Evaluation method for anticancer drug

Abstract

To provide a method that enables an evaluation closer to the evaluation obtained when using a cancer-bearing animal model in the drug efficacy evaluation of an anticancer drug by an assay system using cultured cells.SOLUTION: An evaluation method for an anticancer drug includes: a culture step for culturing cell structures including cancer cells and stromal cells in the presence of one or more anticancer drugs; and an evaluation step for evaluating an anticancer effect of the anticancer drug using, as an index, the number of cells having a cancer cell growth ability in the cell structures after the culture step, wherein a signal transduction pathway in the cancer cells stimulated by a hepatocyte growth factor, a placental growth factor, or a vascular endothelial growth factor exhibits greater activation compared to the signal transduction pathway in normal cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for evaluating the anticancer effect of an anticancer agent with higher reliability in an in vitro system without using an animal model. [Background technology]

[0002] In order to develop anticancer drugs or to select appropriate anticancer drugs for cancer treatment, the effects of anticancer drugs on cancer cells are evaluated using in vitro assay systems. In addition, the drug approval rate at domestic pharmaceutical companies is extremely low at 0.1%, so in order to increase the success rate, it is necessary to quickly determine whether a drug candidate substance is likely to have the desired efficacy, and a reliable drug efficacy evaluation method is required. In particular, the limitations of conventional animal models are said to be one of the reasons why pharmaceutical companies are slow to come up with new drugs, so pharmaceutical companies are looking for drug evaluation models that reproduce the in vivo environment to replace animal models.

[0003] A conventional method for evaluating anticancer drugs in an in vitro system involves administering an anticancer drug to cancer cells cultured on a two-dimensional (flat) surface and examining the effect on the growth of the drug to evaluate its anticancer effect. However, even if an anticancer drug is evaluated as having a high anticancer effect using this evaluation method, it often does not have the expected anticancer effect when actually administered to animals. For this reason, the conventional in vitro evaluation method does not allow the selection of an appropriate anticancer drug for cancer treatment, and there are cases where the results of cancer treatment cannot be improved.

[0004] In an in vitro assay system, it is expected that the anti-cancer effect in the body will be more accurately reflected by using cells in an environment closer to that in the body. For example, Patent Document 1 discloses a method of culturing cancer cells and immune cells in the presence of each other in a collagen gel droplet, and using the obtained spheroids to perform anti-cancer evaluation of drugs. In addition, there is a method of preparing coated cells in which stromal cells are coated alternately with two types of extracellular matrix components, layering and adhering them to each other, and culturing the cell structure constructed by this, and using the cell structure in an in vitro assay system for evaluating drug response (Patent Document 2). In addition, a method has been reported in which a cell mixture of stromal cells suspended in a solution containing at least a cationic buffer, an extracellular matrix component, and a polyelectrolyte is collected on a substrate, and then cultured, and the cell structure constructed by this is used to evaluate anti-cancer drugs (Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2008-11797 A [Patent Document 2] Patent No. 5850419 [Patent Document 3] International Publication No. 2017 / 183673 [Non-patent literature]

[0006] [Non-Patent Document 1] Raghav et al, Translational Lung Cancer Research, 2012, vol.1(3), p.179-193. [Non-Patent Document 2] Owen et al, Cancers, 2019, vol.11, 2002. Summary of the Invention [Problem to be solved by the invention]

[0007] Three-dimensional culture methods such as spheroid culture are said to reproduce the in vivo environment better than conventional two-dimensional culture methods. However, there is not much difference in the sensitivity to drugs, including anticancer drugs, between cell structures obtained by three-dimensional culture and two-dimensional culture cells, and there are many cases where the results are significantly different from those of cancer-bearing animal models.

[0008] An object of the present invention is to provide a method for evaluating the efficacy of an anticancer agent using an assay system that employs cultured cells, which method provides an evaluation closer to that obtained when a cancer-bearing animal model is used. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above-mentioned problems, and have found that the sensitivity of cancer cells to anticancer drugs can be affected by growth factors secreted from interstitial tissues, such as hepatocyte growth factor (HGF), placental growth factor (PIGF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). Therefore, when evaluating the efficacy of cancer cells in which the intracellular signaling pathways downstream of these growth factors are more activated than in normal cells, a three-dimensional cell structure containing the cancer cells and stromal cells can be used to obtain an efficacy evaluation similar to that obtained when using a cancer-bearing animal model, and have completed the present invention.

[0010] The method for evaluating an anticancer agent according to a first aspect of the present invention comprises a culture step of culturing a cell structure containing cancer cells and stromal cells in the presence of one or more types of anticancer agents, and an evaluation step of evaluating the anticancer effect of the anticancer agent using as an index the number of cells having proliferative ability among the cancer cells in the cell structure after the culture step, wherein the signal transduction pathway stimulated by hepatocyte growth factor, placental growth factor, vascular endothelial growth factor, or basic fibroblast growth factor in the cancer cells is more activated than in normal cells. In the first aspect of the present invention, the signal transduction pathway is preferably the ERK / MAPK pathway, the PI3K / AKT pathway, the Jak-STAT pathway, or the WNT / β-Catenin pathway. In the first aspect of the present invention, the cancer cells are preferably one or more types selected from the group consisting of HER2-positive cancer cells, ALK-positive cancer cells, BRAF-positive cancer cells, and ROS1-positive cancer cells. In the first aspect of the present invention, it is preferable that the anticancer agent targets a molecule that constitutes a signal transduction pathway stimulated by hepatocyte growth factor, placental growth factor, or vascular endothelial growth factor, or a molecule that activates the signal transduction pathway. In the first aspect of the present invention, the anticancer agent is preferably a HER2 inhibitor, an ALK inhibitor, a BRAF inhibitor, or a WNT inhibitor. In the first aspect of the present invention, the stromal cells are preferably one or more types selected from the group consisting of fibroblasts, dendritic cells, macrophages, and mast cells. In the first aspect of the present invention, the cell structure preferably has a cancer cell layer on its outer surface. In the first aspect of the present invention, the cell structure may contain, as cells constituting the stroma, one or more types selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells, and fibroblasts, and the total number of vascular endothelial cells and lymphatic endothelial cells in the cell structure may be 0.1% or more of the number of fibroblasts. In the first aspect of the present invention, the cell structure may have a thickness of 5 μm or more. In the first aspect of the present invention, the cell structure may have a vascular network structure. In the first aspect of the present invention, the cancer cells may be cancer cells collected from a cancer patient. In the first aspect of the present invention, the culture time in the culture step may be 24 to 96 hours. In the first aspect of the present invention, the cell structure may be produced by the following steps prior to the culture step: (a) mixing cells, extracellular matrix components, and a polymer electrolyte in a cationic buffer solution to obtain a mixture; (b) seeding the mixture obtained by step (a) in a cell culture vessel; and (c) after step (b), obtaining a cell structure in which cells are stacked in multiple layers in the cell culture vessel. Effect of the Invention

[0011] Although the method for evaluating an anticancer agent according to the present invention is an in vitro evaluation system, it is possible to obtain an efficacy evaluation of an anticancer agent that is close to that obtained when a cancer-bearing animal model is used. [Brief description of the drawings]

[0012] [Figure 1] This is a diagram showing the results of evaluating the sensitivity of non-small cell lung cancer cell line LCC007 to Lapatinib in Example 1. Figure 1(A) shows the results of measuring the relative cell viability (%) of each cell group over time, and Figure 1(B) shows the results of measuring the tumor volume of each mouse group over time. [Diagram 2] 2 shows the results of evaluating the sensitivity of non-small cell lung cancer cell line LCC381 to Lapatinib in Example 1. Fig. 2(A) shows the results of measuring the relative cell viability (%) of each cell group over time, and Fig. 2(B) shows the results of measuring the tumor volume of each mouse group over time. [Diagram 3] FIG. 1 shows the results of measuring the amounts of HGF, VEGF, PIGF, and bFGF in the culture supernatants obtained by 2D culture and 3D culture of NHDFs and HUVECs in Example 2. [Figure 4] These are images of Western blotting performed in Example 3 to examine the activation state of molecules in the signal transduction pathway stimulated by HGF in cells cultured in 2D from non-small cell lung cancer cell line LCC007, cell structures cultured in 3D from LCC007 with NHDF and HUVEC, and tumor tissue in tumor-bearing mice transplanted with LCC007. [Diagram 5] FIG. 5(A) shows the results of evaluating the sensitivity of non-small cell lung cancer cell line LCC028-3 to an ALK inhibitor, and FIG. 5(B) shows the results of evaluating the sensitivity of colon cancer cell line JC215 to a BRAF inhibitor in Example 4. [Figure 6] FIG. 6(A) shows the results of evaluating the sensitivity of colon cancer cell line JC215 to a WNT inhibitor, and FIG. 6(B) shows the results of evaluating the sensitivity of non-small cell lung cancer cell line LCC168 to a WNT inhibitor, in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the present embodiment and the present specification, the term "cell structure" refers to a three-dimensional structure in which multiple cell layers are stacked. The term "cell layer" refers to a layer composed of a group of cells and stroma that exist in a direction perpendicular to the thickness direction and in which the cell nuclei do not overlap in the thickness direction when observed at a magnification at which the cell nuclei can be recognized in a slice image of a cross section of the cell structure in the thickness direction, that is, at a magnification at which the entire thickness of the stained slice is visible. In addition, "layered" means that two or more different cell layers are stacked in the thickness direction.

