JP6956398B2 - Screening method for anticancer drugs using cancer organoids - Google Patents

Description

The present invention relates to a method for constructing a structure (cancer organoid) similar to human cancer tissue in vitro, and an in vitro anticancer drug targeting cancer stem cells using the cancer organoid. Regarding the screening method.

Cancer stem cells are considered to be responsible cells for cancer metastasis, recurrence, and treatment resistance, and are deeply involved in the poor prognosis of cancer patients. Therefore, the development of anticancer drugs targeting cancer stem cells is required, but high-throughput screening (HTS) because cancer stem cells form a very small cell population in cancer tissues. It is difficult to obtain a sufficient amount of cancer stem cells. Cancer cell lines that have been conventionally used as a scale-up capable cell material that can withstand HTS do not have the ability to construct tissues similar to human cancer tissues. Although a certain degree of reality is observed in cancer organoids derived from patient tissues, it is not possible to obtain a sufficient amount of cancer stem cells capable of constructing organoids and non-stem cell cancer cells to be compared and controlled (Non-Patent Document 1, Non-Patent Document 1, 2).

Takebe et al. Have reported that by co-culturing human liver cancer cell lines with endothelial cells and mesenchymal stem cells, cancer organoids can be formed by self-organization (Non-Patent Document 3). The similarity between the obtained tissue and human cancer tissue is not clear, and cancer stem cells, which are the main constituents of tissue construction, have not been obtained.

On the other hand, the present inventors have recently succeeded in inducing cancer stem cell-like cells by introducing OCT3 / 4, SOX2, and KLF4 into human colon cancer or lung cancer cell lines, and induce these cells. It was named type cancer stem cell (iCSC) (Patent Document 1, Non-Patent Document 4). Inducible colorectal cancer stem cells have higher drug excretion capacity than the original cancer cells and well mimic the structure of human colorectal cancer tissue in vivo. However, it is not clear whether induced cancer stem cells can form a structure similar to cancer tissue when cultured in vitro, as in vivo.

International Publication No. 2015/199088

Vaira, V. et al., Proc. Natl. Acad. Sci. USA (2010) 107 (18): 8352-8356 van de Wetering, M. et al., Cell (2015) 161: 933-945 Takebe, T. et al., Cell Stem Cell (2015) 16: 556-565 Oshima, N. et al., PLoS One (2014) 9 (7): e101735

An object of the present invention is to provide an in vitro screening system for an anticancer drug targeting cancer stem cells, which can respond to HTS.

In order to achieve the above object, the present inventors focused on the induced cancer stem cells (iCSC) established by the present inventors, formed spheres from the iCSC by suspension culture, and performed immunohistochemical analysis. , The ability to form cancer tissue-like structures (cancer organoids) in vitro was investigated. As a result, it was possible to form tissue-like structures having immunohistochemical characteristics similar to those of human cancer tissues, that is, cancer organoids, from both iCSCs of colorectal cancer and lung cancer. Furthermore, by co-culturing iCSC and stromal cells (mesenchymal stem cells (MSC) and vascular endothelial cells), not only cancer cells similar to human cancer tissue but also αSMA-positive cells and vascular endothelial cells are contained. We also succeeded in forming a large cell mass.

Next, the present inventors compared the comprehensive gene expression of iCSC and non-stem cell cancer cells differentiated in the maintenance culture process of iCSC using a microarray, and used them as genes showing high expression specifically for iCSC. , The regulator of calcineurin 2 (RCAN2) was identified for colon cancer, and the interleukin (IL) -6 gene was identified for lung cancer. Since RCAN2 is known to inhibit calcineurin, a sphere formation assay was performed by adding the calcineurin inhibitor FK506, and the addition of FK506 promoted the sphere formation ability of induced colorectal cancer stem cells. On the other hand, calcineurin is known to promote the translocation of NFAT protein to the nucleus. Conversely, when GSK3, which is known to promote the translocation of NFAT from the nucleus to the cytoplasm, was inhibited, any GSK3 inhibitor was used. The sphere-forming ability of induced colon cancer stem cells was significantly suppressed even when the drug was used. Since GSK inhibitors are known to have anticancer activity, these results are obtained from spheres obtained by three-dimensional culture of cancer stem cells (that is, cancer tissue-like structures = cancer organoids). ) Can be used as an index to evaluate the efficacy of anticancer drugs, especially anticancer drugs that target cancer stem cells, and cancer stem cells and non-stem cell cancer cells Search for anti-cancer drugs that target cancer stem cells more efficiently by identifying genes whose expression levels fluctuate significantly among them and using drugs that target the genes (products) as test substances. Strongly suggests that is possible.

Therefore, in order to substantiate the above hypothesis, the present inventors exert on the tissue-building ability of lung cancer organoids prepared by co-culturing induced lung cancer stem cells or original lung cancer cells with MSC and vascular endothelial cells. And the effect of anti-IL-6 antibody was investigated. As a result, the addition of IL-6 enriched the cell mass formed from the original lung cancer cells and significantly increased the number of αSMA-positive cells, whereas the addition of anti-IL-6 antibody resulted in induced lung cancer. In the cell mass formed from stem cells, the number of αSMA-positive cells was significantly reduced. In addition, the cell mass formed from induced lung cancer stem cells was cisplatin (CDDP) resistant, but when combined with anti-IL-6 antibody, marked cell loss was observed, and the structure of lung cancer organoids was destroyed. These results support the above hypothesis, and at the same time, lung cancer stem cells promote the differentiation of MSC into αSMA-positive cells by highly expressing IL-6, and contribute to the formation of lung cancer organoids. It is shown that the IL-6 inhibitor can suppress the differentiation of MSC into αSMA-positive cells by blocking the effect of IL-6.

As a result of further research based on these findings, the present inventors have completed the present invention.
That is, the present invention is as follows.
[1] A method for screening an anticancer drug, which comprises the following steps.
(1) Step of contacting a test substance with a cancer organoid obtained by three-dimensionally culturing cancer stem cells (2) Selecting a test substance that has caused deconstruction of the cancer organoid as an anticancer drug Step [2] The method according to [1], wherein the cancer organoid is obtained by co-culturing cancer stem cells with mesenchymal stem / progenitor cells and vascular endothelial cells.
[3] A method for screening an anticancer drug, which comprises the following steps.
(1) Step of culturing cancer stem cells three-dimensionally in the presence of a test substance to induce cancer organoid formation (2) Compared to the case of three-dimensional culturing in the absence of a test substance, cancer organoids Step of selecting a test substance that inhibits the formation of cancer as an anticancer drug [4] In step (1), cancer stem cells are co-cultured with mesenchymal stem / progenitor cells and vascular endothelial cells, [3]. The method described in.
[5] The test substance is a substance capable of regulating the expression or function of a gene whose expression level and / or epigenetic modification is significantly different between cancer stem cells and non-stem cell cancer cells [1] to The method according to any one of [4].
[6] The method according to [5], further comprising identifying genes whose expression levels and / or epigenetic modifications differ significantly between cancer stem cells and non-stem cell cancer cells.
[7] Cancer stem cells are derived by culturing non-stem cell cancer cells into which an exogenous reprogramming factor has been introduced under conditions in which embryonic stem (ES) cells cannot be maintained. The method according to any one of [1] to [6].
[8] Induced cancer stem cells
(A) It has a drug exclusion ability in the presence of an ABC transporter inhibitor at a concentration effective for suppressing the drug exclusion ability of non-stem cell cancer cells into which an exogenous reprogramming factor has not been introduced. Or (b) it does not dissociate from the culture vessel by trypsin treatment.
The method according to [7].
[9] The method according to any one of [1] to [8], wherein the cancer stem cells are colon cancer stem cells or lung cancer stem cells.
[10] A method for producing a cancer organoid, which comprises a step of co-culturing a cancer stem cell with a mesenchymal stem / progenitor cell and a vascular endothelial cell in a three-dimensional culture.
[11] Cancer stem cells are induced by culturing non-stem cell cancer cells into which an exogenous reprogramming factor has been introduced under conditions in which embryonic stem (ES) cells cannot be maintained, and (a). In the presence of an ABC transporter inhibitor at a concentration effective in suppressing the drug-eliminating ability of non-stem cell cancer cells that have not introduced an exogenous reprogramming factor, or has a drug-eliminating ability. (B) The method according to [10], which does not dissociate from the culture vessel by trypsin treatment.
[12] A cancer organoid in which cancer stem cells, mesenchymal stem / progenitor cells, and vascular endothelial cells are self-assembled in vitro.
[13] The cancer organoid according to [12], which is obtained by the method according to [10] or [11].
[14] A regulator of differentiation of mesenchymal stem / progenitor cells into αSMA-positive cells, which comprises an IL-6 regulator.

According to the present invention, it is possible to develop a novel cancer stem cell target therapeutic agent by using a triad of cancer stem cells, non-stem cell cancer cells and cancer organoids derived from the cancer cells. Become. The use of iCSC as a cancer stem cell is superior to the method using a sample derived from patient cancer tissue in that there is no quantitative limitation on the sample, and it is possible to obtain tissue and human cancer tissue. It is also superior to existing technologies in terms of similarity.

