type of actual solid cancer. Reflects CAF and stromal characteristics of pancreatic cancer.

Furthermore, as shown in Figure 3, when the mixing ratio of PCO and CAF is 1:3, CIPCO continues to grow as the culture period increases, and growth occurs even when the culture period is longer than 10 days. Confirmed (A in Figure 3). In addition, CIPCO expresses CK19, a PCO marker, and Vimentin, a CAF marker. In particular, vimentin was confirmed to be expressed evenly around PCO, confirming that CAF is distributed throughout CIPCO (Figure B of 3).

Based on the above results, CIPCO, prepared by mixing pancreatic cancer organoids (PCO) with CAF at a cell number ratio of 1:3 to 5, can simulate the microenvironment of pancreatic cancer, so it is useful as a drug screening and efficacy evaluation system. You can see that it can be done.

Example 2. Organizational similarity of CIPCO

CIPCO prepared in Example 1 was analyzed for similarity to actual human pancreatic cancer tissue.

Specifically, tissue similarity was analyzed by staining with H&E, Masson's Trichrome, etc.

As a result, as can be seen in Figure 4, it was confirmed that CIPCO exhibited histological characteristics very similar to human pancreatic cancer tissue. In addition, in both CIPCO and human pancreatic cancer tissues, it was confirmed that vimentin, a marker of fibroblasts, was expressed in the stromal portion of the cancer, through which it was confirmed that the stromal matrix was derived from CAF (Figure 4A). In addition, the representative matrix that appears in solid tumors such as pancreatic cancer is known to be collagen secreted by CAF, and CIPCO established in Example 1 also confirmed that most of the matrix formed by CAF is collagen (FIG. 4B to D).

Furthermore, the subtypes of CAF observed in CAF, PCO, and CIPCO derived from human pancreatic cancer tissue were analyzed through immunohistochemical analysis. Generally, CAFs can be divided into three subgroups: MyoCAFs (Myofibroblasts) expressing α-SMA, iCAFs (Inflammatory fibroblasts) expressing IL-6, and apCAFs (Antigen pregenting fibroblasts) expressing MHC II, respectively. am.

Specifically, CAF, PCO, and CIPCO were each cultured, and on the 10th day of culture, they were fixed with 4% PFA, then paraffin blocks were created and sectioned. Afterwards, analysis was conducted on the markers of CAF subtype: α-SMA, IL-6, and MHC II.

As a result, as can be seen in Figure 5, CIPCO was confirmed to express markers of the CAF subtype in a pattern very similar to human pancreatic cancer tissue (Figure 5A). In addition, CIPCO confirmed that CAF subtypes were expressed at a similar rate to human pancreatic cancer tissue (Figure 5B). Among the above CAF subtypes, myoCAF is a CAF that is directly attached to or located adjacent to cancer cells, and iCAF is a CAF that is located away from cancer cells. In the in vivo pancreatic cancer microenvironment, several CAF subtypes of myoCAF, iCAF, and ap-CAF move around cancer cells in various ways. It is structured in the form.

On the other hand, when pancreatic cancer cells isolated from pancreatic cancer tissue are formed into organoids (PCO), the organoids are then disassociated to create single cell pancreatic cancer cells, and then fused with CAF, the cancer cells and It was confirmed that only adjacent CAFs exist. Therefore, it was confirmed that using pancreatic cancer organoids in the production of CIPCO can more effectively simulate the cancer microenvironment than using single-cell pancreatic cancer cells.

From the above results, it can be seen that CIPCO prepared in Example 1 can simulate the cancer microenvironment, such as reflecting the CAF characteristics of pancreatic cancer tissue, and thus can be usefully used as a drug screening and efficacy evaluation system. .

Example 3. CIPCO's epithelial to mesenchymal transition (EMT)

For CIPCO prepared in Example 1, the degree of epithelial-mesenchymal transition was analyzed. EMT refers to the transition from epithelial cell properties to mesenchymal cell properties, and is an important process in the migration and metastasis of tumor cells.

