KR102511633B1 - Method for evaluating efficacy of anti-cancer agent using mixture of m1 macrophage and m2 macrophage - Google Patents

Abstract

The present invention relates to a method for evaluating the efficacy of an anti-cancer drug, or a method for screening an anti-cancer drug, including the step of treating a mixture of cancer organoids, M1 macrophages, and M2 macrophages with an anti-cancer drug or anti-cancer drug candidate. The present invention relates to a system for evaluating the efficacy of an anti-cancer drug comprising cancer organoids, M1 macrophages and M2 macrophages, or a screening system for the anti-cancer drug. The mixture of cancer organoids, M1 macrophages and M2 macrophages, according to the present invention, can be mixed in a specific ratio to similarly reproduce the tumor microenvironment in which cancer cells exist in vivo. Therefore, when utilized, the efficacy of a drug, when administered in vivo, can be accurately predicted.

Description

Anticancer drug efficacy evaluation method using M1 macrophage and M2 macrophage mixture

The present invention relates to a method for evaluating the efficacy of an anti-cancer agent or a method for screening an anti-cancer agent, comprising treating a mixture of cancer organoids, M1 macrophages and M2 macrophages with an anti-cancer agent or candidate substance. It also relates to an efficacy evaluation system or an anticancer agent screening system including cancer organoids, M1 macrophages and M2 macrophages.

Organoids are organ analogues prepared by 3-dimensional culture of cells derived from tissues or organs. Organoids have the advantages of long-term culture, cryopreservation, and easy manipulation and observation. At the same time, it is an experimental model that can study physiological phenomena at a higher level than cells by reproducing the cell hierarchical and histological structure that could only be seen in vivo as well as maintaining the original characteristics of the cell as it does not require immortalization. Due to these characteristics, organoids can be used for drug evaluation with high accuracy compared to immortalized cell lines in which the intrinsic characteristics of cells are changed or animal models that have a different structure from the human body. It has the advantage of being able to check the safety as well as efficacy of the drug in advance. However, since cancer organoids previously developed as drug evaluation models have limitations in not reflecting the tumor microenvironment, efforts have been made to develop organoids with various tumor microenvironments and use them as drug evaluation models. need to be

The tumor microenvironment is a cellular environment in which blood vessels, immune cells, fibroblasts, lymphocytes, signal transduction molecules, and extracellular matrix (ECM) surround cancer cells. In the tumor microenvironment, cancer cells affect the microenvironment by inducing extracellular signal release, cancer cell angiogenesis promotion, and peripheral immune tolerance. Therefore, cancer cells change the microenvironment, and the microenvironment affects the growth or metastasis of cancer cells. In particular, the tumor microenvironment related to lymphocytes is highly related not only to cancer development and metastasis, but also to cancer's immune system evasion. It affects the treatment response by anticancer drugs and is a major target for anticancer immunotherapy (Robert L Ferris, J Clin Oncol. 2015 Oct 10;33(29):3293-304).

For example, cancer progression is caused by an imbalance between cancer cell growth and individual immunity. In the tumor microenvironment, immune cells infiltrate into tumor tissue to prevent tumor growth. Accordingly, among various lymphocytes, tumor-infiltrating lymphocytes (TIL) are considered to be the most important lymphocytes that represent an individual's anti-tumor immune response (M J M Gooden et al. Br J Cancer. 2011 Jun 28;105(1 ):93-103). Tumor-associated macrophages (TAMs) are a type of tumor-infiltrating lymphocytes, and are subdivided into M1 macrophages and M2 macrophages. In general, M1 macrophages are known to be associated with tumor growth inhibition and M2 macrophages are known to be associated with tumor growth promotion, and they express different immune markers, metabolic properties, and gene expression profiles to perform different functions. In addition, differentiation into M1 and M2 is determined according to the type and concentration of signaling substances or cytokines present in the tumor microenvironment, and differentiation into M1 and M2 must be in an appropriate balance in order to elicit an immune response against the tumor. (Yuxin Lin et al. J Hematol Oncol. 2019 Jul 12;12(1):76).

