WO2017126616A1 - Cell model for ewing's sarcoma family of tumors, and antitumor agent screening method using same - Google Patents

Abstract

In order to address the problem of establishing a cell model for the Ewing's sarcoma family of tumors that reacts directly with chimeric genes to form tumors and can be used in screening for an effective, novel anticarcinogen for the Ewing's sarcoma family of tumors, the present invention provides an Ewing's sarcoma family of tumors (ESFT) cell derived from bone marrow stromal cells, wherein an EWS chimeric gene controlled by a promoter that includes an inducible transcriptional regulatory sequence is introduced into a chromosome, and the ESFT cell is viable and has unlimited proliferative capacity during expression of the EWS chimeric protein.

Description

Ewing sarcoma family tumor model cell and screening method for antitumor agent using the same

The present invention relates to a cell and a method for screening a drug using the cell, and specifically relates to a screening method for an Ewing sarcoma family tumor model cell and an antitumor agent using the same.

Ewing's sarcoma of family tumors (ESFT) are sarcomas that affect bone and soft tissue from childhood to adolescence. The site of onset varies, and there are many metastatic sites, such as lung, bone marrow, and bone.

Chimeric genes have been detected in most Ewing sarcoma family tumor patients, and the chimeric genes are considered to be the causative genes for carcinogenesis. If a genetic test detects chimeric genes such as EWS-FLI, EWS-ERG, EWS-ETV1, and EWS-FEV, it will be a definitive diagnosis of Ewing sarcoma family tumors.

Chemotherapy that is highly effective against Ewing sarcoma family tumors includes doxorubicin, cyclophosphamide, vincristine, ifosfamide, etoposide, and actinomycin, and multidrug chemotherapy combining these is performed. Although progress in chemotherapy has improved treatment outcomes, there is nothing definitive, and multidisciplinary treatment combining chemotherapy, surgery, and radiation therapy is necessary. In particular, the prognosis is poor when it occurs in a region where it is difficult to perform surgery and radiation irradiation such as the head and neck, spine, and pelvis. In addition, long-term survivors after treatment have increased secondary cancer caused by anticancer drugs and radiation, and long-term follow-up of late complications including secondary cancer is important.

In view of the above background, development of an effective new anticancer substance against Ewing sarcoma family tumors is desired. In particular, development of molecular target-specific inhibitors for chimeric genes that are characteristic of Ewing sarcoma family tumors and that are the root cause of sarcoma development is expected.

To date, efforts have been made to create animal models and cell models of Ewing sarcoma family tumors using chimeric genes, but there are reports of sarcoma formation and cell line production by introducing chimeric genes, but the creation of tumor models that respond directly to chimeric genes. There are no reports. For example, in Non-Patent Documents 1 and 2, the chimeric gene EWS-FLI1 is introduced into a primary-cultured bone-derived cell or bone marrow-derived mesenchymal progenitor cell using a retroviral vector to produce a tumor, but EWS-FLI1 is made constitutively. It is unclear whether tumor cells have acquired cell proliferation and tumorigenicity depending on EWS-FLI1.

Conventional Ewing sarcoma family tumor cell lines do not react directly with chimeric genes, and even if they are used for drug screening, it is unknown whether the compound's site of action is a chimeric gene, and it simply suppresses cell growth. There was a problem that the possibility of obtaining a compound was high.

Therefore, an object of the present invention is to establish a model cell of an Ewing sarcoma family tumor that can be used for screening of an effective new anticancer substance against an Ewing sarcoma family tumor and that forms a tumor by directly reacting with a chimeric gene. To do.

As a result of intensive studies to solve the above problems, the present inventors succeeded in constructing an EWS-FLI1-dependent osteosarcoma model from mouse bone marrow stromal cells using the doxycycline-inducible EWS-FLI1 system. did. Furthermore, using artificial pluripotent stem (iPS) cell technology, we have succeeded in obtaining EWS-FLI1-dependent osteosarcoma-derived iPS cells having cancer-related gene abnormalities. And it discovered that these cells could be used suitably for the screening of the medicine with respect to Ewing sarcoma family tumor, and came to complete this invention.

That is, the present invention provides the following.
[1] Ewing sarcoma family tumor cells derived from bone marrow stromal cells, in which an EWS chimeric gene placed under the control of a promoter containing an inducible transcriptional regulatory sequence is introduced onto the chromosome and grows upon expression of the EWS chimeric protein Ewing sarcoma family tumor cells that are capable and have acquired infinite growth potential (immortalized).
[2] The Ewing sarcoma family tumor cell according to [1], wherein the EWS chimeric gene is the EWS-FLI1 gene.
[3] The Ewing sarcoma family tumor cell according to [1] or [2], wherein the EWS chimeric gene is placed under the control of a promoter containing an inducible transcription control sequence together with a drug resistance gene.
[4] The Ewing sarcoma family tumor cell according to any one of [1] to [3], wherein the inducible transcription control sequence is a Tet operator and the cell expresses a reverse tetracycline-regulated transactivator.
[5] The Ewing sarcoma family tumor cell according to any one of [1] to [4], wherein the EWS chimeric gene is introduced onto a chromosome using a lentiviral vector.
[6] An induced pluripotent stem cell obtained by reprogramming the Ewing sarcoma family tumor cell according to any one of [1] to [5].
[7] Ewing sarcoma family tumor model non-human mammal, which is transplanted with the Ewing sarcoma family tumor cell according to any one of [1] to [5] and develops Ewing sarcoma family tumor depending on the expression of EWS chimeric protein .
[8] A step of adding a test compound to the Ewing sarcoma family tumor cell according to any one of [1] to [5] cultured under the induction of EWS chimeric gene expression, Examining a tumor phenotype and selecting a test compound as a therapeutic drug candidate compound for an Ewing sarcoma family tumor, using as an index the reduction of the proliferative ability of the Ewing sarcoma family tumor cells or the reduction of the tumor phenotype A screening method for an Ewing sarcoma family tumor therapeutic agent comprising a step.
[9] A step of differentiating the induced pluripotent stem cell according to [6] into a bone cell, a step of inducing expression of an EWS chimeric protein, and a test compound on the Ewing sarcoma family tumor cell expressing the obtained EWS chimeric protein The step of adding, the step of determining the proliferative ability or tumor phenotype of the Ewing sarcoma family tumor cells, and reducing the proliferative ability of the Ewing sarcoma family tumor cells or reducing the tumor phenotype, A method for screening an Ewing sarcoma family tumor therapeutic agent, comprising a step of selecting a test compound as a therapeutic drug candidate compound for an Ewing sarcoma family tumor.
[10] A step of culturing the induced pluripotent stem cell according to [6] in a bone differentiation-inducing medium to which a test compound is added in a state where no EWS chimeric protein is expressed to differentiate into an osteocyte, and a degree of differentiation into an osteocyte A screening method for a bone differentiation promoting substance, comprising a step of determining, and a step of selecting a test compound as a bone differentiation promoting substance using as an index an increase in the degree of differentiation into the bone cells.

According to the present invention, sarcoma cells capable of controlling the expression of chimeric genes (such as EWS-FLI1) detected in Ewing sarcoma family tumors by stimulation with drugs and the like, and iPS cells derived from sarcoma cells were obtained. This sarcoma cell differs from conventional cell lines in that cell proliferation activity is induced depending on the expression of the chimeric gene. In addition, sarcoma cell-derived iPS cells can form Ewing sarcoma family tumors by expressing a chimeric gene after induction of bone differentiation. These are considered cell lines that can directly monitor the effects of the chimeric gene.
Cell lines that respond to the Ewing sarcoma family tumor-associated chimeric gene are useful for screening the development of direct molecular target-specific inhibitors against the chimeric gene. Moreover, since ON / OFF of the expression of the chimeric gene can be regulated and the phenotype of the tumor can be reversibly controlled, it can be suitably used for analyzing the mechanism of osteosarcoma generation.

