Cancer organoid and production method therefor

Abstract

Provided is a cancer organoid having high similarity to a tumor microenvironment (TME) of actual cancer tissue, compared to known cancer organoids. Vascular cells, mesenchymal cells, cancer cells, and macrophages are co-cultured in vitro to produce cancer organoids with a TME.

Description

Cancer organoids and their manufacturing method

[0001] The present invention relates to cancer organoids and methods for producing the same.

[0002] Pancreatic ductal adenocarcinoma (PDAC), which is associated with a tumor microenvironment (TME), is an aggressive cancer with a poor prognosis, with a 5-year survival rate of approximately 10% despite the development of various treatments. Although anticancer drugs that have shown tumor-shrinking effects in vitro have been developed for PDAC, no anticancer drugs have yet been developed that are highly effective in clinical settings. Therefore, it is believed that the poor prognosis of PDAC is largely due to the TME rather than the characteristics of the cancer cells themselves. The TME has attracted attention as a factor that influences the acquisition of malignant potential, such as drug resistance and metastatic potential. The TME of PDAC contains a rich extracellular matrix (ECM), which has been suggested to be involved in the progression of cancer malignancy (Non-Patent Document 1). Furthermore, the TME contains a variety of cancer-associated fibroblasts (CAFs), and the diversity of cells derived from these is considered to be one of the factors that make treatment with anticancer drugs more difficult (Non-Patent Documents 2 and 3). Over the past decade, various organoid generation technologies have been established. In particular, cancer organoid models are expected to be used for evaluating the efficacy of cancer drugs and discovering new tumor markers. A method for reproducing the TME within cancer tissue using organoid culture has been reported (Non-Patent Document 4). However, faithful reproduction of the TME presents challenges. Patent Document 1 discloses an invention related to the construction of three-dimensional organs containing functional cells, not limited to cancer. It reports that organoids with a three-dimensional structure were constructed without the use of scaffold materials by co-culturing vascular cells and mesenchymal cells derived from pluripotent stem cells with functional cells. This method may also be an effective culture method for reproducing the cancer TME.

[0003] International Publication No. 2019 / 107535

[0004] Hessmann E.,et al.,Physiol.Rev.Vol.100:1707−1751(2020)Chen Y.,et al.Nat Rev Clin Oncol.Vol.18:792−804(2021)Lavie D.,et al.Nat Cancer.Vol.3:793−807(2022)LeSavage BL.,et al.Nat Mater.Vol.21:143−159(2022)

[0005] The generation of cancer organoids has already been reported. However, it has been revealed that actual cancer tissues contain cancer-associated fibroblasts and tumor-associated macrophages (TAMs) containing multiple subpopulations, and cancer organoids containing such diverse cell populations have not been reported to date. The present invention aims to provide cancer organoids that have properties closer to the TME of actual cancer tissues than known cancer organoids. After extensive research, the present inventors discovered that co-culturing cancer cells, vascular endothelial cells, and mesenchymal cells, as well as cancer-associated macrophages, can form cancer organoids that resemble the TME of actual tissues, thereby completing the present invention. The present invention provides the following embodiments: (1) A method for producing cancer organoids, comprising co-culturing vascular cells, mesenchymal cells, cancer cells, and macrophages in vitro. (2) The method according to (1), comprising inducing macrophages from monocytes and / or pluripotent stem cells before the co-culture. (3) The method according to (1) or (2), comprising inducing vascular cells and mesenchymal cells, respectively, from pluripotent stem cells before the co-culture. (4) The method according to (2) or (3), wherein at least one of the cancer cells, pluripotent stem cells, and monocytes is a human-derived cell. (5) The method according to any one of (2) to (4), wherein the pluripotent stem cells are at least one cell selected from the group consisting of induced pluripotent stem cells and embryonic stem cells. (6) The method according to any one of (1) to (5), wherein the pluripotent stem cells do not comprise the use of a scaffold material. (7) A cancer organoid comprising vascular cells, mesenchymal cells, cancer cells, and macrophages. (8) The cancer organoid according to (7), wherein the macrophages are induced from monocytes and / or pluripotent stem cells. (9) The cancer organoid according to (7) or (8), wherein the vascular cells and mesenchymal cells are each induced from pluripotent stem cells. (10) The cancer organoid according to either (8) or (9), wherein at least one of the cancer cells, pluripotent stem cells, and monocyte cells is of human origin. (11) A cancer organoid produced by the method according to any one of (1) to (6).(12) A drug screening method comprising evaluating the effect of a drug using the cancer organoid according to any one of (7) to (11). (13) A drug screening method comprising evaluating the delivery of a drug to cancer tissue using the cancer organoid according to any one of (7) to (11). According to the present invention, it is possible to provide cancer organoids having properties closer to the TME of cancer tissue than conventional cancer organoids. This specification includes the disclosure of Japanese Patent Application No. 2024-107859, which is the basis of the priority claim of this application.