Therefore, in order to develop cancer organoids equipped with a tumor microenvironment and use them as a platform for drug evaluation, the present inventors shared M1 macrophages and M2 macrophages with cancer organoids at a specific ratio among various cells constituting the tumor microenvironment. A culture system was established. In addition, it was experimentally proven that cancer organoids grew properly and drug efficacy was high in the system, and the present invention was completed.

Each description and embodiment disclosed herein can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

In addition, terms not specifically defined in this specification should be understood to have meanings commonly used in the technical field to which the present invention belongs. In addition, unless otherwise defined in context, singular includes plural, and plural includes singular.

One aspect of the present invention provides a method for evaluating the efficacy of an anticancer agent, comprising the following steps:

(a) co-cultivating a mixture of cancer organoids, M1 macrophages (classically activated macrophages, M1) and M2 macrophages (alternatively activated macrophages, M2);

(b) treating the mixture of cancer organoids, M1 and M2 of step (a) with an anticancer agent; and

(c) determining that the efficacy of the anticancer agent is excellent when the growth inhibition or death of cancer organoids is increased in the group treated with the anticancer agent in step (b) compared to the group not treated with the anticancer agent or the positive control group.

Specifically, step (a) is a step of mixing and co-cultivating cancer organoids M1 and M2, and is a step of reproducing the in vivo tumor microenvironment in vitro.

As used herein, the term "organoid" refers to a mass of cells having a three-dimensional three-dimensional structure, and refers to organ analogues prepared by three-dimensional culture of cells isolated from organs or tissues. Since organoids contain specific cell populations constituting an organ or tissue and are structurally organized in a form similar to an actual tissue or organ, the specific form and function of each tissue or organ can be reproduced.

As used herein, the term "cancer organoid" refers to organoids derived from cells isolated from tumors.

As used herein, the term "classically activated macrophage (M1)" is a type of tumor-associated macrophage (TAM), and includes LPS, IFN-γ, IL-1β, TNF-α, and TLR. It refers to macrophages activated by cytokines such as engagement. M1 secretes pro-inflammatory cytokines or chemokines such as IL-6, IL-12, and TNF-α, and functions as an antigen-presenting cell (APC) to activate T cells or tumor-infiltrating lymphocytes ( It can induce pathogen death or cancer cell death by activating tumor infiltrating lymphocyte (TIL) or NK cells, or it can directly kill cancer cells through phagocytosis.

As used herein, the term "M2 macrophage (alternatively activated macrophage, M2)" is a type of tumor-associated macrophage (TAM), and is a type of cytokines such as IL-4, IL-13, and IL-10. Means a macrophage activated by M2 secretes enzymes such as MMPs or COX-2, or anti-inflammatory cytokines such as IL-10 and TGF-β, thereby performing immune responses related to inflammation reduction, wound healing, and tissue repair. However, enzymes secreted by M2 destroy tissues around tumors to promote tumor cell proliferation and metastasis, or induce tumor growth by participating in the initiation and maintenance of angiogenesis.

In the present invention, the cancer organoids M1 and M2 may be mixed at a cell number ratio of 1:1 to 3:0.5 to 5. The tumor microenvironment in vivo can be simulated only when the cancer organoids, M1 and M2, are mixed and co-cultured in the above ratio. As M1 exhibits tumor cell death promoting properties and M2 exhibits tumor cell growth promoting properties, when co-cultured at ratios other than the above ratios, there is a problem that cancer organoids do not grow or grow excessively even before drug treatment. In addition, since the anticancer effect of the drug cannot be accurately confirmed, it is important to mix the cancer organoids, M1 and M2, in the cell number ratio as described above.

In the method for evaluating the efficacy of an anticancer agent according to the present invention, the anticancer agent is treated with a mixture of cancer organoids, M1 and M2, in the above ratio. That is, the efficacy of anticancer drugs is evaluated through a system that similarly reproduces the tumor microenvironment, which is the form in which cancer cells exist in vivo. Therefore, the efficacy of anticancer drugs when administered in vivo can be accurately predicted.