Schematic diagram of EWS-FLI1 expression system using lentivirus. Lentiviral vectors are introduced into bone marrow stromal cells collected from Rosa26-M2rtTA mice, and EWS-FLI1-expressing neomycin resistant cells are selected. The microscope picture which shows the growth in the Dox containing medium or Dox free medium of the immortalized cell (EFN # 2) in each cell. EFN # 2 grew rapidly in Dox-containing medium. Dox removal caused delayed growth and morphological changes in EWS-FLI1-expressing cells (4 days after removal). The graph which shows the result of qRT-PCR which shows the mRNA expression of EWS-FLI1 with and without Dox in a Dox treatment sample. Data are presented as mean ± SD. The expression level of Dox-untreated cells was set to 1. The figure (photograph) which shows the result of the western blotting which uses an anti- HA antibody. EWS-FLI1 protein was detected in the presence of Dox. The photograph which shows the tumor formation in the presence or absence of Dox of the mouse | mouth which transplanted the EWS-FLI1-dependent immortalized cell (EFN # 2). EFN # 2 developed tumors in immunocompromised mice only in the presence of Dox (10 weeks after transplantation). The figure which shows the tumor weight 10 weeks after the transplantation of EFN # 2 with and without Dox. Tumor development was dependent on Dox administration. The figure which shows the result of the cell proliferation assay of the constructed | assembled EWS-FLI1 dependent sarcoma cell line (SCOS # 2 and SCOS # 12). Sarcoma cell proliferation was dependent on EWS-FLI1 expression, and sarcoma cells that had not received Dox exposure began to lose proliferation 3 days after Dox removal. Gene ontology enrichment analysis showed that extracellular region-related genes and matrix-related genes were upregulated 72 hours after Dox removal in SCOS # 2 cells. Those genes with elevated expression were selected with a hold change greater than 1.5 and a cut-off point with a p-value less than 1.0E-4. The top five enriched clusters are revealed. Scatter plot analysis of osteogenesis related genes and chondrogenesis related genes. It was revealed that osteogenesis-related genes and chondrogenesis-related genes are upregulated 72 hours after Dox removal in SCOS # 2 cells. The figure (photograph) which shows the result of alkaline phosphatase dyeing | staining. Above is a photomicrograph. Five days after the removal of Dox, the sarcoma cells showed alkaline phosphatase activity. Scale bar, 100 μm. A photomicrograph of cells 38 days after Dox removal (bottom with Dox). Heterogeneous cells were observed that grew slowly 38 days after Dox removal. The figure which shows the result of having measured mRNA expression level by qRT-PCR 38 days after the removal of Dox (the left has Dox). 38 days after Dox removal, the cells showed higher expression of bone differentiation related genes. Data are presented as mean ± SD. The expression level of Dox treated cells was set to 1. In qRT-PCR, Sost, Fgf23 and Mepe were not detectable in Dox-treated samples. Therefore, the expression level of Dox-untreated cells was set to 1 instead. The figure (photograph) which shows the result of HE dyeing | staining, Ki67 immunohistochemistry, and Alizarin red dyeing | staining. HE staining shows that Dox removal caused a significant decrease in small blue cell population and increased bone formation. Ki67 immunohistochemistry shows active cell proliferation in sarcomas due to Dox. Scale bar, 50 μm (Dox treated), 200 μm (Dox untreated, top), 50 μm (Dox untreated, bottom). FIG. 6 shows the results of an in vivo tumor formation assay using sarcoma cell line SCOS # 2. Dox treatment was discontinued at 3 weeks and mice were sacrificed at 7 weeks. The micrograph of the obtained iPS cell-like cell. IPS cell-like cells were constructed from sarcoma cells by introducing reprogrammed transcription factors. The graph which shows the result of qRT-PCR which shows the expression level of a pluripotency related gene in a sarcoma origin iPS cell-like cell. It was revealed that the expression level of pluripotency-related genes is equivalent to that of ES cells. Data are presented as mean ± SD. The expression level of ES cells was set to 1. The figure which shows the result of a bisulfite sequencing analysis. It was revealed that the Nanog promoter and Oct3 / 4 distal enhancer region were demethylated in sarcoma-derived iPS cell-like cells. White circles and black circles represent unmethylated cytosine and methylated cytosine at the CpG site, respectively. The photograph which shows the dyeing | staining result of the section | slice of a nerve tissue, a cartilage tissue, and columnar epithelium. Sarcoma iPS cells produced teratomas composed of ectoderm tissue, mesoderm tissue and endoderm tissue in the subcutaneous tissue of immunocompromised mice. Scale bar, 50 μm. Schematic diagram of in vitro bone differentiation. The figure which shows the result of the qRT-PCR analysis of a bone differentiation related gene. Wild type ES cells (V6.5) or EWS-FLI1-inducible ES cells (Rosa-rtTA / Rosa :: tetO-EWS-FLI1-ires-mCherry) were used as controls in bone differentiation experiments. Regarding the expression of bone differentiation-related genes, sarcoma-derived iPS cells and control ES cells on day 0 and day 17 during bone differentiation were examined. Mean values ± SD of three independent experiments are shown. The average expression level of ES cells on day 17 was set to 1. The photograph which shows the result of alizarin red dyeing | staining in an iPS cell-like cell. ES cells were shown for comparison. A bright reddish orange stained extracellular calcium deposit was revealed (28 days after induction of bone differentiation). Scale bar, 20 μm. The photograph which shows the result of the histological analysis of the bone formation area | region which has the osteoid production in teratoma. Ki67 immunohistochemistry revealed that osteoid-producing cells derived from sarcoma iPS cells had higher cell proliferative activity than those derived from control ES cells. Scale bar, 50 μm. The figure which shows the Ki67 positive rate of the bone formation area | region in the teratoma derived from a sarcoma iPS cell or a control ES cell. EWS-FLI1-induced ES cells (Rosa-M2rtTA / Col1a1 :: tetO-EWS-FLI1-ires-mCherry) were used as a control. Shown is the mean ± SD of 6 independent osteogenic regions in 2 independent sarcoma-derived iPS cell teratomas or 9 independent osteogenic regions in 2 independent ES cell teratomas. Mann-Whitney U test was used for statistical analysis. Graph showing proliferation of iPS cells or control ES cells with and without Dox. EWS-FLI1 expression does not promote proliferation of undifferentiated pluripotent stem cells. Schematic diagram of in vitro bone differentiation and EWS-FLI1 induction. Induced osteogenic cells (17 days after induction of bone differentiation) were later treated with Dox for 2 weeks or not. The photograph which shows the proliferation of each cell with and without Dox. Although sarcoma iPS cell-derived osteogenic cells proliferated remarkably by Dox treatment, Rosa-M2rtTA / Rosa :: tetO-EWS-FLI1 ES cell-derived osteogenic cells did not. The graph which shows the result of qRT-PCR analysis regarding EWS-FLI1 expression. EWS-FLI1 expression in induced osteogenic cells was detectable by Dox exposure. The photograph which shows the tumor formation with and without Dox of the mouse | mouth transplanted with the sarcoma iPS cell origin osteogenic cell. Osteogenic cells derived from iPS cells with EWS-FLI1 developed tumors in immunocompromised mice only in the presence of Dox (3-7 weeks after treatment). The photograph which shows the result of HE dyeing | staining of tumor tissue (the left is no Dox). Histologically, the tumor that developed was a sarcoma composed of small round blue cells similar to small cell osteosarcoma. Scale bar, 200 μm (left) and 50 μm (right).