[0014] In this specification and the present application, the term "cell aggregate" refers to a group of cells. The cell aggregate also includes cell precipitates (cell aggregates formed by precipitating cells) obtained by centrifugation, filtration, and the like. In one embodiment, the cell aggregate is a slurry-like viscous body. In this specification, the term "slurry-like viscous body" refers to a gel-like cell aggregate as described in Akihiro Nishiguchi et al., Macromol Biosci. 2015 Mar;15(3):312-7.

[0015] The method for evaluating an anticancer agent according to the first embodiment of the present invention will be described. The method for evaluating an anticancer agent according to a first embodiment of the present invention (hereinafter sometimes referred to as the "evaluation method according to the present embodiment") comprises a culture step of culturing a cell structure containing cancer cells and cells constituting the stroma (stromal cells) in the presence of one or more anticancer agents, and an evaluation step of evaluating the anticancer effect of the anticancer agent using as an index the number of cells having the ability to proliferate among the cancer cells in the cell structure after the culture step.

[0016] The evaluation method according to the present embodiment is characterized in that the anticancer effect is evaluated using a cell structure (hereinafter, sometimes referred to as the "cell structure according to the present embodiment") containing cancer cells and cells constituting the stroma (stromal cells). The cell structure according to the present embodiment is a cell structure containing cancer cells for evaluating the efficacy of an anticancer drug inside or on the surface of a three-dimensional structure formed from stromal cells. The stroma is an important component in the cancer microenvironment in a living body. In particular, cancer cells in a living body form a microenvironment by interacting with the surrounding stromal cells, and the microenvironment affects the sensitivity to anticancer drugs. In other words, by coexisting cancer cells in a cell structure (layered artificial stromal tissue) having a three-dimensional structure formed by layering stromal cells, the environment of the cancer cells in a living body can be reproduced, and the anticancer effect in a living body can be appropriately evaluated. Therefore, by using the cell structure, it is possible to evaluate an anticancer drug that more closely reflects human clinical results, even in an in vitro evaluation system, and a highly reliable evaluation can be obtained.

[0017] In the cell structure according to the present embodiment, the interstitial cells are formed into a three-dimensional structure like the interstitial tissue in a living body, and therefore various components are secreted, as in the case of the interstitial tissue in a living body. The drug sensitivity of the cancer cells is affected by these secretions, but how they are affected depends on the type of cancer cells and drug. The interstitial cells in the cell structure according to the present embodiment secrete HGF, PIGF, VEGF, and bFGF, and therefore the cancer cells in the cell structure are under the influence of these growth factors, as are the cancer cells in the interstitial tissue in a living body. Therefore, the drug sensitivity of the cancer cells in the cell structure according to the present embodiment becomes closer to that of the cancer cells in a living body, and the evaluation method according to the present embodiment can provide a drug efficacy evaluation close to that obtained when a cancer-bearing animal model is used.

[0018] The evaluation method according to the present embodiment is preferably applied to cancer cells in which the signal transduction pathway stimulated by HGF, PIGF, VEGF, or bFGF is more activated than in normal cells. That is, the cancer cells contained in the cell structure according to the present embodiment are preferably cancer cells in which the signal transduction pathway stimulated by HGF, PIGF, VEGF, or bFGF is more activated than in normal cells. In cancer cells in which the signal transduction pathway stimulated by these growth factors is activated, the sensitivity to anticancer drugs is significantly different in the presence and absence of these growth factors. For example, in cancer cells in which activation of the signal transduction pathway causes resistance (decreased sensitivity) to anticancer drugs, the sensitivity to anticancer drugs is reduced in the presence of a growth factor that stimulates the signal transduction pathway compared to the absence of the growth factor. Conversely, in cancer cells in which activation of the signal transduction pathway causes sensitivity (enhanced sensitivity) to anticancer drugs, the sensitivity to anticancer drugs is increased in the presence of a growth factor that stimulates the signal transduction pathway compared to the absence of the growth factor. For this reason, in an in vitro assay system in which these growth factors are insufficient, the deviation from the evaluation using an animal model is very large. By using the cell structure according to this embodiment, it is possible to obtain a more reliable drug efficacy evaluation for such cancer cells.

[0019] The signal transduction pathway stimulated by HGF, PIGF, VEGF, or bFGF is not particularly limited. In addition, when there are two or more signal transduction pathways stimulated by one type of growth factor, at least one of them may be activated. For example, HGF is a ligand of hepatocyte growth factor receptor (MET), and MET activated by binding with HGF activates the ERK / MAPK pathway, PI3K / AKT pathway, and WNT / β-Catenin pathway in cells (Non-Patent Document 1). In addition, VEGF is a ligand of vascular endothelial growth factor receptor (VEGFR), and VEGFR activated by binding with VEGF activates the Jak-STAT pathway in cells (Non-Patent Document 2).

[0020] Whether or not a signal transduction pathway stimulated by HGF, PIGF, VEGF, or bFGF is activated can be determined, for example, by using as an index the amount of activated molecules (e.g., phosphorylated molecules, molecules that have undergone genetic mutations, etc.) present in the cells for any of the molecules that constitute each signal transduction pathway.

[0021] Among the molecules constituting the signal transduction pathway, examples of molecules activated by phosphorylation include ERK, MAPK, etc. in the ERK / MAPK pathway, Akt, S6, etc. in the PI3K / AKT pathway, and Jak, STAT, etc. in the Jak-STAT pathway.

[0022] Among the molecules that constitute the signal transduction pathway, when the phosphorylated molecule pA of molecule A that is activated by phosphorylation is not detected in normal cells, but the phosphorylated molecule pA is detected in cancer cells, the cancer cells can be said to be cancer cells in which the signal transduction pathway is activated. In addition, when [amount of phosphorylated molecule pA in cancer cells (relative value)] / [amount of phosphorylated molecule pA in normal cells (relative value)] is 1.1 or more, preferably 1.2 or more, more preferably 1.5 or more, the cancer cells can be said to be cancer cells in which the signal transduction pathway is activated. The amount of phosphorylated molecule can be measured by a known method such as Western blotting using an antibody that specifically recognizes the molecule.

[0023] Among the molecules constituting the signal transduction pathway, examples of molecules that are constitutively activated by gene mutation include, for example, Ras mutation, Raf mutation, etc. in the ERK / MAPK pathway; Akt mutation, PI3K mutation, PTEN mutation, etc. in the PI3K / AKT pathway; β-Catenin and Wnt, etc. in the WNT / β-Catenin pathway; and Jak and STAT, etc. in the Jak-STAT pathway.

[0024] Specific examples of cancer cells in which the signal transduction pathway stimulated by HGF, PIGF, VEGF, or bFGF is activated include HER2-positive cancer cells (cancer cells in which the expression level of HER2 is increased due to genetic abnormality of the HER2 gene), ALK-positive cancer cells (cancer cells having an ALK fusion gene), BRAF-positive cancer cells (cancer cells having a genetic mutation of the BRAF gene), and ROS1-positive cancer cells (cancer cells having a ROS1 fusion gene). In the evaluation method according to this embodiment, it is preferable to evaluate the sensitivity of these cancer cells to one or more types of anticancer drugs.

[0025] <Cell structure> The cell structure used in this embodiment (the cell structure according to this embodiment) is constructed by cancer cells and stromal cells. The stromal cells contained in the cell structure according to this embodiment may be of one type or of two or more types. The cancer cells contained in the cell structure according to this embodiment may be of one type or of two or more types. Note that cancer cells are cells that are derived from somatic cells and have acquired the ability to proliferate indefinitely.

[0026] The cells including stromal cells and cancer cells constituting the cell structure according to the present embodiment are not particularly limited, and may be cells collected from an animal, cells obtained by culturing cells collected from an animal, cells obtained by subjecting cells collected from an animal to various treatments, or cultured cell lines. In the case of cells collected from an animal, the site of collection is not particularly limited, and the cells may be somatic cells derived from bone, muscle, internal organs, nerves, brain, bone, skin, blood, etc., germ cells, or embryonic stem cells (ES cells). In addition, the species from which the cells constituting the cell structure according to the present embodiment are derived is not particularly limited, and cells derived from animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats can be used. The cells cultured from cells collected from animals may be primary culture cells or subculture cells. In addition, examples of cells subjected to various treatments include induced pluripotent stem cells (iPS cells) and cells after differentiation induction. In addition, the cell structure according to the present embodiment may be composed of only cells derived from the same species of organism, or may be composed of cells derived from multiple species of organisms.

[0027] Examples of interstitial cells include endothelial cells, fibroblasts, nerve cells, mast cells, epithelial cells, cardiomyocytes, hepatocytes, pancreatic islet cells, tissue stem cells, smooth muscle cells, etc. The interstitial cells contained in the cell structure according to this embodiment may be of one type or of two or more types. The type of interstitial cells contained in the cell structure according to this embodiment is not particularly limited, and can be appropriately selected in consideration of the origin and type of cancer cells to be contained, the type of anticancer agent to be used for evaluation, the in vivo environment in which the desired anticancer activity is exerted, etc.