In the method for evaluating the efficacy of an anticancer agent according to the present invention, step (b) is a step of treating a mixture in which cancer organoids, M1 and M2 are co-cultured according to step (a), with an anticancer agent.

As used herein, the term "anti-cancer agent" refers to a substance that exhibits a preventive or therapeutic effect on cancer, and specifically means a substance capable of killing cancer cells, cancer organoids, or tumors, or inhibiting their growth.

In the present specification, the 'anti-cancer agent' may be used interchangeably with 'drug', and the 'treatment' may be used interchangeably with 'addition' or 'administration'.

Specifically, the anticancer agent may target cancer organoids, M1 and/or M2. For example, the anticancer agent targets only cancer organoids, only M1, only M2, targets both cancer organoids and M1, or simultaneously targets cancer organoids and M2. It may be to target M1 and M2 at the same time, or to target cancer organoids, M1 and M2 at the same time. Preferably, the anticancer agent may target M1, and a specific example of the anticancer agent targeting M1 may be an anti-CD47 antibody, but is not limited thereto. The "anti-CD47 antibody" can improve phagocytosis of tumor cells by macrophages by directly blocking the binding between CD47 expressed in tumor cells and SIRPα expressed in macrophages.

In the present invention, the anticancer agent is a compound, protein, fusion protein, compound-protein complex, drug-protein complex, antibody, compound-antibody complex, drug-antibody complex, amino acid, peptide, virus, carbohydrate, lipid, nucleic acid, extract, Fractions and the like are included without limitation.

For example, the anticancer agent may include a compound, peptide, peptide mimetics, fusion protein, antibody, aptamer, antibody-drug conjugate (ADC; Antibody Drug Conjugate), etc., but is not limited thereto. Preferably, the anti-cancer agent may be an antibody or an immuno-anticancer agent.

The term "antibody" includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and antibodies already known or commercially available in the art other than novel antibodies. Also included. The antibodies include functional fragments of antibody molecules as well as full-length forms comprising two heavy chains and two light chains. The functional fragment of the antibody molecule refers to a fragment having at least an antigen-binding function, which may include Fab, F(ab'), F(ab') ₂ , Fv, etc., but is not limited thereto. . The term “Peptide Mimetics” refers to peptides, or modified peptides, that biologically mimic the active ligands of hormones, cytokines, enzyme substrates, viruses or other biological molecules. The term "Aptamer" refers to a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity.

As another example, the anticancer agent may include antisense nucleic acid, siRNA, shRNA, miRNA, ribozyme, etc. that complementarily bind to DNA or mRNA, but is not limited thereto.

The term "antisense nucleic acid" refers to DNA, RNA, or fragments or derivatives thereof containing a nucleic acid sequence complementary to a specific mRNA sequence, and complementarily binds or hybridizes to the sequence of mRNA to form a protein of mRNA. It acts to impede the translation of The term "small interfering RNA (siRNA)" refers to a short double-stranded RNA capable of inducing RNA interference (RNAi) through cleavage of a specific mRNA. siRNA includes a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can suppress the expression of a target gene, it is used in a gene knockdown method or a gene therapy method. The term "short hairpin RNA (shRNA)" refers to single-stranded RNA, which is divided into a stem portion forming a double-stranded portion by hydrogen bonding and a loop portion having a ring shape. It is processed by proteins such as Dicer and converted into siRNA and can perform the same function as siRNA. The term "miRNA (micro RNA)" refers to a non-coding RNA of 21 to 23 nt that regulates gene expression after transcription by promoting degradation of target RNA or inhibiting their translation. The term "ribozyme" refers to an RNA molecule having an enzyme-like function that recognizes a specific nucleotide sequence and cleave it itself. A ribozyme is composed of a region that binds with specificity to a complementary nucleotide sequence of a target messenger RNA strand and a region that cleaves the target RNA.