<Ewing sarcoma family tumor cells>
The Ewing sarcoma family tumor cells of the present invention are derived from bone marrow stromal cells, and an EWS chimeric gene placed under the control of a promoter containing an inducible transcriptional regulatory sequence is introduced onto the chromosome, and during expression of the EWS chimeric protein, It is a cell that can grow and has infinite proliferation ability.

The Ewing sarcoma family tumor cell of the present invention introduces an EWS chimera gene that is placed under control of a promoter containing an inducible transcription control sequence into bone marrow stromal cells derived from a mammal, It can be obtained by selecting immortalized cells that can grow in a state in which the protein is expressed.

Bone marrow stromal cells can be obtained from a variety of sources. Sources of bone marrow stromal cells and methods for obtaining bone marrow stromal cells from those sources and methods for culturing them have been described in the prior art (eg Friedenstein et al., 1987, Cell Tissue Kinetics 20: 263- 272; Castro-Malaspina et al., 1980, Blood 56: 289-301; Mets et al., 1981, Mech. Ageing Develop. 16: 81-89; Piersma et al., 1985, Exp. Hematol. 13: 237 -243; Caplan, 1991, J.Orthoped.Res. 9: 641-650; Prockop, 1997, Science 276: 71-74; Beresford et al., 1992, J.Cell Sci. 102: 341-351; Cheng et al., 1994, Endocrinology 134: 277-286; Rickard et al., 1994, Develop. Biol. 161: 218-228; Clark et al., 1995, Ann. N. Y. Acad. Sci. 770: 70- 78).

Bone marrow stromal cells can be obtained from any bone marrow, including, for example, cells from the bone marrow obtained by aspiration of the iliac crest of a human donor. Methods for obtaining bone marrow from a donor are well known techniques. In addition, bone marrow stromal cells can also be obtained commercially, for example, bone marrow stromal cells isolated from humans, mice, rats, rabbits, dogs, goats, sheep, pigs and horses can be obtained from Cognate® Bioservices (Baltimore , MD).

Examples of EWS chimeric genes include EWS-FLI, EWS-ERG, EWS-ETV1, and EWS-FEV as described in Cancer Res. 2000 Mar 15; 60 (6): 1536-40 etc. EWS-FLI1 is preferable.
EWS-FLI1 is preferably human-derived EWS-FLI1, for example, a fusion protein of EWS (SEQ ID NO: 2) and FLI1 (SEQ ID NO: 3) encoded by the nucleotide sequence of base numbers 1-1494 of SEQ ID NO: 1. Examples include proteins containing amino acid sequences. However, an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more identity with the above-mentioned amino acid sequence is included as long as the function of inducing cells into cancer and inducing the Ewing sarcoma family tumor is maintained. It may be a protein.

Examples of inducible transcription control sequences include sequences that initiate transcription from a promoter in response to stimuli such as drugs. Examples include Tet operator sequences, Cumate operator sequences (System Biosciences: SparQ Cumate Switch Inducible System), γ An operator sequence (Special Table 2006-526991) and the like can be mentioned.

The Tet operator sequence is a sequence that binds a reverse tetracycline-regulated transactivator (tetracycline response factor: TRE), and reverse tetracycline expressed from the host cell depending on reverse tetracycline such as doxycycline (Dox) added to the cell. A regulatory transactivator binds and induces expression of a gene linked downstream thereof.

Examples of the reverse tetracycline-regulated transactivator include a fusion protein composed of a mutant Tet repressor protein (tTetR) and a VP16 activation domain (AD) included in Clonetech's Tet-On® System. When using an operator sequence, a cell expressing the protein is used as a host cell into which a gene is introduced.

An EWS chimeric gene linked to a promoter capable of functioning in mammalian cells is arranged downstream of the inducible transcription control sequence. Examples of promoters that can function in mammalian cells include β-actin promoters, CMV promoters, CAG (CAGGS) promoters, etc., but many types of promoters are known and are not particularly limited thereto.

The expression construct for gene transfer includes a tag sequence (for example, Flag tag: SEQ ID NO: 4, HA tag: SEQ ID NO: 5), drug in addition to the above-described inducible transcription control sequence, promoter, and EWS chimeric gene. Selection marker sequences such as resistance genes, ribosome binding sequences, photoprotein coding sequences and the like may be included.

By placing the EWS chimeric gene and the drug resistance gene under the control of the Tet operon, it becomes possible to induce the EWS chimeric gene and the drug resistance gene simultaneously by administration of doxycycline (Dox). Although the EWS chimeric gene is an oncogene, it contributes to carcinogenesis only in certain cell types and causes toxicity in many cell tumors. By culturing tumor cells in the presence of Dox + drugs (such as neomycin), it is ensured. Cells that are positively controlled by the EWS chimeric gene can be selected. This makes it possible to obtain tumor cells that depend on the EWS chimeric gene more reliably.

The method of gene transfer onto the chromosome is not particularly limited, and examples thereof include a method using a viral vector, a method using a plasmid vector, a method using an artificial chromosome, and the like. A method using a viral vector is preferable, and a lentiviral vector is used. The method is more preferred.

As a lentiviral vector, a simian immunodeficiency virus vector, an equine infectious anemia virus (EIAV) vector, a human immunodeficiency virus (HIV, for example, HIV1 or HIV2) vector, a feline immunodeficiency virus (FIV) vector, etc. can be used. A commercially available lentiviral vector can also be used.

An EWS chimeric gene placed under the control of a promoter containing an inducible transcription control sequence is introduced into a bone marrow stromal cell derived from a mammal and then added to a chromosome, and then a stimulating substance for gene expression induction is added. The EWS chimeric protein of the present invention can be obtained by expressing an EWS chimeric protein, and culturing and growing cells in that state, and selecting immortalized cells. Ewing sarcoma family tumor cells are characterized by chromosomal abnormalities, tumor-specific genes, and expression of cell cycle markers (eg, Ki67) in addition to their proliferative ability. The Ewing sarcoma family tumor cells of the present invention may be established.

By transplanting the Ewing sarcoma family tumor cells of the present invention into a non-human mammal such as a mouse or a rat, an Ewing sarcoma family tumor model animal that produces an Ewing sarcoma family tumor depending on the expression of the EWS chimeric protein can be obtained. Note that the transplant site and the amount of cells used for transplantation may be appropriately determined according to the body weight of the animal, the desired phenotype, and the like.

<Artificial pluripotent stem (iPS) cells>
By initializing the Ewing sarcoma family tumor cells of the present invention, it is possible to obtain iPS cells that can differentiate into cells exhibiting the Ewing sarcoma family tumor phenotype depending on the expression of the EWS chimeric protein.

iPS cells introduce certain nuclear reprogramming substances into the Ewing sarcoma family tumor cells of the present invention in the form of DNA or protein, or increase the endogenous mRNA and protein expression of the nuclear reprogramming substances by drugs. (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676, K. Takahashi et al. (2007) Cell, 131: 861-872, J. Yu et al. (2007) Science, 318: 1917-1920, M. Nakagawa et al. (2008) Nat. Biotechnol., 26: 101-106, International Publication WO 2007/069666 and International Publication WO / 2010/068955). The nuclear reprogramming substance is not particularly limited as long as it is a gene specifically expressed in ES cells, a gene that plays an important role in maintaining undifferentiation of ES cells, or a gene product thereof. For example, Oct3 / 4 , Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPB28 E7 Examples are Lin28b, Nanog, Esrrb, or Esrrg. These reprogramming substances may be used in combination when iPS cells are established. For example, a combination including at least one, two, or three of the above-described initialization substances, preferably a combination including four.