[0028] A vascular network structure and a lymphatic network structure are important for the proliferation and activity of cancer cells. For this reason, the cell structure according to this embodiment is preferably one having a vascular network structure. That is, the cell structure according to this embodiment is preferably one in which a vascular network structure such as lymphatic vessels and / or blood vessels is three-dimensionally constructed inside a laminate of cells that does not form vessels, constructing a tissue closer to that in a living body. The vascular network structure may be formed only inside the cell structure, or may be formed so that at least a part of the vascular network structure is exposed on the surface or bottom surface of the cell structure. In this embodiment and this specification, the term "vascular network structure" refers to a net-like structure such as a vascular network or lymphatic network in a living tissue.

[0029] The vascular network structure can be formed by including endothelial cells that constitute blood vessels as interstitial cells. The endothelial cells included in the cell structure according to this embodiment may be vascular endothelial cells or lymphatic endothelial cells. In addition, the cell structure may include both vascular endothelial cells and lymphatic endothelial cells.

[0030] When the cell structure according to this embodiment has a vascular network structure, the cells other than endothelial cells in the cell structure are preferably cells that constitute the tissue surrounding blood vessels in a living body, since endothelial cells are likely to form a vascular network that retains its original function and shape, and since the cells are more likely to resemble the cancer microenvironment in a living body, cells containing at least fibroblasts are more preferable as the cells other than endothelial cells, and cells containing vascular endothelial cells and fibroblasts, cells containing lymphatic endothelial cells and fibroblasts, or cells containing vascular endothelial cells, lymphatic endothelial cells and fibroblasts are even more preferable. Note that the cells other than endothelial cells contained in the cell structure may be cells derived from the same biological species as the endothelial cells, or may be cells derived from a different biological species.

[0031] The number of endothelial cells in the cell structure according to the present embodiment is not particularly limited as long as it is a sufficient number for forming a vascular network structure, and can be appropriately determined in consideration of the size of the cell structure, the cell types of endothelial cells and cells other than endothelial cells, and the like. For example, a cell structure in which a vascular network structure is formed can be prepared by setting the abundance ratio (cell number ratio) of endothelial cells to the total cells constituting the cell structure according to the present embodiment to 0.1% or more. When fibroblasts are used as cells other than endothelial cells, the number of endothelial cells in the cell structure according to the present embodiment is preferably 0.1% or more of the number of fibroblasts, and more preferably 0.1 to 5.0%. When both vascular endothelial cells and lymphatic endothelial cells are included as endothelial cells, the total number of vascular endothelial cells and lymphatic endothelial cells is preferably 0.1% or more of the number of fibroblasts, and more preferably 0.1 to 5.0%.

[0032] The number of cancer cells in the cell structure according to this embodiment is not particularly limited, but in order to more closely resemble the cancer microenvironment in a living body, it is preferable that the ratio of the number of endothelial cells to the number of cancer cells in the cell structure ([number of endothelial cells] / [number of cancer cells]) is more than 0 (greater than 0) and 1.5 or less. Furthermore, when using a cell structure containing endothelial cells, fibroblasts, and cancer cells, the ratio of the number of fibroblasts to the number of cancer cells in the cell structure ([number of fibroblasts] / [number of cancer cells]) is preferably 0.6 to 100, and more preferably 50 to 100.

[0033] The total number of cells constituting the cell structure according to the present invention is not particularly limited, but it is preferable that the number of cells results in a theoretical number of cell layers of 15 to 25 layers, because it is easy to form a structure in which a layer of vascular endothelial cells contains other cell populations, and because it is easy to observe the formed cell structure. The theoretical number of cell layers is represented by the following formula. In the formula, "N A ” is the number of all cells that make up the cell structure, “N S " represents the number of cells per layer when a cell structure is constructed using the same type of cells on the same type of substrate, and "L" represents the theoretical number of cell layers.

[0034] [L]=[NA ] / [N S ]

[0035] N S can be experimentally determined. For example, a 96-well plate is used as the substrate, and 6 NHDF and 0.0135 × 10 6 When a cell suspension was prepared using 10 HUVECs, the constructed cell structure had about 20 nuclei stacked vertically. Therefore, in the case of a cell structure constructed using NHDFs and HUVECs on a 96-well plate, N A is 0.45 × 10 5 1 piece.

[0036] The cancer cells contained in the cell structure according to the present embodiment may be cultured cells established or may be cancer cells collected from a cancer patient. The cancer cells collected from a cancer patient may be cells that have been cultured and grown in advance. Specifically, primary cancer cells collected from a cancer patient, artificially cultured cancer cells, iPS cancer stem cells, cancer stem cells, and established cancer cells prepared in advance for use in research on cancer treatment and development of anticancer drugs may be mentioned. The cancer cells may be human-derived or may be cancer cells derived from animals other than humans. In addition, when the cell structure according to the present embodiment contains cancer cells collected from a cancer patient, it may also contain cells other than the cancer cells collected from the cancer patient together with the cancer cells. Examples of cells other than the cancer cells include one or more types of cells contained in solid tissue removed after surgery.

[0037] Examples of cancers from which the cancer cells contained in the cell structure according to this embodiment are derived include breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer, etc.), prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (e.g., pancreatic ductal carcinoma, etc.), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.), colon cancer (e.g., gastrointestinal stromal tumor, etc.), rectal cancer (e.g., gastrointestinal stromal tumors, etc.), colon cancer (e.g., familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumors, etc.), small intestine cancer (e.g., non-Hodgkin's lymphoma, gastrointestinal stromal tumors, etc.), esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer (e.g., nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, etc.), head and neck cancer, salivary gland cancer, brain tumors (e.g., pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, etc.), neurilemmoma, liver cancer (e.g., primary liver cancer, extrahepatic bile duct cancer, etc.), kidney Cancer of the liver (e.g., renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, etc.), gallbladder cancer, bile duct cancer, pancreatic cancer, liver cancer, endometrial cancer, cervical cancer, ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low malignant potential tumor, etc.), bladder cancer, urethral cancer, skin cancer (e.g., intraocular (eye) melanoma, Merkel cell carcinoma, etc.), hemangioma, malignant lymphoma (e.g., reticulum cell sarcoma, lymphosarcoma, Hodgkin's disease, etc.), melanoma, thyroid cancer (e.g., medullary thyroid cancer, etc.), and parathyroid Examples of cancers that may be present include, but are not limited to, cancer of the nasal cavity, cancer of the paranasal sinuses, bone tumors (e.g., osteosarcoma, Ewing's tumor, uterine sarcoma, soft tissue sarcoma, etc.), metastatic medulloblastoma, angiofibroma, dermatofibrosarcoma protuberans, retinal sarcoma, penile cancer, testicular tumor, pediatric solid tumors (e.g., Wilms' tumor, pediatric renal tumor, etc.), Kaposi's sarcoma, Kaposi's sarcoma caused by AIDS, maxillary sinus tumor, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, chronic myeloproliferative disorder, leukemia (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, etc.), and the like.

[0038] The cell structure according to this embodiment may be a cell structure in which cancer cells are dispersed throughout the structure, or a cell structure in which cancer cells are present only in a specific cell layer, or a cell structure in which an interstitial cell layer and a cancer cell layer are separated by a semipermeable membrane.

[0039] In the cell structure according to the present embodiment, when cancer cells are present only in a specific cell layer, the position of the cell layer (cancer cell layer) containing the cancer cells in the cell structure is not particularly limited. Since the effects of immune cells and / or anticancer drugs can reach the cell structure sufficiently, the position of the cancer cell layer in the thickness direction in the cell structure is preferably within a range from the top surface (upper surface) of the cell structure to half the height in the thickness direction. In particular, when the cell structure is cultured in the presence of immune cells, the anticancer effect can be evaluated including the ability of immune cells to infiltrate and reach the cancer cells in the cell structure by providing the cancer cell layer not on the top surface of the cell structure but inside the cell structure. In this specification, the thickness of the cell structure is the length in the direction of the tissue's own weight. The direction of gravity is the direction in which gravity is applied, and is also called the thickness direction.

[0040] The cell structure according to this embodiment may contain cells other than cancer cells and interstitial cells, such as immune cells, nerve cells, hepatic cells, pancreatic cells, cardiac muscle cells, smooth muscle cells, bone cells, alveolar epithelial cells, and spleen cells.

[0041] The size and shape of the cell structure according to this embodiment are not particularly limited. Since a vascular network structure closer to the blood vessels formed in tissues in vivo can be formed and more accurate evaluation is possible, the thickness of the cell structure is preferably 5 μm or more, more preferably 10 μm or more, even more preferably 50 μm or more, and even more preferably 100 μm or more. The thickness of the cell structure is also preferably 500 μm or less, more preferably 400 μm or less, and even more preferably 300 μm or less. The number of cell layers in the cell structure according to this embodiment is preferably about 2 to 60 layers, more preferably about 5 to 60 layers, and even more preferably about 10 to 60 layers.

[0042] The number of cell layers constituting a cell structure is measured by dividing the total number of cells constituting the three-dimensional structure by the number of cells per layer (the number of cells required to constitute one layer). The number of cells per layer can be examined by culturing cells confluently in advance in a planar manner in a cell culture vessel used to form the cell structure. Specifically, the number of cell layers of a cell structure formed in a certain cell culture vessel can be calculated by counting the total number of cells constituting the cell structure and dividing it by the number of cells per layer of the cell culture vessel.