Anticancer agents for which efficacy is evaluated or screened according to the method of the present invention are, for example, biliary tract cancer, gastric cancer, lung cancer, liver cancer, colon cancer, colon cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, scleroderma, 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, leukemia, lymphoma, fibroadenoma, etc. may be prevented or treated, but is limited thereto no.

In the method for evaluating the efficacy of an anticancer agent according to the present invention, step (c) is performed when the growth inhibition or death of cancer organoids is increased in the group treated with the anticancer agent in step (b) compared to the group not treated with the anticancer agent or the positive control group. This is a step in which the efficacy of the anticancer drug is judged to be excellent.

As used herein, the term "positive control group" refers to a group treated with a drug used as an anticancer agent in the art.

Specifically, growth inhibition or death of the cancer organoid may be confirmed by increasing or decreasing the area of the cancer organoid. In addition, when the area of the cancer organoid decreases, it may be determined that growth inhibition or death of the cancer organoid is increased. In addition, the increase or decrease in the area of the cancer organoid can be confirmed by a method known in the art, and for example, it can be confirmed through an analysis method such as HCS (High-Content Screening) or flow-cytometer. it is not going to be

Another aspect of the present invention provides a method for screening an anticancer agent comprising the following steps:

(a) co-cultivating a mixture of cancer organoids, M1 and M2;

(b) treating the mixture of cancer organoids, M1 and M2 of step (a) with an anticancer drug candidate; and

(c) determining the candidate substance as an anticancer drug when growth inhibition or death of cancer organoids is increased in the group treated with the candidate anticancer drug in step (b) compared to the group not treated with the candidate anticancer drug.

In the method for screening anticancer agents according to the present invention, each term is the same as described in the method for evaluating the efficacy of the anticancer agent unless otherwise specified.

Specifically, step (a) is a step of mixing and co-cultivating cancer organoids M1 and M2, and is a step of reproducing the in vivo tumor microenvironment in vitro.

In the present invention, the cancer organoids M1 and M2 may be mixed at a cell number ratio of 1:1 to 3:0.5 to 5. The tumor microenvironment in vivo can be simulated only when the cancer organoids, M1 and M2, are mixed and co-cultured in the above ratio. As M1 exhibits tumor cell death promoting properties and M2 exhibits tumor cell growth promoting properties, when co-cultured at ratios other than the above ratios, there is a problem that cancer organoids do not grow or grow excessively even before drug treatment. In addition, since the anticancer effect of the drug cannot be accurately confirmed, it is important to mix the cancer organoids, M1 and M2, in the cell number ratio as described above.

In the anticancer drug screening method according to the present invention, a mixture of cancer organoids, M1 and M2, in the above ratio is treated with an anticancer drug candidate. That is, the efficacy of anticancer drug candidates is evaluated through a system that similarly reproduces the tumor microenvironment, which is the form in which cancer cells exist in vivo. Therefore, it is possible to accurately predict the efficacy of an anticancer agent when administered in vivo, and to screen an anticancer agent capable of exhibiting excellent efficacy when administered in vivo.

In the method for screening anticancer drugs according to the present invention, step (b) is a step of treating a mixture in which cancer organoids, M1 and M2 are co-cultured according to step (a), with an anticancer drug candidate.

As used herein, the term "anti-cancer agent" refers to a substance that exhibits a preventive or therapeutic effect on cancer, and specifically means a substance capable of killing cancer cells, cancer organoids, or tumors, or inhibiting their growth.

In the present specification, the 'anti-cancer agent' may be used interchangeably with 'drug', and the 'treatment' may be used interchangeably with 'addition' or 'administration'.

As used herein, the term "cancer agent candidate" refers to a substance expected to exhibit a preventive or therapeutic effect on cancer, and specifically, can kill cancer cells, cancer organoids, or tumors, or inhibit their growth. material that is expected to

In the present specification, the 'anti-cancer agent' may be used interchangeably with 'drug', and the 'treatment' may be used interchangeably with 'addition' or 'administration'.