[0006] 1 is a schematic diagram showing the culture procedure for FPCO, MO-FPCO, and iMac-FPCO. (A) A schematic diagram showing the culture procedure for MO-FPCO and iMac-FPCO at the start of culture (day 1). (B) A schematic diagram showing the culture schedule for FPCO, MO-FPCO, and iMac-FPCO. A schematic diagram showing the schedule for extended culture and analysis of FPCO and MO-FPCO, and the schedule for extended culture and analysis of iMac-FPCO. (A) Schedule for extended culture and analysis of FPCO and MO-FPCO. (B) Schedule for extended culture and analysis of iMac-FPCO. Arrows in the figure indicate the timing of sampling. Graph showing the relative expression levels of macrophage-related genes (ITGAM, CD36, CD68) in THP-1 cells, macrophages, and these KuO-labeled cells (n=6). The vertical axis indicates the relative value to the 18S rRNA level. Expression data are shown as mean ± standard deviation. Data from each group were compared using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. In the figure, "*" indicates p<0.05, "**" indicates p<0.01, "****" indicates p<0.0001, and "ns" indicates no significant difference. This graph shows the relative expression levels of M1 marker genes (TNFA, CXCL10) and M2 marker genes (CCL17, CCL22) in M0 macrophages (M0-Mac), macrophages induced to M1 type with LPS and IFNγ (M1-Mac), and macrophages induced to M2 type with IL4 and IL13 (M2-Mac). The vertical axis indicates the relative value to the 18S rRNA level. Expression data are shown as mean ± standard deviation. Data from each group were compared using one-way ANOVA followed by Tukey's multiple comparison test. In the figure, "*" indicates p<0.05, "**" indicates p<0.01, "***" indicates p<0.001, and "ns" indicates no significant difference. Fluorescence micrographs of PCO, FPCO, MO-FPCO, and iMac-FPCO are shown. The green areas represent GFP-transfected cancer cells, and the orange areas represent KuO-transfected macrophages. The scale bar indicates 100 μm.Graphs showing the anticancer drug resistance of PCO, FPCO, MO-FPCO, and iMac-FPCO. (A) Graph showing the results of quantifying cancer cells in PCO, FPCO, and MO-FPCO in the presence of gemcitabine. The vertical axis shows relative values, with the number of cells in the presence of 0 μM gemcitabine set to 1. "ns" in the figure indicates no significant difference. (B) Graph showing the results of quantifying cancer cells in PCO, FPCO, and iMac-FPCO in the presence of gemcitabine. The vertical axis shows relative values, with the number of cells in the presence of 0 μM gemcitabine set to 1. Graphs showing fluorescent micrographs of immunofluorescent staining of CD163, CD80, and CD11b in human PDAC tissue and MO-FPCO, and quantification of M1-like TAM and M2-like TAM on the images. (A) Photographs of immunofluorescent staining. The scale bar indicates 50 μm. Arrow a indicates CD80. ＋ CD11b ＋ M1-Mac, arrow b indicates CD163 ＋ CD11b ＋ (B) M1-like TAMs (CD80) in human PDAC tissues and M0-FPCO. ＋ CD11b ＋ ) and M2-like TAMs (CD163 ＋ CD11b ＋) quantification. Data are presented as mean ± standard deviation. Comparisons between groups were performed using an unpaired Student's t-test (n = 5). In the figure, "*" indicates p<0.05, and "****" indicates p<0.0001. This graph shows flow cytometry of cells constituting FPCO and M0-FPCO. The vertical axis shows the fluorescence intensity of KuO, and the horizontal axis shows the fluorescence intensity of GFP. The boxes in the figure indicate the gate areas used to select macrophages in M0-FPCO and cancer cells in FPCO and M0-FPCO. This graph shows the quantification of M1 / M2 markers by RT-qPCR for macrophages in THP-1 cells, M0-Mac, and M0-FPCO, as well as cytokine (LPS and IFNγ / IL4 and IL13)-induced M1 / M2 macrophages (n = 3). The vertical axis shows the relative value to the amount of 18S rRNA. Expression data are presented as mean ± standard deviation (SD) and were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. In the figure, "*" indicates p<0.05, "**" indicates p<0.01, "****" indicates p<0.0001, and "ns" indicates no significant difference. This is a continuation of Figure 9-1. This is a schematic diagram showing the procedure for obtaining M0-Mac, M1-Mac, M2-Mac, and FPCO-Mac from THP-1 cells. M1 induction was performed using LPS and IFNγ, while M2 induction was performed using IL4 and IL13. This is a principal component analysis plot of data from bulk RNA sequencing. FPCO-Mac exhibits distinct properties from M0-Mac and cytokine-induced M2-Mac. This is the result of GSEA of FPCO-Mac. FPCO-Mac showed enhanced angiogenesis and hypoxia, and reduced inflammatory characteristics (IFNγ, inflammatory response). Graph showing the results of GO analysis of M0-Mac and FPCO-Mac. Heat map showing the results of analysis of differentially expressed genes (DEGs) between M0-Mac and FPCO-Mac. Results of cytokine array of FPCO and M0-FPCO. (A) Image of cytokine array. (B) Graph comparing the expression levels of various cytokines in FPCO and M0-FPCO.CHI3L1: chitinase-3-like protein 1; MMP9: matrix metallopeptidase 9; OPN: osteopontin; EGF: epidermal growth factor; and IGFBP2: insulin-like growth factor binding protein 2. (A) Fluorescence micrographs showing the results of whole-mount immunofluorescence staining of FPCO, MO-FPCO, and iMac-FPCO, and a graph showing the length and volume of the CD31-positive area in each organoid. (B) Fluorescence micrographs showing the results of whole-mount immunofluorescence staining. The upper row shows images stained with CD31 (magenta) and Sytox green (cyan). The lower row shows images stained with CD31 (magenta) alone. The scale bar indicates 100 μm. (C) Graph showing the length of the CD31-positive area in each organoid. Measurement data are shown as mean ± standard deviation. Comparisons between groups were performed using an unpaired Student's t-test (n = 5). In the figure, "*" indicates p<0.05, and "****" indicates p<0.0001. (C) Graph showing the volume of CD31-positive areas in each organoid. Measurement data are shown as mean ± standard deviation. Comparisons between groups were performed using an unpaired Student's t-test (n=5). "****" in the figure indicates p<0.0001. These are micrographs showing the results of immunofluorescence staining of FPCO and M0-FPCO for CD31 and CD11b. It was shown that the microvasculature was maintained in FPCO but not in M0-FPCO. These are the results of GSEA of PDAC cells in FPCO and M0-FPCO. It was confirmed that gene expression related to the G2M checkpoint, E2F targets, and epithelial-mesenchymal transition was increased in PDAC cells compared with M0-FPCO. This is a heat map showing the results of DEG analysis between cancer cells in M0-FPCO and FPCO. Three independent samples were tested for each condition, and the average data was used. A fold change of >2.5 was defined. This graph shows the results of luciferase assays of FPCO and MO-FPCO from days 7 to 14 after the start of culture (extended culture in GFR). Validation of the luciferase assay data was performed using various organoids derived from two patients. Measurement data are shown as the mean ± standard deviation.Comparisons between groups were performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. In the figure, "*" indicates p<0.05, and "***" indicates p<0.001. Images of proliferating PDAC cells in FPCO and MO-FPCO detected by immunofluorescence staining of Ki67 and CK19. Ki67 in CK19-positive cells. ＋ Cell ratio and pMLKL ＋ (A) Ki67 ＋ (B) pMLKL ＋The figure shows the cell ratio of FPCO to M0-FPCO. Measurement data are presented as mean ± standard deviation (n = 6). Comparisons between groups were performed using an unpaired Student's t-test. In the figure, "**" indicates p<0.01. (A) Flow cytometry data for cells under GFR conditions and the number of sorted cancer cells on the 14th day after the start of culture. The vertical axis shows the fluorescence intensity of KuO-Mac, and the horizontal axis shows the fluorescence intensity of cancer cells. (B) Graph comparing the number of cancer cells in FPCO and M0-FPCO. Measurement data are presented as mean ± standard deviation (n = 3). Comparisons between groups were performed using an unpaired Student's t-test. In the figure, "*" indicates p<0.05. (C) Graph showing the results of GO analysis of PDAC cells in FPCO and M0-FPCO on the 14th day after the start of culture. Increased expression of cell cycle-related genes was observed in M0-FPCO. GSEA results for FPCO and M0-FPCO cancer cells on day 14 after the start of culture. Venn diagram showing genes with increased expression levels in M0-FPCO PDAC cells compared to FPCO on days 7 and 14 after the start of culture (extended culture with GFR). 32 overlapping genes were confirmed. UMAP plot of single-cell RNA sequencing results for M0-FPCO. UMAP plot of single-cell RNA sequencing results for M0-FPCO macrophages. It was shown that macrophages can be classified into five subclusters. M2 and M1 signatures are plotted within macrophage clusters. CD163 and CD80 genes are plotted within macrophage clusters. UMAP plot of gene expression of SPP1. SPP1 ＋ This figure shows cells plotted within macrophage clusters. This figure shows the results of measuring osteopontin (OPN) in differentiation media for M0-FPCO and FPCO from day 13 to day 14 after the start of culture. Osteopontin was measured by ELISA. Measurement data show mean ± standard deviation (n = 3). Comparisons between groups were performed using an unpaired Student's t-test. In the figure, "**" indicates p<0.01. Human PDAC data and CD11b in M0-FPCO ＋This figure shows the results of an integrated analysis of myeloid cell populations. Publicly available human PDAC data was used. This figure shows the clustering results of cancer cell populations within FPCO and M0-FPCO. Eight clusters were identified on UMAP. This figure shows the results of single-cell RNA sequencing of iMac-FPCO on UMAP. This figure shows the results of single-cell RNA sequencing of iMac-FPCO TAMs and human PDAC TAMs, as well as the results of integrating and extracting these data. (A) shows single-cell RNA sequencing of iMac-FPCO, (B) shows single-cell RNA sequencing of human PDAC TAMs, (C) shows the integrated data of (A) and (B), and (D) shows the results of extracting single-cell RNA sequencing of iMac-FPCO on days 7 and 14 of culture from the results of (C). Figure 1 shows the results of single-cell DNA sequencing of cancer cells in iMac-FPCO on days 7 and 14 of culture, plotted using UMAP. Figure 2 shows the results of GO analysis of PDAC cells in iMac-FPCO on days 7 and 14 after the start of culture.