[0043] Generally, the cell structure according to this embodiment is constructed in a cell culture vessel. The cell culture vessel is not particularly limited as long as it is a vessel in which a cell structure can be constructed and the constructed cell structure can be cultured. Specific examples of the cell culture vessel include dishes, cell culture inserts (e.g., Transwell (registered trademark) inserts, Netwell (registered trademark) inserts, Falcon (registered trademark) cell culture inserts, Millicell (registered trademark) cell culture inserts, etc.), tubes, flasks, bottles, plates, etc. In constructing the cell structure according to this embodiment, dishes or various cell culture inserts are preferred because evaluation using the cell structure can be more appropriately performed.

[0044] The cell structure according to the present embodiment may be a structure formed from multiple cell layers including cancer cells and stromal cells, and the method of constructing the cell structure is not particularly limited. For example, the cell structure may be constructed by sequentially stacking one layer at a time, or may be constructed by simultaneously stacking two or more cell layers, or may be constructed by appropriately combining both construction methods to construct multiple cell layers. The cell structure according to the present embodiment may be a multi-layer structure in which the cell types constituting each cell layer are different for each layer, or may be constructed by a common cell type for all layers of the structure. For example, the cell structure may be constructed by forming a layer for each cell type and sequentially stacking the cell layers for each cell type, or may be constructed by previously preparing a cell mixture containing multiple types of cells and simultaneously constructing a multi-layered cell structure from the previously prepared cell mixture containing multiple types of cells.

[0045] As a method of constructing layers one by one and stacking them in sequence, for example, there is a method described in Japanese Patent No. 4919464, that is, a method of stacking cell layers continuously by alternately repeating a step of forming a cell layer and a step of contacting the formed cell layer with a solution containing ECM (extracellular matrix) components. For example, when carrying out this method, a cell mixture in which all cells constituting the cell structure are mixed is prepared in advance, and each cell layer is formed with this cell mixture, thereby constructing a cell structure in which a vascular network structure is formed throughout the structure and cancer cells are scattered throughout the structure. In addition, by forming each cell layer for each cell type, a cell structure in which a vascular network structure is formed only in the layer formed from endothelial cells and cancer cells are present only in a specific cell layer can be constructed.

[0046] An example of a method for constructing two or more cell layers at once is the method described in Japanese Patent No. 5850419. In this method, the entire surface of a cell is coated in advance with a polymer containing an arginine-glycine-aspartic acid (RGD) sequence to which integrins bind and a polymer that interacts with the polymer containing the RGD sequence, and the coated cells coated with this adhesive film are placed in a cell culture vessel, and then the coated cells are accumulated by centrifugation or the like to construct a cell structure formed from multiple cell layers. For example, when carrying out this method, a cell mixture in which all the cells constituting the cell structure are mixed is prepared in advance, and coated cells prepared by adding an adhesive component to this cell mixture are used. This allows a cell structure in which cancer cells are scattered throughout the structure to be constructed by a single centrifugation process. Also, for example, coated cells coating endothelial cells, coated cells coating fibroblasts, and coated cells coating a group of cells collected from a cancer patient are each prepared separately, and after forming a multilayer composed of coated cells of fibroblasts, a layer formed of coated cells of endothelial cells is laminated on top of that, a multilayer formed of coated cells of fibroblasts is laminated on top of that, and a layer formed of coated cells of cells containing cancer cells is laminated on top of that. This allows the construction of a cell structure that has a vascular network structure sandwiched between thick layers of fibroblasts and has a layer containing cancer cells collected from a cancer patient on the top surface.

[0047] The cell structure according to this embodiment can also be constructed by a method including the following steps (a) to (c). (a) mixing cells and extracellular matrix components in a cationic buffer to obtain a mixture; (b) seeding the mixture obtained by step (a) into a cell culture vessel; and (c) after step (b), obtaining a cell structure in which cells are stacked in multiple layers in the cell culture vessel.

[0048] In step (a), cells are mixed with a buffer solution containing a cationic substance (cationic buffer solution) and an extracellular matrix component, and a cell aggregate is formed from this cell mixture, thereby obtaining a three-dimensional cell tissue with few large internal voids. Furthermore, since the obtained three-dimensional cell tissue is relatively stable, it can be cultured for at least several days, and the tissue is unlikely to collapse even when the medium is replaced. Furthermore, in this embodiment, step (b) may include allowing the cell mixture seeded in the cell culture vessel to settle in the cell culture vessel. The cell mixture may be sedimented actively by centrifugation or the like, or may be allowed to settle naturally.

[0049] The cationic substance used in this embodiment may be any positively charged substance as long as it does not adversely affect cell growth and cell aggregate formation. Examples of the cationic substance include cationic buffers such as Tris-HCl buffer, Tris-maleic acid buffer, Bis-Tris buffer, and HEPES, as well as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, and polyarginine.

[0050] The concentration and pH of the cationic substance in the cationic buffer (e.g., Tris in Tris-HCl buffer) are not particularly limited as long as they do not adversely affect cell growth and cell structure construction. For example, the concentration of the cationic substance in the cationic buffer can be 10 to 100 mM, preferably 40 to 70 mM, and more preferably 50 mM. The pH of the cationic buffer can be 6.0 to 8.0, preferably 6.8 to 7.8, and more preferably 7.2 to 7.6.

[0051] In step (a), it is preferable to further mix the cells with a polyelectrolyte. By mixing the cells with a cationic substance, a polyelectrolyte, and an extracellular matrix component, a thick, three-dimensional cell tissue with few voids can be obtained even when the cells are allowed to settle naturally without the need for a process for actively aggregating the cells, such as centrifugation, in step (b).

[0052] In the present invention and the present specification, the term "polymer electrolyte" refers to a polymer having a dissociable functional group in the polymer chain. As the polymer electrolyte used in this embodiment, any polymer electrolyte can be used as long as it does not adversely affect the growth of cells and the formation of a cell structure. Examples of the polymer electrolyte include glycosaminoglycans such as heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid; dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, and polyacrylic acid, or derivatives thereof, but are not limited thereto. The mixture prepared in step (a) may contain only one type of polymer electrolyte, or may contain a combination of two or more types. In the construction of the cell structure according to this embodiment, the polymer electrolyte is preferably a glycosaminoglycan. It is more preferable to use at least one of heparin, dextran sulfate, chondroitin sulfate, and dermatan sulfate.The polymer electrolyte used in this embodiment is more preferably heparin.

[0053] The amount of the polyelectrolyte mixed in the cationic buffer is not particularly limited as long as it does not adversely affect cell growth and construction of a cell structure. For example, the concentration of the polyelectrolyte in the cationic buffer can be more than 0 mg / mL (higher than 0 mg / mL) and less than 1.0 mg / mL, preferably 0.025 to 0.1 mg / mL, and more preferably 0.05 to 0.1 mg / mL. In the present embodiment, the mixture can be prepared without mixing the polyelectrolyte, and construction of a cell structure can also be performed.

[0054] The extracellular matrix component used in this embodiment may be any component constituting the extracellular matrix (ECM) as long as it does not adversely affect cell growth and cell aggregate formation. Examples of the extracellular matrix component include collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan, glycosaminoglycan, or modified or variant thereof. Examples of the proteoglycan include chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan. Examples of the glycosaminoglycan include hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin. The mixture prepared in step (a) may contain only one type of extracellular matrix component, or may contain a combination of two or more types. In the production of the cell structure according to the present invention, it is preferable to use one or more selected from the group consisting of collagen, laminin, and fibronectin, and among these, collagen is preferable. Modified and variant forms of the above-mentioned extracellular matrix components may also be used as long as they do not adversely affect cell growth and cell structure formation.

[0055] The amount of the extracellular matrix component mixed in the cationic buffer is not particularly limited as long as it does not adversely affect cell growth and the production of cell structures. For example, the concentration of the extracellular matrix component in the cationic buffer may be more than 0 mg / mL, preferably 0.010 mg / mL or more, more preferably 0.020 mg / mL or more, even more preferably 0.025 mg / mL or more, and even more preferably 0.05 mg / mL or more. The concentration of the extracellular matrix component in the cationic buffer is preferably less than 1.0 mg / mL, more preferably 0.75 mg / mL or less, even more preferably 0.5 mg / mL or less, even more preferably 0.25 mg / mL or less, and particularly preferably 0.1 mg / mL or less.

[0056] The blending ratio of the polymer electrolyte to the extracellular matrix component mixed in the cationic buffer is 1:2 to 2:1. In the production of the cell structure according to the present invention, the blending ratio of the polymer electrolyte to the extracellular matrix component is preferably 1:1.5 to 1.5:1, and more preferably 1:1.

[0057] A cell structure of sufficient thickness can be constructed by repeating steps (a) to (c), specifically, by seeding the mixture prepared in step (a) as step (b) on the cell structure obtained in step (c), and then repeating step (c). The cell composition of the mixture newly seeded on the cell structure obtained in step (c) may be the same as or different from the cell composition constituting the already constructed cell structure.