Nucleotide sequences of mouse and human cDNAs of each nuclear reprogramming substance and amino acid sequence information of the protein encoded by the cDNA can be obtained by referring to NCBI accession numbers described in WO 2007/079666. A person skilled in the art can prepare a desired nuclear reprogramming substance by a conventional method based on the cDNA sequence or amino acid sequence information.

These nuclear reprogramming substances may be introduced into tumor cells to be reprogrammed in the form of proteins, for example, by lipofection, binding to cell membrane permeable peptides, microinjection, or in the form of DNA. For example, it can be introduced into tumor cells by techniques such as vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, and microinjection. Examples of viral vectors include retroviral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009) and the like.
Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). As a plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that a nuclear reprogramming substance can be expressed. Examples of the promoter used include EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV- A TK (herpes simplex virus thymidine kinase) promoter or the like is used. Among them, EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, SRα promoter and the like can be mentioned. Furthermore, if necessary, drug resistance genes (for example, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidine kinase gene, diphtheria toxin gene and other selectable marker sequences, green fluorescent protein (GFP), β-glucuronidase ( GUS), reporter gene sequences such as FLAG, and the like. In addition, after introduction into a tumor cell, the above vector contains a LoxP sequence before and after the gene or promoter encoding the nuclear reprogramming substance and the gene encoding the nuclear reprogramming substance that binds to it. You may have. In another preferred embodiment, there is a method for completely removing a transgene from a chromosome by incorporating a transgene into a chromosome using a transposon and then allowing a transferase to act on the cell using a plasmid vector or an adenovirus vector. Can be used. Preferred transposons include, for example, piggyBac, a transposon derived from lepidopterous insects (Kaji, K. et al., (2009), Nature, 458: 771-775, Woltjen et al., (2009), Nature, 458: 766-770, WO 2010/012077). In addition, the vector replicates without chromosomal integration and is present episomally, so that the origin and replication of lymphotrophic herpes virus, BK virus, and bovine papillomavirus The arrangement | sequence which concerns on may be included. Examples include EBNA-1 and oriP or Large T and SV40ori sequences (WO 2009/115295, WO 2009/157201 and WO 2009/149233). Moreover, in order to simultaneously introduce a plurality of nuclear reprogramming substances, an expression vector for polycistronic expression may be used. For polycistronic expression, the gene-encoding sequence may be linked by an IRES or foot-and-mouth disease virus (FMDV) 2A coding region (Science, 322: 949-953, 2008 and WO 2009/092042, WO 2009/152529).

In order to increase the induction efficiency of iPS cells upon nuclear reprogramming, in addition to the above factors, for example, histone deacetylase (HDAC) inhibitors [for example, valproic acid (VPA) (Nat. Biotechnol., 26 (7 ): 795-797 (2008)), small molecule inhibitors such as trichostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (e.g., HDAC1 siRNA (Smartpool® (Millipore), HuSH 29mer shRNA Nucleic acid expression inhibitors such as Constructs against HDAC1 (OriGene) etc.], 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 siRNA (human) (Santa Cruz Biotechnology) Etc.), L-channel］ calcium］ agonist (eg B ayk8644) (Cell Stem Cell, 3, 568-574 (2008)), p53 inhibitors (eg 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)), growth factors such as LIF or bFGF, ALK5 inhibitors (eg, SB431542) (Nat. Methods, 6: 805-8 (2009)), mitogen-activated protein kinase signaling inhibitor, glycogen synthase kinase-3 inhibitor (PloS Biology, 6 (10), 2237-2247 (2008)), miR 291-3p, miR-294, miR-295 and other miRNA (RL Judson et al., Nat. Biotech., 27: 459-461 2009 (2009)), etc. can be used.

Examples of the drug in the method for increasing the expression of the endogenous protein of the nuclear reprogramming substance by the drug include 6-bromoindirubin-3'-oxime, indirubin-5-nitro-3'-oxime, valproic acid, 2- (3- (6-methylpyridin-2-yl) -lH-pyrazol-4-yl) -1,5-naphthyridine, 1- (4-methylphenyl) -2- (4,5,6,7-tetrahydro-2-imino- 3 (2H) -benzothiazolyl) ethanone HBr (pifithrin-alpha), prostaglandin J2, and prostaglandin E2 are exemplified (WO 2010/068955).

Examples of the culture medium for iPS cell induction include (1) DMEM, DMEM / F12 or DME medium containing 10-15% FBS (these media include LIF, penicillin / streptomycin, puromycin, L-glutamine). , (2) ES cell culture medium containing bFGF or SCF, such as mouse ES cell culture medium (for example, TX-WES medium, thrombos. X) or primate ES cell culture medium (for example, primate (human & monkey) ES cell culture medium (Reprocell, Kyoto, Japan), mTeSR-1).

As an example of the culture method, for example, a tumor cell and a nuclear reprogramming substance (DNA or protein) are contacted in DMEM or DMEM / F12 medium containing 10% FBS in the presence of 5% CO ₂ at 37 ° C. Culture for ~ 7 days, then re-spread cells onto feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.), and bFGF-containing primate ES cells approximately 10 days after contact between tumor cells and nuclear reprogramming substance The cells can be cultured in a culture medium and ES cell-like colonies can be generated about 30 to about 45 days or more after the contact. Further, in order to increase the induction efficiency of iPS cells, the cells may be cultured under conditions of an oxygen concentration as low as 5-10%.

Alternatively, as an alternative culture method, 10% FBS-containing DMEM medium (including LIF, penicillin / streptomycin, puromycin, L-glutamine, etc.) on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.) Non-essential amino acids, β-mercaptoethanol, etc. can be included as appropriate.) And ES-like colonies can be formed after about 25 to about 30 days or more. During the culture, the medium is replaced with a fresh medium once a day from the second day after the start of the culture. The number of tumor cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ^{2 of} culture dish.

When a gene containing a drug resistance gene is used as the marker gene, marker gene-expressing cells can be selected by culturing in a medium (selective medium) containing the corresponding drug. In addition, when the marker gene is a fluorescent protein gene, the marker gene-expressing cells can be obtained by observing with a fluorescence microscope, by adding a luminescent substrate in the case of a luminescent enzyme gene, Can be detected.

Obtaining iPS cells can be confirmed by expression of pluripotency-related genes such as Nanog and Oct3 / 4, demethylation of Nanog promoter and Oct3 / 4 distal enhancer, or teratoma formation.

Method of inducing differentiation of iPS cells into bone cells <br/> The iPS cells obtained as described above are cultured and differentiated into bone cells, so that the EWS chimeric protein expression depends on the expression of Ewing sarcoma family tumors. Presenting cells can be obtained.

Known methods can be used for culturing iPS cells. Adhesive culture, suspension culture, or culture using feeder cells may be used.

A medium for culturing iPS cells can be prepared using a medium used for culturing animal cells as a basal medium. Examples of the basal medium include IMDM medium, Medium 、 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, and Fischer's medium. Media and the like are included. ΑMEM medium is preferable. Furthermore, the medium may contain serum or may be serum-free. If necessary, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), fatty acid, insulin, collagen precursor, trace element, 2-mercaptoethanol, 3'-thiol May contain one or more serum replacements such as glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, antibiotics, antioxidants, pyruvate, buffers, inorganic salts , N2 supplement (Invitrogen), B27 supplement (Invitrogen), and one or more cytokines. What is necessary is just to add a component required for the bone differentiation illustrated to triiodothyronine to this. The method of bone differentiation is described, for example, in Nature 467, 285-290. 2010.

The culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is preferably about 2 to 5%. is there.

Bone differentiation can be confirmed by expression of bone differentiation markers such as bone differentiation-related genes such as Runx2, Sp7, Pth1r, Col1a1 and Dmp1, and cell morphology.