[0007] 1. Method for Producing Cancer Organoids The first embodiment of the present invention is a method for producing cancer organoids. This method is characterized by the in vitro co-culturing of vascular cells, mesenchymal cells, cancer cells, and macrophages. As used herein, "organoid" refers to an artificial organ produced in vitro. More specifically, it refers to an organ-specific cell aggregate, a three-dimensional structure self-organized through cell-cell interactions. Conventionally, in the production of organoids, scaffolding materials have been used to form the three-dimensional structure. As scaffolding materials, one or more combinations of materials selected from the group consisting of Matrigel, laminin, collagen, various proteoglycans, fibronectin, entactin, and vitronectin are typically used. It is known that the extracellular matrix is ​​involved in the malignant transformation of cancer tissues. Therefore, it has been found that in order to reproduce the malignant transformation of actual cancer tissues in organoids, it is necessary to produce organoids under conditions that do not contain exogenous extracellular matrices such as Matrigel. On the other hand, it is known that various cancer-associated fibroblasts (CAFs) exist in cancer tissues. It has been desired to reproduce such various CAFs in matrix formation. The present inventors have found that organoids having a three-dimensional structure can be generated without using a scaffold material by co-culturing vascular cells, mesenchymal cells, and tissue or organ cells (Patent Document 1). Furthermore, the present inventors have found that pancreatic cancer organoids can be generated by converting tissue or organ cells into pancreatic cancer cells (particularly, PDAC cells). It has been reported that the TME of PDAC contains significantly more myeloid cells, including macrophages, than normal cells (Panni RZ, et al., Sci. Transl. Med. Vol. 587 11: eaau9240 (2019), Liu X, et al., Cell, Vol. 41: 1073-1090 (2023)). Based on these findings, the present inventors attempted to generate pancreatic cancer organoids incorporating macrophages as a major component of the TME.As a result, it has become possible to construct an organoid model with properties closer to human PDAC tissue than organoids that do not contain macrophages. As used herein, the term "vascular cells" refers to a concept that includes cells differentiated into cells that constitute blood vessels or undifferentiated cells that can differentiate into such cells. Undifferentiated cells include stem cells, progenitor cells, mesodermal cells, and the like. Undifferentiated cells are preferably cells whose differentiation fate into vascular cells has been determined but which have not yet differentiated into vascular cells. Examples of vascular cells include vascular endothelial cells, vascular endothelial progenitor cells, endocardial progenitor cells, and hemangioblasts, with vascular endothelial cells being preferred. Vascular endothelial cells can be identified by the presence or absence of expression of their marker proteins, TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and / or CD41. Vascular endothelial cells used in the present invention may be differentiated or undifferentiated. The presence or absence of differentiation of vascular endothelial cells can be confirmed using CD33 and CD144 as indicators. Among terms used by those skilled in the art, the vascular endothelial cells of the present invention include endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial precursor cells, vasculogenic progenitors, and hemangioblasts (Joo H.J., et al., Blood, 118(8):2094-104. (2011)). Vascular system cells may be any of those collected from blood vessels, particularly umbilical veins, and those prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods. In particular, it is preferable to use pluripotent stem cells. Vascular cells can be derived from humans or non-human animals (e.g., animals used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically, mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.), but it is particularly preferable to use vascular cells derived from humans.As used herein, the term "mesenchymal cells" refers to cells that exist primarily in connective tissue derived from the mesoderm and have differentiated into connective tissue cells that form a support structure for cells that function in tissues, or undifferentiated cells that can differentiate into such cells. Undifferentiated cells include stem cells, progenitor cells, mesodermal cells, etc. Preferably, undifferentiated cells are cells whose differentiation fate into mesenchymal cells has been determined but which have not yet differentiated into mesenchymal cells. Undifferentiated mesenchymal cells can be identified by the presence or absence of expression of their marker proteins, Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, and / or Nestin. Mesenchymal stem cells that do not express any of the above markers can be determined to be differentiated mesenchymal cells. Among terms used by those skilled in the art, mesenchymal stem cells, mesenchymal progenitor cells, mesenchymal cells (Peters R., et al. PLoS One, 5(12): e15689 (2010)), etc., are included in the mesenchymal cells of the present invention. Mesenchymal cells may be bone marrow-derived mesenchymal cells (particularly mesenchymal stem cells), such as those collected from bone marrow, adipose tissue, placental tissue, umbilical cord tissue, dental pulp, or other tissues. Alternatively, mesenchymal cells may be prepared from pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ES cells) according to known methods. It is particularly preferable to use pluripotent stem cells. The mesenchymal cells may be derived from humans or non-human animals (e.g., animals used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically, mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.), but it is particularly preferable to use mesenchymal cells derived from humans. In the present invention, "cancer cells" may be cells isolated from cancer tissue collected from a patient, or may be cells obtained from a cell bank, etc.The cancer type is not particularly limited, and includes leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL), lymphomas such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma, as well as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, gastric cancer, testicular cancer, gallbladder cancer and biliary tract cancer, thyroid cancer, thymic cancer, bone tumors, and brain tumors (glioma, astrocytoma, glioblastoma), etc. In particular, cancer types with high immunosuppressive properties are preferred because of the usefulness of investigating the characteristics of their therapeutic targets and screening for therapeutic agents. Examples of such cancer types include pancreatic cancer, bile duct cancer, some colon cancers, and brain tumors (particularly glioblastoma). Pancreatic cancer is particularly preferred. Pancreatic cancer is broadly divided into three types: invasive ductal carcinoma, acinar cell carcinoma, and mucus-producing pancreatic cancer, but any of these may be used. In particular, invasive ductal carcinoma is preferred. Known markers for pancreatic cancer include CEA, CA19-9, Span-1, and DUPAN-2. The following describes exemplary methods for producing pancreatic cancer organoids, but the present invention is not intended to limit the scope of the present invention to the method for producing pancreatic cancer organoids. In this specification, "macrophage" refers to a large, amoeba-like cell that is distributed in almost all tissues of animal cells and has the function of ingesting and digesting foreign matter, senescent cells, etc., and refers to one type of immune cell. It is known that a large number of macrophages are present around cancer cells in the TME, and such macrophages are called tumor-associated macrophages (TAM). Macrophages are classically broadly divided into two types, M1 type macrophages and M2 type macrophages, based on their activation. M1 type macrophages are known to produce inflammatory cytokines in response to pathogenic infections and the like. On the other hand, M2 type macrophages are known to have the effect of suppressing the host immune response and to produce growth factors and anti-inflammatory cytokines.In cancer tissues, activation of these M2 macrophages is thought to suppress antitumor immunity and / or promote angiogenesis, which is thought to contribute to the malignant progression of cancer. In the present invention, it is preferable to use macrophages (M0 macrophages (M0-Mac)) before they are induced into M1 and M2 types. Macrophages observed in organoids generated by co-culture of M0-Mac, pancreatic cancer cells, vascular cells, and mesenchymal cells were classified into multiple subpopulations and were confirmed to have characteristics different from M1-Mac and M2-Mac. That is, it was shown that in the TME of pancreatic cancer tissues, macrophages undergo unique induction due to interactions with pancreatic cancer cells and other factors. Therefore, to more reliably reproduce this, it was considered preferable to use macrophages before M1 / M2 induction. In the present invention, macrophages may be directly collected from a human, or may be prepared by inducing monocytes (peripheral blood monocytes (PBMCs)) in blood collected from a human. Alternatively, they may be prepared by inducing pluripotent stem cells. Alternatively, they may be a mixture of monocytes and pluripotent stem cells. While monocytes are peripheral blood cells derived from bone marrow, many organs, including the pancreas, contain tissue-resident macrophages differentiated from fetal yolk sac (yolk sac)-derived monocytes. A method for inducing differentiation of yolk sac-derived monocytes from pluripotent stem cells, particularly iPCS, has been reported. Previously reported single-cell RNA sequencing has shown that resident macrophage-derived and peripheral blood monocyte-derived TAMs are present in PDAC tissues (Oh K. et al., Nat. Commun., 2023). Therefore, it has been suggested that in order to more accurately understand the cellular diversity within human PDAC TAMs, it is necessary to closely examine the behavior of both bone marrow-derived and yolk sac-derived macrophages. As used herein, "monocytes" refer to a type of agranular leukocyte that develops from bone marrow-derived hematopoietic cells and is a mononuclear cell that accounts for 4-8% of peripheral blood leukocytes. Macrophages are known to be derived from monocytes.The monocytes used in the present invention can be derived from humans or non-human animals (e.g., animals used for laboratory tests, pets, working animals, racehorses, fighting dogs, etc., specifically, mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.), but it is particularly preferable to use monocytes derived from humans. As used herein, examples of "pluripotent stem cells" include pluripotent cells obtained from a living organism (e.g., ES cells), pluripotent cells obtained by induction through reprogramming (e.g., iPSCs (induced pluripotent stem cells), MUSE cells (multilineage-differentiating stress-ending cells), iMPCs (induced multipotent progenitor cells)), and combinations thereof. Pluripotent stem cells can be derived from humans or non-human animals (e.g., animals used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.), but it is particularly preferable to use pluripotent stem cells derived from humans. 1-1 Cell induction step Below, the method of this embodiment will be explained step by step. The method of this embodiment may include a step of inducing at least one type of vascular cells, mesenchymal cells, or macrophages. (1) Induction of vascular cells Vascular cells can be induced from pluripotent stem cells. Any known method can be used to induce differentiation of pluripotent stem cells, such as iPSCs, into vascular endothelial cells (iPSC-ECs). For example, the method described in Patent Document 1 can be used. Specifically, iPSCs are dispersed and seeded in the presence of Rho kinase, and then cultured in Medium 1 (DMEM / F12 medium, 1-2% B27, 1% Glutamax, 25 ng / mL BMP4, 8 uM CHIR 99021) for 3 days, followed by priming culture in Medium 2 (StemPro34-SFM, 200 ng / mL VEGF, 2 uM Forskolin) for 3-4 days, or in Medium 3 (StemPro34-SFM, 50 ng / mL VEGF) for 7 days.In one aspect, the above-mentioned pluripotent stem cells can be cultured in the presence of a ROCK inhibitor, a β-catenin activator, and a factor belonging to the TGFβ superfamily (e.g., for 1 to 2 days), then cultured in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily (e.g., for 2 to 3 days), and further cultured in the presence of a vascular endothelial growth factor receptor (VEGFR) activator and an adenylate cyclase activator (e.g., for 4 to 8 days). In another aspect, pluripotent stem cells can be cultured (for example, for 1 to 2 days) in the presence of a ROCK inhibitor, a vascular endothelial growth factor receptor (VEGFR) activator, a factor belonging to the transforming growth factor β family, a β-catenin activator, and a factor belonging to the TGFβ superfamily, followed by culture (for example, for 2 to 4 days) in the presence of a vascular endothelial growth factor receptor (VEGFR) activator, a factor belonging to the transforming growth factor β family, a β-catenin activator, and a factor belonging to the TGFβ superfamily, and further cultured (for example, for 4 to 7 days) in the presence of a vascular endothelial growth factor receptor (VEGFR) activator and an inhibitor of the type I TGF-β receptor. In yet another embodiment, pluripotent stem cells can be cultured