Specifically, the anticancer drug or anticancer drug candidate may target cancer organoids, M1 and/or M2. For example, the anticancer drug or anticancer drug candidate targets only cancer organoids, only M1, only M2, targets both cancer organoids and M1, or both cancer organoids and M2 may be simultaneously targeted, M1 and M2 may be simultaneously targeted, or cancer organoids M1 and M2 may be simultaneously targeted. Preferably, the anticancer agent may target M1, and a specific example of the anticancer agent targeting M1 may be an anti-CD47 antibody, but is not limited thereto. The "anti-CD47 antibody" can improve phagocytosis of tumor cells by macrophages by directly blocking the binding between CD47 expressed in tumor cells and SIRPα expressed in macrophages.

In the present invention, the anticancer drug or anticancer drug candidate is a compound, protein, fusion protein, compound-protein complex, drug-protein complex, antibody, compound-antibody complex, drug-antibody complex, amino acid, peptide, virus, carbohydrate, lipid, Nucleic acids, extracts, fractions and the like are included without limitation.

For example, the anticancer drug or anticancer drug candidate may include a compound, peptide, peptide mimetics, fusion protein, antibody, aptamer, antibody-drug conjugate (ADC), etc., but is not limited thereto. . Preferably, the anti-cancer agent or candidate anti-cancer agent may be an antibody or an immuno-anticancer agent.

In the anticancer drug screening method according to the present invention, step (c) is performed when the growth inhibition or death of cancer organoids is increased in the group treated with the anticancer drug candidate in step (b) compared to the group not treated with the anticancer drug candidate. This is the step of determining a candidate substance as an anticancer drug.

Another aspect of the present invention provides an efficacy evaluation system for an anticancer agent including cancer organoids, M1 and M2, as a system for evaluating the efficacy of an anticancer agent using the method for evaluating the efficacy of an anticancer agent.

In the efficacy evaluation system of the anticancer agent according to the present invention, each term is as described in the method for evaluating the efficacy of the anticancer agent unless otherwise specified.

The anticancer drug efficacy evaluation system of the present invention simulates a tumor microenvironment, and includes cancer organoids as a tumor constituting the tumor microenvironment in vivo, and M1 and M2 as immune cells.

Specifically, the system for evaluating the efficacy of the anticancer agent may include cancer organoids, M1 and M2, in a cell number ratio of 1:1 to 3:0.5 to 5. The tumor microenvironment in vivo can be simulated only when the cancer organoids, M1 and M2, are mixed and co-cultured in the above ratio. As M1 exhibits tumor cell death promoting properties and M2 exhibits tumor cell growth promoting properties, when co-cultured at ratios other than the above ratios, there is a problem that cancer organoids do not grow or grow excessively even before drug treatment. In addition, since the anticancer effect of the drug cannot be accurately confirmed, it is important to mix the cancer organoids, M1 and M2, in the cell number ratio as described above.

Since the system for evaluating the efficacy of an anticancer agent according to the present invention includes a mixture in which cancer organoids, M1 and M2 are mixed in the above ratio, the tumor microenvironment in which cancer cells exist in vivo can be similarly reproduced. Therefore, since the efficacy of an anticancer agent when administered in vivo can be accurately predicted, it can be usefully used in a method for evaluating the efficacy of an anticancer agent.

Another aspect of the present invention provides an anticancer agent screening system for using the above anticancer agent screening method, including cancer organoids M1 and M2.

In the anticancer agent screening system according to the present invention, each term is the same as described in the method for evaluating the efficacy of the anticancer agent unless otherwise specified.

The anticancer drug screening system of the present invention simulates a tumor microenvironment, and includes cancer organoids as a tumor constituting the tumor microenvironment in vivo, and M1 and M2 as immune cells.

Specifically, the system for evaluating the efficacy of the anticancer agent may include cancer organoids, M1 and M2, in a cell number ratio of 1:1 to 3:0.5 to 5. The tumor microenvironment in vivo can be simulated only when the cancer organoids, M1 and M2, are mixed and co-cultured in the above ratio. As M1 exhibits tumor cell death promoting properties and M2 exhibits tumor cell growth promoting properties, when co-cultured at ratios other than the above ratios, there is a problem that cancer organoids do not grow or grow excessively even before drug treatment. In addition, since the anticancer effect of the drug cannot be accurately confirmed, it is important to mix the cancer organoids, M1 and M2, in the cell number ratio as described above.

