WO2011049099A1 - Method for producing cancer stem cell - Google Patents

Method for producing cancer stem cell Download PDF

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WO2011049099A1
WO2011049099A1 PCT/JP2010/068406 JP2010068406W WO2011049099A1 WO 2011049099 A1 WO2011049099 A1 WO 2011049099A1 JP 2010068406 W JP2010068406 W JP 2010068406W WO 2011049099 A1 WO2011049099 A1 WO 2011049099A1
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gene
cancer
cancer stem
stem cell
stem cells
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French (fr)
Japanese (ja)
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正樹 森
健一 永井
慶人 富丸
範克 三吉
宏光 星野
秀始 石井
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国立大学法人大阪大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0695—Stem cells; Progenitor cells; Precursor cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/30—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cancer cells, e.g. reversion of tumour cells

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  • the present invention relates to a cancer stem cell and a method for producing the same.
  • the present invention also relates to various screening methods using cancer stem cells, methods for evaluating the efficacy of cancer therapeutics, and the like.
  • Cancer cells have the property of being capable of self-proliferation and being able to wet to surrounding tissues or metastasize to distant tissues. However, not all cancer cells forming a cancer tissue have these characteristics, and there are very few cancer cells that develop cancer or advance cancer. It is known to be a cancer stem cell. Cancer stem cells exhibit undifferentiated surface characteristics like normal stem cells, have self-renewal ability and differentiation ability, and have the property of producing all cancer cells in various differentiation stages constituting cancer tissue. That is, cancer stem cells are thought to be responsible for generating the majority of cancer cells by differentiation while maintaining the same cells as themselves by self-replication in cancer tissues.
  • cancer stem cells are useful as a development tool for drug discovery and diagnostics, and the establishment of their preparations is eagerly desired.
  • cancer stem cells are only a few percent or less of the cancer cells that make up cancer tissue, and the purification process is extremely difficult. The current situation is that the product has not been established.
  • An object of the present invention is to provide a technique for artificially producing cancer stem cells. Furthermore, an object of the present invention is to provide various methods for cancer treatment and anticancer drug discovery using artificially produced cancer stem cells.
  • the present inventors did not isolate a cancer stem cell from a cancer tissue, but conducted a diligent study from a new and unprecedented viewpoint of newly producing a cancer stem cell. It should be said that for cancer cells, the group consisting of (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS At least one undifferentiation-inducing factor gene selected from and / or at least one tumor suppressor gene expression inhibitor selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 Thus, the present inventors have found that the cancer cells can be induced into cancer stem cells that have self-replicating ability and have acquired undifferentiation. The present invention has been completed by further studies based on this finding.
  • this invention provides the invention of the aspect hung up below.
  • Item 1. (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS A cancer comprising a step of introducing into a cancer cell an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 A method for producing stem cells.
  • Item 2. The production method according to Item 1, wherein the KLF family gene is a KLF4 gene.
  • Item 3. Item 3.
  • Item 1 or 2 wherein the OCT family gene is an OCT3 / 4 gene.
  • Item 4. The production method according to any one of Items 1 to 3, wherein the SOX family gene is a SOX2 gene.
  • Item 5. (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS It is obtained by introducing into a cancer cell an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1, Cancer stem cells.
  • Item 6. Item 6.
  • Item 7 An antibody that specifically binds to the cancer stem cell of Item 5.
  • Item 7. A screening method for a substance that induces differentiation of cancer stem cells, comprising the following steps: 6. A step of bringing a test substance into contact with the cancer stem cell according to Item 5 and measuring the presence or absence of differentiation of the cancer stem cell, and a step of selecting the test substance obtained by differentiating the cancer stem cell as a substance that induces differentiation of the cancer stem cell.
  • a screening method for a substance that can suppress the growth of cancer stem cells or a substance that promotes the growth of cancer stem cells comprising the following steps: A step of contacting a test substance with the cancer stem cell according to Item 5 and measuring the degree of proliferation of the cancer stem cell, and a test substance that suppresses the growth of the cancer stem cell, or a test substance that promotes the growth of the cancer stem cell. The process of selecting as a substance which can suppress the proliferation of a cancer stem cell, or a substance which promotes the proliferation of a cancer stem cell.
  • a screening method for a therapeutic agent for cancer comprising the following steps: 6.
  • Item 10 A method for evaluating the efficacy of a therapeutic agent for cancer, comprising the following steps: The step of bringing a cancer therapeutic agent into contact with the cancer stem cell according to Item 5 to determine the degree of inhibition of the growth of the cancer stem cell, and the greater the degree of inhibition of the growth of the cancer stem cell, the higher the efficacy of the therapeutic agent for cancer is determined. Process. Item 11.