The characteristics of iCSC-derived spheres are shown. (A) parental SW480 cells, showing 1 ^st V50-OKS cell-derived non-V50 cells, and the spheres forming ability in 2 ^nd V50-OKS cells. Substantial number of spheres are formed in a 2 ^nd V50-OKS cells. The number of spheres in each experiment is shown (n = 3). * P <0.05, showing a histological and immunohistochemical analysis of spheres of ** p <0.01 (B) parental SW480 cells and 2 ^nd V50-OKS cells. The spheres derived from the parent SW480 cells were negative for CK20 and CK7 and positive for CDX2. 2 ^nd V50-OKS cell derived spheres, CK20, CDX2 is positive, CK7 were negative. (A) Spheres derived from iCSC, not the parent strain SW480, became colonies of collective cells in co-culture of HUVEC and MSC. (B) Histological and immunohistochemical analysis of co-cultured spheres is shown. The spheres derived from iCSC, HUVEC and MSC were positive for α-SMA and CD31, and only the outside of the sphere was Ki67 positive. The spheres derived from the parents SW480, HUVEC and MSC were negative for α-SMA and CD31 and were totally positive for Ki67. Mock-SW480 cells, shows a comparison of gene profile in non-V50-OKS cells and 2 ^nd V50 cells. (A) Mock-SW480 and 2 ^nd V50-OKS in gene expression between cells, 3914 probesets were identified as having a significant difference (t-test, the false positive rate (FDR) <0.05 and 2 Double difference, indicated by gray dots). (B) in gene expression between non-V50 cells from 1 ^st V50-OKS cells and 2 ^nd V50-OKS cells, 56 probes were identified as having a significant difference (t-test, the false discovery rate ( FDR) <0.05 and 2x difference, indicated by gray dots). (C) Non-V50, the probe is expressed higher in 2 ^nd V50 than mock, and non-V50, shows a Venn diagram of a probe to lower expression in 2 ^nd V50 than mock. In the Venn diagram, each of the four probes overlapped. (D) The Venn diagram shows the mRNA expression levels of the eight overlapping probes (n = 3). (E) shows the 5 days after selection, parental SW480 cells, 1 ^st V50-OKS cell-derived non-V50 cells and 2 ^nd V50-OKS SEMA6A in a cell, the total transcript levels of qRT-PCR of FAM105A and RCAN2 ( n = 3). * p <0.05 FIG. 4 shows a schematic diagram showing a series of flows from the introduction of the reprogramming factor to the start of the addition of FK506 in the FK506 addition experiment. (A) The cell numbers of parent SW480 cells and 2nd V50-OKS cells with or without addition of FK506 were counted on the 5th day after seeding (n = 3). * P <0.05, in the case of addition or without ** P <0.01 (B) FK506 , shows a change in the form of the parent SW480 cells and 2 ^nd V50-OKS cells. (C) in the case of addition or without FK506, shows a sphere-forming ability of the 2 ^nd V50-OKS cells. In 2 ^nd V50-OKS cells supplemented with FK506, the number of spheres formed is increased. (D) at 10 days after sowing, in the case of addition or without FK506, the number of spheres of parental SW480 cells or 2 ^nd V50-OKS cells (n = 3). * p <0.05, ** p <0.01 (E) The histological and immunohistochemical analysis of iCSC spheres with or without the addition of FK506 is shown. Both spheres were positive for CK20, CDX2 and negative for CK7. GSK3 inhibition is shown to suppress the ability of induced colorectal cancer stem cells. (A) The effect of siRNA addition on GSK3α and GSK3β in planar adhesive culture is shown. (B) Shows the effect of adding valproic acid (VPA) or CHIR99021 in planar adhesive culture. (C) Shows the effect of addition of valproic acid (VPA) or CHIR99021 (CHIR) on the ability to form spheres. Shows the ability of induced lung cancer stem cells to form spheres. (A) Shows the sphere-forming ability of lung cancer cells (A549), derived lung cancer stem cells (OKS-A549) and transfection control (EGFP-A549). (B) Phase difference images (top) and HE-stained images (bottom) of spheres derived from lung cancer cells (A549) and lung cancer stem cells (OKS-A549-Colony) derived from them are shown. The schematic diagram (top) of the preparation of lung cancer-like tissue (lung cancer organoid) in vitro and the result of immunostaining of the prepared lung cancer cell (A549) -derived and lung cancer stem cell-derived tissue (bottom) are shown. In the figure below, the right column shows the actual human lung cancer tissue. αSMA is a marker for cancer associated fibroblast (CAF) found in human lung cancer tissue, and CK7 is a marker for epithelium (ie, cancer cancer cells). It is shown that a tissue similar to human lung cancer tissue could be constructed by co-culture of induced lung cancer stem cells and stromal cells. (A) Alcian blue-PAS staining of tissues derived from lung cancer cells (A549) and lung cancer stem cells is shown. Tissues made from induced lung cancer stem cells (OSK-A549-Colony) showed a polar duct-like structure with mucin secretion. (B) Cancer cells (induced lung cancer stem cells or parent cell lines) were colored with GFP, vascular endothelial cell line HUVEC was colored with m-cherry, and the produced organoids were observed under a fluorescence microscope. (C) The change in the fluorescence intensity (mcherry-positive cell number) of mCherry observed in (B) is shown. (D) HUVEC-derived CD31-positive cells in lung cancer cell (A549) -derived and lung cancer stem cell (OSK-A549-Colony) -derived tissues are detected by immunohistochemical analysis. (E) The distribution of Ki67-positive cells in tissues derived from lung cancer cells (A549), lung cancer stem cells (OSK-A549-Colony), and human lung cancer tissue specimens is shown. (F) In tissues derived from lung cancer cells (A549) and lung cancer stem cells (OSK-A549-Colony), the number of Ki67-positive cells present inside and outside the tissues is shown. (G) Quantitative RT-PCR shows that lung cancer stem cells specifically express IL-6. (H) It is shown that the addition of IL-6 increases the fluorescence intensity in tissues derived from fluorescently labeled lung cancer cells (A549). (I) It is shown that the addition of anti-IL-6 antibody does not attenuate the fluorescence intensity in tissues derived from fluorescently labeled lung cancer cells (A549). It is shown that IL-6 is highly expressed specifically for lung cancer stem cells, and that the combined use of anti-IL-6 antibody and cisplatin (CDDP) destroys inducible cancer stem cell-derived tissues. (A) The results of microarray comparison of gene expression in A549 and inducible lung cancer stem cells prepared from it are shown. (B, C, D) Response to CDDP of tissues prepared by co-culture of induced lung cancer stem cells and stromal cells, and response to CDDP when anti-IL-6 antibody is used in combination with lung cancer stem cell-derived tissues. Shows the change of. It is shown that IL-6 has a positive effect on lung cancer tissue formation by converting MSC into αSMA-positive cells, and anti-IL-6 antibody blocks this. (A, B, C) The effect of IL-6 addition on the cell clumps formed by co-culture using a normal lung cancer cell line (A549) is shown. (D, E, F) The effect of anti-IL-6 antibody addition on cancer organoids formed by co-culture using lung cancer stem cells is shown. (G) It is shown that αSMA-positive cells appear when IL-6 is added to the adhesive culture of MSC alone.

[I] Screening Method for Anticancer Drugs The present invention provides a method for screening a novel anticancer drug that targets cancer stem cells (hereinafter, also referred to as “method of the present invention”). The method of the present invention is characterized by including the following steps.
(1) Step of contacting a test substance with a cancer organoid obtained by three-dimensionally culturing cancer stem cells (2) Selecting a test substance that has caused deconstruction of the cancer organoid as an anticancer drug Steps (The method including steps (1) and (2) is also hereinafter referred to as "method A of the present invention").
or,
(1') Step of three-dimensionally culturing cancer stem cells in the presence of a test substance to induce cancer organoid formation (2') Compared to the case of three-dimensional culture in the absence of a test substance, The step of selecting a test substance that inhibits the formation of organoids as an anticancer drug (the method including steps (1') and (2') is hereinafter also referred to as "method B of the present invention").

1. 1. Cancer Organoids As used herein, the term "cancer organoid" means an in vitro induced tissue structure having a structure similar to that normally observed in natural cancer tissues. For example, cancer stem cells and non-stem cell cancer cells differentiated from the cancer stem cells may be included and organized in a manner similar to that observed in natural cancer tissues. .. In a preferred embodiment, the cancer organoids of the invention are αSMA derived from stromal cells (eg, mesenchymal stem / progenitor cells (MSC / MPC)) in addition to cancer stem cells and non-stem cell cancer cells. It can include non-cancer cells that make up natural cancer tissue, such as positive cells and / or vascular endothelial cells).

Having the same histological characteristics as natural cancer tissue should be confirmed by immunohistochemical analysis, for example, using the expression of markers specific to cancer stem cells and / or non-stem cell cancer cells as an index. Can be done. For example, in the case of colorectal cancer organoids, it can be confirmed by showing immunohistochemical findings such as CK20 positive, CK7 negative, and CDX2 positive as a typical staining pattern of colorectal cancer tissue. In the case of lung cancer organoids, it can be confirmed by showing immunohistochemical findings such as CK7 positive, TTF1 positive, and Napsin A positive. In addition, when cancer organoids are obtained by co-culture of cancer stem cells and MSC / MPC, αSMA positive, which is a marker of MSC / MPC-derived myofibroblasts, and cancer organoids are co-cultured with vascular endothelial cells. When obtained by culture, it can also be confirmed by showing immunohistochemical findings positive for CD31, which is a vascular endothelial cell marker.

The type of cancer organoid used in the method of the present invention is not particularly limited, and examples thereof include those corresponding to any natural cancer. For example, it can be a cancer derived from epithelial cells, but it can also be a non-epithelial sarcoma or a hematological cancer. More specifically, for example, head and neck cancer (eg, maxillary cancer, pharyngeal cancer, laryngeal cancer, tongue cancer, thyroid cancer), chest cancer (eg, breast cancer, lung cancer (non-small)). Cellular lung cancer, small cell lung cancer)), gastrointestinal cancer (eg, esophageal cancer, gastric cancer, duodenal cancer, colon cancer (colon cancer, rectal cancer), liver cancer (hepatocellular cancer, bile duct) Cellular cancer), bile sac cancer, bile duct cancer, pancreatic cancer, anal cancer), urinary cancer (eg, renal cancer, urinary tract cancer, bladder cancer, prostate cancer, penis cancer, Testis cancer (testicle cancer), genital cancer (eg, uterine cancer (cervical cancer, uterine body cancer), ovarian cancer, genital cancer, vaginal cancer), skin cancer (eg, cervical cancer) (Bass cell carcinoma, spinous cell carcinoma), but not limited to these. In one preferred embodiment, colorectal cancer organoids, lung cancer organoids and the like can be mentioned.

The animal species of the cancer organoid is not particularly limited, but it is desirable that the animal species is the same as the target of administration of the anticancer drug intended by the method (1) of the present invention. For example, mammals (eg, humans, mice, rats, monkeys, dogs, cats, cows, horses, etc.) can be mentioned, but in light of the above object of the method (1) of the present invention, humans are preferable.

2. Cancer stem cells Cancer organoids can be obtained by culturing cancer stem cells in three dimensions. The cancer stem cells used for producing cancer organoids are not particularly limited as long as they have the ability to reconstruct cancer tissue (hereinafter, also referred to as "cancer tissue remodeling ability"). The ability to reconstruct cancer tissue can be confirmed by a method known per se. For example, as described in Patent Document 1, by transplanting cancer stem cells into mice and evaluating the tumorigenicity in vivo, the tissue remodeling ability can be evaluated. Moreover, since the sphere formation ability by three-dimensional culture can be used as an index of the cancer tissue remodeling ability, the cancer tissue remodeling ability can also be evaluated by the sphere formation assay.

Further, in the present invention, cancer stem cells can be obtained using the expression of cancer stem cell markers, cell proliferation rate, resistance to anticancer drugs, and / or drug excretion ability as indicators. For example, in the case of colon cancer stem cells, one or more markers previously reported as colon cancer stem cell markers, specifically at least one marker selected from the group consisting of CD133, CD44, CD26, ABCG2 and LGR5. Positive cells can be used as colon cancer stem cells. In the case of lung cancer stem cells, for example, CD133-positive cells previously reported as lung cancer stem cell markers can be used as lung cancer stem cells. Here, "positive for cancer stem cell marker" can be determined by the expression of the cancer stem cell marker mRNA or the expression of the cancer stem cell marker protein in the cells. .. The mRNA of the cancer stem cell marker is not particularly limited, but can be confirmed by a method known per se, such as RT-PCR method and Northern blotting method. The protein of the cancer stem cell marker is not particularly limited, but can be confirmed by a method known per se, such as Western blotting and immunostaining. When the cancer stem cell marker is a cell surface antigen marker, it can be confirmed that the cancer stem cell marker is positive by measuring with a flow cytometer.

On the other hand, when the cell proliferation rate, resistance to anticancer drug, and / or drug excretion ability are used as indicators, the cell proliferation rate is slower and the resistance to anticancer drug is higher than that of normal cancer cells. And / or cells having characteristics such as high drug excretion ability can be used as cancer stem cells. Further, for example, in the case of lung cancer stem cells, cells that do not dissociate from the culture vessel when treated with trypsin at a constant concentration for a certain period of time are selected as lung cancer stem cells, using the dissociation resistance to trypsin treatment as an index in adhesive culture, as described later. be able to.