[0058] For example, first, in step (a), a mixture containing only fibroblasts as cells is prepared, and steps (b) and (c) are performed to obtain a cell structure formed of 10 fibroblast layers in a cell culture vessel. Next, in step (a), a mixture containing only vascular endothelial cells as cells is prepared, and steps (b) and (c) are performed to laminate one vascular endothelial cell layer on the fibroblast layer in the cell culture vessel. Furthermore, in step (a), a mixture containing only fibroblasts as cells is prepared, and steps (b) and (c) are performed to laminate 10 fibroblast layers on the vascular endothelial cell layer in the cell culture vessel. Furthermore, in step (a), a mixture containing cancer cells collected from a cancer patient is prepared, and steps (b) and (c) are performed to laminate one cancer cell layer on the fibroblast layer in the cell culture vessel. This allows the construction of a cell structure in which layers are laminated in order for each cell type, such as 10 fibroblast layers - 1 vascular endothelial cell layer - 10 fibroblast layers - 1 cancer cell layer. By adjusting the number of cells seeded in step (b), the thickness of the cell layer laminated in step (c) can be adjusted. The more the number of cells seeded in step (b), the more the number of cell layers laminated in step (c). In addition, in step (a), a mixture of fibroblasts for 20 fibroblast layers and vascular endothelial cells for one vascular endothelial cell layer is prepared, and steps (b) and (c) are performed. By laminating a mixture containing cancer cells collected from a cancer patient and prepared in the same manner on the formed multi-layered structure, a cell structure having a thickness of 21 layers and having a vascular network structure scattered inside the structure and having cancer cell layers laminated on the structure can be constructed. Furthermore, in step (a), a mixture of fibroblasts equivalent to 20 fibroblast layers, vascular endothelial cells equivalent to one vascular endothelial cell layer, and cells derived from a cancer patient equivalent to one cancer cell layer is prepared, and steps (b) and (c) are carried out to construct a cell structure having a thickness of 22 layers, in which both cancer cells and vascular network structures are independently scattered within the structure.

[0059] When steps (a) to (c) are repeated, the obtained cell structure may be cultured after step (c) and before step (b). Culture conditions such as the composition of the culture medium used for culture, culture temperature, culture time, and atmospheric composition during culture are suitable for culturing the cells that constitute the cell structure. Examples of culture media include D-MEM, E-MEM, MEMα, RPMI-1640, and Ham's F-12.

[0060] After step (a), (a'-1) a step of removing the liquid portion from the obtained mixture to obtain a cell aggregate, and (a'-2) a step of suspending the cell aggregate in a solution may be performed, and then the process may proceed to step (b). Although the desired tissue can be obtained by performing the above-mentioned steps (a) to (c), a more homogeneous tissue can be obtained by performing (a'-1) and (a'-2) after step (a) and then performing step (b).

[0061] As a means for removing the liquid portion in step (a'-1), a method known to those skilled in the art can be used. For example, the liquid portion may be removed by centrifugation or filtration. The conditions of centrifugation are not particularly limited as long as they do not adversely affect cell growth and cell aggregate formation. For example, the microtube containing the mixture is centrifuged at room temperature and 400 x g for 1 minute to separate the liquid portion from the cell aggregates, thereby removing the liquid portion. Alternatively, the cells may be collected by natural sedimentation, and then the liquid portion may be removed.

[0062] The solution used in step (a'-2) is not particularly limited as long as it does not adversely affect cell growth and cell aggregate formation. For example, a cell culture medium or buffer suitable for the cells used may be used.

[0063] Moreover, after step (a), the following steps (b'-1) and (b'-2) may be performed instead of step (b). A more homogeneous tissue can also be obtained by performing steps (b'-1) and (b'-2). Like step (b), step (b'-2) may also include settling the cell mixture seeded in the cell culture vessel in the cell culture vessel. The settling of the cell mixture may be performed by actively settling the cells by centrifugation or the like, or by allowing them to naturally settle. In this embodiment and this specification, the term "cell viscous body" refers to a gel-like cell aggregate as described in Non-Patent Document 4. (b'-1) seeding the mixture obtained in step (a) into a cell culture vessel, and then removing liquid components from the mixture to obtain a viscous cell mass; (b'-2) A process of suspending the cell viscous body in a solvent in a cell culture vessel.

[0064] The solvent for preparing the cell suspension is not particularly limited as long as it is non-toxic to cells and does not impair proliferation or function, and water, a buffer solution, a cell culture medium, etc. can be used. Examples of the buffer solution include physiological saline phosphate (PBS), HEPES, Hanks buffer, etc. Examples of the culture medium include D-MEM, E-MEM, MEMα, RPMI-1640, Ham's F-12, etc. When a cell culture medium is used as the solvent for preparing the cell suspension, the cells can be cultured without removing the liquid components in the step (c) described below.

[0065] Instead of the above step (c), the following step (c') may be carried out. (c'): A process for forming a layer of cells on a substrate.

[0066] In steps (c) and (c'), liquid components may be removed from the seeded mixture. The method of removing liquid components in steps (c) and (c') is not particularly limited as long as it does not adversely affect cell growth and cell structure construction, and can be appropriately performed by a method known to those skilled in the art as a method for removing liquid components from a suspension of liquid components and solid components. Examples of such methods include aspiration, centrifugation, magnetic separation, and filtration. For example, when a cell culture insert is used as a cell culture vessel, the cell culture insert seeded with the mixture is centrifuged at 10°C and 400 x g for 1 minute, whereby the cell mixture settles, and the liquid components can be removed by aspiration.

[0067] The culture medium used for the culture in step (c) is not particularly limited as long as it is a medium in which the cells that constitute the three-dimensional construct can grow, but a medium with a low content or no content of growth factors such as epidermal growth factor (EGF), VEGF, fibroblast growth factor (FGF), insulin-like growth factor (IGF), etc., or a medium with a low content or no content of serum is preferred. When the content of various growth factors, etc. is high, angiogenesis may be promoted. In this embodiment, a medium without added growth factors is preferred, and a serum-free medium is more preferred.

[0068] <Anti-cancer drugs> In the evaluation method according to this embodiment, the anticancer drug whose efficacy is to be evaluated may be any drug used in cancer treatment, and includes not only drugs that act directly on cancer cells, such as cytotoxic drugs, but also drugs that are not cytotoxic but suppress the proliferation, etc. of cancer cells.

[0069] Examples of anticancer drugs without cytotoxicity include drugs that do not directly attack cancer cells, but rather act in cooperation with immune cells or other drugs in the body to suppress the proliferation of cancer cells, slow down the activity of cancer cells, or kill cancer cells, and drugs that suppress the proliferation of cancer cells by damaging cells or tissues other than cancer cells. The anticancer drug used in this embodiment may be a drug known to have an anticancer effect, or may be a candidate compound for a novel anticancer drug.

[0070] Examples of cytotoxic anticancer agents include, but are not limited to, molecular targeted drugs, alkylating agents, metabolic antagonists such as 5-FU anticancer agents, plant alkaloids, anticancer antibiotics, platinum derivatives, hormones, topoisomerase inhibitors, microtubule inhibitors, and compounds classified as biological response modifiers.

[0071] Examples of anticancer drugs that do not have cytotoxicity include, but are not limited to, angiogenesis inhibitors, prodrugs of anticancer drugs, drugs that adjust intracellular metabolic enzyme activity related to the metabolism of anticancer drugs or their prodrugs (hereinafter referred to as "intracellular enzyme regulators" in the specification), and immunotherapeutic drugs (drugs that obtain anticancer effects by improving immune function through activation of immune function or motility of immune cells, etc.). Other examples include drugs that ultimately participate in anticancer action by enhancing the function of anticancer drugs or improving immune function in the body. Note that prodrugs of anticancer drugs are drugs that are converted into active forms having anticancer action by intracellular enzymes in organs such as the liver or cancer cells. The cytokine network increases the enzymatic activity of intracellular enzymes, thereby increasing the amount of active form and enhancing the antitumor effect, and therefore, they are listed as drugs that participate in anticancer action.

[0072] The anticancer drug to be evaluated in the evaluation method according to the present embodiment is preferably an anticancer drug that targets a molecule that constitutes a signal transduction pathway stimulated by HGF, PIGF, VEGF, or bFGF or a molecule that activates the signal transduction pathway, since the effect of the present invention, that is, an evaluation closer to that obtained when a cancer-bearing animal model is used, can be more fully demonstrated. Examples of such anticancer drugs include HER2 inhibitors, ALK inhibitors, BRAF inhibitors, and WNT inhibitors.

[0073] The anticancer drug whose efficacy is evaluated in the evaluation method according to the present embodiment may be one type, or may be a combination of two or more types of anticancer drugs. In addition, the anticancer drug may be combined with a drug other than the anticancer drug. For example, in actual clinical practice, the evaluation method according to the present embodiment is carried out for an anticancer drug that is administered in combination with other drugs, and the drug efficacy expected in actual treatment can be evaluated with higher reliability and accuracy, which is preferable.

[0074] <Culture process> In the evaluation method according to the present embodiment, first, in the culture step, the cell structure according to the present embodiment is cultured in the presence of an anticancer drug to be evaluated for efficacy. Specifically, the cell structure according to the present embodiment is cultured in a culture medium mixed with an anticancer drug. The anticancer drug may be added to the medium in which the cell structure is cultured at the same time as the start of culture, or may be added at an appropriate time after the start of culture. Furthermore, when evaluating two or more types of drugs (two or more types of anticancer drugs, or a combination of one or more types of anticancer drugs and one or more types of non-anticancer drugs), the two or more types of drugs may be added simultaneously or independently to the culture medium for the cell structure.