in the presence of a ROCK inhibitor and a factor belonging to the transforming growth factor β family (e.g., for 1-2 days), then cultured in the presence of a factor belonging to the transforming growth factor β family (e.g., for 1-3 days), further cultured in the presence of FGF and a factor belonging to the TGFβ superfamily (e.g., for 1-3 days), and then cultured in the presence of a vascular endothelial growth factor receptor (VEGFR) activator (e.g., for 2-7 days). In yet another embodiment, pluripotent stem cells can be cultured in the presence of a ROCK inhibitor and a factor belonging to the TGFβ superfamily (e.g., for 1-2 days), then cultured in the presence of a factor belonging to the TGFβ superfamily (e.g., for 2-4 days), and further cultured in the presence of a factor belonging to the TGFβ superfamily, a vascular endothelial growth factor receptor (VEGFR) activator, and FGF (e.g., for 2-7 days). In any of the above embodiments, the method may include a step of selecting or sorting CD31-positive and CD144 (VE-cadherin)-positive cells after culturing.Additional culture steps may be performed before or after each culture step to induce differentiation of pluripotent stem cells into CD31-positive and CD144-positive cells. Furthermore, CD31-positive and CD144-positive cells may be reseeded and cultured in expansion medium to increase the CD31 and / or CD144 positivity rate. The expansion medium may be replaced with another medium after a certain period of time. The expansion medium used for reseeding is not particularly limited, but StemPro-34SFM supplemented with VEGF-A is suitable, and Miracell (registered trademark) EC (Takara Bio) is suitable as the replacement medium. Furthermore, a ROCK inhibitor may be added for one day during reseeding. The vascular cells obtained in this process are preferably CD31-positive and CD144-positive. Furthermore, it is preferable that the expression of at least one gene selected from the group consisting of PECAM1, CDH5, KDR, and CD34 in the vascular cells is increased compared to that in the pluripotent stem cells before differentiation induction. (2) Induction of Mesenchymal Cells Mesenchymal cells can be induced from pluripotent stem cells. Any known method can be used to induce differentiation of pluripotent stem cells, such as iPS cells, into mesenchymal cells (iPSC-MCs). For example, the method described in Patent Document 1 can be used. Specifically, iPSCs were dispersed and seeded in the presence of Rho kinase, then cultured for 3 days in Medium 1 (DMEM / F12 medium, 1-2% B27, 1% Glutamax, 25 ng / mL BMP4, 8 μM CHIR 99021), followed by priming culture in Medium 2 (StemPro34-SFM, 200 ng / mL VEGF, 2 μM Forskolin) for 3-4 days, or in Medium 3 (StemPro34-SFM, 50 ng / mL VEGF) for 7 days.In one aspect, pluripotent stem cells can be cultured in the presence of a ROCK inhibitor, a β-catenin activator, and a factor belonging to the TGFβ superfamily (e.g., for 1-2 days), followed by culture in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily (e.g., for 3-5 days), culture in the presence of a PDGF receptor activator and a factor belonging to the transforming growth factor β family (e.g., for 1-4 days), and further culture in the presence of FGF and a factor belonging to the transforming growth factor β family (e.g., for 2-6 days), followed by maintenance culture in a mesenchymal cell medium (e.g., for 3-20 days). After culture, the method may include a step of selecting or sorting CD166-positive, CD31-negative cells. Additional culture steps may be included before or after each culture step in inducing differentiation of pluripotent stem cells into CD166-positive, CD31-negative cells. Examples of mesenchymal cell media include, but are not limited to, MSCGM. In another embodiment, the mesenchymal cells may be septum transversum mesenchyme (STM) cells. STM cells may be LHX2-positive and WT1-positive. STM cells have activated transcription of FOXF1, HLX1, COL4A, and ALCAM, and may be LHX2-positive, WT1-positive, and MIIA-positive. In this embodiment, pluripotent stem cells may be cultured in the presence of a ROCK inhibitor, a β-catenin activator, and a factor belonging to the TGFβ superfamily (e.g., for 1-2 days), then cultured in the presence of a β-catenin activator and a factor belonging to the TGFβ superfamily (e.g., for 3-5 days), cultured in the presence of a PDGF receptor activator and a factor belonging to the transforming growth factor β family (e.g., for 1-4 days), and further cultured in the presence of FGF and a factor belonging to the transforming growth factor β family (e.g., for 2-6 days). After the culture, a step of selecting or sorting LHX2-positive and WT1-positive cells may be included. Additional culture steps may be included before or after each culture step up to inducing differentiation of LHX2-positive and WT1-positive cells from pluripotent stem cells. (3) Induction of Macrophages Macrophages can be induced from monocytes and / or pluripotent stem cells.Monocytes may be isolated from human blood, but commercially available leukemia-derived cells such as THP-1 monocytes (available from ATCC (registered trademark)) can also be used. When inducing macrophages from monocytes, for example, undifferentiated macrophages (MO-Mac) can be obtained by culturing the monocytes in Roswell Park Memorial Institute (RPMI)-1640 medium containing 200 nM phorbol 12-myristate 13-acetate (PMA). Any known method can be used to induce macrophages from pluripotent stem cells. For example, the method described in non-patent literature (Cao X. et al., Stem Cell Rep. 2019) can be used. That is, iPSCs were dispersed and seeded using Accutase, then cultured for two days in Medium 1 (IF9S medium, 50 ng / mL BMP4, 15 ng / mL ActA, 1.5 uM CHIR, 5% PVA, 1× Lipid mix (Gibco), 1× ITS (Gibco), 0.0064% ascorbic acid, 0.004% MTG, 1% Glutamax, 1% NEAA), and then cultured for three days in Medium 2 (IF9S medium, 50 ng / mL bFGF, 50 ng / mL SCF, 50 ng / mL VEGF, 10 uM SB431542). The cells are then cultured for 4 days in Medium 3 (IF9S medium, 50 ng / mL bFGF, 50 ng / mL SCF, 50 ng / mL VEGF, 50 ng / mL EPO, 50 ng / mL IL6, 10 ng / mL IL3). After all the floating and adherent cells are collected, they are cultured for 9 to 10 days in Medium 4 (IF9S medium, 50 ng / mL IL6, 10 ng / mL IL3, 80 ng / mL MCSF). The monocytes obtained here are seeded on FBS-coated plates and further cultured for 4 days in Medium 5 (IF9S medium, 80 ng / mL MCSF) to induce macrophages. Regardless of whether the culture conditions are for monocytic cells or pluripotent stem cells, a step of selecting or sorting CD11b-positive cells after culture may be included. An additional culture step may be inserted before or after each culture step until differentiation of monocytes or pluripotent stem cells into CD11b-positive cells is induced.1-2 Co-Culture The method of this embodiment involves in vitro co-culturing of vascular cells, mesenchymal cells, pancreatic cancer cells, and macrophages. The vascular cells, mesenchymal cells, and macrophages can be the cells induced as described in 1-1(1) to (3) above. The mixing ratio of the four types of cells at the start of co-culture is not particularly limited as long as it is within the range that allows organoids to be generated. For example, the ratio of vascular cells:mesenchymal cells:pancreatic cancer cells:macrophages can be 3-15:10-40:10:5-20. In particular, the ratio of pancreatic cancer cells:macrophages is 1:0.5-2.0, preferably 1:0.7-1.5, and most preferably 1:1. It has been demonstrated that the ratio of pancreatic cancer cells (PDAC) to TAM in the organoids obtained by co-culture under the above conditions is approximately 5:1 (data not shown). The culture medium used for culturing is not particularly limited as long as it can produce organoid, but can use the culture medium for vascular cells (for example, vascular endothelial cells), the culture medium for pancreatic cancer cells, or the mixture of these two culture media.The culture medium for vascular endothelial cells can be used in any case, but for example, can use the medium containing at least one of hEGF (recombinant human epidermal growth factor), VEGF (vascular endothelial growth factor), hydrocortisone, bFGF, ascorbic acid, IGF1, FBS, antibiotics (for example, gentamicin, amphotericin B, etc.), heparin, L-Glutamine, Phenolred, BBE. Alternatively, as a medium for culturing vascular endothelial cells, EGM-2 Bullet Kit (Lonza), EGM Bullet Kit (Lonza), VascuLife EnGS Comp Kit (LCT), Human Endothelial-SFM Basal Growth Medium (Invitrogen), or Human Microvascular Endothelial Cell Growth Medium (Toyobo) may be used.Any medium for culturing pancreatic cancer cells can be used, including, for example, DMEM / F12 medium containing B27, nicotinamide, N-acetyl-L-cysteine, EGF, noggin, A83-01, gastrin-1, FGF10, Wnt3a, and R-spondin-1 (Boj S.F., et al., Cell 160, 324-338 (2015), https: / / doi.org / 10.1016 / j.cell.2014.12.021). It is preferable not to use a scaffold material when culturing organoids. The temperature during culture is not particularly limited, but is preferably 30 to 40°C, and more preferably 37°C. The culture period is not particularly limited, but is preferably 1 to 60 days, and more preferably 3 to 10 days. Most preferably, the culture period is approximately 7 days. 2. Cancer Organoids A second embodiment of the present invention is a cancer organoid. The cancer organoids of this embodiment are characterized by comprising vascular cells, mesenchymal cells, cancer cells, and macrophages. Preferably, the cancer organoids of this embodiment are organoids produced by the method described in Section "1. Method for Producing Cancer Organoids." In this embodiment, the definitions of terms, production conditions, properties of various cells, etc. are the same as those described in Section "1. Method for Producing Cancer Organoids" unless otherwise specified. The vascular cells are preferably cells induced from pluripotent stem cells. In particular, vascular endothelial cells are preferably preferred. The mesenchymal cells are preferably cells induced from pluripotent stem cells. When mesenchymal cells are cultured as organoids, they can have cancer-associated fibroblast (CAF)-like functions within the organoids and the ability to produce extracellular matrix. The macrophages are preferably induced from monocytes or pluripotent stem cells. When macrophages are cultured as organoids, they can have TAM-like functions within the organoids. The cell ratios of cancer cells (e.g., PDAC cells), CAFs, and TAMs contained in cancer organoids are not particularly limited, but are preferably within the ranges of 50-75%, 15-30%, and 3-40%, respectively.In particular, the TAM cell ratio in human PDAC is known to be approximately 5-36%. To more closely reproduce human PDAC, the TAM cell ratio in organoids is preferably also approximately 5-36%. The vascular cells, mesenchymal cells, cancer cells, and macrophages contained in cancer organoids can be derived from humans or non-human animals (e.g., laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically, mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.). It is preferable that at least one of the vascular cells, mesenchymal cells, cancer cells, and macrophages is derived from humans. Most preferably, the vascular cells, mesenchymal cells, cancer cells, and macrophages are all derived from humans. The cancer organoids of this embodiment have characteristics closer to those of actual human cancer tissue than conventional organoids, making them useful as cancer treatment models. Applications of cancer treatment models include the search for therapeutic target factors and screening for therapeutic drugs (antitumor effects, drug delivery). It can also be used to study unknown functions and cellular behaviors of cancer tissues, such as the migration of T cells within cancer tissues. 3. Drug Screening Method A third embodiment of the present invention is a drug screening method. The method of this embodiment is characterized by evaluating the effects of a drug using the cancer organoids described in Section "2. Cancer Organoids." In this embodiment, the definitions of terms, production conditions, and properties of various cells are the same as those described in Sections "1. Method for Producing Cancer Organoids" and "2. Cancer Organoids" unless otherwise specified. Drugs evaluated by the method of this embodiment are not particularly limited, but anticancer drugs and the like can be suitably applied. In particular, it is difficult to find drugs that provide sufficient therapeutic effects for pancreatic cancer, bile duct cancer, some colon cancers, and brain tumors (particularly glioblastomas), which are known to have high immunosuppressive properties. The drugs evaluated here, particularly anticancer drugs, may be drugs that target cancer cells themselves, or drugs that target TME-constituting cells (TAM, CAF, etc.).By using cancer organoids that are highly reproducible with respect to various characteristics of human cancer tissue and TME, it is possible to select drugs that have efficient anti-tumor effects in vitro. Another aspect of the method of this embodiment is characterized by including the use of the cancer organoids described in Section "2. Cancer Organoids" to evaluate the deliverability of drugs to cancer tissue. Cancer tissue, particularly pancreatic cancer, is known to have poor drug deliverability due to the large number of stromal cells, so it is also useful to select drugs with high drug deliverability in the development of therapeutic drugs. Cancer organoids that reproduce the TME of cancer tissue can also be used as an evaluation model for drug deliverability.