The source of the cancer stem cell used in the present invention is not particularly limited, and may be a cancer stem cell (induced cancer stem cell; iCSC) induced by introducing a reprogramming factor into the cancer cell, a cultured cancer cell, or a cultured cancer cell. It may be a cell isolated from a cancer tissue in the living body, but in light of the purpose of the present invention of establishing an HTS system of an anticancer drug, a large amount of cancer stem cells and / or cancer organoids can be easily obtained. Inducible cancer stem cells (iCSCs) are preferred in that they can be provided.

(2-1) Inducible cancer stem cells (iCSC)
The iCSC used in the present invention can be produced by a known method (for example, the method described in Patent Document 1, WO 2011/049099). For example, cancer cells introduced with an exogenous reprogramming factor can be induced by culturing under conditions in which embryonic stem (ES) cells cannot be maintained. It can be suitably used for the method of the present invention.

Examples of the reprogramming factors used in the preparation of iCSC include combinations of Oct3 / 4, Sox2 and Klf4. In this case, Oct3 / 4 is replaced by another member of the Oct family, such as Oct1A, Oct6, etc. Can also be used. Also, instead of Sox2, other members of the Sox family, such as Sox1, Sox3, Sox15, Sox17, Sox18, can be used. You can also use other Klf family members, such as Klf1, Klf2, and Klf5, instead of Klf4. Furthermore, in addition to Oct3 / 4, Sox2 and Klf4, any substance may be further contained. Any substance added is a substance (group) that shifts the somatic cell to a more undifferentiated state by introducing it into the somatic cell, for example, a gene or ES specifically expressed in the ES cell. Examples include, but are not limited to, genes that play an important role in maintaining undifferentiated cells or their gene products. Genes that are specifically expressed in ES cells or genes that play an important role in maintaining undifferentiated ES cells or their gene products include, for example, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T. Examples include antigen, HPV16 E6, HPV16 E7, Bmi1, Lin28, Lin28b, Nanog, Esrrb or Esrrg. Alternatively, any substance added upon introduction can be a substance (group) that increases the efficiency of transition of the somatic cell to a more undifferentiated state by introducing it into the somatic cell, for example, establishment of iPS cells. Substances (groups) that promote efficiency include, but are not limited to. Substances (groups) that promote iPS cell establishment efficiency include, but are not limited to, the following substances (groups): histone deacetylase (HDAC) inhibitors [eg, valproic acid (VPA). (Nat. Biotechnol., 26 (7): 795-797 (2008)), small molecule inhibitors such as tricostatin A, sodium butyrate, MC 1293, M344, siRNAs and shRNAs against HDACs (eg, HDAC1 siRNA Smartpool (registered) Nucleic acid expression inhibitors such as (Trademarks) (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene)], DNA methyltransferase inhibitors (eg 5'-azacytidine) (Nat. Biotechnol., 26 (7) :. 795-797 (2008)), G9a histone methyltransferase inhibitors [eg, small molecule inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), siRNA and shRNA against G9a (eg, G9a) Nucleic acid expression inhibitors such as siRNA (human) (Santa Cruz Biotechnology))], L-channel calcium agonist (eg Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)), p53 inhibitors (eg Proliferation of siRNA and shRNA against p53) (Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling activator (eg soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), LIF or bFGF Factors, ALK5 inhibitors (eg SB431542) (Nat. Methods, 6: 805-8 (2009)), mitogen-activated protein kinase signaling inhibitors, glycogen synthase kinase-3 inhibitors (PloS Biology, 6 (10), 2237-2247 (2008)), miRNAs such as miR-291-3p, miR-294, miR-295 (RL Judson et al. , Nat. Biotech., 27: 459-461 (2009)).

Reprogramming factors can be introduced in the form of DNA or in the form of proteins. When introduced in the form of DNA, for example, vectors such as viruses, plasmids, artificial chromosomes (eg, human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC), etc.), lipofection, liposomes, etc. , Can be introduced into somatic cells by techniques such as microinjection. Viral vectors include retroviral vectors and lentiviral vectors (Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007. ), Adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector (Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009) and the like. The vector can include regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that the reprogramming factor can be expressed. Promoters used include, for example, EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Molony mouse leukemia virus) LTR, HSV- A TK (simple herpesvirus thymidine kinase) promoter or the like is used. In addition, the vector replicates without chromosomal integration and is the origin and replication of lymphotrophic herpes virus, BK virus and Bovine papillomavirus, as present episomal. May include the sequence according to. For example, it may contain EBNA-1 and oriP or Large T and SV40 ori sequences (WO 2009/115295, WO 2009/157201 and WO 2009/149233).

Moreover, in order to introduce a plurality of reprogramming factors at the same time, an expression vector expressed polycistronically may be used. For polycistronic expression, the genes encoding sequences may be linked by the IRES, the foot-and-mouth disease virus (FMDV) 2A coding region or the Thosea asigna virus 2A coding region (T2A) ( Science, 322: 949-953, 2008 and WO 2009/092042, WO 2009/152529, PLoS One. 6 (4): e18556, 2011). From the viewpoint of uniform expression of the reprogramming factor, an expression vector that is polycistronically expressed is preferable.

On the other hand, when introducing in the form of a protein, for example, a method using a protein transfer reagent, a method using a protein transfer domain (PTD) fusion protein, a microinjection method, and the like can be mentioned. Protein transfer reagents include cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systmes), Pro-Ject ^TM Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX), and lipid-based Project-1 (Targeting Systems). ), Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif) based on membrane-permeable peptides, GenomONE (Ishihara Sangyo) using HVJ envelope (inactivated Sendai virus), etc. are commercially available. The introduction can be performed according to the protocol attached to these reagents, but the general procedure is as follows. Dilute the reprogramming factor in a suitable solvent (for example, buffer solution of PBS, HEPES, etc.), add the introduction reagent, and incubate at room temperature for about 5 to 15 minutes to form a complex, which is replaced with serum-free medium. Add to the cells and incubate at 37 ° C for 1 to several hours. Then, the medium is removed and replaced with a serum-containing medium.

PTDs include AntP from Drosophila, TAT from HIV (Frankel, A. et al, Cell 55, 1189-93 (1988); Green, M. & Loewenstein, PM Cell 55, 1179-88 (1988)), Penetratin (Derossi, D. et al, J. Biol. Chem. 269, 10444-50 (1994)), Buforin II (Park, CB et al. Proc. Natl Acad. Sci. USA 97, 8245-50 (2000)) ), Transportan (Pooga, M. et al. FASEB J. 12, 67-77 (1998)), MAP (model amphipathic peptide) (Oehlke, J. et al. Biochim. Biophys. Acta. 1414, 127-39 ( 1998)), K-FGF (Lin, YZ et al. J. Biol. Chem. 270, 14255-14258 (1995)), Ku70 (Sawada, M. et al. Nature Cell Biol. 5, 352-7 (2003)) )), Prion (Lundberg, P. et al. Biochem. Biophys. Res. Commun. 299, 85-90 (2002)), pVEC (Elmquist, A. et al. Exp. Cell Res. 269, 237-44 ( 2001)), Pep-1 (Morris, MC et al. Nature Biotechnol. 19, 1173-6 (2001)), Pep-7 (Gao, C. et al. Bioorg. Med. Chem. 10, 4057-65 ( 2002)), SynBl (Rousselle, C. et al. MoI. Pharmacol. 57, 679-86 (2000)), HN-I (Hong, FD & Clayman, GL. Cancer Res. 60, 6551-6 (2000)) )), Those using the cell transit domain of proteins such as VP22 derived from HSV have been developed. Examples of CPPs derived from PTD include polyarginines such as 11R (Cell Stem Cell, 4: 381-384 (2009)) and 9R (Cell Stem Cell, 4: 472-476 (2009)). A fusion protein expression vector incorporating the cDNA of the reprogramming factor and the PTD sequence or CPP sequence is prepared and recombinantly expressed, and the fusion protein is recovered and used for introduction. The introduction can be carried out in the same manner as described above except that the protein introduction reagent is not added. It is suitable for introducing deletion mutants having a relatively small molecular weight.

Microinjection is a method in which a protein solution is put into a glass needle having a tip diameter of about 1 μm and punctured and introduced into cells, and the protein can be reliably introduced into cells. In addition, electroporation method, semi-intact cell method (Kano, F. et al. Methods in Molecular Biology, Vol. 322, 357-365 (2006)), introduction method by Wr-t peptide (Kondo, E. et al. , Mol. Cancer Ther. 3 (12), 1623-1630 (2004)) and other protein transfer methods can also be used.

The protein introduction operation can be performed any number of times (for example, 1 time or more and 10 times or less, or 1 time or more and 5 times or less), and preferably the introduction operation is performed 2 times or more (for example, 3 times or 4 times). ) Can be repeated. Examples of the interval when the introduction operation is repeated include 6 hours to 7 days, preferably 12 to 48 hours or 7 days.

The cancer cells used to prepare iCSC may be primary cultured cells isolated from an individual, or cell lines that have acquired the ability to proliferate indefinitely (immortalized) in vitro. Examples of the colorectal cancer cell line include HT29, HCT8, HCT116, W620, SW480, SW837, DLD-1, CACO-2, LoVo, etc., and SW480 cells are preferable. Examples of lung cancer cell lines include A549, A427, Calu-1, Calu-6, CLS-54, DMS-79, GCT, HEL-299 and the like, but A549 cells are preferable.

In the present invention, the medium for inducing cancer stem cells can be prepared using the medium used for culturing animal cells as the basal medium. The basal medium includes, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen). , And mixed media thereof and the like are included. The medium may contain serum or may be serum-free. If desired, the medium may be, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), amino acids, insulin, collagen precursors, trace elements. , 2-Mercaptoethanol (2ME), may contain one or more serum substitutes such as thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules It may also contain one or more substances such as compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts.

In the present invention, the cancer cells into which the reprogramming factor has been introduced are preferably cultured under conditions other than the maintenance culture conditions for ES cells. Here, "ES cell maintenance culture conditions" are, for example, conditions for culturing in a medium containing bFGF or SCF, and extracellular matrices used for the purpose of assisting maintenance culture (for example, Matrigel, Laminin 511, Laminin 332, etc. Conditions using (or fragments thereof), conditions using feeder cells used for the purpose of assisting maintenance culture (for example, mouse embryo-derived fibroblasts (MEF), STO cells (ATCC, CRL-1503)), or the feeder cells. Conditions such as using the cultured culture supernatant can be mentioned, but it is obvious to those skilled in the art that under what culture conditions the ES cells can be maintained and cultured. In the present invention, cancer cells into which a reprogramming factor has been introduced are cultured without using the maintenance culture conditions for these ES cells. However, in the present invention, since it is desirable to culture under conditions other than the maintenance culture condition of ES cells only in the final step of obtaining cancer stem cells, it is prevented from culturing under the maintenance culture conditions of ES cells in the intermediate step. It's not a thing.

The medium for culturing cancer cells into which the reprogramming factor has been introduced in the present invention can preferably be DMEM, DMEM / F12 or DMEM medium containing FBS.