<Screening method>
One embodiment of the screening method of the present invention includes a step of adding a test compound to the Ewing sarcoma family tumor cell of the present invention cultured under the induction of EWS chimeric protein expression, the proliferative ability of the Ewing sarcoma family tumor cell, or Examining a tumor phenotype and selecting a test compound as a therapeutic drug candidate compound for an Ewing sarcoma family tumor, using as an index the reduction of the proliferative ability of the Ewing sarcoma family tumor cells or the reduction of the tumor phenotype Process. If the growth ability or tumor phenotype of the tumor cell line is reduced or decreased as compared with the absence of the compound or the addition of the control compound, the test compound can be selected as a tumor therapeutic drug candidate compound.

Other aspects of the screening method of the present invention include the step of differentiating the EPS sarcoma family tumor cell-derived iPS cells of the present invention into bone cells, the step of inducing the expression of EWS chimeric protein, and the expression of the obtained EWS chimeric protein. Adding a test compound to an Ewing sarcoma family tumor cell, determining a growth ability or tumor phenotype of the Ewing sarcoma family tumor cell, and reducing the growth ability of the Ewing sarcoma family tumor cell, or tumor expression Selecting a test compound as a candidate drug for a Ewing sarcoma family tumor using the reduction in type as an index. A compound may be added simultaneously with the induction of EWS chimeric protein expression.

The proliferation ability of tumor cells can be examined by a known method. The tumor cell phenotype can be examined by cell morphology, expression of tumor marker genes and cell cycle-related genes such as Ki67, expression of tumor proteins, etc. The expression level of genes and proteins is determined by known methods. can do.

In addition, screening for therapeutic drugs for Ewing sarcoma family tumors can be performed using a non-human mammal transplanted with the Ewing sarcoma family tumor cells of the present invention.
For example, the expression of EWS chimeric protein is induced in a non-human mammal transplanted with the Ewing sarcoma family tumor cell of the present invention, and the obtained EWS chimeric protein is expressed and tested on an Ewing sarcoma family tumor model animal that has produced a tumor. A compound is administered to determine the size or tumor phenotype of the Ewing sarcoma family tumor cell, and to reduce the size of the Ewing sarcoma family tumor or to decrease the tumor phenotype as an index The mode of selecting as a therapeutic drug candidate compound for a family tumor is exemplified.

Furthermore, in another aspect of the screening method of the present invention, the EPS sarcoma family tumor cell-derived iPS cell of the present invention is cultured in a bone differentiation-inducing medium supplemented with a test compound in a state where no EWS chimeric protein is expressed. And a step of determining the degree of differentiation into bone cells, and a step of selecting a test compound as a bone differentiation promoting substance using as an index the increase in the degree of differentiation into bone cells. Since the EPS sarcoma family tumor cell-derived iPS cells of the present invention have suppressed bone differentiation compared to control cells, screening for a bone differentiation promoting substance can be performed using bone differentiation as an index. If bone differentiation is promoted compared to when no compound is added or when a control compound is added, the test compound can be selected as a bone differentiation promoting substance. Bone differentiation promoting substances can be therapeutic agents for bone-related diseases such as osteoporosis, and since bone differentiation failure has been shown to be involved in sarcoma formation, bone differentiation promoting substances can also be candidates for sarcoma inhibitory substances. The degree of differentiation into bone cells can be confirmed by the expression of bone differentiation markers such as bone differentiation-related genes such as Runx2, Sp7, Pth1r, Col1a1 and Dmp1, and the morphology of cells.

In the screening method of the present invention, any test substance can be used, for example, cell extract, cell culture supernatant, microbial fermentation product, marine organism-derived extract, plant extract, purified protein or crude protein. , Peptides, non-peptide compounds, synthetic low molecular weight compounds, and natural compounds. In the present invention, the test substance may also be (1) a biological library method, (2) a synthetic library method using deconvolution, and (3) a “one-bead one-compound” library method. And (4) can be obtained using any of a number of approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography sorting. Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des . 12: 145-67). Examples of methods for the synthesis of 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 are available in solution (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. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89) : 1865-9) or phage (Scott and Smith (1990) Science 249: 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).

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following modes.

<Experimental materials and methods>
Rosa26 targeting vector, ES cell targeting, and generation of chimeric mice The EWS-FLI1 fusion gene was cloned from the Ewing sarcoma cell line TC135 (Int J Cancer 128, 216-226. 2011). For the Rosa-M2rtTA / Rosa :: tetO-EWS-FLI1 system using the Red / ET BAC recombination system, TetOP-EWS-FLI1-FLAG-HA-ires-mCherry-pA and the selection cassette (SA-rox-PGK -EM7-BsdR-pA-rox-2pA) was introduced into intron 1 of Rosa26 BAC. The obtained vector was introduced into KH2 ES cells (Genesis 44, 23-28. 2006) having the Rosa26-M2rtTA allele by electroporation.
ES cells were cultured in an ES medium containing 15 μg / ml blasticidin S (Bsd, Funakoshi Co., Ltd.). Bsd resistant colonies were collected and expanded. It was confirmed by Southern blotting that the ES clone was correctly targeted. For the Rosa-M2rtTA / Col1a1 :: tetO-EWS-FLI1 system, the EWS-FLI1-FLAG-HA-ires-mCherry-pA sequence was inserted into pBS31 and introduced into KH2 ES cells by electroporation as described above. did. Chimeric mice were obtained by blastocyst injection in both systems.

Construction of lentiviral vector, lentiviral infection and cell culture To construct a doxycycline (Dox) inducible lentiviral vector, pEN-TmiRC3 and pSLIK-Neo lentiviral vector plasmids obtained from Addgene were modified. First, pEN-TmiRC3 was digested with SpeI and XhoI, and EWS-FLI1-FLAG-HA was ligated downstream of the tetOP-mCMV promoter. Thereafter, the ires-NeoR cassette was ligated to the 3 ′ side of the HA tag, and then the UbiC-rtTA3-ires-NeoR sequence was excised from pSLIK-Neo. After LR recombination between pEN-TmiRC3 (tetO-EWS-FLI1-ires-Neo) and pSLIK (no UbiC-rtTA3-ires-Neo), the pSLIK-TetO-EWS-FLI1-ires-Neo vector is obtained. It was.

Bone marrow stromal cells were obtained from 3-6 week old Rosa26-M2rtTA mice (Genesis 44, 23-28. 2006) as reported in Nat Protoc 4, 102-106. 2009. Non-adherent cells (hematopoietic cells) were removed by exchanging the medium 3 to 4 days after collection of bone marrow cells, and the adherent cells were infected with lentivirus. The cells were then placed in DMEM (Nacalai Tesque) containing 10% FBS (Gibco), penicillin, streptomycin, 200 μg / ml G418 (Nacalai Tesque) and 2 μg / ml Dox (Sigma) for 2 months. Cultured and EWS-FLI1-dependent immortalized cells were selected. The same medium was used to maintain the osteosarcoma cell lines, SCOS # 2 and SCOS # 12.

In Vivo Experiments Rosa-M2rtTA / Rosa :: tetO-EWS-FLI1 mice and immunocompromised mice inoculated with sarcoma cells were treated with 2 mg / ml Dox-containing water and 10 mg / ml sucrose. Because of early lethality, Rosa-M2rtTA / Col1a1 :: tetO-EWS-FLI1 mice were treated with lower concentrations of Dox (100 μg / ml to 2 mg / ml).