[0008] The present invention will be described in more detail below using examples, but the scope of the present invention is not intended to be limited to the scope of the examples. 1. Materials, Reagents, etc. PDAC tissue samples were collected from human PDAC patients at the Kanagawa Cancer Center. This study was approved by the Kanagawa Cancer Center Ethics Committee (Approval Number A160128003, 28 55, 2019-60716). The composition of each medium used in the examples is as follows: - The organoid basal medium was DMEM / Ham's F-12 medium (Nacalai Tesque) supplemented with 1% penicillin / streptomycin (Nacalai Tesque) and Glutamax (Thermo Fisher Scientific). Organoid medium was prepared by adding B-27 (Thermo Fisher Scientific), 10 mM nicotinamide (Sigma-Aldrich), 1 mM N-acetyl-L-cysteine ​​(Sigma-Aldrich), 50 ng / mL EGF (Sigma-Aldrich), 0.1 mg / mL Noggin (PeproTech), 0.5 mM A83-01 (Selleck), 0.1 mg / mL FGF10 (PeproTech), 10 nM Gastrin I (Sigma-Aldrich), 50 ng / mL Wnt3A (R&D Systems), and 0.1 mg / mL R-spondin 1 (PeproTech) to organoid basal medium. - Growth factor reduced FPCO medium (GFR medium) is a 1:1 (volume ratio) mixture of organoid basal medium and EGM (manufactured by Lonza). - FPCO medium is a 1:1 (volume ratio) mixture of organoid medium and EGM. 2 Test procedure 2-1 Preparation of fluorescently labeled PDAC The firefly luciferase gene was subcloned into the CSII-EF-MCS-EGFP plasmid (RIKEN BioResource Center) to construct the CSII-EF-Luc-IRES-EGFP plasmid. PDAC cells were transduced with CSII-EF-Luc-IRES-EGFP using a lentivirus containing the plasmid, and Luc-GFP was expressed. ＋PDAC cells were used. 2-2 Preparation of fluorescently labeled macrophages 2-2-1 Preparation from monocytes THP-1 cells were obtained from the American Type Culture Collection (ATCC (registered trademark)). CSII-EF-KuO plasmid (a plasmid obtained from the RIKEN BioResource Center into which the KuO gene had been inserted) was transfected into a lentivirus, which was then used to infect THP-1 cells and transduce the Kusabira-Orange (KuO) gene. The KuO gene was then transduced using a cell sorter. ＋ THP-1 cells were isolated. ＋THP-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol (Gibco), and 1% penicillin / streptomycin (Nacalai Tesque). THP-1 cells were cultured in RPMI-1640 medium containing 200 nM PMA (Sigma-Aldrich) for 72 hours to differentiate into M0-macrophages (M0-Mac). After differentiation, macrophages were harvested using 0.25% trypsin-EDTA and a cell scraper. M0-Mac cells were induced into M1-macrophages (M1-Mac) in a medium containing 100 nM lipopolysaccharide (LPS) (Sigma-Aldrich) and 20 ng / mL IFNβ (Biotech), and into M2-macrophages (M2-Mac) in a medium containing 20 ng / mL IL4 (Biotech) and IL13 (Biotech). Flow cytometric analysis and sorting of THP-1 cells and macrophages were performed under the following conditions: THP-1 cells and macrophages were incubated in flow cytometry buffer containing Fc block (422302, BioLegend) at 4°C for 25 minutes. After washing, the cells were resuspended in flow cytometry buffer containing a secondary antibody against anti-CD11b (M1 / 70, BioLegend) for 20 minutes at 4°C, followed by 0.1% propidium iodide (PI) staining. A BD Celesta™ flow cytometer was used, and data were analyzed using FlowJo software, version 10. 2-2-2 Preparation from hiPSCs. KuO-labeled hiPSCs were prepared by introducing a KuO knock-in reporter into the AAVS1 region of hiPSCs (RIKEN BioResource Center CELL BANK) (see Takebe T. et al., Nature protocol, Vol. 9, No. 2: 396-409 (2014)).KuO-labeled hiPSCs were isolated using Accutase and cultured in Medium 1 (IF9S medium, 50 ng / mL BMP4, 15 ng / mL ActA, 1.5 μM CHIR, 5% PVA, 1× Lipid mix (Gibco), 1× ITS (Gibco), 0.0064% ascorbic acid, 0.004% MTG, 1% Glutamax, 1% NEAA) for 2 days, followed by culture in Medium 2 (IF9S medium, 50 ng / mL bFGF, 50 ng / mL SCF, 50 ng / mL VEGF, 10 μM SB431542) for 3 days. The cells were then cultured for 4 days in Medium 3 (IF9S medium, 50 ng / mL bFGF, 50 ng / mL SCF, 50 ng / mL VEGF, 50 ng / mL EPO, 50 ng / mL IL6, 10 ng / mL IL3). After collecting all the floating and adherent cells, they were cultured for 9–10 days in Medium 4 (IF9S medium, 50 ng / mL IL6, 10 ng / mL IL3, 80 ng / mL MCSF). The resulting cells were seeded onto FBS-coated plates and further cultured for 4 days in Medium 5 (IF9S medium, 80 ng / mL MCSF) to obtain pluripotent stem cell-derived macrophages (iMacs). 2-3 Induction of hiPSC-ECs. hiPSCs were isolated using Accutase, suspended in StemFit® containing 10 μM Y-27632, and plated on laminin 511 E8 fragment (iMatrix-511™, Nippi). The next day, the medium was replaced with a priming medium consisting of B27 medium (a 1:1 mixture (volume ratio) of DMEM and F12 medium, containing 1% Glutamax and 1% B27 (Life Technologies)), 8 μM CHIR99021 (Tocris Bioscience), and 25 ng / mL BMP4 (R&D Systems). After another 3 days, the priming medium was replaced with an EC induction medium consisting of StemPro-34 SFM medium (Life Technologies) supplemented with 200 ng / mL VEGF (Life Technologies) and 2 μM forskolin (Sigma-Aldrich). The EC induction medium was replaced every day.On day 7 after the initiation of differentiation, ECs were detached with 0.05% trypsin and subjected to flow cytometry analysis, which confirmed typical endothelial morphology with junctional localization of CD144 and CD31. ２ ECs were cultured at a density of 50,000 cells / cm in an EC expansion medium consisting of StemPro-34 SFM supplemented with 50 ng / mL VEGF-A on a dish coated with 100 μg of fibronectin (Sigma-Aldrich). ２The hiPSCs were seeded at a density of 10 μM and cultured. EC expansion medium was changed every other day. 2-4 Induction of hiPSC-MCs. hiPSCs were dissociated using Accutase and plated on laminin-511 E8 fragments in StemFit® containing 10 μM Y-27632 for 4-6 days. During the MC induction phase, the maintenance medium was replaced with MC induction medium (a 1:1 mixture of DMEM:F12 containing 1% Glutamax and 1% B27, containing 8 μM CHIR99021 and 25 ng / mL BMP4), followed by exposure to 2 ng / mL activin A and 10 ng / mL PDGFBB (R&D Systems) for 3 days. After 3 days, the MC induction medium was replaced with MC induction medium consistent with StmePro-34 MC medium supplemented with 10 ng / mL FGF2 and 10 ng / mL PDGFBB, and cultured for 3 days. 2-5 Construction of Fused Pancreatic Cancer Organoids (FPCO) Containing Macrophages 2-5-1 Preparation of FPCO Using Monocyte-Derived Macrophages (M0-Mac) Fluorescently labeled PDAC cells (cell ratio 21%) constructed in 2-1 and KuO-labeled M0-Mac (KuO-M0-Mac) (cell ratio 21%) constructed in 2-2-1 were suspended in GRM medium containing 10 μM Y-27632 together with hiPSC-ECs (cell ratio 15%) and hiPSC-MCs (cell ratio 43%), and the suspension was seeded into a 24-well ultra-low attachment plate (Corning Elplasia) and co-cultured for 24 hours. The multicellular spheroids formed by co-culture were collected and resuspended in 5 mL of the same medium. They were then dropped onto cell culture inserts (Greinar) for air-liquid interface culture. MO-FPCO cells were cultured in FPCO medium for 24 hours, then cultured in GRM medium for 5 days to form MO-FPCO cells (Figure 1). As a control, FPCO cells were cultured under the same conditions except for the absence of KuO-MO-Mac.2-5-2 Preparation of FPCO using hiPSC-derived macrophages (iMac). Fluorescently labeled PDAC cells (21% cell ratio) constructed in 2-1 and iMACs (21% cell ratio) constructed in 2-2-2 were suspended in GRM medium containing 10 μM Y-27632 together with hiPSC-ECs (15% cell ratio) and hiPSC-MCs (43% cell ratio). The suspension was seeded into a 24-well ultra-low attachment plate (Corning Elplasia) for 24 hours. The multicellular spheroids formed by the co-culture were collected, resuspended in 5 mL of the same medium, and then placed dropwise onto cell culture inserts (Greinar) for air-liquid interface culture. After culturing iMac-FPCO in FPCO medium for 24 hours, they were cultured in GRM medium for 5 days to form iMac-FPCO (Figure 1). 2-5-3 Preparation of Macrophage-Free FPCO As a control, fluorescently labeled PDAC cells (cell ratio 27%), hiPSC-ECs (cell ratio 19%), and hiPSC-MCs (cell ratio 54%) were cultured in the absence of macrophages, and cell culture was performed under the same conditions as in 2-5-1 to construct FPCO. 2-5-4 Preparation of MC- and EC-Free Pancreatic Cancer Organoids (PCO) As a control, pancreatic cancer organoids (PCO) free of macrophages, MCs, and ECs were prepared using the following procedure. Pancreatic cancer surgical specimens were minced into 1-2 mm pieces using surgical scissors. The minced tumor fragments were subjected to enzymatic reaction and cell dispersion using a Tumor Dissociation Kit (Miltenyi Biotech) in combination with gentleMACS™ Dissociators (Miltenyi Biotech). The resulting cell suspension was treated with MACS™ SmartStrainers, washed with DMEM / F12 (Thermo Fisher Scientific) containing 1x Glutamax and 1% Primocin (Invivogen), and then suspended and seeded in Matrigel growth factor reduced (Corning). These were cultured in organoid medium for 3-14 days to generate PCO. The PCO generated by the above method was maintained in organoid medium and passaged approximately every 7 days.The following treatments were performed during the passaging of PCO and the preparation of FPCO. The Matrigel / PCO suspension was detached from the culture vessel using a cell scraper and incubated in TrypLE Express (Thermo Fisher Scientific) for 8 minutes at 37°C to dissociate the gel. The cells were then further incubated in Accutase at 37°C for 1 hour to disperse them into aggregates of 3-4 cells. During this time, the cells were dispersed by pipetting every 10 minutes. After cell dispersion, the PCO was either suspended in Matrigel and passaged, or suspended in organoid basal medium and used to prepare FPCO, MO-FPCO, and iMac-FPCO. 2-6 Immunofluorescence Staining and Histological Analysis FPCO, MO-FPCO, and iMac-FPCO were fixed with 4% paraformaldehyde (PFA) and either paraffin-embedded or frozen in phosphate-buffered saline (PBS) containing 15% sucrose and 7.5% gelatin. Each block was cut into 5 μm-thick sections and stained with hematoxylin-eosin (H&E) and immunofluorescent markers using standard histological methods. Antigen retrieval was performed in citrate buffer (pH 6.0) at 120°C for 12 minutes. After permeabilization with PBS containing 0.05% Tween 20 (Nacalai Tesque) and blocking with serum-free liquid blocking protein (Dako), primary antibodies were added and incubated for 16 hours at 4°C. After washing, secondary antibodies were added and incubated at room temperature for 1 hour. After the secondary antibody reaction, the samples were washed and mounted with Apati mounting medium (Fujifilm) containing 0.1% DAPI (Nacalai Tesque).The following antibodies were used as primary antibodies for immunofluorescence staining: anti-cytokeratin 19 (MBS423230, 1:200, MyBiosource), anti-CD11b (101,201, 1:100, BioLegend), anti-CD163 (ab156769, 1:200, Abcam), anti-CD80 (ab134120, 1:400, Abcam), anti-CD31 (M0823, 1:100, Dako), anti-Ki67 (ab16667, 1:200, Abcam), and anti-cleaved caspase 3 (CST-9661, 1:200, Cell Signaling). The primary antibodies used were anti-pMLKL (MAB9187-SP, 1:200, R&D Systems), and anti-pMLKL (MAB9187-SP, 1:200, R&D Systems). The primary antibodies were diluted 100-400 times with Protein Block Serum-Free Ready-to-use (#X0909, Dako). The following antibodies were used as secondary antibodies: goat anti-mouse IgG1 cross-adsorbed secondary antibody, Alexa Fluor™ 555 (Alexa Fluor™ A21127); goat anti-mouse IgG2a cross-adsorbed secondary antibody, Alexa Fluor™ 488 (Alexa Fluor™ A211008); goat anti-rat IgG2b cross-adsorbed Alexa Fluor™ 647 (anti-rat IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor™ 488 (Alexa Fluor, A21208). The secondary antibodies were prepared by pre-cleaving the above antibodies using Protein Block Serum-Free. The sample was diluted 400-fold with Ready-to-use (#X0909, Dako) and used. Quantification was performed using ImageJ software (https: / / ImageJ.nih.gov / ij / ). As controls, formalin-fixed, paraffin-embedded human PDAC tissue samples provided by medical institutions and a commercially available human pancreatic cancer tissue array (PA1003, Biomax) were used, and immunofluorescence staining and histological analysis were performed in the same manner as for M0-FPCO.2-7 Whole-mount immunofluorescence staining. Organoids (FPCO, M0-FPCO, and iMac-FPCO) were fixed overnight with 4% paraformaldehyde (PFA). During this time, the organoids were delipidated and enhanced in clarity by shaking for 24 hours at 37°C in a solution containing 10% CHAPS (Nacalai Tesque) and 25% N-methyldiethanolamine (NMDEA) (Sigma-Aldrich). After washing three times with 0.1% Tween-PBS (PBST), the organoids were immersed in serum-free blocking protein solution (Dako) and incubated at room temperature for 2 hours. Primary antibody, anti-CD31 antibody (M0823, 1:100, Dako), was added and incubated for 5 days at 4°C on a shaker. The organoids were then washed for 24 hours in PBST. Subsequently, a secondary antibody, goat anti-mouse IgG1 cross-adsorbed secondary antibody, Alexa Fluor™ 555 (A21127, 1:500), was added and incubated on a shaker at 4°C for 2 days. The sections were then washed with PBST three times on ice for 60 minutes each. Sytox Green (S34862, 1:1000, Invitrogen) was added to detect cell nuclei. The sections were dehydrated by serial dilution with methanol, and the tissue was permeabilized using the BABB method (see, e.g., Yokomizo T., et al., Nat. Protoc. 