The concentration of FBS in the medium may be any concentration as long as it is used by those skilled in the art in normal cell culture, and can be in the range of, for example, 1 to 30%, preferably 1 to 20%. The concentration of FBS in the medium is, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%. , 15%, 16%, 17%, 18%, 19%, 20%, preferably 10%.

The medium used in the present invention may further contain penicillin for the purpose of preventing bacterial infection, and the concentration of penicillin may be any concentration as long as it is used in normal cell culture by those skilled in the art, for example, 1 to 1. It can be in the range of 500 Units / ml, preferably 1 to 200 Units / ml. The concentrations of penicillin in the medium are, for example, 1 Units / ml, 25 Units / ml, 50 Units / ml, 60 Units / ml, 70 Units / ml, 80 Units / ml, 90 Units / ml, 100 Units / ml, 110. Units / ml, 120 Units / ml, 130 Units / ml, 140 Units / ml, 150 Units / ml, 175 Units / ml, 200 Units / ml, preferably 100 Units / ml.

The medium used in the present invention may further contain streptomycin for the purpose of preventing bacterial infection, and the concentration of streptomycin may be any concentration as long as it is used in normal cell culture by those skilled in the art, for example, 1 to 1. It can be in the range of 500 μg / ml, preferably 1 to 200 μg / ml. The concentrations of streptomycin in the medium are, for example, 1 μg / ml, 25 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 110 μg / ml, 120 μg / ml, 130 μg. It is / ml, 140 μg / ml, 150 μg / ml, 175 μg / ml, 200 μg / ml, preferably 100 μg / ml.

The culture temperature is preferably 30 to 40 ° C, more preferably 37 ° C. The CO ₂ concentration is preferably 2 to 5%. As an example of the culture method, after introducing the reprogramming factor into cancer cells, the cells are cultured in DMEM medium containing 10% FBS, penicillin and streptomycin _{, for example, in the presence of 5% CO 2 at 37 ° C.} The method can be mentioned. The culture period is not particularly limited, and examples thereof include 4 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more. At this time, it is preferable to replace the medium with a fresh medium after a lapse of a certain period of time. Particularly preferably, the medium is replaced 24 hours after contact between the cancer cells and the reprogramming factor.

The number of cancer cells used to induce cancer stem cells is not particularly limited as long as a reprogramming factor can be introduced, but a cell number that is 70% to 100% confluent in a culture dish is exemplified.

In a preferred embodiment, the iCSC used in the present invention is an ATP binding cassette (ABC) transporter inhibitor at a concentration effective in suppressing the drug exclusion ability of cancer cells into which an exogenous reprogramming factor has not been introduced. It is a cell that has the ability to eliminate drugs even in the presence of. A cell having a drug-excluding ability is, for example, a Side population (SP) cell. SP cells are different from normal cells (cells other than undifferentiated cells) that fluoresce at 405 nm and 600 nm when Hoechst 33342, a fluorescent dye, is taken up by cells and excited by UV in flow cytometric analysis. It is a cell population that appears at a position (dark part of fluorescence, that is, "Hoechst Blue weakly positive and Hoechst Red weakly positive"). Therefore, the preferred excreted drug in the present invention is Hoechst33342, and cancer stem cells having a drug-eliminating ability can be extracted using Hoeschst33342 as an index. Therefore, in the above embodiment, iCSC is a drug under the condition that an ABC transporter inhibitor at a concentration effective for suppressing the drug exclusion ability of colon cancer cells into which an exogenous reprogramming factor has not been introduced is added. It is preferable that the ^{cells have the drug exclusion ability (hereinafter, abbreviated as "1 st} iCSC") obtained by the step of extracting the cells having the exclusion ability (hereinafter, abbreviated as "primary extraction step"). the 1 ^st ICSC after a certain period cultured further (hereinafter abbreviated as "secondary extraction step".) similar to the above extraction process performed, cells with drug elimination capacity obtained by the process (hereinafter "2 ^nd ICSC It is more preferable that it is abbreviated as.). The period from the introduction of the reprogramming factor to the primary extraction step includes, for example, 6 to 12 days, preferably 10 days. The period from the primary extraction step to the secondary extraction step is, for example, 10 to 20 days, preferably 17 days. When using the 1 ^st ICSC or 2 ^nd ICSC to the present invention, the extraction step may be used cells immediately after, but may be used cells for a period of time the culture.

The ABC transporter in the present invention is, for example, a transporter that transports using the hydrolysis energy of ATP, and is preferably involved in extracellular transport of an anticancer drug, for example, P-glycoprotein (Pgp /). MDR1 / ABCB1), MDR-asscociated protein 1 (MPR1), ABCG2 (BCRP / ABCP / MXR) are exemplified.

In the present invention, the ABC transporter inhibitor is not particularly limited as long as it inhibits the function of the ABC transporter, and is, for example, VX-710, GF120918, XR9576, fumitremorgin C, Ko143, pantoprazole, flavonoids, estrogens, antiestrogens, Dofequidar. Fumarate (dofekidal fumarate) (MS-209) (Cancer Science Volume 100, Issue 11, pages 2060-2068, November 2009), verapamil, reserpine, zoskidal (LY335973), cyclospoli A, tamoxifen, quinidine, d-α tocopheryl Examples include polyethylene glycol 1000 succinate, PSC833, phenothiazine, SDZ PSC 833, TMBY, MS-073, S-9788, SDZ 280-446, and XR9051. ABC transporter inhibitors are preferably fumitremorgin C, Ko143, Dofequidar Fumarate, verapamil, reserpine, and particularly preferably verapamil.

The concentration of the ABC transporter inhibitor used in the present invention is a concentration effective for suppressing the drug exclusion ability of cancer cells into which a reprogramming factor has not been introduced. For example, an ABC transporter inhibitor can be used. 10 μM or more for fumitremorgin C, 1 μM or more for Ko143, 5 μM or more for Dofequidar Fumarate, 50 μM or more for verapamil, and 10 μM or more for reserpine. be. More preferably, in the case of Bellapamil, 50 μM or more and less than 250 μM (for example, 240 μM or less, 230 μM or less, 220 μM or less, 210 μM or less, 200 μM or less, 190 μM or less, 180 μM or less, 170 μM or less. , 160 μM or less, 150 μM or less).

In another preferred embodiment, the iCSC used in the present invention is a cell that does not dissociate from the culture vessel by trypsin treatment. Here, the "trypsin treatment" means a treatment for dissociating the cell-cell adhesion and the cell-culture vessel adhesion in the adhesion culture. The conditions for trypsin treatment are not particularly limited as long as they are sufficient conditions for normal cancer cells to dissociate from the culture vessel, but the trypsin concentration is, for example, 0.1 to 1% (eg, 0.1%, 0.15). %, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%), preferably 0.25%. The processing time is, for example, 1 minute or more (eg, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 6 minutes or more, 7 minutes or more, 8 minutes or more, 9 minutes or more. , 10 minutes or more), preferably 5 minutes or more, more preferably 6 to 10 minutes. Contact between the cells and trypsin may be under normal culture conditions of the cells to be treated, eg, 35-42 ° C, preferably 36-40 ° C, more preferably 37-39 ° C, 2-5% CO. It can be done in an atmosphere of ₂ , 5 to 20% O _2.

After the trypsin treatment is completed, the trypsin and the detached cells are removed, washed with an isotonic buffer such as PBS, and the cells remaining without dissociation from the culture vessel can be recovered as cancer stem cells.

(2-2) Other Cancer Stem Cell Cultures As a method for isolating colon cancer stem cells from cancer cells or colon cancer tissues in vivo, for example, spheres are formed by suspension-cultured cells without serum. Examples include a sphere formation separation method for concentrating cancer stem cells, a separation method using an SP fraction, and a separation method using a surface marker of stem cells. These methods can be performed in the same manner as the methods described above. In addition, as described in WO2013 / 035824 A1, cancer tissues derived from colon cancer patients are transplanted into immunodeficient mice, passaged, and then cancer stem cells are selected from the tumor tissues using LGR5 as an index. The method may be used.

The cancer stem cells used in the present invention may be in the form of a heterogeneous cell population in which differentiated cancer cells other than cancer stem cells are mixed, but are preferably a uniform cell population consisting only of cancer stem cells. .. Since it is difficult to maintain and culture cancer stem cells by self-renewal in vitro after isolation, in order to obtain a uniform cell population consisting only of cancer stem cells, for example, cells having the above-mentioned drug exclusion ability are used. It is more desirable to perform an extraction step or a step of extracting cells that do not dissociate by trypsin treatment.

3. 3. Formation of cancer organoids (three-dimensional culture of cancer stem cells)
As described above, cancer organoids can be obtained by culturing cancer stem cells in three dimensions. Three-dimensional culture uses a low-adhesive culture vessel and scaffolds (scaffolds) such as porous membranes and hydrogels to form cell aggregates (spheres, spheroids) to make cells more in vivo. It means culturing in a close three-dimensional state. Depending on the presence or absence of scaffolding, it is roughly divided into scaffold type and scaffold-free type. The former is subdivided into hydrogel type, inert matrix type and the like depending on the type of scaffold. Examples of hydrogels include animal-derived matrigels, collagen, laminin, etc., plant-derived alginate hydrogels, and other synthetic compounds (eg, OGel ^TM MT 3D Matrix (Ogel SA), 3-D Life Biomimetic (Cellendes), Puramatrix. (3D MATRIX) etc.) can be used. As the inert matrix, for example, alvetex (reinnavate), 3D Insert (3D Biotek), VECELL-3D Insert (iwaki) and the like can be used. Alternatively, a 96 or 384-well plate or the like can be loaded with a porous polystyrene disc for scaffold culture. The scaffold-free type is also subdivided into a low-adhesion plate, a micropattern surface plate, a hanging drop method, etc., depending on the type of culture vessel used. A low-adhesion plate is a plate having a bottom surface coated with a hydrophilic polymer and processed to suppress cell adhesion. For example, PrimeSurface (Sumitomo Bakelite), Ultra-Low Attachment (Corning), Nunclon Sphera ( Thermoscientific) and the like. A micropattern surface plate is a plate having a bottom surface processed into a micropattern that affects proliferation. For example, since only a part of the bottom surface is adhesive, cells accumulate there and agglomerates are formed. Examples thereof include those that form cells and those that suppress cell adhesion by laying nanofibers and nanolattices on the bottom surface and form cell clusters by suppressing planar spread. The hanging drop method is a method of forming a cell mass in a droplet. For example, a medium drop containing cells is formed at the tip of a chip passed through a hole in a dish, and the chip is pulled out from the hole to keep the drop in the hole. There is a method of agglutinating cells at the bottom of the drop by gravity.

In the present invention, the three-dimensional culture method can be preferably carried out by suspension culture. Suspension culture means culturing the target cells or cell mass without adhering it to the bottom surface of the incubator. Even if the cells or cell mass are in contact with the bottom surface, the cells or cell mass can be shaken gently by shaking the culture medium. Culturing in a state where the cells float in the culture solution is also included in the suspension culture. During suspension culture, the culture vessel was coated with a plastic dish with an untreated bottom surface or an adhesion-preventing coating agent (poly (2-hydroxyethyl methacrylate), etc.) to prevent the cells from adhering to the substrate. It is preferable to use one.