Since the anticancer drug screening system according to the present invention includes a mixture of cancer organoids, M1 and M2 in the above ratio, it is possible to similarly reproduce the tumor microenvironment in which cancer cells exist in vivo. Therefore, since the efficacy of an anticancer agent when administered in vivo can be accurately predicted, it can be usefully used in a screening method for an anticancer agent.

The mixture of cancer organoids, M1 macrophages and M2 macrophages according to the present invention can be mixed in a specific ratio to similarly reproduce a tumor microenvironment in which cancer cells exist in vivo. Therefore, when using this, the efficacy of the drug when administered in vivo can be accurately predicted.

1A is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 macrophages (classically activated macrophages, hereinafter referred to as 'M1') and M2 macrophages (hereinafter referred to as 'M2'), wherein M1 is It relates to the results of co-cultivation under conditions without ("1:3:0") or without ("1:0:5") M2.
1B is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and results of co-cultivation under mixed conditions in which cancer organoids and M1 were fixed at a cell number ratio of 1:0.5. It is about.
1C is a graph showing the growth rate of cancer organoids according to the co-cultivation ratio of cancer organoids, M1 and M2, and results of co-cultivation under mixed conditions in which cancer organoids and M1 were fixed at a cell number ratio of 1:1. It is about.
1D is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and results of co-cultivation under mixed conditions in which cancer organoids and M1 were fixed at a cell number ratio of 1:3. It is about.
Figure 2a is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, under a condition without M1 ("1:3:0") or without M2 ("1:0:5").'Non' means a drug-untreated group, and 'CD47' means a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The mortality rate was analyzed based on the results of measuring the growth rate after 72 hours of drug treatment (72h).
Figure 2b is a graph showing the mortality rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, under conditions without M1 ("1:3:0") or without M2 ( It relates to the result of co-culture in "1:0:5"). 'Non' means a drug-untreated group, and 'CD47' means a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The mortality rate was analyzed based on the results of measuring the growth rate after 72 hours of drug treatment (72h).
Figure 3a is a graph showing the growth rate of cancer organoids according to the co-cultivation ratio of cancer organoids, M1 and M2, and results of co-cultivation under mixed conditions in which cancer organoids and M1 were fixed at a cell number ratio of 1:0.5. It is about. 'Non' means a drug-untreated group, and 'CD47' means a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The mortality rate was analyzed based on the results of measuring the growth rate after 72 hours of drug treatment (72h).
3B is a graph showing the death rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, in which cancer organoids and M1 were co-cultured at a fixed cell number ratio of 1:0.5 under mixed conditions. It's about results. 'Non' means a drug-untreated group, and 'CD47' means a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The mortality rate was analyzed based on the results of measuring the growth rate after 72 hours of drug treatment (72h).
Figure 4a is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and results of co-cultivation under mixed conditions in which cancer organoids and M1 were fixed at a cell number ratio of 1:1. It is about. 'Non' means a drug-untreated group, and 'CD47' means a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The mortality rate was analyzed based on the results of measuring the growth rate after 72 hours of drug treatment (72h).
3B is a graph showing the death rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, in which cancer organoids and M1 were co-cultured at a fixed cell number ratio of 1:1 under mixed conditions. It's about results. 'Non' means a drug-untreated group, and 'CD47' means a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The mortality rate was analyzed based on the results of measuring the growth rate after 72 hours of drug treatment (72h).
5A is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and results of co-cultivation under mixed conditions in which cancer organoids and M1 were fixed at a cell number ratio of 1:3. It is about. 'Non' means a drug-untreated group, and 'CD47' means a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The mortality rate was analyzed based on the results of measuring the growth rate after 72 hours of drug treatment (72h).
5B is a graph showing the death rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, wherein cancer organoids and M1 were co-cultured at a fixed cell number ratio of 1:3 under mixed conditions. It's about results. 'Non' means a drug-untreated group, and 'CD47' means a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The mortality rate was analyzed based on the results of measuring the growth rate after 72 hours of drug treatment (72h).