7:421-31 (2012)) using a 1:1 mixture of benzyl benzoate (Sigma-Aldrich) and benzyl alcohol (Tokyo Chemical Industry Co., Ltd.). Tissue imaging was performed using a Leica SP8 (Leica) confocal microscope and an Olympus FVMPE-RS (Olympus) multiphoton microscope. 3D rendering was performed using Imaris software. 2-8 Comparison of anticancer drug resistance of organoids. PCO, FPCO, MO-FPCO, and iMac-FPCO were cultured in organoid medium until day 7, and then cultured for 3 days with the addition of 0, 0.3, 1.0, or 3.0 μM gemcitabine. The number of viable cancer cells in each medium was then quantified. 2-9 Sorting by flow cytometry. KuO in MO-FPCO or FPCO. ＋ Macrophages and GFP ＋Cancer cells were separated by flow cytometry. MO-FPCO cells were detached by treatment with Accumax (M&S TechnoSystems) for 2 hours and incubated with SYTOX™ Blue (Invitrogen). Using the expression of KuO and GFP as markers, KuO was detected using a flow cytometry Aria III Special Order Research Product (BD Bioscience). ＋ Macrophages and GFP ＋ Cancer cells were selected. 2-10 Real-time quantitative polymerase chain reaction (qPCR) Total RNA was extracted from macrophages in M0-FPCO using the PureLink™ RNA Mini Kit (Invitrogen) and the PureLink™ RNA Micro Kit (Invitrogen). cDNA was prepared using the total RNA as a template with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). qPCR was performed using the THUNDER BIRD Probe qPCR Mix (Toyobo) and the Universal ProbeLibrary System (Roche). qPCR was performed in a 96-well plate on a CFX96 Real-Time System (Bio-Rad) using a program provided by Eurofins Genomics and custom-designed primers. The expression level of each gene was calculated as a relative value normalized to the signal of 18S ribosomal RNA (18S rRNA) expression under the same conditions. For control, qPCR was also performed under the same conditions on THP-1 cells and macrophages before FPCO fusion. Table 1 shows the sequences of all primers used in RT-qPCR. 2-11 Enzyme-linked immunosorbent assay (ELISA) and cytokine array. To reduce FBS bias, FPCO and MO-FPCO differentiation media were cultured in GRM medium for 1 week before switching to organoid basal medium. After 24 hours, the differentiation media were assayed using a human osteopontin enzyme immunoassay kit (K021H1, Arbor Assays) and a human XL cytokine array kit (ARY022B, R&D Systems). ELISA data were analyzed using Arigo's ELISA Calculator (https: / / www.arigobio.com / ELISA-calculator). After 24 hours, cytokine array data for the differentiation media of FPCO and MO-FPCO were further acquired using a ChemiDoc™ MP Imaging System (Bio-Rad). 2-12 Extended Culture: FPCO, MO-FPCO, and iMac-FPCO prepared in 2-5 were extended cultured. FPCO and MO-FPCO were cultured in organoid medium until day 7, and then in growth factor-reduced FPCO medium (GFR) without EGF or FBS from day 7 to day 14 (Figure 2A). iMac-FPCO was cultured in organoid medium until day 14 (Figure 2B). 2-13 Luciferase Assay: MO-FPCO was sampled for luciferase assay on days 7, 10, and 14 after the initiation of extended culture. MO-FPCO was completely lysed in 1x Glo Lysis Buffer (Promega) using a QIAGEN Tissue Lyser LT (Qiagen). Luciferase assays were performed using the One-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions.2-14 Bulk RNA Sequencing. RNA-seq libraries were generated using 8 ng of total RNA from cancer cells in FPCO, MO-FPCO, and iMac-FPCO, and 40 ng of THP-1, MO-Mac, and M1 / M2 macrophages, as well as macrophages in MO-FPCO and iMac-FPCO, using the Ion Ampliseq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. For iMac-FPCO on day 7 of culture, cancer cells and macrophages on day 14 of culture, and MO-FPCO and iMac-FPCO on day 14 after construction, 40 ng and 100 pg of total RNA were generated, respectively. Data were analyzed using GeneSpring (Agilent Technologies). Genes that were significantly differentially expressed among THP-1 cells, M1 / M2 macrophages, M0 macrophages, M0 macrophages (day 7), and FPCO macrophages were identified by one-way analysis of variance (ANOVA), followed by Tukey's honestly significant difference test and Benjamini-Hochberg false discovery rate. RNA analysis was also performed on cancer cells in FPCO and M0-FPCO, and on cancer cells in iMac-FPCO at days 7 and 14 of culture. Gene Set Enrichment Analysis (GSEA) was performed using software from the Broad Institute (https: / / www.GSEA-msigdb.org / GSEA / index.jsp), and Gene Ontology (GO) analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (https: / / david.ncifcrf.gov / ). 2-15 Library Construction for Single-Cell RNA Sequencing For single-cell RNA sequencing, MO-FPCO and iMac-FPCO were digested in a single-cell suspension containing collagenase (Sigma-Aldrich) and Pronase (Roche).RNA libraries were constructed using the Chromium NEXT GEM Single Cell 3' Kit v3.1 (PN-1000130, 10x Genomics), the Chromium NEXT GEM Chip G Single Cell Kit (PN-1000127), and the Chromium NEXT GEM Single Cell 3' v3.1 Gel Beads (PN-1000129, 10x Genomics). High-sensitivity quality control was performed using a D5000 ScreenTape (Agilent Technologies), and sequencing was performed on a NovaSeq6000 (Illumina) with a 150-bp paired-end read. Raw data processing was performed using Cell Ranger (10x Genomics). 2-16 Data Processing for Single-Cell RNA-Seq Data Single-cell RNA-seq data were analyzed using R v4.3.1 and the Seurat v4.3.0.1 package. FPCO single-cell RNA-seq data were filtered by the number of detected features (>500), the number of detected counts (>1500), and the proportion of mitochondrial genes (<25%). Data were processed using UMAP at a size of 1:20, a resolution of 0.5, and visualization was performed using ITGAM for FPCO-macrophage clusters. ＋ F4 / 80 ＋Cells were extracted using Cell Selector, an arbitrary Seurat object. The FPCO-macrophage subset was processed and reclustered using UMAP with a scale of 1:20 and a resolution of 0.5. 2-17 Analysis using public PDAC datasets. Kaplan-Meier log-rank tests for patient survival were obtained using Kaplan-Meier Plotter (https: / / www.kmplot.com). The human PDAC dataset and corresponding clinical data were obtained from The Cancer Genome Atlas (TCGA) (177 cases). Data on myeloid cells in human PDAC were downloaded from the National Center of Biotechnology Institute Gene Expression Omunibus (NCBI GEO) (Accession No. GSE205013) and used for integrated analysis. From the GSR0005013 dataset, data from P04 (GSM6204112), P05 (GSM6204113), P08 (GSM6204116), and P23 (GSM6204131), which represent characteristics of resected, non-preoperative chemotherapy, classical subtype tissue samples, were used. The TAM RNA dataset in human PDAC was published by Carroni N. et al., Nature, 623(7986), pp. 211-218. 415-422 (2023). Data were integrated using UMAP after removing batch effects, filtered, and then clustered. 2-18 Statistical Analysis All quantitative data from the above tests were performed with n = 3 or more, and data were expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software). Pairwise tests between more than two groups of data were performed using one-way ANOVA with Tukey's post-hoc test. For comparisons of data between two groups, an unpaired Student's t-test was performed. In all cases, "*" means p < 0.05, "**" means p < 0.01, "***" means p < 0.001, and "****" means p < 0.0001; ns = not significant.2-19 Data Deposition The raw data from bulk and single-cell RNA sequencing of M0-FPCO were deposited with GEO under accession number GSE249673. 3 Results 3-1 Example 1 Preparation of PDAC Organoids Containing Macrophages (1) Macrophage Induction Before co-culture of macrophages with PDAC cells, THP-1 cells were labeled with KuO using lentivirus, followed by macrophage induction. KuO-labeled THP-1 cells were isolated by fluorescence-activated cell sorting (flow cytometry). THP-1 cells were cultured in RPMI-1640 medium containing 200 nM PMA for 72 hours to differentiate into M0-macrophages (M0-Mac). Differentiation into M0-Mac was confirmed by RT-PCR, which measured the increased expression of macrophage markers ITGAM / CD11b and CD36 (Figure 3). Differentiated M0-Mac cells were harvested using 0.25% trypsin-EDTA and a cell scraper. M0-Mac cells were induced to M1-macrophages (M1-Mac) in a medium containing 100 nM LPS and 20 ng / mL IFNβ (Biotech), and to M2-macrophages (M2-Mac) in a medium containing 20 ng / mL IL4 (Biotech) and IL13 (Biotech), as confirmed by quantitating the expression levels of the respective marker genes using RT-PCR (Figure 4). (2) Preparation of Macrophage-Containing Organoids. M0-Mac and iMac were labeled with KuO and then co-cultured with PDAC cells (labeled with luciferase and GFP), hiPSC-derived mesenchymal cells, and endothelial cells (hiPSC-MC and hiPSC-EC) in ultra-low attachment plates for 24 hours to form multicellular spheroids. The cells were then cultured at an air-liquid interface for 6 days. H&E staining confirmed that both FPCOs effectively reproduced the dense stroma structure characteristic of PDAC. Furthermore, fluorescence microscopy confirmed that KuO-M0-Mac and KuO-iMac-FPCOs were distributed throughout the FPCO, based on the dispersion of the KuO signal (Figure 5).