Known methods for forming spheres from cancer stem cells (eg, Ricci-Vitiani L. et al., Nature 2007; 445: 111-51, Sato T. et al., Gastroenterology 2011; 141: 1762-72) ) Can be used. Specifically, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin, transferrin and / or BSA are added to a serum-free medium, and the cells are suspended-cultured. be able to. The plate used at this time is preferably an Ultra Low Attachment plate (Corning).

In addition, when performing three-dimensional culture of cancer stem cells, co-culture with stromal cells such as mesenchymal stem / progenitor cells (MSC / MPC) and vascular endothelial cells makes the natural tissue more faithful. It is possible to form mimicking cancer organoids. Therefore, in one preferred embodiment, the three-dimensional culture step of cancer stem cells is performed by co-culture with stromal cells containing at least mesenchymal stem / progenitor cells and preferably further containing vascular endothelial cells. Examples of MSC / MPC include stem / progenitor cells derived from bone marrow, adipose tissue, synovial tissue, muscle tissue, peripheral blood, placenta tissue, menstrual blood, umbilical cord blood and the like. Examples of the vascular endothelial cells include umbilical vein venous endothelial cells, neonatal capsule / adult skin-derived microvascular endothelial cells, pulmonary arterial vascular endothelial cells, and aortic vascular endothelial cells, and umbilical vein vascular endothelial cells (particularly, particularly). Human umbilical vein vascular endothelial cells (HUVEC)).

The animal from which MSC / MPC and vascular endothelial cells are derived is not particularly limited, but it is preferably derived from the same species as the animal from which cancer stem cells are derived. More preferably, it is human. MSC / MPC and vascular endothelial cells can be obtained by methods known per se, and are generally commercially available.

The ratio of the amount of MSC / MPC and vascular endothelial cells to cancer stem cells is not particularly limited, but for example, the ratio of cancer stem cells: MSC / MPC: vascular endothelial cells = 10: 4: 1 to 5: 4: 4. Can be co-cultured.

As the basal medium and medium additives for three-dimensional culture of cancer stem cells, the above-mentioned media for inducing iCSC can be used in the same manner. Preferably, DMEM, DMEM / F12 or DMEM medium containing FBS is used as the basal medium. The concentration of FBS added is the same as above.

The culture temperature is, for example, 35 to 42 ° C, preferably 36 to 40 ° C, and more preferably 37 to 39 ° C. Culturing can be carried out in an atmosphere of 2-5% CO ₂ and 5-20% O _2. An example of the culturing method is a method of culturing cancer stem cells in DMEM medium containing 10% FBS, penicillin and streptomycin at _{37 ° C. in the presence of 5% CO 2.} Not limited.

4. Contact between a cancer organoid or a cancer stem cell and a test substance In the method A of the present invention and the method B of the present invention, the former contacts a cancer organoid previously formed from a cancer stem cell with a test substance. On the other hand, the latter differs in that the test substance is brought into contact with the cancer stem cells in the process of forming the cancer organoid from the cancer stem cells. Therefore, in the former, whether or not the test substance has the ability to deconstruct cancer organoids, that is, to destroy similar structures of cancer tissues is used as an index, and in the latter, the test substance is a cancer organoid. The index is whether or not the formation is suppressed.

The test substance used in the method of the present invention may be any known compound or a novel compound, for example, cell extract, cell culture supernatant, microbial fermentation product, marine organism-derived extract, plant extract. , Purified protein or crude protein, peptide, non-peptide compound, synthetic low molecular weight compound, natural compound and the like. In the method of the present invention, the test substance is also (1) a biological library method, (2) a synthetic library method using a deconvolution, and (3) "one-bead one-compound". It can be obtained using any of the many approaches in combinatorial library methods known in the art, including (4) synthetic library methods using affinity chromatography sorting. Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches are applicable to small molecule compounds of peptides, non-peptide oligomers, or compounds (Lam (1997)). Anticancer Drug Des. 12: 145-67). Examples of methods for synthesizing molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem . 37: 1233-51). Compound libraries include solutions (see Houghten (1992) Bio / Techniques 13: 412-21) or beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555- 6), bacteria (US Pat. No. 5,223,409), spores (US Pat. No. 5,571,698, No. 5,403,484, and No. 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9) or Phage (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87 : 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; US Patent Application No. 2002103360).

Contact between the cancer organoid or cancer stem cell and the test substance can be performed by adding the test substance to the culture medium of the cancer organoid or cancer stem cell. The concentration of the test substance added may be appropriately selected depending on the type thereof within a range that does not adversely affect the growth of cells, but is usually 0.1 nM to 10 mM, preferably 1 nM to 1 mM. The contact period is, for example, 1 to 14 days, preferably 3 to 10 days.

5. Drug efficacy evaluation In the method of the present invention, cancer stem cells can form cancer organoids similar to natural cancer tissues by three-dimensional culture, whereas non-stem cell cancer cells are naturally cultured even if they are three-dimensionally cultured. Based on the discovery that it does not form spheres that exhibit the histological properties typical of cancerous tissues. That is, since the ability to form cancer organoids and the ability to maintain the structure of cancer organoids depends on the maintenance of cancer stem cells in the cancer organoids, the deconstruction of cancer organoids (that is, the destruction of structures similar to cancer tissues). Alternatively, an anticancer drug that targets cancer stem cells can be selected using the suppression of the formation of cancer organoids as an index. For example, in step (2) of method A of the present invention, the test substance that caused deconstruction of cancer organoids is used as a candidate drug for an anticancer drug, particularly an anticancer drug that targets cancer stem cells. You can choose. Further, in the step (2') of the method B of the present invention, the test substance that inhibited the formation of cancer organoids was used as an anticancer drug, as compared with the case of three-dimensional culture in the absence of the test substance. In particular, it can be selected as a candidate drug for an anticancer drug that targets cancer stem cells.

In Method A of the present invention, the deconstitution of a cancer organoid is a sphere in which the cancer organoid consists of a cancer stem cell and a non-stem cell cancer cell differentiated from the cancer stem cell, for example, a decrease in the number of spheres, a decrease in the sphere diameter, and a sphere. The morphological change of the sphere, the immunostaining pattern of the sphere (for example, CK20, CK7 and CDX2 in the case of colon cancer, CK7, TTF1 and NapsinA in the case of lung cancer) can be used as an index for evaluation. In addition, when cancer organoids further contain stromal cells such as MSC / MPC and vascular endothelial cells, for example, morphological changes in cell clumps (eg, in the case of lung cancer organoids, disruption of mutin-secreting duct-like structures). Etc.), decrease in the number of cells in the cell clump, immunostaining pattern of cancer organoids (eg, cancer cells (for colon cancer, for example, CK20 positive, for lung cancer, for example, CK7 positive), derived from MSC / MPC It can also be evaluated using cells (eg, αSMA positive), vascular endothelial cells (eg, CD31 positive), shedding, changes in the tissue distribution of Ki67-positive cells, etc.) as indicators.

In Method B of the present invention, the cancer organoid is a sphere consisting of cancer stem cells and non-stem cell cancer cells differentiated from the cancer stem cells, for example, as compared with the case of three-dimensional culture in the absence of a test substance. Small number of spheres, small sphere diameter, abnormal sphere morphology, natural sphere immunostaining patterns (eg, CK20, CK7 and CDX2 for colon cancer, CK7, TTF1 and NapsinA for lung cancer) It can be evaluated that the formation of cancer organoids is inhibited when the cancer tissue of the cancer is not reflected. In addition, when the cancer organoid further contains stromal cells such as MSC / MPC and vascular endothelial cells, the morphology of the cell clumps is abnormal as compared with the case where the cells are three-dimensionally cultured in the absence of the test substance, for example. (For example, in the case of lung cancer organoids, mutin-secreting duct-like structures are not formed, etc.), the number of cells in the cell clumps is small, and the immunostaining pattern of cancer organoids reflects natural cancer tissue. Not (eg, constituent cells [cancer cells (for colon cancer, for example, CK20 positive, for lung cancer, for example, CK7 positive), MSC / MPC-derived cells (eg, αSMA positive), vascular endothelial cells (eg, CD31) It can be evaluated that the formation of cancer organoids is inhibited when the number of positive cells is small, the Ki67-positive cells have a uniform distribution in the tissue, etc.).

In addition, when the cancer organoid is resistant to an anticancer drug (eg, CDDP, 5-fluorouracil, etc.), it is brought into contact with a test substance in the coexistence of the anticancer drug, and the anticancer drug is used. A test substance with reduced resistance to cancer can also be selected as a candidate drug for an anticancer drug that targets cancer stem cells.

In the method A of the present invention, the test substance having a more enriched structure of the cancer organoid and the test substance in which the formation of the cancer organoid was promoted in the method B of the present invention are the maintenance and amplification of cancer stem cells. And / or can be used as an induction promoter for cancer organoids.

6. Triple Screening System In a preferred embodiment, the test substance used in the methods of the invention is a gene of genes whose expression levels and / or epigenetic modifications differ significantly between cancer stem cells and non-stem cell cancer cells. A substance that can regulate expression or function.

As described above, the cancer stem cells used for the formation of cancer organoids in the present invention can preferably maintain embryonic stem (ES) cells as non-stem cell cancer cells into which an exogenous reprogramming factor has been introduced. It is an iCSC induced by culturing under no conditions. Not only does iCSC have no quantitative restrictions on itself or the cancer organoids derived from it, but it also differentiates into non-stem cell cancer cells during maintenance culture, so non-stem cell cancers that are genetically synonymous with iCSC. Cells can also be obtained without quantitative restrictions. In addition, when a strained cell is used as a cancer cell for iCSC induction, the parent strain (including the empty vector-introduced parent strain (mock) used for introducing the reprogramming factor) is also iCSC. It is a non-stem cell cancer cell that is genetically synonymous with, and can be obtained without quantitative restrictions.

Therefore, comprehensive gene expression between iCSC and genetically syngeneic non-stem cell cancer cells is compared using a microarray, or comprehensive DNA methylation by the WGBS method or PBAT method. By comparing the states, genes whose expression is increased or decreased specifically in cancer stem cells can be reliably identified. Since the expression and / or function of these genes is likely to be involved in the tissue remodeling ability of cancer stem cells, substances capable of regulating the expression or function of the genes target cancer stem cells. It can be said that it is a promising candidate drug that can be an anticancer drug.

Therefore, in a preferred embodiment of the invention, the method of the invention further comprises identifying genes with significantly different expression levels and / or epigenetic modifications between cancer stem cells and non-stem cell cancer cells. .. In this step, cancer stem cells and non-stem cell cancer cells genetically synonymous with them (eg, non-stem cell cancer cells differentiated from cancer stem cells in the maintenance culture process, parent cell line used for iCSC induction, etc.) ), For example, a comprehensive gene expression analysis using a microarray, or a comprehensive DNA methylation analysis by the WGBS method or the PBAT method, and comparing both data can be carried out.