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

Example 1. Establishment of anticancer drug efficacy evaluation and screening system

An attempt was made to establish an in vitro efficacy evaluation and screening system capable of accurately predicting efficacy when a drug is administered in vivo by similarly reproducing an environment in which cancer exists in vivo.

Specifically, in order to establish a co-culture system in which cancer organoids, M1 macrophages (classically activated macrophages, hereinafter referred to as 'M1') and M2 macrophages (hereinafter referred to as 'M2') coexist, co-culture When (i) cancer organoids grow properly and (ii) drug efficacy is high, these mixing ratios were derived that satisfy the observed conditions. As shown in Table 1 below, after the cancer organoids were fixed at a ratio of 1, M1 macrophages and M2 macrophages were set at a ratio of 0 to 5. At this time, the number of each organoid or cell was 5.0×10 ³ and set to 1.

group cancer organoids M1 M2 One One 3 0 2 0 5 3 0.5 0.5 4 One 5 3 6 5 7 One 0.5 8 One 9 3 10 5 11 3 0.5 12 One 13 3 14 5

Cancer organoids were prepared to have a 3D structure by isolating lung cancer tissues obtained from patients into single cells and culturing them together with ECM (extracellular matrix). The formed cancer organoids were confirmed to be tumors through pathological analysis, and then separated into single cells using Tryp-LE and then used.

M1 macrophages and M2 macrophages were differentiated from iPSCs, and differentiated into M1 macrophages or M2 macrophages from the H9 cell line using a general method known in the art.

Example 1.1. Conditions for proper growth of cancer organoids

A mixing ratio of cancer organoids, M1 and M2, which allows cancer organoids to grow properly even when immune cells are co-cultured, was derived.

Specifically, after co-cultivation at the mixing ratio according to Table 1, the area of cancer organoids immediately after the start of co-culture (Oh), after 24 hours (24h), after 48 hours (48h), and after 72 hours (72h) Each was measured and the growth rate was analyzed with the following formula.

^* Cancer organoid growth rate (%) = (cancer organoid area after 24, 48 or 72 hours of co-culture) / (cancer organoid area immediately after co-culture begins) × 100

As a result, as can be seen in FIGS. 1a to 1c, the growth of cancer organoids was not inhibited at all mixing ratios, and the growth rate of cancer organoids tended to increase as the ratio of M2 increased.

Example 1.2. Conditions in which drug efficacy is high

A mixing ratio of cancer organoids, M1 and M2, which makes the efficacy of the anticancer agent high, was derived.

Specifically, for the co-cultures of cancer organoids, M1 and M2 according to the mixing ratio in Table 1, those not treated with the drug were set as the control group (hereinafter referred to as 'drug-untreated group'), and the experimental group (hereinafter referred to as 'drug-treated group') ) was treated with an anti-CD47 antibody targeting M1 as an exemplary drug. After co-cultivating them for 72 hours, the growth rate was measured 72 hours after drug treatment (72h), respectively, and the mortality rate was analyzed by the following method through the measured growth rate.

Cancer organoid death rate (%) = [1 - (cancer organoid growth rate of drug-treated group ^* )/(cancer organoid growth rate of drug-untreated group ^** )] × 100

^* Cancer organoid growth rate (%) of drug-treated group = (cancer organoid area after 72 hours of drug treatment) / (cancer organoid area immediately after the start of co-culture (0h)) × 100

^** Growth rate of cancer organoids in the drug-untreated group (%) = (cancer organoid area after 72 hours without drug treatment)/(cancer organoid area immediately after the start of co-culture (0h)) × 100

As a result, as can be seen in FIGS. 2A and 2B , among cancer organoids, M1 and M2, except for M2, only cancer organoids and M1 are co-cultured at a mixing ratio of 1 : 3, cancer caused by anti-CD47 antibody. The death rate of organoids was as high as about 25%. On the other hand, among cancer organoids, M1 and M2, when only cancer organoids and M2, except for M1, are co-cultured at a mixing ratio of 1:5, the killing rate of cancer organoids by anti-CD47 antibody is about 5%. appeared at a significantly lower level.