(3) Comparison of organoid anticancer drug resistance For PCO, FPCO, and M0-FPCO, the number of viable PDAC cells was counted after 3 days of culture in the presence of 0, 1, 3, 10, and 30 μM gemcitabine. Similarly, for PCO, FPCO, and iMac-FPCO, the number of viable PDAC cells was counted after 3 days of culture in the presence of 0, 0.3, 1, and 3 μM gemcitabine. Each relative value was calculated, with the number of viable cells at 0 μM gemcitabine set to 1. The results for M0-FPCO are shown in Figure 6A, and the results for iMac-FPCO are shown in Figure 6B. In PCO, the number of viable cells decreased in a gemcitabine concentration-dependent manner, whereas in FPCO and M0-FPCO, no significant decrease in the number of viable cells was observed, and the number of viable cells in FPCO and M0-FPCO was almost the same (Figure 6A). Similarly, the number of viable cells in iMac-FPCO was comparable to that in FPCO (Figure 6B). This indicates that FPCO, M0-FPCO, and iMac-FPCO all have higher resistance to anticancer drugs compared to PCO. (4) Comparison of M0-FPCO and Human PDAC Tissue The characteristics of macrophages were compared based on the results of immunofluorescence staining of M0-FPCO and human PDAC tissue obtained from patients. Macrophages are classified into an inflammatory type known as "M1-like" and an immunosuppressive type known as "M2-like." Here, we focused on CD11b. ＋ CD80 ＋ Cells were differentiated into M1-like TAMs, CD11b ＋ CD163 ＋The cells were identified as M2-like TAMs. The results confirmed that M2-like TAMs were more abundant than M1-like TAMs. This was also confirmed in human PDAC tissues (Figure 7). These results indicated that M0-Mac differentiated into M2 type in M0-FPCO, and that M0-FPCO closely reproduced the infiltration of M2-like TAMs in human PDAC tissues. (5) Gene Expression Analysis of KuO-Labeled M0 Macrophages. For gene expression analysis, KuO-labeled macrophages (FPCO-Macs) were isolated from FPCO and M0-FPCO by flow cytometry. RT-qPCR revealed that macrophages in M0-FPCO expressed lower levels of M1 markers, such as CD80, CXCL10, and TNFA, compared with cytokine-induced M1-Mac, and higher levels of M2 markers, such as CD163, MRC1 / CD206, IL10, and CCL17, compared with M0-Mac (Figure 9). On the other hand, FPCO-Mac did not display the gene expression pattern typically seen in cytokine-induced M2-Mac. These results suggest that FPCO-Macs represent a unique cell population distinct from either M1- or M2-Mac. (6) Analysis of Cellular Proportions. The constituent cell profiles of the formed M0-FPCO were analyzed using single-cell RNA sequencing data. The results showed that the abundance ratios of PDAC cells, CAFs, and TAMs were 65%, 23%, and 12%, respectively. This was within the range of TAM cell ratios typically found in human pancreatic cancer tissue (5-36%). 3-2 Example 2: Functional Analysis of Macrophages in FPCO (1) Analysis of Bulk RNA Sequencing Data. To confirm that FPCO-Macs possess the characteristics of cytokine-induced M2-Macs, the gene expression profile of FPCO-Macs obtained by bulk RNA sequencing was compared with that of cytokine-induced M1-Macs, M2-Macs, M0-Macs, and THP-1 cells (Figure 10). A principal component analysis plot generated from the bulk RNA sequencing data indicated that FPCO-Macs possessed distinct characteristics from M2-Macs and M0-Macs (Figure 11).As a control, M0-Mac was exposed to organoid culture medium for 7 days, and it was confirmed that the gene expression profile was similar to that of M0-Mac. This indicated that FPCO-Mac possesses the characteristics of FPCO-TME. GSEA of FPCO-Mac confirmed that the expression of angiogenesis and hypoxia was increased in FPCO-Mac, while inflammatory responses such as INFγ responses were decreased (Figure 12). Furthermore, GO analysis confirmed that, compared to M0-Mac, angiogenesis genes were increased, while inflammatory genes were decreased (Figure 13). Furthermore, analysis of differentially expressed genes (DEGs) comparing FPCO-Mac and M0-Mac showed increased expression of vascular development genes and decreased expression of immune response-related genes in FPCO-Mac (Figure 14). (2) Cytokine Array Cytokine array results confirmed that chitinase 3-like protein 1 (CHI3L1) and osteopontin (OPN) were increased in M0-FPCO compared to FPCO (Figure 15), suggesting that CHI3L1 and OPN are secreted from TAMs. These cytokines have been reported to correlate with angiogenesis and cancer cell proliferation in tumors (Zhao T, et al., Signal Transduct Target Ther. 2020; Vol. 5: 201, Zhao H, et al., Cell Death Dis. 2018; 9: 356). The results of GO analysis and cytokine array revealed that FPCO-Mac is a cell with enhanced angiogenic function. (3) Immunofluorescence staining and whole-mount immunofluorescence staining. Because FPCO-Mac suggested enhanced angiogenesis, we examined the vascular structure in M0-FPCO and iMac-FPCO using immunofluorescence staining and whole-mount immunofluorescence staining. Compared to FPCO, M0-FPCO and iMac-FPCO contained more CD31-positive cells, revealing the formation of an interconnected network (Figure 14). On the other hand, compared to FPCO, the microvascular structure in M0-FPCO was not maintained as a tubular structure (Figure 17).Transmission electron microscopy also showed an increase in incomplete vascular-like structures in M0-FPCO compared to FPCO. It has been reported that increased expression of angiogenesis-related genes promotes angiogenesis in PDAC tissue, but the structure of tumor microvessels is fragile (DuFort CC, et al., Gastroenterology, Vol. 150: 1545-1557 (2016), Annese T, et al., Cancers, Vol. 11: 381 (2019)). This suggests that the characteristics of PDAC-TME may be more faithfully reproduced in M0-FPCO. 3-3 Example 3: Evaluation of angiogenesis and cancer cell survival function of FPCO-Mac (1) Gene expression analysis of cancer cells in M0-FPCO. We investigated the possibility that TAM affects the characteristics or survival of cancer cells. GFP-labeled cancer cells were isolated from FPCO and MO-FPCO cells using flow cytometry (Figure 8). GSEA based on bulk RNA sequencing data revealed increased expression of G2M checkpoint, E2F-related genes, and epithelial-mesenchymal transition genes in MO-FPCO cells (Figure 18). DEG analysis comparing FPCO and MO-FPCO cancer cells revealed increased expression of cell proliferation-related genes and endothelial cell-derived genes in the presence of macrophages (Figure 19). The increased expression of endothelial cell-derived genes in cancer cells may be due to a direct effect of macrophages, as well as an indirect mechanism of angiogenesis promotion via cancer cells. Furthermore, PDAC cells were shown to be more proliferative in the presence of TAMs. To examine whether PDAC cells acquire enhanced proliferation potential in the presence of macrophages, MO-FPCO cells were cultured in organoid medium for 1 week, followed by another week in cytokine-free GFR medium. In the absence of exogenous cytokines (days 7-14), the appearance of FPCO and MO-FPCO was similar, but the number of cancer cells in FPCO decreased between days 10 and 14, whereas no decrease was observed in MO-FPCO (Figure 20).The same study was performed using PDAC cells derived from two donors (patients 1 and 2), and it was confirmed that similar results were obtained in both cases. (2) Analysis by immunofluorescence staining Immunofluorescence staining revealed that Ki67 was significantly higher in M0-FPCO cells than in FPCO cells. ＋ CK19 ＋It was clear that many PDAC cells were present (Figure 21A). In FPCO, PDAC cells were sparsely present in the center, confirming the occurrence of cell death (necroptosis). Cleaved caspase 3-positive cells were observed in both FPCO and MO-FPCO, but the number of PDAC cells positive for phosphorylated mixed lineage kinase domain-like protein (pMLKL), a necrosis marker, was observed to be reduced in MO-FPCO compared to FPCO (Figure 21B). These results indicated that FPCO-Mac contributes to both PDAC cell proliferation and survival. 3-4 Example 4: Analysis of the Expression of Cell Survival-Related Factors in PDAC Cells in MO-FPCO. GFP-labeled cancer cells were analyzed using flow cytometry for MO-FPCO and FPCO on day 14 after the start of culture. The results confirmed that the number of PDAC cells isolated from MO-FPCO was higher than that of FPCO (Figure 22). Comparison of FPCO and M0-FPCO PDAC cells by GO analysis showed increased expression of cell cycle-related genes in M0-FPCO (Figure 23). Meanwhile, GSEA showed increased expression of Myc-target_V1 and MTORC1-related genes in M0-FPCO PDAC cells (Figure 24). These results demonstrated that TAM influences PDAC cell proliferation and survival in M0-FPTO. Comparison of gene expression profiles between M0-FPCO and FPCO PDAC cells on days 7 and 14 after the start of culture revealed increased expression of 61 genes on day 7 and 378 genes on day 14 in the presence of TAM. Of these, 32 genes whose expression levels increased on both days 7 and 14 were identified (Figure 25). TCGA datasets confirmed that 13 of these genes were associated with poor prognosis in PDAC patients. 3-5 Example 5 Diversity of tumor-associated macrophages in M0-FPCO M0-FPCO on day 14 after the start of culture was analyzed by flow cytometry to determine the diversity of CD11b ＋Cells were sorted as TAMs and subjected to cluster analysis, revealing that they could be classified into five subpopulations, including SPP1, C1QC, TREM2, proliferative, and epithelial-like TAMs (Figures 26 and 27). These subpopulations have also been identified in patient-derived PDAC tissues. All clusters, except for SPP1-TAM, expressed the M2 marker CD163. Meanwhile, the M1 marker CD80 was expressed only in a small number of cells, preventing classification of these clusters using an M1 / M2 signature (Figures 28 and 29). SPP1 expression was primarily observed in macrophages (Figure 30), particularly in cluster 2 (Figure 31). ELISA analysis of osteopontin levels in the culture media of FPCO and M0-FPCO confirmed that osteopontin levels were significantly higher in M0-FPCO (Figure 32). Because osteopontin is a translation product of SPP1, our results support the hypothesis that SPP1-TAM is highly expressed in MO-FPCO. This finding is consistent with the cytokine array results (Figure 17). Furthermore, we performed an integrated analysis of myeloid cells using a human PDAC database (Werba G, et al., Nat. Commun. Vol. 14: 797 (2023)) and the MO-FPCO analysis results. Both PDAC-TAM and MO-FPCO were divided into eight clusters (Figure 33). On the other hand, PDAC-TAM also contained dendritic cells and myeloid suppressor cell populations not present in MO-FPCO. GSEA results confirmed that SPP1-TAM and C1QC-TAM exhibited gene expression patterns similar to those reported in PDAC. These results confirmed that the formed organoids accurately reproduced the diversity of TAMs present in human PDAC tissue. To investigate the effects of diverse TAMs on cancer cells, cluster analysis of PDAC cell populations in M0-FPCO and FPCO was performed. In M0-FPCO, the proportion of cluster 4 increased, and clusters 3 and 6 emerged (Figure 34). GO analysis revealed that cluster 4 was associated with cell proliferation. Ki67 ＋Cells were observed in all clusters, but were particularly concentrated in cluster 4. GO analysis suggested that clusters 3 and 6 were cell populations with high expression of genes involved in the regulation of RAS protein signaling and small GTPase-mediated signaling, respectively. This has previously been reported to be related to the development and progression of PDAC (Yoshimachi S., et al., Cancer Sci., 112: 3064-3073 (2021); Razidlo GL, et al., J. Biol. Chem., 293: 11143-11153 (2018)). These results suggest that PDAC clusters 3, 4, and 6, which increase or appear in the presence of FPCO-Mac, may contribute to the proliferation and survival of PDAC cells. 3-6 Example 6 Analysis of Tumor-Associated Macrophages in iMac-FPCO Single-cell RNA sequencing results for iMac-FPCO on day 14 after the start of culture were plotted using UMAP (Figure 35). The RNA data expressed by TAMs in iMAC-FPCO (Figure 36A) was integrated with the RNA dataset of TAMs in human PDAC (Carroni N. et al., Nature, 623(7986), pp. 415-422(2023)) (Figure 36B) (Figure 36C). RNAs expressed in iMAC-FPCO on days 7 and 14 of culture were extracted (Figure 36D). In Figure 36, the area surrounded by a red dashed line indicates the expression of RNAs characteristic of tissue-resident macrophages (TRM). As shown in Figure 36D, expression of RNA characteristic of TRM (e.g., FOLR2) was observed in many of the iMac-FPCO cells after culture. Furthermore, it was confirmed that a cell population containing a variety of cells was formed by day 14 of culture compared to day 7 of culture. 3-7 Example 7 Analysis of Cancer Cells in iMac-FPCO The results of single-cell DNA sequencing of cancer cells in iMac-FPCO cells on days 7 and 14 of culture were plotted using UMAP (Figure 37). It was confirmed that the abundance ratio of Cluster 3 increased from 1% to 19% between days 7 and 14 of culture. GO analysis confirmed that expression of cell proliferation regulatory genes was reduced in Cluster 3 (Figure 38).Furthermore, it was confirmed that the expression of KRT19, EGLN3, SLC2A1, LDHA, and MUC5B was enhanced compared to other clusters, indicating that the cancer cells forming cluster 3 form a cancer stem cell-like (basal type PDAC cell-like) cell population.