Induction and maintenance of iPS cells Induction of iPS cells by utilizing retroviral vectors (pMX-hOCT3 / 4, pMX-hSOX2, pMX-hKLF4 and pMX-h-cMYC; Addgene) containing reprogramming factors went. Reprogramming factor-induced sarcoma cells are cultured in ES cell medium supplemented with human recombinant LIF (Wako Pure Chemical Industries, Ltd.), 2-mercaptoethanol (Invitrogen) and 50 μg / ml L-ascorbic acid (Sigma). The constructed iPS cells were maintained in ES cell medium supplemented with LIF, 1 μM PD0325901 (Stemgent) and 3 μM CHIR99021 (Stemgent).

RT-PCR and quantitative real-time RT-PCR
RNA was extracted using RNeasy plus mini kit (QIAGEN). Up to 1 μg of RNA was used for reverse transcription to cDNA. RT-PCR and quantitative real-time PCR were performed using Go-Taq Green Master Mix and Go-Taq qPCR Master Mix (Promega), respectively. Transcript levels were normalized by β-actin.

Western blot analysis Cultured cells were harvested in 500 μl RIPA lysis buffer and protein concentration was determined. The protein was denatured at 95 ° C. for 5 minutes using 2 × SDS. A total of 20 μg of denatured protein was loaded onto a 10% SDS / PAGE gel and transferred to a PVDF membrane (Amersham Hybond-P PVDF membrane, GE Healthcare). Proteins were detected by immunoblotting using the following antibodies: anti-HA (Cell Signaling; dilution 1: 600), anti-β-actin (Santa Cruz; dilution 1: 1000). Pierce ECL plus Western blotting substrate (Thermo Scientific) was used for visualization and LAS4000 (GE Healthcare) was used for detection.

Histological analysis and immunohistochemistry All tissue and tumor samples were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin using standard protocols. For immunohistochemistry, the antibodies used were anti-HA (Cell signaling; dilution 1: 200) and anti-Ki67 (SP6) (Abcam; dilution 1: 150).

The cultured cells were washed with immunocytochemistry PBS and fixed with 2% paraformaldehyde for 10 minutes at room temperature. For immunocytochemistry, the antibodies used were anti-p53 (Abcam; dilution 1: 200) and anti-p21 (HUGO291) (Abcam; dilution 1: 500).

Cell proliferation assay Sarcoma cells and ES / iPS cells were seeded in 12-well culture plates at a density of 5 x 10 ⁴ cells / well and 1 x 10 ⁵ cells / well, respectively. The experiment was performed three times and each sample was measured twice. The number of cells was measured with an automatic cell counter (TC10 (trademark), Bio-Rad).

Xenograft assay A total of 3 × 10 ⁶ EWS-FLI1-dependent immortalized cells, EWS-FLI1-dependent in NOD / ShiJic-scid Jcl mice or BALB / cSLC-nu / nu mice purchased from CLEA Japan and Japan SLC, respectively Sarcoma cells, ES cells or iPS cells were transplanted.
NOD / ShiJic-scid Jcl mice were inoculated with EWS-FLI1-dependent immortalized cells and the mice were sacrificed 10 weeks after transplantation. The subcutaneous tissue of BALB / cSLC-nu / nu mice was inoculated with EWS-FLI1-dependent osteosarcoma cells. Tumor size was measured weekly with a digital caliper and tumor volume was calculated as follows: volume = width 2 × length ÷ 2. ES cells / iPS cells were transplanted into BALB / cSLC-nu / nu mice, and teratomas were obtained 3 to 4 weeks later.

In vitro differentiation of ES cells / iPS cells into osteogenic lineage
The in vitro bone differentiation protocol described in Nature 467, 285-290. 2010 was used with slight modifications. Specifically, 96-well plate (Nunclon ™ Sphere, Thermo Scientific) containing ES differentiation medium (IMDM, 15% FBS, penicillin / streptomycin, L-glutamine, L-ascorbic acid, transferrin, thioglycerol) Inside, 5000 ES cells or iPS cells were cultured for 2 days. On the second day, retinoic acid was added (final concentration, 10 ⁻⁶ M). On day 5, embryoid bodies were collected, transferred to 6-well tissue culture dishes, and cultured in bone differentiation medium (αMEM, 10% FBS, penicillin / streptomycin, L-glutamine, 2 nM triiodothyronine, ITS). did. The medium was changed every other day. On day 17, RNA was extracted, and the expression of osteogenic genes in the induced osteogenic cells was confirmed by quantitative real-time RT-PCR. On day 28, alizarin red staining was performed.

Cultured cells were washed with ALP stained PBS, fixed, and stained with ALP staining kit (Sigma) according to manufacturer's protocol.

Senescence-related β-gal stained PBS were washed with PBS, fixed, and stained with an aging β-galactosidase staining kit (# 9860S, Cell Signaling) according to the manufacturer's protocol.

The cultured cells were washed with alizarin red-stained PBS and fixed with 4% paraformaldehyde for 5 minutes at room temperature. Fixed cells were washed several times with deionized water and stained for 5 minutes in alizarin red staining solution (alizarin red (Sigma, A5533) 2%, adjusted to pH 4.2 with NH ₄ OH). Similarly, deparaffinized sections were stained in alizarin red staining solution for 5 minutes.

Detection of lentivirus integration site
The lentiviral integration site was examined by a slightly modified method described in Stem Cells 27, 300-306. Genomic DNA extracted from SCOS # 2 was digested into 500-800 bp fragments using an ultrasonicator (Covaris E210). The linker cassettes obtained by annealing LC1 and LC2 were ligated to their digested genomic DNA fragments. Then, the first PCR was performed using the primer set of AP1_F and pSLIK1_R, and the nested PCR was performed using the primer set of AP2_F and pSLIK2_R. The PCR product was cloned into the pCR4-TOPO vector (Invitrogen) by the TA cloning method, and the DNA sequence of the inserted fragment was analyzed by 3500 × L Genetic Analyzer (Applied Biosystems) using the seq_LTR_R primer. The resulting sequence was examined by BLAST.

Array comparative genomic hybridization Genomic DNA was extracted using the PureLink® Genomic DNA Mini Kit (Invitrogen). Array comparative genomic hybridization analysis was performed using SurePrint G3 mouse genomic CGH microarray kit (Agilent), and analysis was performed using Agilent Genomic Workbench 7.0.

Microarray analysis 200 ng of total RNA prepared using the RNeasy mini kit was subjected to cDNA synthesis using the WT expression kit (Ambion), the resulting cDNA was fragmented, and the mouse gene 1.0ST array (Affymetrix) ). Following hybridization, the GeneChip array was washed according to the manufacturer's standard protocol, stained with GeneChip Fluidic Station 450 (Affymetrix), and detected with the Scanner 3000TG system (Affymetrix). Data obtained by using GeneSpring GX software (version 13.0, Agilent Technology) was analyzed.

Bisulfite genome sequencing Bisulfite treatment was performed using EZ DNA Methylation-Gold Kit ™ (ZYMO RESEARCH) according to the manufacturer's protocol. The PCR primers used are indicated in the additional information. The amplification product was cloned into pCR4-TOPO vector (Invitrogen) and transformed into DH5α. Colonies were randomly selected and sequenced using M13 forward and reverse primers for each gene.

<Reference example>
Construction of EWS-FLI1-inducible ES cells and mice First, an attempt was made to construct an EWS-FLI1-inducible mouse model by the locus targeting method. Use KH2 mice with tet-inducible cassette and Rosa26 targeting vector (Cell 156, 663-677. 2014; J Clin Invest 123, 600-610. 2013; Genesis 44, 23-28. 2006) Two types of ES cells containing the Dox-inducible EWS-FLI1 allele were constructed. In these ES cells, the reverse tetracycline-regulated transactivator (rtTA) is expressed from the Rosa26 locus, while the Tet operator-EWS-FLI1-ires-mCherry construct is integrated into the 3′UTR of the Col1a1 locus. (Rosa-M2rtTA / Col1a1 :: tetO-EWS-FLI1), and the other is incorporated into intron 1 of the Rosa26 locus (Rosa-M2rtTA / Rosa :: tetO-EWS-FLI1). Both ES cells expressed mCherry fluorescence when treated with Dox in vitro. The inducible EWS-FLI1 expression in ES cells was also confirmed by qRT-PCR and Western blotting.