On the other hand, as shown in FIGS. 3a and 3b, when cancer organoids and M1 were co-cultured at a ratio of 1:0.5, the mortality rate of cancer organoids according to the increase in the ratio of M2 did not show a significant difference between the mixing ratios. In addition, the death rate of cancer organoids in all drug-treated groups treated with anti-CD47 antibody was only about 10% at maximum, confirming that the efficacy of the drug was not high.

On the other hand, as shown in FIGS. 4a and 4b, when cancer organoids and M1 are co-cultured at a ratio of 1:1, and M2 is mixed at a ratio of 0.5 to 5, the killing rate of cancer organoids by the drug is observed in all cases. It was confirmed that this increased to a high level of up to about 23%.

In addition, as shown in FIGS. 5A and 5B, when cancer organoids and M1 are co-cultured at a ratio of 1:3 and M2 is mixed at a ratio of 0.5 to 5, the killing rate of cancer organoids by the drug is observed in all cases. It was confirmed that this increased to a high level of up to about 25%.

The above results show that the co-culture ratio of cancer organoids, M1 and M2, of 1: 1 to 3: 0.5 to 5 is the most optimal ratio for an anticancer drug efficacy evaluation or screening system, and this system is a system in which macrophages are involved in drug action. It suggests that it is suitable for efficacy evaluation or screening of anticancer drugs.

Claims (8)

A method for evaluating the efficacy of an anticancer agent, comprising the following steps:
(a) mixing and co-cultivating cancer organoids, M1 macrophages (classically activated macrophages) and M2 macrophages (alternatively activated macrophages) at a cell number ratio of 1:1 to 3:0.5 to 5;
(b) treating the cancer organoid, M1 macrophage, and M2 macrophage mixture of step (a) with an anticancer agent; and
(c) determining that the efficacy of the anticancer agent is excellent when the growth inhibition or death of cancer organoids is increased in the group treated with the anticancer agent in step (b) compared to the group not treated with the anticancer agent or the positive control group. A method for screening an anticancer agent comprising the following steps:
(a) mixing and co-cultivating cancer organoids, M1 macrophages and M2 macrophages at a cell number ratio of 1:1 to 3:0.5 to 5;
(b) treating the cancer organoid, M1 macrophage, and M2 macrophage mixture of step (a) with an anticancer drug candidate; and
(c) determining the candidate substance as an anticancer drug when growth inhibition or death of cancer organoids is increased in the group treated with the candidate anticancer drug in step (b) compared to the group not treated with the candidate anticancer drug. According to claim 1 or 2,
The anticancer agent is a compound, peptide, peptide mimetics, fusion protein, antibody, aptamer, antibody-drug conjugate (ADC; Antibody Drug Conjugate), antisense nucleic acid that binds complementary to DNA or mRNA, siRNA, shRNA, miRNA, and At least one method selected from the group consisting of ribozymes. According to claim 1 or 2,
The cancer is biliary tract cancer, stomach cancer, lung cancer, liver cancer, colon cancer, colon cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, scleroderma, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, kidney At least one selected from the group consisting of cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, hematological cancer, lymphoma, and fibroadenoma. According to claim 1 or 2,
The growth inhibition or death of the cancer organoid is confirmed by increasing or decreasing the area of the cancer organoid. A system for evaluating the efficacy of an anticancer agent using the method for evaluating the efficacy of an anticancer agent according to claim 1, wherein the system includes cancer organoids, M1 macrophages and M2 macrophages. An anticancer agent screening system for using the anticancer agent screening method according to claim 2, wherein the anticancer agent screening system includes cancer organoids, M1 macrophages and M2 macrophages.
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