[0075] The amount of anticancer drug to be mixed into the culture medium can be determined experimentally taking into consideration conditions such as the type and number of cells constituting the cell structure, the type and amount of cancer cells contained therein, the type of culture medium, the culture temperature, and the culture time. For example, the culture time is not particularly limited and can be 24 to 96 hours, preferably 48 to 96 hours, and more preferably 48 to 72 hours. In addition, hydrodynamic stress such as reflux can be applied as necessary to the extent that the culture environment is not significantly changed.

[0076] <Evaluation process> The number of cells having the ability to proliferate among the cancer cells in the cell structure after the culture step is used as an index to evaluate the anticancer effect of the anticancer agent. The anticancer effect means the effect of suppressing the proliferation of cancer cells or killing cancer cells.

[0077] Specifically, when the number of cells having the ability to proliferate among the cancer cells in the cell structure is smaller than that when cultured in an environment without the anticancer drug, the anticancer drug used is evaluated as having an anticancer effect on the cancer cells contained in the cell structure, whereas when the number of cells having the ability to proliferate among the cancer cells is the same or significantly larger than that when cultured in an environment without the anticancer drug, the anticancer drug is evaluated as not having an anticancer effect on the cancer cells contained in the cell structure.

[0078] The number of cells having the ability to proliferate in cancer cells can be evaluated using a signal that is correlated with the number of cancer cells having the ability to proliferate or the amount of cancer cells having the ability to proliferate. It is sufficient to be able to measure the number of cancer cells having the ability to proliferate at the time of evaluation, and it is not necessary to measure them in a living state. For example, cancer cells can be labeled to distinguish them from other cells, and the signal from the label can be used as an index for examination. For example, after fluorescently labeling cancer cells, the live / dead state of the cells can be determined, and the number of cells determined to be live cells can be directly counted as the cancer cells having the ability to proliferate in the cell structure. In this case, image analysis technology can also be used. The live / dead state of cells can be determined by a known method for determining the live / dead state of cells, such as trypan blue staining or PI (propidium iodide) staining. Note that the fluorescent labeling of cancer cells can be performed by a known method, such as an immunostaining method, in which an antibody against a substance specifically expressed on the cell surface of cancer cells is used as a primary antibody, and a fluorescently labeled secondary antibody that specifically binds to the primary antibody is used. The live / dead state of cells and the measurement of the number of live cells may be performed in the state of the cell structure, or may be performed in a state in which the cell structure is destroyed to the single cell level. For example, after labeling cancer cells and dead cells and destroying the three-dimensional structure of the cell structure, it is possible to directly count only the cancer cells that were alive at the time of evaluation using FACS (fluorescence activated cell sorting) using the label as an indicator.

[0079] The number of cancer cells in the cell structure having the ability to proliferate can be measured over time by labeling the cancer cells in the cell structure while they are still alive and detecting the signal from the label over time. The cancer cells in the cell structure may be labeled after the cell structure is constructed, or the cancer cells may be labeled in advance before the cell structure is constructed. For example, when a cell structure containing a cell group including cancer cells derived from a cancer patient is used, the cancer cells can be labeled in advance before the cell structure is constructed. In addition, other cells derived from the cancer patient may be similarly labeled together with the cancer cells. In addition, when cancer cells that constitutively express a fluorescent dye are used, the number of live cancer cells can also be evaluated by measuring the fluorescence intensity of the lysate obtained by dissolving the cell structure using a microplate reader or the like.

[0080] The evaluation method according to the present embodiment uses a cell structure comprising stromal cells secreting growth factors such as HGF, similar to the structure of tissues surrounding cancer cells in an actual living body, and performs evaluation in an in vitro environment that is closer to in vivo, so that a highly reliable evaluation of drug efficacy can be obtained. An anticancer drug evaluated to have an anticancer effect by the evaluation method according to the present embodiment is expected to have a sufficient anticancer effect even when actually administered to a cancer patient. Therefore, the evaluation method according to the present embodiment can be used as an unprecedented in vitro drug evaluation tool in screening of anticancer drug candidate compounds and drug repositioning screening in drug discovery sites, and in selection and determination of anticancer drug treatment methods (single drug and combination drug) in clinical sites (anticancer drug sensitivity tests). In particular, an anticancer drug evaluated to have an anticancer effect by the evaluation method according to the present embodiment using a cell structure containing cancer cells collected from a cancer patient is expected to have an appropriate anticancer effect when actually administered to the cancer patient. EXAMPLES

[0081] The present invention will be described in more detail and specifically below by showing examples, but the scope of the present invention is not limited to these examples.

[0082] <Reagents, etc.> In the subsequent experiments, the following reagents were used.

[0083] [Table 1]

[0084] [Table 2]

[0085] [Table 3]

[0086] [Example 1] The efficacy of anticancer drugs against cancer cells was evaluated using 2D cultured cancer cells, 3D cultured cell structures, and cancer-bearing model mice, and the results were compared.

[0087] <Drug susceptibility assessment (3D culture)> Human umbilical vein endothelial cells (HUVEC) (manufactured by Lonza) were used as vascular endothelial cells, human neonatal dermal fibroblasts (NHDF) (manufactured by Lonza) were used as interstitial cells, and two strains of human non-small cell lung cancer cells with HER2 mutations (LCC007 strain and LCC381 strain) were used as cancer cells.A cell structure was constructed in which a layer of cancer cells was stacked on top of a layer of HUVEC and NHDF with a thickness of 5 μm or more, and the anti-cancer effect of the HER2 inhibitor lapatinib was evaluated.

[0088] (Construction of cell structures) The NHDF was mixed with HUVECs at a ratio of 1.5% (cell number / cell number) and centrifuged. After removing the supernatant, Hep / Col solution (a mixture of heparin and collagen) was added. After removing the supernatant by centrifugation, the cells were suspended in culture medium. The total volume of the suspension was 0.9 x 10 NHDF. 6The cells were suspended in the culture medium and plated onto a 96-well plate (0.4 μm polyester membrane) equipped with a fibronectin-coated Transwell insert. The cells were pelleted by plate centrifugation (400 G for 2 minutes) and incubated at 37°C, 5% CO 2 The cell structure was obtained by culturing in an incubator under atmospheric conditions. On the day following the start of culturing, an appropriate amount of cancer cells were seeded on the top surface of the cell structure and cultured to construct a cell structure containing cancer cells, NHDF, and HUVEC. On the fifth day from the day when NHDF and HUVEC were seeded (the day when the cell structure was constructed), a drug (anticancer drug) solution was added to the culture medium to perform drug treatment. After culturing for three days from the addition of the anticancer drug (the eighth day from the day when the cell structure was constructed), the cell structure was fixed by 10% formalin treatment. The cell structure was cultured in the same manner except that only the solvent of the solution was added instead of the anticancer drug solution, and used as a control.

[0089] (Fluorescent Immunostaining) After formalin fixation, the cell structure was immersed in 1% BSA / 0.2% TritonX-containing PBS(-) for 1 hour at room temperature for membrane permeabilization and blocking. Next, primary antibodies and DAPI diluted to the designated concentrations in 1% BSA / 0.2% TritonX-containing PBS(-) were added and the structure was left to stand overnight at 4℃ for the primary antibody reaction. After washing three times with PBS(-), secondary antibodies diluted to the designated concentrations in 1% BSA / 0.2% TritonX-containing PBS(-) were added and the structure was left to stand at room temperature for 1 hour in the dark for the secondary antibody reaction. After washing with PBS(-), the structure was dehydrated and cleared by immersing in 99.5% ethanol.

[0090] (Fluorescence image acquisition and analysis) Fluorescent staining images of the samples after immunostaining were obtained using a confocal imaging system (Operetta CLS, manufactured by PerkinElmer). The obtained images were analyzed using image analysis software (Harmony). After setting a first ROI (Region of Interest) in a circular shape around the area where the cancer cells were present, the CK8 / 18 expression region was recognized. The CK8 / 18 recognition site was used as the second ROI to recognize the Ki-67 expression region. The CK8 / 18 staining area confluency and the number of Ki-67 recognition objects for the first ROI were calculated. The number of cancer cells in the control without the addition of an anticancer drug was set as 100%, and the relative number (%) of surviving cancer cells after treatment with each concentration of anticancer drug for 3 days was taken as the relative cancer cell survival rate (%).

[0091] <Drug sensitivity evaluation (spheroid culture)> The drug sensitivity of the spheroid-cultured cells was evaluated by ATP assay. First, cancer cells were seeded on an ultra-low adhesion surface round-bottom black plate (transparent bottom) at an appropriate seeding number determined in advance. The next day or 3 days after seeding, it was confirmed using a phase contrast microscope that the cancer cells had aggregated to form spherical cell masses (spheroids). Then, an anticancer drug solution (a solution containing an anticancer drug in 1 / 3 the amount of culture medium at the time of seeding) was added to the culture medium to perform drug treatment. After culturing for 3 days from the addition of the anticancer drug, a bioluminescence assay kit "CellTiter-Glo (registered trademark)" was added to the culture medium, and the medium was shaken at room temperature for 10 minutes, and chemiluminescence was detected.