Next, blastocyst injection of the ES cells was performed to obtain chimeric mice. When treated with Dox, EWS-FLI1 was expressed in a wide variety of organs and tissues of the mouse, including bone marrow and bone cortex where Ewing sarcoma often occurs. Some mice (Rosa-M2rtTA / Col1a1 :: tetO-EWS-FLI1) died shortly after EWS-FLI1 induction, which was accompanied by atypical changes in intestinal cells due to abnormal differentiation (14 mice) 8 of them). However, despite long-term induction of EWS-FLI1 (up to 13 months), no EWS-FLI1-dependent tumor formation was observed in either system (Rosa-M2rtTA / Col1a1 :: tetO-EWS- FLI1 mice: n = 14, Rosa-M2rtTA / Rosa :: tetO-EWS-FLI1 mice: n = 9).

<Example>
Construction of EWS-FLI1-dependent immortalized cells by Dox-inducible EWS-FLI1 lentiviral system Our results suggested that induction of EWS-FLI1 in mature mice is not sufficient for sarcoma development. Therefore, a lentiviral EWS-FLI1 expression vector having a Dox-inducible expression system was prepared. The TetO-EWS-FLI1-ires-Neo cassette (FIG. 1) was transduced with lentivirus into bone marrow stromal cells of Rosa26-M2rtTA (3-4 weeks old). Transduced bone marrow cells were cultured with Dox and G418. Thereafter, viable cells were cultured for 2 months in a medium containing Dox and G418. Although most cells with the EWS-FLI1 inducible allele did not survive, we still obtained three immortalized cell lines (EFN # 2, EFN # 12 and EFV # 4; FIG. 2). Three of these strains expressed EWS-FLI1 mRNA and protein in response to Dox (FIGS. 3 and 4), and constantly grew cells under Dox-containing culture conditions (FIG. 2). When Dox was removed, the morphology of the two cell lines (EFN # 2 and EFN # 12) gradually changed to a flat shape and cell growth was inhibited. These observations indicate that we obtained two EWS-FLI1-dependent immortalized cell lines from mouse mature bone marrow stromal cells in vitro.

In order to confirm whether EWS-FLI1-dependent immortalized cells that have formed osteosarcoma in vivo have tumorigenic potential in vivo, we immunoreduced EFN # 2 and EFN # 12 The mice were transplanted subcutaneously. Mice transplanted 10 weeks after inoculation developed tumors from both cell lines when administered Dox (16/16 for EFN # 2, 2/4 for EFN # 12; FIGS. 5 and 6 On the other hand, in the absence of Dox administration, no tumor formation was observed in mice (0/16 for EFN # 2, 0/4 for EFN # 12; FIGS. 5 and 6). Histological analysis revealed that these tumors consisted of small round blue cells similar to Ewing sarcoma. Many tumor cells showed osteoid formation and were considered small cell osteosarcoma. Furthermore, immunohistochemistry showed that tumor cells expressed EWS-FLI1 and were positive for Ki67, a marker for proliferating cells (data not shown).

Construction of EWS-FLI1-dependent osteosarcoma cell line In order to investigate the characteristics of EWS-FLI1-induced osteosarcoma in more detail, we investigated the subcutaneous bones of immunocompromised mice inoculated with EFN # 2 and EFN # 12 cells. EWS-FLI1-dependent osteosarcoma cell lines were constructed from sarcomas (SCOS # 2 and SCOS # 12, respectively). As observed in the EWS-FLI1-dependent immortalized cells, the constructed osteosarcoma cell lines expressed EWS-FLI1 in a Dox concentration-dependent manner and actively proliferated in the presence of Dox (Fig. 7). SCOS # 2 and SCOS # 12 changed their morphology after Dox removal and stopped cell growth. We simultaneously observed increased expression of p53 and p21, but no increase in senescence-related β-gal (SAβgal) activity. When Dox was re-administered, cells that had stopped growing regained their cell growth ability. This reversible phenotype suggested that the loss of EWS-FLI1 arrested the cell cycle of these osteosarcoma cells.

Considering that lentiviral genomic insertion may play a role in osteosarcoma development, we determined the site of viral integration of the EWS-FLI1-induced osteosarcoma cell line (SCOS # 2). We have identified a single integration in the intergenic region 13 kb downstream of Cd14, a position that is unlikely to act as a genetic driver of sarcoma development.

To investigate the target of EWS-FLI1 whose osteosarcoma cell bone differentiation was promoted by loss of EWS-FLI1 expression , we next used SCOS # 2 and SCOS # 12 to identify EWS-FLI1-expressing sarcoma cells. Gene expression profiles were compared among EWS-FLI1 non-expressing sarcoma cells. Interestingly, extracellular matrix-related genes and extracellular region-related genes, often including osteogenesis-related genes and chondrogenesis-related genes, compared to Dox-treated EWS-FLI1-expressing sarcoma cells by GO enrichment analysis in both cell lines Was significantly enriched in Dox-untreated sarcoma cells (72 hours) (FIGS. 8, 9). In Ewing sarcoma cells, long-term knockdown of EWS-FLI1 by shRNA causes cell differentiation into osteogenic, adipogenic, and chondrogenic series, which is suggested by Cancer Cell 11, 421-429 2007, etc. Consistent with the origin of leaf stem cells (MSC). Similarly, in this study, the short-term loss of EWS-FLI1 in SCOS # 2 and SCOS # 12 caused the promotion of bone differentiation along with increased alkaline phosphatase activity (FIG. 10). Fraction sarcoma cells after long-term loss of EWS-FLI1 grew slowly and showed heterogeneous morphology (FIG. 11). Sarcoma cells in which EWS-FLI1 expression was discontinued expressed higher levels of bone differentiation marker genes (FIG. 12), as well as chondrogenic and adipogenic genes.

SCOS # 2 and SCOS # 12 formed the aforementioned small cell osteosarcoma in immunocompromised mice receiving Dox (FIG. 13). These sarcoma cells had high cell proliferating activity according to Ki67 immunohistochemistry (FIG. 13). Consistent with the in vitro findings that growth of both SCOS # 2 and SCOS # 12 is dependent on EWS-FLI1 expression, the subcutaneous tumors stopped or retarded growth after removal of Dox in vivo (FIGS. 13 and 14). Histological analysis revealed that the tumor from which Dox was removed was composed of osteoid tissue, mature bone tissue and a small number of blue cells (FIG. 13). These results indicated that loss of EWS-FLI1 promotes bone differentiation of osteosarcoma cells in vivo. Based on the above, our results revealed the role of EWS-FLI1 expression in the inhibition of terminal differentiation of osteosarcoma cells.

Construction of iPS cells from EWS-FLI1-induced osteosarcoma cells Considering that additional genetic abnormalities may be required for EWS-FLI1-induced sarcoma development, multi-potency from EWS-FLI1-induced sarcoma cells Construction of sex stem cells should provide a unique tool to study the effects of genetic abnormalities other than EWS-FLI1 expression on sarcoma development. We tried to construct iPS cells from SCOS # 2 and SCOS # 12. After single cell cloning from these sarcoma cells, OCT3 / 4, SOX2, KLF4, and cMYC were introduced into the sarcoma cells to obtain iPS cell-like colonies in the absence of EWS-FLI1 expression (colony formation efficiency). Was 0.0009% (FIG. 15). These iPS cell-like cells expressed pluripotency-related genes such as Nanog and Oct3 / 4 at the same level as ES cells (FIG. 16). Similarly, the overall gene expression pattern of iPS cell-like cells was similar to that of normal ES cells and control iPS cells.