As an example, by comparing the comprehensive gene expression of inducible colon cancer stem cells and genetically syngeneic non-stem cell colon cancer cells, as one of the genes whose expression is specifically increased in colon cancer stem cells. RCAN2 has been identified. Since RCAN2 has been reported to negatively regulate calcineurin (Cao X., et al., Biochem J 2002; 367: 459-66), we added the calcineurin inhibitor FK506. When the effects on the maintenance and amplification of colon cancer stem cells and the ability to form cancer organoids were investigated, FK506 promoted the maintenance and amplification of colon cancer stem cells and the formation of cancer organoids. On the other hand, since RCAN2 is known to promote the nuclear translocation of NFAT (nuclear factor of activated T cells) proteins, it is a GSK3 inhibitor (GSK3α) that promotes the translocation of NFAT from the nucleus to the cytoplasm, contrary to RCAN2. And siRNA, valproic acid, CHIR99021) on GSK3β were added to investigate the effects on the maintenance and amplification of colon cancer stem cells and the ability to form cancer organoids. It suppressed the formation of cancer organoids.

As another example, IL-6 is one of the genes whose expression is specifically increased in lung cancer stem cells by comparing the comprehensive gene expression of induced lung cancer stem cells and non-stem cell lung cancer cells genetically synonymous with it. Identified. Therefore, the present inventors investigated the effects of IL-6 and anti-IL-6 antibodies on the tissue reconstruction of lung cancer stem cells and lung cancer stem cells, and found that the addition of IL-6 resulted in the tissue reconstruction of the original lung cancer cells. While the ability was improved, the addition of anti-IL-6 antibody reduced the tissue remodeling ability of lung cancer stem cells. In addition, the cell mass formed from induced lung cancer stem cells was cisplatin (CDDP) resistant, but when combined with anti-IL-6 antibody, marked cell loss was observed, and the structure of lung cancer organoids was destroyed.

7. Cancer Organoids The present invention also provides cancer organoids that are derived from iCSC and can be supplied without quantitative restrictions. The cancer organoid can be obtained by co-culturing iCSC with mesenchymal stem / progenitor cells and vascular endothelial cells in a three-dimensional culture. The various conditions for three-dimensional culture are the same as above. More specifically, iCSC, which is used to produce cancer organoids, cultures non-stem cell cancer cells into which exogenous reprogramming factors have been introduced under conditions in which embryonic stem (ES) cells cannot be maintained. Drug elimination in the presence of an ABC transporter inhibitor at a concentration effective in suppressing the drug exclusion capacity of non-stem cell cancer cells thus induced and (a) not introducing organoid reprogramming factors. It is capable or does not dissociate from the culture vessel by (b) trypsin treatment.

The above-mentioned cancer organoids can also be provided as a kit in combination with the original iCSC. Furthermore, non-stem cell cancer cells differentiated from the iCSC and a parent cell line for inducing the iCSC can also be included in the kit. In the former case, the user can fractionate and obtain what is generated in the process of maintaining and culturing iCSC. In addition, the kit may include a substance that promotes maintenance and amplification of iCSC and / or induction of cancer organoids (for example, in the case of colorectal cancer stem cells, a calcineurin inhibitor such as FK506) as a component.

8. New Uses of IL-6 Modulators As shown in the examples below, according to the present invention, lung cancer stem cells promote the differentiation of MSC / MPC into αSMA-positive cells by highly expressing IL-6, thereby promoting lung cancer organoids. On the other hand, it was revealed that IL-6 inhibitors can suppress the differentiation of MSC / MPC into αSMA-positive cells by blocking the effect of IL-6.
Therefore, the present invention also provides an agent for controlling differentiation of MSC / MPC into αSMA-positive cells, which comprises an IL-6 regulator. Examples of the IL-6 enhancer include IL-6 itself (which may be in the form of recombinant cells expressing it). On the other hand, examples of IL-6 inhibitors include, but are not limited to, anti-IL-6 antibodies, aptamers against IL-6, siRNA against IL-6, antisense nucleic acids against IL-6, and the like.

The IL-6 regulator can be used as a differentiation regulator in addition to the medium in the differentiation or maintenance culture of MSC / MPC. It can also be used for the treatment and / or prevention of diseases associated with abnormal differentiation of MSC / MPC into αSMA-positive cells. Examples of such diseases include cancer, idiopathic interstitial pneumonia, idiopathic pulmonary fibrosis, drug-induced pulmonary fibrosis, and chronic kidney disease with renal fibrosis. When the IL-6 regulator is used for pharmaceutical purposes, it can be formulated as a pharmaceutical composition together with a pharmaceutically acceptable carrier. The IL-6 regulator is preferably prepared as a preparation for parenteral administration (eg, intravenous administration, intramuscular administration, subcutaneous administration, etc.). The administration method and dose of the IL-6 regulator are not particularly limited, but for example, in the case of an anti-IL-6 antibody, it can be appropriately selected according to the dosage and administration used in the existing drug.

Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited thereto.

In the examples described later, the experiment was performed as follows.
<Cell culture>
Human colorectal cancer cell lines (SW480) and Plat-A anhotropic retrovirus packaging cells were obtained from the ATCC Collection and Cell Biolabs (San Diego, CA, USA), respectively. In addition, a human lung cancer cell line (A549; RCB0098) was obtained from the RIKEN BioResource Center. Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies), penicillin (100 Units / ml) and streptomycin (100 μg / ml) (Life Technologies) (Nacalai Tesque, Kyoto, Japan) ), These cells were cultured in a humidified 5% CO _{2 incubator at 37 ° C.} In Plat-A cultures, 1 μg / ml puromycin (Nacalai Tesque) and 10 μg / ml Blasticidin (Funakoshi) were added. HUVEC (Lonza) was maintained in endothelial growth medium (Lonza) at 37 ° C. in a humidified 5% CO _{2 incubator.} Human MSC (Lonza) was maintained in endothelial growth medium (Lonza) (at the time of colorectal cancer organoid preparation) or MSC growth medium (Lonza) (at the time of lung cancer organoid preparation) at 37 ° C. in a humidified 5% CO _{2 incubator.} ..

<Retrovirus infection>
Retroviral vectors (pMXs-OCT3 / 4, pMXs-SOX2, pMXs-KLF4) that separately encode OCT3 / 4, SOX2 or KLF4 in pMX-based vectors were obtained from Addgene. For the production of colorectal cancer stem cells, we designed a polycistronic retroviral vector (pMXs-OKS) encoding OCT3 / 4, KLF4 and SOX2. Briefly, using each of the above vectors as a template, human OCT3 / 4, KLF4 and SOX2 are amplified by polymerase chain reaction (PCR) using primers containing the 2A sequence (T2A) of Thosea asigna virus and in-fusion. It was cloned into the EcoRI site of the pMX vector using an HD cloning system (Clontech).
One day prior to transfection, Plat-A packaging cells were seeded at a density of ^{1 × 10 6} cells or 1.2 × 10 ^{6 cells per 60 mm dish.} The next day, according to the manufacturer's instructions, with 3 μg of pMX-OKS (for SW480 cells) vector or pMXs-OCT3 / 4, pMXs-SOX2 or pMXs-KLF4 (for A549 cells) using Fugene HD transfection reagent (Promega). , Plat-A cells were transfected. After 24 hours of transfection, the Plat-A medium was replaced and after an additional 24 hours of culture, these virus-containing supernatants were filtered through a 0.45 mm cellulose acetate filter (Whatman) and 4 μg / ml polybrene (Nacalai Tesque). Was replenished. Equal amounts of pMXs-OCT3 / 4, pMXs-SOX2 and pMXs-KLF4 containing supernatants were mixed. These virus-containing supernatants were promptly added to SW480 or A549 cells seeded on a 60 mm dish the day before. 24-36 hours after infection, the virus-containing medium was replaced with new medium.

<Culture of cells after introduction of reprogramming factor, isolation of iCSC-enriched cell population and induction of cancer organoids>
Culture of SW480 cells after introduction of reprogramming factors, isolation of induced colon cancer stem cells (hereinafter, also referred to as “colon iCSC”) enriched cell population, and induction of colon cancer organoids are scheduled as shown in FIG. It was carried out in. The establishment culture of colon iCSC and the sorting of the colon iCSC enriched cell population were performed according to the method described in Non-Patent Document 4 (flow cytometry utilizing Hoechst 33342 exclusion ability in the presence of 50 μM verapamil). Below, the colon iCSC-enriched cell population (cell population unlabeled by Hoechst 33342 in the presence of 50 μM verapamil; V50 cells) obtained by the first sorting is divided into 1st V50 cells (or 1st V50-OKS cells) and the second sorting. The obtained V50 cells may be abbreviated as 2nd V50 cells (or 2nd V50-OKS cells). Induction of colorectal cancer organoids was performed by the method described in the sphere formation assay described later.
On the other hand, A549 cells after introduction of the reprogramming factor were maintained in DMEM supplemented with 10% FBS, 0.5% penicillin and 0.5% streptomycin at _{37 ° C. and 5% CO 2 atmosphere.} Colonies appearing after 10-15 days were treated with 0.25% trypsin for 6 minutes to remove dissociated cells and trypsin, after which the cells remaining on the dish were washed twice with PBS. Cell colonies were collected, gently pipetted and then transferred to a new 60 mm dish. About 5 colonies can be subcultured per dish. Larger colonies appeared 7 to 10 days after passage, and spindle-shaped cells were observed around them. The colonies were treated with 0.25% trypsin for 6 minutes and the cells dissociated in the supernatant were collected as OSK-A549-SN cells. The cells remaining on the dish were washed twice with PBS. Cell colonies were collected as inducible lung cancer stem cells (lung iCSC; OSK-A549-Colony cells), gently pipetted and then transferred to a new dish. Lung cancer organoids were induced by the method described in the sphere formation assay described below.

<Sphere formation assay>
Serum-free DMEM (sphere) containing 10 ng / ml bFGF (WAKO), 10 μg / ml human insulin (CSTI), 100 μg / ml human transferase (Roche) and 100 μg / ml BSA (Nacalai Tesque). The cells were transferred to an Ultra Low Attachment plate (Corning) supplemented with (forming medium) and _{incubated in a 5% CO 2} incubator at 37 ° C. for 6 or 10 days. The number of spheres was calculated based on the size of spheres greater than 100 μm.

<Calcineurin inhibition and GSK3 inhibition>
SW480 cells and 2nd V50 cells with calcineurin inhibitor (FK506 (Sigma, 25 μM)) or GSK3 inhibitor (valproic acid (1 mM) or CHIR99021 (3 μM)) for 5 days (in the case of adhesive culture) or 10 days, respectively. After treatment (in the case of suspension culture), the number of cells (spheres) was counted using the Countess (Invitrogen) system.
In addition, Duplexed Stealth siRNA (Invitrogen) was used in SW480 cells and 2nd V50 cells, respectively, and the following siRNAs were introduced into SW480 cells and adherently cultured according to the manufacturer's protocol.
GSK3α siRNA 5'-CCA AGG CCA AGU UGA CCA UCC CUA U-3'(SEQ ID NO: 1)
GSK3β siRNA 5'-GCU CCA GAU CAU GAG AAA GCU AGA U-3'(SEQ ID NO: 2)
SCRAMBLED siRNA 5'-AAU UCU CCG AAC GUG UCA CGU GAG A-3'(SEQ ID NO: 3)
On the 5th day after siRNA introduction, the number of cells was counted using the Countess (Invitrogen) system.