Specifically, when EMT progresses, the expression of mesenchymal cell marker genes Vimentin, Fibronectin, and ZEB-1 increases. Accordingly, the expression level of the gene was analyzed.

As a result, as can be seen in A of Figure 6, it was confirmed that CIPCO increased the expression of vimentin compared to PCO. In particular, it was confirmed that the expression changes of EMT-related genes in CIPCO showed a pattern very similar to that in human pancreatic cancer tissue.

In order to confirm whether the changes in the expression patterns of EMT-related factors as described above are due to the influence of CAFs, ATRA (all-trans-retinoic acid), a drug that inactivates CAFs, was treated with CAFs before co-culture, and ATRA-treated CAFs were treated with ATRA. CIPCO was produced by co-culturing with PCO. Afterwards, single cells were isolated from CIPCO using FACS, and the expression levels of EMT-related genes were analyzed through qRT-PCR.

As a result, as can be seen in Figure 6B, CIPCO confirmed that the expression of all EMT-related genes (ZEB-1, fibronectin, vimentin) was reduced by ATRA treatment, and the EMT of cancer cells increased in co-culture. It was confirmed that the difference in marker expression was due to CAF.

Based on the above results, CIPCO prepared in Example 1 can induce EMT involved in cancer movement and metastasis, and can perfectly mimic the microenvironment of pancreatic cancer, making it useful as a drug screening and efficacy evaluation system. You can see that it can be used.

Example 4. EMT and metastatic ability of CIPCO using Xenograft mouse model

For CIPCO prepared in Example 1, EMT and metastatic ability were analyzed using a Xenograft mouse model.

As a result, as can be seen in Figure 7A, it was confirmed that when CIPCO was implanted, the survival rate of mice decreased sharply compared to when PCO was implanted.

In addition, as can be seen in Figure 7B, it was confirmed that human nuclei and human CK19 were expressed in PCO- and CIPCO-derived cancer cells, which showed that the cancer cells were not native to the mouse, but were transplanted from the transplanted PCO and CIPCO. It was confirmed that it originated from .

In addition, CIPCO-derived cancer cells were confirmed to have increased expression of the α-SMA gene compared to PCO-derived cancer cells. The expression of α-SMA/vimentin represents fibroblasts like CAF, and the expression level was higher in CIPCO-derived cancer cells than in PCO-derived cancer cells, confirming that CAFs form tissues together in CIPCO-derived cancer cells. It was confirmed that the formation of my ECM was further increased.

In addition, CIPCO-derived cancer cells were confirmed to have a higher amount of Twist-1 expressed when the tumor metastasizes compared to PCO-derived cancer cells, and through this, it was confirmed that EMT increases in CIPCO.

Based on the above results, CIPCO prepared in Example 1 can aggressively induce cancer growth, migration, and metastasis in vivo, and can perfectly mimic the microenvironment of pancreatic cancer, making it suitable for drug screening and efficacy evaluation. It can be seen that it can be usefully utilized as a system.

Example 5. Anticancer drug resistance of CIPCO

Anticancer drug resistance of CIPCO prepared in Example 1 was analyzed. Gemcitabine is mainly used as a treatment for pancreatic cancer, but when applied to actual pancreatic cancer patients, the anticancer effect may be reduced due to resistance to the drug. The mechanism for anticancer drug resistance has been suggested to be 1) inefficient drug delivery due to physical obstacles caused by the tumor microenvironment or 2) molecular defense caused by EMT of tumor cells.

Specifically, anticancer drug resistance was analyzed by confirming the degree of survival of CIPCO by anticancer drug treatment. Immunohistochemical analysis was performed by staining the nuclei of cells with PI (propidium iodide), and the ratio of PI area was analyzed.