The sarcoma-derived iPS cell-like cells showed demethylation of both Nanog promoter and Oct3 / 4 distal enhancer (FIG. 17). This indicates that these cells have acquired pluripotency via epigenetic reconstitution. Silencing of the exogenous 4 factor expression occurring in the late stage of cell reprogramming was observed in several iPS cell-like clones. This suggests that these cells have been fully reprogrammed. Next, we performed array comparative genomic hybridization (array CGH) and found that the single cell-derived sarcoma cells had extensive chromosomal abnormalities. Several sarcoma-derived iPS cell-like cells have several identical chromosomal abnormalities, confirming that these iPS cell-like clones were obtained from the parent sarcoma cells. These sarcoma-derived iPS cell-like cells have lost the ability to become mature chimeric mice upon blastocyst injection, presumably due to the widespread genetic abnormalities observed in CGH analysis. However, sarcoma-derived iPS cell-like cells formed teratomas composed of cells that differentiated into three different germ layers when inoculated into subcutaneous tissues of immunocompromised mice (FIG. 18). This indicates that these cells are pluripotent. These results indicate that iPS cells were successfully produced from EWS-FLI1-induced osteosarcoma cells.

Sarcoma-derived iPS cells suggest that EWS-FLI1-dependent osteosarcoma may arise from osteogenic cells due to increased bone differentiation of sarcoma cells when EWS-FLI1, which shows abnormal bone differentiation independent of EWS-FLI1 expression, disappears It was done. Therefore, in the absence of EWS-FLI1 expression, an attempt was made in vitro to induce osteogenic cells, which are the assumed sarcoma of the sarcoma, from pluripotent stem cells (FIG. 19) (Method of Nature 467, 285-290. 2010). reference). In control ES cells, bone differentiation stimulation induced bone differentiation-related genes such as Runx2, Sp7, Pth1r, Col1a1 and Dmp1 (day 17) (FIG. 20). Bone differentiation stimulation also induced the expression of Runx2, which is an important transcription factor for bone differentiation, in sarcoma-derived iPS cells, but the induction of bone morphogenetic genes downstream of Runx2 decreased even without EWS-FLI1 expression ( Day 17) (Figure 20). When bone differentiation induction was prolonged (day 28), calcified areas were detected in all samples as assessed by alizarin red staining (FIG. 21). However, the calcified area was greater in control ES cells than in sarcoma-derived iPS cells (FIG. 21). We used an in vivo differentiation method of sarcoma-derived iPS cells to generate teratomas in immunocompromised mice. Both sarcoma-derived iPS cells and control ES cells formed teratomas, and these teratomas contained osteogenic regions in the absence of EWS-FLI1 expression (FIG. 22). The Ki67 positive rate of sarcoma iPS cell-derived osteogenic cells was significantly higher than that of control ES cell-derived osteogenic cells (P <0.01) (FIG. 23). In summary, sarcoma-derived iPS cells show abnormal bone differentiation independent of EWS-FLI1 expression, which is also inhibited by differentiation other than EWS-FLI1 fusion and maintains proliferative progenitor cell state It suggests.

EWS-FLI1 expression induced rapid sarcoma development from sarcoma-iPS cell-derived osteogenic cells. Analysis of the cooperative action between EWS-FLI1 expression and differentiation abnormalities related to genetic abnormalities on sarcoma development. Tried. In both sarcoma-derived iPS cells and control ES cells (Rosa-M2rtTA / Rosa :: tetO-EWS-FLI1), EWS-FLI1 expression has no promoting effect on cell proliferation under undifferentiated culture conditions (FIG. 24). Next, bone differentiation of sarcoma-derived iPS cells and control ES cells was induced in vitro, followed by EWS-FLI1 expression (FIG. 25). On day 17 of the bone differentiation protocol, osteogenic precursor cells derived from sarcoma-derived iPS cells and control ES cells were treated with Dox (FIG. 25). As a result, only sarcoma-derived osteogenic cells showed significant cell proliferation in vitro in response to Dox on day 31 (FIGS. 26 and 27). Tumor development occurred only in mice administered Dox by xenotransplantation of these cells (FIG. 28). Histological analysis revealed that these xenograft tumors were sarcomas composed of small round blue cells (FIG. 29). Osteogenic cells derived from control ES cells did not show clear EWS-FLI1-dependent proliferation in vivo (data not shown). This confirms that sarcoma development requires additional abnormalities. Taken together, these results suggest that the differentiation potential associated with the sarcoma genome contributes to the rapid malignant transformation of osteogenic cells during EWS-FLI1 expression.

Claims (10)

1. Ewing sarcoma family tumor cells derived from bone marrow stromal cells,
   An Ewing sarcoma family tumor cell in which an EWS chimeric gene placed under the control of a promoter containing an inducible transcriptional regulatory sequence is introduced onto a chromosome and can grow upon expression of the EWS chimeric protein and has infinite proliferation ability.
2. The Ewing sarcoma family tumor cell according to claim 1, wherein the EWS chimeric gene is the EWS-FLI1 gene.
3. The Ewing sarcoma family tumor cell according to claim 1 or 2, wherein the EWS chimeric gene is placed under the control of a promoter containing an inducible transcription control sequence together with a drug resistance gene.
4. The Ewing sarcoma family tumor cell according to any one of claims 1 to 3, wherein the inducible transcription control sequence is a Tet operator and the cell expresses a reverse tetracycline-regulated transactivator.
5. The Ewing sarcoma family tumor cell according to any one of claims 1 to 4, wherein the EWS chimeric gene is introduced onto a chromosome using a lentiviral vector.
6. An induced pluripotent stem cell obtained by reprogramming the Ewing sarcoma family tumor cell according to any one of claims 1 to 5.
7. An Ewing sarcoma family tumor model non-human mammal, which is transplanted with the Ewing sarcoma family tumor cell according to any one of claims 1 to 5 and develops the Ewing sarcoma family tumor depending on the expression of the EWS chimeric protein.
8. 6. A step of adding a test compound to Ewing sarcoma family tumor cells according to any one of claims 1 to 5 cultured under the induction of expression of an EWS chimeric gene, the proliferative ability or tumor phenotype of the Ewing sarcoma family tumor cells And a step of selecting a test compound as a therapeutic drug candidate compound for Ewing sarcoma family tumors using as an index the reduction of the proliferative ability of the Ewing sarcoma family tumor cells or the reduction of the tumor phenotype , Screening method for Ewing sarcoma family tumor therapeutic agent.
9. The step of differentiating the induced pluripotent stem cells according to claim 6 into bone cells, the step of inducing the expression of EWS chimeric protein, and the step of adding a test compound to the Ewing sarcoma family tumor cells expressing the obtained EWS chimeric protein Determining the growth ability or tumor phenotype of the Ewing sarcoma family tumor cell, and reducing the growth ability of the Ewing sarcoma family tumor cell or reducing the tumor phenotype as an index, A screening method for a therapeutic agent for Ewing sarcoma family tumor, comprising a step of selecting the candidate compound as a therapeutic agent for Ewing sarcoma family tumor.
10. A step of culturing the induced pluripotent stem cell according to claim 6 in a bone differentiation-inducing medium to which a test compound is added in a state in which no EWS chimeric protein is expressed to differentiate into a bone cell, a step of determining the degree of differentiation into a bone cell And a method for screening a bone differentiation promoting substance, comprising the step of selecting a test compound as a bone differentiation promoting substance using as an index the increase in the degree of differentiation into the bone cells.

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