<Co-culture with HUVEC and MSC>
5 × 10 ⁵ parental SW480 cells or 2nd V50 cells were ^{resuspended in sphere formation medium with 5 × 10 4} HUVEC and 2 × 10 ⁵ MSC to create a low-adhesion 24-well flat plate (Prime Surface® 24F, Sumitomo). Bakelite) was sown. In addition, A549 cells or OSK-A549-Colony cells were similarly ^{co-cultured with HUVEC and 2 × 10 5} MSC (at this time, cancer (stem) cells: HUVEC: MSC = 5: 1: 4-5: Mixed at a ratio of 4: 4). After 6-12 days, self-assembled spheres were photographed with BZ8000 (Keyence) and pathologically analyzed.
5 μM CDDP, 1 μg / ml anti-IL-6 antibody (R & D, MAB206) and 10 ng to investigate CDDP sensitivity in A549 or OSK-A549-Colony cell-derived spheres and the function of IL-6 in forming spheres. The sphere was cultured in the presence or absence of / ml IL-6. The pictures taken were analyzed and the fluorescence intensity was calculated using the ImageJ software program.

<Histological and immunohistochemical analysis of spheres>
The sphere was embedded in a paraffin block and sectioned to a thickness of 5 μm. Deparaffinized sections, hematoxylin and eosin (HE), anti-human cytokeratin 20 (CK20) mouse monoclonal antibody (clon: Ks20.8, diluted 1:50, Dako), anti-human cytokeratin 7 (CK7) mouse monoclonal Antibodies (clone: OV-TL 12/30, diluted 1:50, Dako), anti-CDX2 mouse monoclonal antibody (CM226, diluted 1:50, Biocare Medical), anti-Ki67 mouse monoclonal antibody (clone: MIB-1, Stained with 1:50 diluted Dako), anti-αSMA mouse monoclonal antibody (clone: 1A4, diluted 1:50, Dako) and anti-CD31 mouse monoclonal antibody (clone: JC70A, diluted 1:50, Dako). Immunohistochemical analysis was performed using the XT ultraView Universal DAB detection kit (Ventana Medical Systems, Inc) and the Benchmark XT (Roche) autostainer. The proportion of Ki67- and αSMA-positive cells was calculated by counting the number of immunohistochemically positive cells and the total number of nuclei in the three high-power fields.

<RNA isolation and quantitative reverse transcriptase polymerase chain reaction>
Total RNA of cells was extracted using Trizol (Life Technologies). 500 ng of total RNA was reverse transcribed into cDNA using the Prime Script ^TM II 1st strand cDNA Synthesis Kit (Takara) and LightCycler® 480 real-time PCR using ^{SYBR® Premix Ex Taq TM II (Takara).} Quantitative PCR analysis was performed on the system (Roche). The PCR primers used are listed in Table 1.

<Microarray analysis>
Mock-SW480 cells (cells transfected with SW480 cells with an empty vector), a non-V50 cells and 2 ^nd V50-OKS cellular RNA from 1 ^st V50-OKS cells were harvested 5 days after sorting. RNA was also recovered from parent A549 cells, OSK-A549-Colony cells and OSK-A549-SN cells obtained from three independent retrovirus introduction experiments. Gene expression profiling was performed using SurePrint G3 human GE microarray (Agilent Technologies) according to the manufacturer's protocol. Data were analyzed using the GeneSpring 13.0 software program (Agilent Technologies). The data processing was performed as follows: (i) the threshold raw signal was set to 1.0, (ii) log-based 2 conversion was performed, and (iii) 75th percentile normalization was selected as the standardization algorithm (http: /). /genespringsupport.com/faq/normalization). The flags were set as follows: Features were not positive and significant (not detected), not uniform (compromised), not above background (not detected), saturated (compromised), or population outlier (compromised). bottom. The control probe was removed and only the "detected" probe present in at least one of all samples was used for further analysis. The number of probes used in the analysis is 50,739 (Mock-SW480 cells or 1 ^st V50-OKS-cell non-V50 cells vs 2 ^nd V50-OKS cells derived) or 30 886 (A549 cells or OSK-A549-SN cells vs OSK-A549 -Colony cells).

<Statistical analysis>
All data was analyzed using the jstat software program. Data values were expressed as the mean ± standard error (SEM) of three independent experiments. Differences in mean values between the two groups in FIGS. 1A, 3E, 5A, 5D, 6A-6C, 9H, 9I, 11C and 11F were analyzed using a two-sided t-test. The Bonferroni's test was used for data analysis in FIGS. 7A and 9G. Repeated measures ANOVA test was used for data analysis in Fig. 9C. Dunnet's test was used for multiple comparisons in Figure 10C. Differences between the data were considered statistically significant when the P values were <0.05 (*) and <0.01 (**).

Example 1: Verification of in vitro tissue remodeling ability of large intestine iCSCs It has been previously reported that CSCs have a high sphere-forming ability when cultured in a low-adhesion culture dish using a serum-free medium (Ricci). -Vitiani L. et al., Nature 2007; 445: 111-51, Sato T. et al., Gastroenterology 2011; 141: 1762-72). A sphere formation assay was performed to examine the sphere formation ability of these cells.

O + S + K-SW480 previous reports in the large intestine iCSC with (non-patent document 4) consistent with, while the number of spheres in the 2 ^nd V50-OKS cells increased obviously, the parent SW480 spheres Was difficult to find (Fig. 1A). The number of spheres was at an intermediate level in non-V50 cells derived from ^{1 st V50-OKS cells (Fig. 1A).}
Previous xenograft experiments demonstrated that colorectal iCSC, rather than the parental cell line, can reconstruct tissue similar to actual human colorectal cancer tissue in vivo in terms of immunohistological findings (non-patented). Document 4). However, it remains unclear whether colorectal iCSC can exhibit the same phenomenon in vitro. Therefore, we evaluated the parental SW480 cells and 2 ^nd V50-OKS-cell-derived spheres immunohistochemistry. 2 ^nd V50-OKS cell derived spheres, CK20 and CDX2 were positive, CK7 was negative (Fig. 1B). This is consistent with the staining pattern for typical colorectal cancer tissues (Bayrak R et al., Diagn Pathol 2012; 7: 9), but spheres derived from parental SW480 cells are CK20 negative (Fig. 1B). This indicates that colorectal iCSC-derived tissue can reconstruct human colorectal cancer tissue-like structure (colorectal cancer organoid) not only in vivo but also in vitro. Therefore, it is considered that the sphere forming ability can be evaluated as an index of the tissue restructuring ability of these iCSCs.

Human colon cancer tissue is known to consist of not only cancer cells but also stromal cells such as blood vessel and mesenchymal cells (Takebe T. et al., Cell Stem Cell 2015; 16: 556-65, Plaks V. et al., Cell Stem Cell 2015; 16: 225-38). Therefore, when colon iCSC is cultured with mesenchymal stem cells (MSC) and human umbilical vein endothelial cells (HUVEC), more realistic colon cancer organoids can be constructed together with stromal cells in vitro. I checked whether it was.

2 ^nd V50-OKS cells co-cultured with MSC and HUVEC, unlike parental SW480 cells resulted in a large aggregated set cells (Figure 2A). Was subjected to immunohistochemical analysis, in spheres from ^{2 nd V50-OKS, α-} SMA and CD31 (respectively a myofibroblast marker derivable from MSC and vascular cells (Quante M. et al., Cancer Cell 2011; 19: 257-72)) Positive cells were observed, but not in spheres derived from parental SW480 cells (Fig. 2B). This indicates that only colorectal iCSC can build tissue with HUVEC and MSC and lead colorectal cancer organoids to a more mature stage.

Example 2: Mock-SW480 cells, comparison of gene expression profiles in a non-V50 cells and 2 ^nd V50-OKS cells
To identify the molecular mechanisms that promote properties of CSCs, Mock-SW480 cells after 5 days of sorting, an exhaustive gene expression patterns in non V50 cells and 2 ^nd V50-OKS cells from 1 ^st V50-OKS cells, Compared by microarray. First, Mock-SW480 and compared gene expression between 2 ^nd V50-OKS cells, probes 3914 have been identified to have significant differences in their expression (t-test, the false positive rate (FDR) <0.05 and Fold Change> 2) (Fig. 3A). Then, the gene expression profile of 2 ^nd V50-OKS cells, compared to non-V50 cell gene expression profiles from 1 ^st V50-OKS cells (Figure 3B). We identified 56 probes with FDR <0.05 and Fold Change> 2. Then, non-V50 cells, draw a Venn diagram of a probe to lower expression in 2 ^nd V50-OKS cells than higher expression probe, and non-V50, Mock-SW480 cells in 2 ^nd V50 than mock overlap in Venn Eight probes were selected (Figure 3C). Of these eight probes, semaphorin 6A (SEMA6A), FAM105A (family with a sequence similarity 105 member A), and RCAN2 (regulator of calcineurin), in which the expression level in each cell and the sphere forming ability are in a parallel relationship. We narrowed down to 3 probes including 2) (Fig. 3D, 3E).

SEMA6A is a member of the semaphorin family that plays a role in many developmental processes outside the nervous system (Luo Y. et al., Cell 1993; 75: 217-27). In addition to these normal functions of semaphorins, many semaphorins have been found to have functional activity associated with tumor progression (Neufeld G. et al., Cold Spring Harb Perspect Med 2012). 2: a006718, Worzfeld T. et al., Nat Rev Drug Discov 2014; 13: 603-21). At present, few reports have been made on FAM105A, but FAM105A has a conserved protein domain of Rho-GAP (Marchler-Bauer A. et al., Nucleic Acids Res 2015; 43: D222-6). May regulate RhoG protein. RCAN2 was originally identified as the human fibroblast-derived thyroid hormone response gene ZAKI-4 (Miyazaki T. et al., J Biol Chem 1996; 271: 14567-71) and subsequently functions as a negative regulator of calcineurin. Has been reported (Cao X., et al., Biochem J 2002; 367: 459-66).

Next, we decided to conduct experiments using SEMA6A, FAM105A or RCAN2, or small molecule compounds that act on their target molecules. However, neither agonists nor antagonists of SEMA6A were commercially available as far as we were looking for, and the function of FAM105A was unknown. In contrast, RCAN2 is known to negatively regulate calcineurin, and calcineurin-NFAT (nuclear factor of activated T cells) such as FK506, which is theoretically presumed to have the same effect as RCAN2 on colorectal iCSC. Signal transduction inhibitors have been commercially available. Therefore, we focused on assessing the effect of FK506 on the characteristics of colorectal iCSC.

Example 3: Verification of the effect of calcineurin inhibition on planar adhesive culture and sphere forming ability of large intestine iCSC
FK506 significantly reduced the number of cells in parental SW480 cultures, consistent with reports that inhibition of calcineurin inhibits the growth of colon cancer cell lines in vitro (Peuker K. et al.,, Nat Med 2016; 22: 506-15). Meanwhile, in the 2 ^nd V50-OKS culture, significant effects on cell number of FK506 was observed (Figure 5A) (passaged ± FK506 added 5 days later). Furthermore, significant observed form or domed colonies in the 2 ^nd V50-OKS cells, but was more pronounced with the use of FK506, cellular form of the parent SW480 did not change with FK506 (FIG. 5B) (passage ± 5 days after addition of FK506). These data suggest that FK506 has different effects on colon iCSC and parent SW480 cells.
Next, the sphere forming ability was evaluated as an index of the tissue remodeling ability of the large intestine iCSC with or without FK506. FK506 has been significantly increased the number of spheres in the 2 ^nd V50-OKS cells did not increase in the parental SW480 cells (Fig. 5C, 5D). Immunohistochemical analysis, spheres of 2 ^nd V50-OKS cells undergoing FK506 treatment, CK20 and CDX2 were positive, CK7 is negative, was the same pattern as the spheres using no FK506 (FIG. 5E) ..