As a result, as can be seen in Figures 8A and B, it was confirmed that the PI area of CIPCO did not decrease even when treated with a higher concentration of anticancer drugs than that of PCO. Through this, anticancer drug resistance increased and the number of surviving CIPCOs was maintained. Confirmed. However, it was confirmed that when collagenase, which decomposes collagen, one of the main extracellular matrices of CIPCO, is treated with anticancer drugs, the PI area decreases sharply, and through this, anticancer drug resistance decreases (i.e. It was confirmed that the death of CIPCO significantly increased (due to increased anticancer drug sensitivity).

Based on the above results, CIPCO manufactured in Example 1 can simulate the microenvironment of pancreatic cancer that exhibits anticancer drug resistance, making it useful for customized treatment that can determine in advance the drug resistance or drug effect that exists in the patient. It can be used as an effective in vitro model in the development of combination agents for anticancer drug evaluation and sensitivity control.

Claims (12)

1. Method for producing a cancer microenvironment-mimicking pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid) comprising the following steps:

   (a) obtaining pancreatic cancer cells from pancreatic ductal adenocarcinoma (PDAC) tissue isolated from an individual;

   (b) culturing the separated cells into pancreatic cancer organoids (PCO) and cancer-associated fibroblasts (CAF), respectively; and

   (c) Mixing and culturing the pancreatic cancer organoids and cancer-related fibroblasts.
2. According to paragraph 1,

   The preparation method wherein step (a) is performed using a separation buffer containing collagenase II, HEPES, and GlutaMAX.
3. According to paragraph 1,

   The culture of the pancreatic cancer organoid in step (b) is performed by mixing pancreatic cancer cells and Matrigel at a volume ratio of 0.5 to 1.5:0.5 to 1.5.
4. According to paragraph 1,

   Culture of pancreatic cancer organoids in step (b) includes Wnt, R-spondin, B-27, Nicotinamide, HEPES, GlutaMAX, FGF10, and N-acetylcysteine ( A manufacturing method performed in a culture medium containing N-Acetylcysteine), EGF, Gastrin 1, and Plasmocin.
5. According to paragraph 1,

   The preparation method of step (b), wherein the culture of cancer-related fibroblasts is performed with a culture medium containing FBS and GlutaMAX.
6. According to paragraph 1,

   The culture in step (c) includes Wnt, R-spondin, B-27, Nicotinamide, HEPES, GlutaMAX, FGF10, and N-Acetylcysteine. , a manufacturing method performed in a culture medium containing EGF, Gastrin 1, and Plasmocin.
7. According to paragraph 1,

   The mixing in step (c) is performed by mixing pancreatic cancer organoids and cancer-related fibroblasts at a cell number ratio of 1:3 to 5.
8. According to paragraph 1,

   A manufacturing method wherein the cancer microenvironment-mimicking pancreatic cancer organoid expresses CK19 and Vimentin.
9. According to paragraph 1,

   The cancer microenvironment-simulating pancreatic cancer organoid has increased epithelial to mesenchymal transition (EMT), proliferation ability of the cancer organoid, metastatic ability of the cancer organoid, and anticancer drug resistance compared to the pancreatic cancer organoid.
10. A pancreatic cancer organoid simulating a cancer microenvironment manufactured by the manufacturing method of claim 1.
11. Method for evaluating the efficacy of an anticancer agent, comprising the following steps:

    (a) treating the cancer microenvironment-simulating pancreatic cancer organoid (CIPCO; CAF-integrated pancreatic cancer organoid) of item 10 with an anticancer drug candidate;

    (b) Select from the group consisting of epithelial to mesenchymal transition (EMT), proliferative ability of cancer organoids, metastatic ability of cancer organoids, and anticancer drug resistance in pancreatic cancer organoids simulating the cancer microenvironment treated with the above anticancer drug candidate. measuring one or more levels of and

    (c) If the level measured in the pancreatic cancer organoid simulating the cancer microenvironment according to step (b) is lower than the level measured in the control group, determining the anticancer drug candidate as an anticancer drug.
12. According to clause 11,

    The control group is a pancreatic cancer organoid simulating a cancer microenvironment that is not treated with an anticancer drug candidate or a pancreatic cancer organoid treated with an anticancer drug candidate.

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