[0009] The present invention relates to a cancer organoid that can be used as a model for pancreatic ductal carcinoma (PDAC), and can be used for PDAC treatment methods, predicting the effectiveness of therapeutic drugs, screening, etc. The present invention can be used primarily in the medical and pharmaceutical manufacturing fields. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for producing cancer organoids, comprising co-culturing vascular cells, mesenchymal cells, cancer cells and macrophages in vitro.

2. The method of claim 1, further comprising inducing macrophages from monocytes and / or pluripotent stem cells prior to the co-culture.

3. The method according to claim 1, further comprising inducing vascular cells and mesenchymal cells from pluripotent stem cells, respectively, prior to the co-culture.

4. The method according to claim 2 or 3, wherein at least one of the cancer cells, pluripotent stem cells and monocyte cells is derived from a human.

5. The method according to claim 2 or 3, wherein the pluripotent stem cells are at least one cell selected from the group consisting of induced pluripotent stem cells and embryonic stem cells.

6. The method of claim 1, which does not involve the use of a scaffold material.

7. Cancer organoids containing vascular cells, mesenchymal cells, cancer cells and macrophages.

8. The cancer organoid of claim 7, wherein the macrophages are derived from monocytes and / or pluripotent stem cells.

9. The cancer organoid of claim 7, wherein the vascular cells and mesenchymal cells are each induced from pluripotent stem cells.

10. The cancer organoid of claim 8 or 9, wherein at least one of the cancer cells, pluripotent stem cells, and monocyte cells is derived from a human.

11. A cancer organoid produced by the method according to any one of claims 1 to 6.

12. A drug screening method, comprising evaluating the effect of a drug using the cancer organoid described in any one of claims 7 to 9.

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