[0092] <Drug susceptibility evaluation (2D culture)> The drug sensitivity of cancer cells cultured in two dimensions (2D culture) was evaluated by ATP assay. First, cancer cells were seeded on a black plate (transparent bottom) with an appropriate seeding number previously examined. On the day after seeding, an anticancer drug solution (a solution containing an anticancer drug in 1 / 3 the amount of culture medium at the time of seeding) was added to the culture medium to perform drug treatment. Cells cultured in 2D in the same manner except that only the solvent of the solution was added instead of the anticancer drug solution were used as a control. After culturing for 3 days from the addition of the anticancer drug, a bioluminescence assay kit "CellTiter-Glo (registered trademark)" was added to the culture medium, and the culture medium was shaken for 10 minutes at room temperature, after which chemiluminescence was detected. The amount of chemiluminescence of the control cells to which no anticancer drug was added was set to 100%, and the relative amount of chemiluminescence (%) of the cells after treatment with each concentration of anticancer drug for 3 days was used as the relative cancer cell survival rate (%).

[0093] <Drug sensitivity evaluation (2D culture with addition of 3D cell tissue supernatant)> The culture supernatant from the 3D culture was added to the culture medium for 2D culture to evaluate drug sensitivity. Specifically, the same procedure as in <Drug sensitivity evaluation (2D culture)> was used as the anticancer drug solution, except that a solution in which an anticancer drug was added to the culture supernatant from the 3D culture in an amount equivalent to 1 / 3 of the amount at the time of seeding was used.

[0094] <Drug susceptibility evaluation (in vivo)> Cancer cells (LCC007 or LCC381 strain) were subcutaneously transplanted into nude mice to generate tumor-bearing mice, which were then administered anticancer drugs to evaluate drug sensitivity.

[0095] (Creation of tumor-bearing mice) Nude mice (strain: BALB / c-nu (nu / nu), sex: female, age: 5 weeks) purchased from Jackson Laboratory Japan were inoculated with 3–10 × 10 cancer cells. 6 The tumor cells were cultured in advance to a transplantable amount, harvested, and then suspended in Matrigel at 50 μL per mouse.

[0096] (Weight and tumor size measurements, grouping) Prior to the administration of anticancer drugs, the short and long diameters of the subcutaneous tumors were measured, and the tumor volume was calculated using the following formula. After grouping and the start of administration (day 0), the tumor diameter and body weight were measured at least twice a week. 3 When the tumor volume reached approximately 100%, the mice were divided into groups so that the average tumor volume in each group was close to each other, and six mice were assigned to each of the vehicle group and the drug treatment group.

[0097] [Tumor volume (mm 3 )]=1 / 2×L×W×W L: tumor long axis (mm), W: tumor short axis (mm)

[0098] (Drug preparation and administration) The drug (Lapatinib) was orally administered to tumor-bearing mice, and tumor volume and body weight were measured over time. Table 4 shows the solvent, dose, and administration cycle of the drug administered to tumor-bearing mice transplanted with the LCC007 strain. Table 5 shows the solvent, dose, and administration cycle of the drug administered to tumor-bearing mice transplanted with the LCC381 strain. The dosage was 10 μL / g (body weight), and the liquid volume was calculated from the body weight for each individual. On the 14th day after the start of administration, the mice were euthanized by central nervous system destruction (cervical dislocation).

[0099] [Table 4]

[0100] [Table 5]

[0101] Figure 1 shows the results of the sensitivity evaluation of the LCC007 strain to Lapatinib, and Figure 2 shows the results of the sensitivity evaluation of the LCC381 strain to Lapatinib. Figure 1 (A) and Figure 2 (A) show the results of the evaluation in a cell experiment (in vitro), i.e., the results of measuring the relative cell viability (%) of each cell group over time, and Figure 1 (B) and Figure 2 (B) show the results of the evaluation in an animal experiment (in vivo), i.e., the results of measuring the tumor volume of each mouse group over time. As shown in Figure 1 (A) and Figure 2 (A), in vitro, in both cancer cell lines, the relative cell viability decreased in spheroid culture and 2D culture depending on the concentration of the anticancer drug added, but in 3D culture, the viability hardly decreased even when the concentration of the anticancer drug increased. In 2D culture when 3D cell tissue supernatant was added (in the figure, "3D supernatant added"), the cell viability was slightly higher than in 2D culture. On the other hand, as shown in Figures 1(B) and 2(B), in vivo, the tumor volume in the lapatinib-administered group was slightly smaller than that in the vehicle group, but no significant anticancer effect was confirmed. These results confirmed that 2D culture and spheroid culture may not provide the same evaluation as that obtained when using a cancer-bearing animal model, and that the use of the cell structure of this embodiment provides an evaluation closer to that obtained when using a cancer-bearing animal model.

[0102] [Example 2] Growth factors in the culture supernatant of cell constructs containing stroma were examined.

[0103] (Construction of cell structures and collection of culture supernatant) The NHDF was mixed with HUVECs at a ratio of 1.5% (cell number / cell number) and centrifuged. After removing the supernatant, Hep / Col solution (a mixture of heparin and collagen) was added. After removing the supernatant by centrifugation, the cells were suspended in culture medium. The total volume of the suspension was 0.9 x 10 NHDF. 6The cells were suspended in the culture medium and plated onto a 96-well plate (0.4 μm polyester membrane) equipped with a fibronectin-coated Transwell insert. The cells were pelleted by plate centrifugation (400 G for 2 minutes) and incubated at 37°C, 5% CO 2 The cells were cultured in an incubator under atmospheric conditions to obtain a cell structure consisting of NHDF and HUVEC. On the day following the start of culture, an appropriate amount of cancer cells were seeded on the top surface of the cell structure and cultured to construct a cell structure containing cancer cells, NHDF, and HUVEC. Two or five days after the day the NHDF and HUVEC were seeded (the day the cell structure was constructed), the culture medium was replaced with D-MEM without FBS. When changing the medium, the cells were washed twice with PBS before changing the medium. On the third day after the medium change, the culture supernatant was collected and solid matter such as cells was removed through a filter to prepare a sample.

[0104] (2D culture and collection of culture supernatant) NHDF and HUVEC were seeded in a 90φ dish at the same ratio as the 3D cell tissue. Two days after seeding, the culture medium was replaced with D-MEM without FBS. When changing the medium, the cells were washed twice with PBS before changing the medium. Three days after the medium change, the culture supernatant was collected and filtered to remove solid matter such as cells, and the sample was prepared.

[0105] (Culture supernatant analysis) As a control sample, D-MEM without FBS was used, and the sample obtained above was subjected to the growth factor analysis array kit "Growth factor array" to detect the contained proteins, and the amount of contained proteins was measured using various ELISA kits. Growth factor array and various ELISA were performed according to the instructions attached to the kit.

[0106] The results of the "Growth factor array" analysis showed that the 2D culture supernatant did not contain any growth factors, whereas the 3D culture supernatant contained HGF and PIGF on both days 5 and 8 of culture, and the culture supernatant on day 5 also contained VEGF. Furthermore, there was no difference in the amount of growth factors in the 3D culture supernatant between the cell construct consisting of NHDF and HUVEC and the cell construct consisting of NHDF, HUVEC, and cancer cells (LCC007 or LCC0381 strains).

[0107] The amounts of HGF, VEGF, PIGF, and bFGF in each culture supernatant were also measured. The results are shown in Figure 3. In Figure 3, "CNT" represents the results for the culture medium (D-MEM), "2D" represents the results for the 2D culture supernatant, and "3D" represents the results for the 3D culture supernatant. In the figure, "day5" represents the results for the culture supernatant on the 5th day of culture, and "day8" represents the results for the culture supernatant on the 8th day of culture. As a result, almost none of the growth factors were contained in the 2D culture supernatant, but they were contained in the 3D culture supernatant. In addition, the concentrations of HGF (Figure 3(A)) and PIGF (Figure 3(C)) were lower in the culture supernatant of the cell construct containing cancer cells than in the culture supernatant of the cell construct not containing cancer cells, while the concentrations of VEGF (Figure 3(B)) and bFGF (Figure 3(D)) were higher in the culture supernatant of the cell construct containing cancer cells than in the culture supernatant of the cell construct not containing cancer cells. Thus, when co-cultured with cancer cells, the concentration of each growth factor tended to decrease or increase.

[0108] [Example 3] We investigated the activation of intracellular signaling pathways stimulated by HGF in cells of the stroma-containing cell structure. We used the non-small cell lung cancer cell line LCC007 as the cancer cell.

[0109] (Cell structure construction, culture and sample preparation) The NHDF was mixed with HUVECs at a ratio of 1.5% (cell number / cell number) and centrifuged. After removing the supernatant, Hep / Col solution (a mixture of heparin and collagen) was added. After removing the supernatant by centrifugation, the cells were suspended in culture medium. The total volume of the suspension was 0.9 x 10 NHDF. 6 The cells were suspended in the culture medium and plated onto a 96-well plate (0.4 μm polyester membrane) equipped with a fibronectin-coated Transwell insert. The cells were pelleted by plate centrifugation (400 G for 2 minutes) and incubated at 37°C, 5% CO 2 The cell structure was obtained by culturing in an incubator under atmospheric conditions. On the day after the start of the culture, an appropriate amount of cancer cells (LCC007 strain) was seeded on the top surface of the cell structure and cultured to construct a cell structure containing cancer cells, NHDF, and HUVEC. Drug (Lapatinib) treatment was performed 7 or 8 days after the day of seeding NHDF and HUVEC (the day of construction of the cell structure). The cells treated with the drug on the 7th day after the construction of the cell structure were treated with trypsin 24 hours after the treatment, and the cells treated with the drug on the 8th day after the construction of the cell structure were treated with trypsin 6 hours after the treatment, and the cell structure was detached from the polyester membrane and collected. The collected cell structure was treated with a dephosphorylation inhibitor, and then the cancer cells were concentrated using a Tumor isolation kit. Lysis Buffer was added to the concentrated cancer cells, heated at 100°C for 3 minutes, and then ultrasonicated until the liquid lost its viscosity. The cells were then centrifuged (13,500 rpm, 10 minutes) to collect the supernatant. The amount of protein contained in the collected supernatant was quantified, and the solution was diluted with sample buffer so that the amount of protein per lane was constant (2 to 4 μg / lane), and the solution was left to stand at 100°C for 3 minutes for reduction to prepare a sample.