Example 4: Verification of the effect of GSK3 inhibition on planar adhesive culture and sphere-forming ability of large intestine iCSC Calcineurin has been reported in other cells to promote nuclear translocation of NFAT. Conversely, GSK3 was known as a molecule that promotes the translocation of NFAT from the nucleus to the cytoplasm. Therefore, we conducted this experiment based on the hypothesis that inhibition of GSK3 on colorectal cancer stem cells has the opposite effect to the calcineurin inhibitor FK506.
First, GSK3 was inhibited using siRNA against GSK3α and GSK3β. As a result, by adding both siRNA against GSK3α and siRNA against GSK3β in the planar adhesive culture, the morphological characteristics (dome-shaped colonies) of iCSC were suppressed and flattened, and the number of cells also decreased (Fig. 6A). .. No effect was seen with siRNA alone, suggesting that there is a functional redundancy between GSK3α and GSK3β in colorectal cancer.
Next, it was investigated whether the addition of valproic acid (VPA) or CHIR99021 (CHIR), which are inhibitors of both GSK3α and β, had the same effect as siRNA. As a result, as shown in FIG. 6B, all GSK3 inhibitors were found to have the same effect as siRNA. In the same manner as in Example 3, it was examined the effect of these inhibitors on the sphere forming ability, any of GSK3 inhibitors were also dominantly suppressed sphere formation ability of large intestine iCSC (2 ^nd V50).

Example 5: Preparation of lung cancer-like tissue (lung cancer organoid) in vitro (1)
OCT3 / 4, SOX2 and KLF4 were introduced into a human lung cancer cell line (A549) having a G12S mutation in KRAS using a retroviral vector (OSK-A549 cells), and cultured in DMEM containing 10% FBS. After 2 weeks, the proliferation rate of OSK-A549 cells was reduced compared to parent A549 cells and EGFP-A549 cells (pMx-EGFP introduced into A549 cells as a transfection control). To examine the ability to form spheres, which is considered to be a CSC characteristic, these cells were transferred to low-adhesion plates on days 10, 20 and 30 after transfection and cultured in suspension. Parental A549 and EGFP-A549 cells formed less than 3 spheres under either condition, whereas the number of spheres formed from OSK-A549 cells increased significantly (Fig. 7A). In particular, the ability to form spheres was high in cells 20 days after gene transfer. Domed colonies appeared only on OKS-A549 cells 10 to 15 days after gene transfer. When the colonies were picked up and passaged, the colonies became larger during the week, giving rise to spindle-shaped cells around them. Using the difference in dissociation resistance to trypsin, the dome-shaped colonies (OSK-A549-Colony cells) remaining on the dish and the dissociated spindle-shaped cells (OKS-A549-SN cells) were isolated separately. We were able to.
Next, the sphere-forming ability of OSK-A549-Colony cells was examined. As a result of phase-contrast microscopy, clear cell aggregation was observed in OSK-A549-Colony cells, whereas such cell aggregation was not observed in parent A549 cells (Fig. 7B, Phase). As a result of HE staining, OSK-A549-Colony cells formed nectar-like spheres, whereas parent A549 cells were unable to form such spheres (Fig. 7B, HE). .. Alcian blue-PAS staining was used to examine mucin secretion in tissues prepared from OSK-A549-Colony cells. As a result, a polar duct-like structure with mucin secretion was observed in the tissue (Fig. 9A).

Example 6: Preparation of lung cancer-like tissue (lung cancer organoid) in vitro (2)
Lung iCSC (OSK-A549-Colony cells) prepared from a normal lung cancer cell line (A549) or A549 and stromal cells (HUVEC, and MSC) were co-cultured on a low-adhesion plate (Fig. 8, top). ). As a result, a more realistic tissue similar to human lung cancer tissue could be constructed only by co-culture of lung iCSC and stromal cells (Fig. 8, bottom). That is, in co-culture using a normal cell line (A549), spindle-shaped αSMA-positive cells were not observed, and sparse cell clumps consisting only of CK7-positive cancer cells were formed, whereas lung iCSC (OSK) was formed. In co-culture using -A549-Colony cells), solid tissues similar to human lung cancer tissue including not only CK7-positive lung cancer cells but also αSMA-positive cancer associated fibroblast (CAF) -like cells were formed. These findings suggest that lung iCSC has some effect on non-cancer cells (αSMA-positive cells are probably derived from mesenchymal stem cells).

Next, in order to visualize the cell composition of this lung cancer organoid, the cancer cells (lung iCSC or parent cell line) were colored with GFP, and the vascular endothelial cell line HUVEC was colored with m-cherry. Microscopic observation was performed. As a result, mcherry-positive cells were amplified in lung iCSC, but mcherry-positive cells were decreased in the cell mass prepared in the parent cell line (Figs. 9B and 9C). In addition, in the cell mass prepared using lung iSCS, the number of CD31-positive cells, which is an endothelial cell marker, was increased (Fig. 9D). This suggests that lung iCSC acts on vascular endothelial cells in some way.

In human lung cancer tissue specimens, Ki67-positive cells are more frequently detected outside than inside the cancer cell mass (Fig. 9E, right panel), while similar peripheral dominant Ki67-positive cells are lung iCSC-derived organoids. It was found, but not found in parental A549 cell-derived organoids (Figs. 9E, 9F).

Example 7: Verification of the effect of IL-6 inhibition on lung iCSC-derived cancer organoids
Comprehensive gene expression in A549 cells and lung iCSC (OSK-A549-Colony cells) prepared from them was compared by microarray, and the expression fluctuated significantly in OSK-A549-Colony cells (P <0.05, Fold Change>. 2) 575 genes were identified. Next, the expression of these genes was compared between OSK-A549-Colony cells and non-stem cell lung cancer cells (OSK-A549-SN cells) differentiated from the iCSC, and the results are shown in a volcano plot (volcano plot). Figure 10A). 54 genes with significantly altered expression (P <0.05, Fold Change> 2) were identified in OSK-A549-Colony cells. Among them, IL-6 was highly specifically highly expressed in OSK-A549-Colony cells. As a result of quantitative RT-PCR, it was confirmed that IL-6 expression was remarkably upregulated in OSK-A549-Colony cells as compared with A549 cells and OSK-A549-SN cells (Fig. 9G). Therefore, it was suggested that IL-6 is important in the characteristics of lung iCSC (Fig. 10A).

Cell clumps formed by co-culture with A549 (labeled with green fluorescence) are CDDP-sensitive, whereas tissues formed with lung iCSC (labeled with green fluorescence) are CDDP-resistant. Therefore, it was investigated whether the addition of anti-IL-6 antibody changes the CDDP resistance of lung iCSC (OSK-A549-Colony cells) -derived tissues. As a result, a marked decrease in cells was observed when the anti-IL-6 antibody was used in combination with CDDP (Figs. 10B, 10C, 10D).

Example 8: Verification of effect of IL-6 regulation on lung cancer cells or lung iCSC-derived cancer-like tissue IL in cell clumps formed by co-culture using normal lung cancer cell line A549 (labeled with green fluorescence) The effect was verified by adding -6. As a result, the addition of IL-6 made the cell clumps more solid, and the EGFP fluorescence intensity and αSMA-positive cells were significantly increased (FIGS. 11A and 9H, 11B and 11C). On the other hand, when an anti-IL-6 antibody was added to a lung cancer organoid formed by co-culture using lung iCSC (labeled with green fluorescence) and its effect was verified, the EGFP fluorescence intensity was verified by adding the anti-IL-6 antibody. Was not attenuated (FIGS. 9I and 11D), but αSMA-positive cells were significantly reduced (FIGS. 11E and 11F). In addition, when IL-6 was added to the adherent culture of MSC alone, αSMA-positive cells appeared (Fig. 11G). From these results, IL-6 has a positive effect on lung cancer tissue formation by converting MSC to αSMA-positive cells, and anti-IL-6 antibody destroys cancer tissue by blocking this. It was suggested to get.

The screening system of the present invention using cancer stem cells, non-stem cell cancer cells, and a triad of cancer organoids derived from the cancer cells can be used in any sample if iCSC is used as the cancer stem cells. Since there are no specific restrictions, it is extremely useful as an HTS system of anti-cancer drugs that can be put to practical use.

Claims (6)

Dissociated cancer stem cells and mesenchymal stem / progenitor cells and vascular endothelial cells are cancer organoids that are self-assembled in vitro.
(A) The cancer stem cells are cancer stem cells derived from colon cancer or lung cancer cells into which an exogenous reprogramming factor has been introduced.
(B) The cancer organoid includes cancer stem cells, non-stem cell cancer cells genetically syngeneic to the cancer stem cells, αSMA-positive cells derived from mesenchymal stem / progenitor cells, and vascular endothelial cells.
(C) The following features:
(I) Immunohistochemical characteristics of natural colorectal cancer or lung cancer tissue, ie, colorectal cancer organoids are CK20 positive, CK7 negative and CDX2 positive, and lung cancer organoids are CK7 positive, TTF1 positive and Napin A. Positive;
(Ii) In the case of lung cancer organoids, Ki67-positive cells are frequently present outside the inside of the cell mass; and
(Iii) In the case of lung cancer organoids, it has a mucin-secreting duct-like structure;
A cancer organoid having at least one of . Cancer stem cells are induced by culturing non-stem cell cancer cells into which an exogenous reprogramming factor has been introduced under conditions in which embryonic stem (ES) cells cannot be maintained, and (a) exogenous. In the presence of an ABC transporter inhibitor at a concentration effective in suppressing the drug exclusion ability of non-stem cell cancer cells into which a reprogramming factor has not been introduced, or has the drug exclusion ability (b). by trypsinization those that do not dissociate from the culture vessel, cancer organoid according to claim 1. A method for screening an anticancer drug, which comprises the following steps.
(1) Step of contacting the test substance with the cancer organoid according to claim 1 or 2 (2) Step of selecting the test substance that caused deconstruction of the cancer organoid as an anticancer drug. A method for screening an anticancer drug, which comprises the following steps.
(1) Dissociated cancer stem cells and mesenchymal stem / progenitor cells and vascular endothelial cells are three-dimensionally cultured in the presence of a test substance to induce the formation of cancer organoids according to claim 1 or 2. Step (2) A step of selecting a test substance that inhibits the formation of cancer organoids as an anticancer drug as compared with the case of three-dimensional culture in the absence of the test substance. The substance according to claim 3 or 4 , wherein the test substance is a substance capable of regulating the expression or function of a gene whose expression level and / or epigenetic modification is significantly different between cancer stem cells and non-stem cell cancer cells. the method of. The method of claim 5, further comprising identifying genes with significantly different expression levels and / or epigenetic modifications between cancer stem cells and non-stem cell cancer cells.

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