[0110] (Sample preparation of 2D cultured cells) Cancer cells were seeded in a 90φ dish. Drug treatment was performed 7 or 8 days after seeding. The cells treated with drugs on the 7th day after seeding were treated with trypsin 24 hours after treatment, and the cells treated with drugs on the 8th day after seeding were treated with trypsin 6 hours after treatment. Lysis Buffer was added to the collected cells, heated at 100°C for 3 minutes, and then ultrasonicated until the liquid lost its viscosity. The cells were then centrifuged (13,500 rpm, 10 minutes) to collect the supernatant. The amount of protein contained in the collected supernatant was quantified, and the sample was diluted with sample buffer so that the amount of protein per lane was constant (2 to 4 μg / lane), and the sample was left to stand at 100°C for 3 minutes and reduced to obtain a sample.

[0111] (Sample preparation of cells from tumor-bearing mice (in vivo)) After the completion of the test in <Drug susceptibility evaluation (in vivo)> in Example 1, the mice were euthanized by central destruction (cervical dislocation). Thereafter, the subcutaneous tumor was excised and stored at -80°C. The stored tumor was dissolved in an appropriate amount of lysis buffer, heated at 100°C for 3 minutes, and then subjected to ultrasonic treatment until the liquid lost its viscosity. Then, the supernatant was collected by centrifugation (13,500 rpm, 10 minutes). The amount of protein contained in the collected supernatant was quantified, and the sample was diluted with sample buffer so that the amount of protein per lane was constant (2 to 4 μg / lane), and the sample was left to stand at 100°C for 3 minutes and subjected to reduction treatment to obtain a sample.

[0112] (Western Blotting) The prepared samples and molecular weight markers were loaded onto a gel for SDS-PAGE and electrophoresis was performed. Proteins were transferred from the gel after electrophoresis to a PVDF membrane. The transferred membrane was immersed in 4% BSA / PBS and blocked by shaking at room temperature for 1 hour, and then the primary antibody reaction was performed. The primary antibody reaction was performed by shaking at room temperature for 1 hour or by leaving the membrane at 4°C overnight. After washing the membrane, the secondary antibody reaction was performed by shaking at room temperature for 1 hour. After washing the membrane, it was luminesced using a detection reagent and photographed with an imager (AI600, Cytiva). The photographed images were appropriately processed using the image analysis software Photoshop.

[0113] Figure 4 shows the image of the membrane detected by Western blotting. As a result, in the samples from the tumor-bearing mice, phosphorylated Akt (pAkt), phosphorylated ERK (pERK), and phosphorylated S6 (pS6) were detected in both the samples from the mice treated with Lapatinib (lane "Lapa" in "Animal Experiment" in the figure) and the samples from the untreated mice (lane "CNT" in "Animal Experiment" in the figure). In the samples from the 2D cultured cells, the bands of pAkt, pERK, and pS6 were detected in the untreated sample (lane "CNT" in "2D" in the figure), but the bands of pAkt and pERK were not detected in the cells treated with Lapatinib (lanes "6h" and "24h" in "2D" in the figure), and the band of pS6 was much fainter than that of the untreated sample. In contrast, in the samples derived from the 3D-cultured cell structures, bands for pAkt, pERK, and pS6 were detected not only in the untreated sample (lane marked "CNT" under "3D" in the figure) but also in the sample treated with Lapatinib and cultured for 24 hours (lane marked "24h" under "3D" in the figure), as in the samples derived from the cancer-bearing mice. In the sample derived from the 3D-cultured cell structures treated with Lapatinib and cultured for 6 hours, bands for pS6 were detected, but bands for pAkt and pERK were not detected. These results confirmed that in the 3D-cultured cell structures, growth factors such as HGF are secreted from the interstitial tissue, as in the bodies of cancer-bearing mice, and this activates downstream signaling pathways, while in the 2D culture, growth factors are not secreted, and therefore downstream signaling pathways are not activated.

[0114] [Example 4] The efficacy of anticancer drugs against cancer cells was evaluated using two-dimensionally cultured cancer cells and three-dimensionally cultured cell structures, and the results were compared.

[0115] As cancer cells, EML4-ALK positive non-small cell lung cancer cell line LCC028-3, BRAF positive colon cancer cell line JC215, and CD74-ROS1 positive non-small cell lung cancer cell line LCC168 were used. As anticancer drugs, ALK inhibitor (Alectinib), BRAF inhibitor (Davrafenib), and WNT inhibitor (FH535) were used. Sensitivity evaluation by 2D culture and 3D culture was performed in the same manner as in Example 1.

[0116] The results of the sensitivity evaluation of the EML4-ALK positive non-small cell lung cancer cell line LCC028-3 to an ALK inhibitor are shown in Figure 5 (A), the results of the sensitivity evaluation of the BRAF positive colon cancer cell line JC215 to a BRAF inhibitor are shown in Figure 5 (B), the results of the sensitivity evaluation of the BRAF positive colon cancer cell line JC215 to a WNT inhibitor are shown in Figure 6 (A), and the results of the sensitivity evaluation of the CD74-ROS1 positive non-small cell lung cancer cell line LCC168 to a WNT inhibitor are shown in Figure 6 (B). The cell structures of the LCC028-3 and JC215 strains (in the figure, "3D") had higher relative cell viability and smaller anticancer effects against each anticancer drug than the cells cultured in 2D (in the figure, "2D"). This was presumably due to the activation of the ERK / MAPK pathway, PI3K / AKT pathway, and Jak-STAT pathway by HGF and VEGF secreted from the stromal cells of the cell structures. On the other hand, the JC215 and LCC168 cell constructs (3D in the figure) had lower relative cell viability and higher anti-cancer effects than the 2D cultured cells (2D in the figure). This was presumably due to the activation of the WNT / β-Catenin pathway by HGF secreted from the stromal cells of the cell constructs.

Claims

1. A culturing step of culturing a cell structure containing cancer cells and stromal cells in the presence of one or more anticancer agents; an evaluation step of evaluating the anticancer effect of the anticancer agent using as an index the number of cells having proliferation ability among the cancer cells in the cell structure after the culture step; having A method for evaluating an anticancer agent, wherein a signal transduction pathway stimulated by hepatocyte growth factor, placenta growth factor, or vascular endothelial growth factor is more active in the cancer cells than in normal cells.

2. The method for evaluating an anticancer agent according to claim 1, wherein the signal transduction pathway is an ERK / MAPK pathway, a PI3K / AKT pathway, a Jak-STAT pathway, or a WNT / β-Catenin pathway.

3. The method for evaluating an anticancer agent according to claim 1 , wherein the cancer cells are one or more types selected from the group consisting of HER2-positive cancer cells, ALK-positive cancer cells, BRAF-positive cancer cells, and ROS1-positive cancer cells.

4. The method for evaluating an anticancer agent according to claim 1, wherein the anticancer agent targets a molecule that constitutes a signal transduction pathway stimulated by hepatocyte growth factor, placenta growth factor, vascular endothelial growth factor, or basic fibroblast growth factor, or a molecule that activates the signal transduction pathway.

5. The method for evaluating an anticancer drug according to claim 4 , wherein the anticancer drug is a HER2 inhibitor, an ALK inhibitor, a BRAF inhibitor, or a WNT inhibitor.

6. The method for evaluating an anticancer agent according to claim 1 , wherein the stromal cells are one or more types selected from the group consisting of fibroblasts, dendritic cells, macrophages, and mast cells.

7. The method for evaluating an anticancer agent according to claim 1 , wherein the cell structure has a cancer cell layer on its outer surface.

8. the cell structure comprises, as cells constituting the stroma, at least one type selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells, and fibroblasts; The method for evaluating an anticancer agent according to claim 1 , wherein the total number of vascular endothelial cells and lymphatic endothelial cells in the cell structure is 0.1% or more of the total number of fibroblasts.

9. The method for evaluating an anticancer agent according to claim 1 , wherein the cell structure has a thickness of 5 μm or more.

10. The method for evaluating an anticancer agent according to claim 1 , wherein the cell structure comprises a vascular network structure.

11. The method for evaluating an anticancer agent according to claim 1 , wherein the cancer cells are cancer cells collected from a cancer patient.

12. The method for evaluating an anticancer agent according to claim 1, wherein the culture time in the culture step is 24 to 96 hours.

13. Prior to the culturing step, (a) mixing cells, extracellular matrix components, and polyelectrolytes in a cationic buffer to obtain a mixture; (b) seeding the mixture obtained in step (a) into a cell culture vessel; (c) after the step (b), obtaining a cell structure in which cells are stacked in multiple layers in the cell culture vessel; The method for evaluating an anticancer agent according to claim 1 , wherein the cell structure is produced by the following method.

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