  • KLF family gene (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS Cancer stem cell induction characterized by comprising an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 Agent.
  • cancer stem cells that could not be established conventionally can be artificially produced.
  • the cancer stem cells produced according to the present invention can be used for screening of therapeutic drugs for cancer, evaluation of the efficacy of cancer therapeutic drugs, etc., and can contribute to the improvement of cancer treatment technology.
  • by clarifying the characteristics of cancer stem cells produced by the present invention it is possible to elucidate the mechanisms of cancer development, progression, metastasis, etc., and thus innovative drug discovery and treatment methods that lead to the fundamental treatment of cancer Can contribute to the development of
  • the results of measuring the expression level of the Nanog gene 20 days after transfection of the PLC cells into which the combination of each gene (Example 1-6) was introduced are shown.
  • the vertical axis (NANOG / GAPDH) in the figure indicates the ratio of the NANOG gene expression level to the GAPDH gene expression level.
  • FIG. 2-3 The results of measuring the expression level of Nanog gene 12 days after transfection of HuCCT cells into which a combination of genes (Examples 1, 2 and 7-12) was introduced are shown.
  • the results of measuring the expression level of Nanog gene 12 days after transfection of DLD-1 cells into which a combination of genes (Examples 3 and 7-11) has been introduced are shown.
  • cultivated for 20 days is shown.
  • cancer stem cell means a cell that has self-replicating ability and maintains an undifferentiated state and can produce a cancer cell by differentiation.
  • the method for producing cancer stem cells of the present invention comprises: (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and Expression of at least one undifferentiation inducer gene selected from the group consisting of ERAS and / or at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1
  • the method includes a step of introducing an inhibitory factor.
  • the manufacturing method of this invention is explained in full detail.
  • the types of cancer cells induced by cancer stem cells are not particularly limited, and cancer cells derived from any cancer can be used.
  • cancer cells used in the present invention include colorectal cancer cells, colon cancer cells, esophageal cancer cells, gastric cancer cells, pancreatic cancer cells, liver cancer cells, bile duct cancer cells and the like.
  • these cancer cells from the viewpoint of efficiently inducing cancer stem cells, colon cancer cells, pancreatic cancer cells, liver cancer cells, cholangiocarcinoma cells, and more preferably colon cancer cells are exemplified.
  • the said cancer cell what was extracted from the cancer patient may be used, and a commercial item may be used.
  • the cancer cells are appropriately selected from those derived from mammals such as humans, mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, etc., depending on the intended use of cancer stem cells.
  • mammals such as humans, mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, etc.
  • those derived from humans are preferable.
  • human-derived somatic cells they may be derived from any of fetuses, infants, children, and adults.
  • KLF family genes as factors for reprogramming cancer cells into cancer stem cells, (1) KLF family genes, (2) OCT family genes, (3) SOX family genes, and (4) specific undifferentiation inducer genes and Use of a specific tumor suppressor gene suppression factor.
  • KLF family genes as factors for reprogramming cancer cells into cancer stem cells.
  • OCT family genes as factors for reprogramming cancer cells into cancer stem cells.
  • SOX family genes as factors for reprogramming cancer cells into cancer stem cells.
  • specific undifferentiation inducer genes Use of a specific tumor suppressor gene suppression factor.
  • KLF family genes include KLF1, KLF2, KLF4, and KLF5. These KLF family genes may be used alone or in combination of two or more. Among these KLF family genes, the KLF4 gene is preferably used from the viewpoint of efficient induction into cancer stem cells.
  • the base sequence of the KLF4 gene is known (NCBI accession Number NM_010637 (human), NM_004235 (Mouse)).
  • nucleotide sequence of the KLF1 gene (NCBI accession Number NM_006563 (human), NM_010635 (Mouse)), the nucleotide sequence of the KLF2 gene (NCBI accession Number NM_016270 (human), NM_008452 (Mouse)), and the nucleotide sequence of the KLF5 gene (NCBI accession Number NM_001730 (human), NM_009769 (Mouse)) is also known.
  • NCBI NCBI accession Number NM_006563 (human), NM_010635 (Mouse)
  • nucleotide sequence of the KLF2 gene NCBI accession Number NM_016270 (human), NM_008452 (Mouse)
  • nucleotide sequence of the KLF5 gene NCBI accession Number NM_001730 (human), NM_009769 (Mouse)
  • OCT family genes include OCT3 / 4, OCT1A, and OCT6. These OCT family genes may be used alone or in combination of two or more. Among these OCT family genes, the OCT3 / 4 gene is preferably used from the viewpoint of efficient induction into the cancer stem cells.
  • the base sequence of OCT3 / 4 is known (NCBI accession Number NM_002701 (human), NM_013633 (Mouse)).
  • the base sequence of the OCT1A gene NCBI accession Number NM_002697 (human), NM_198934 (Mouse)
  • the base sequence of the OCT6 gene NCBI accession Number NM_002699 (human), NM_011141 (Mouse) are also known.
  • SOX family genes include SOX1, SOX2, SOX3, SOX7, SOX15, SOX17, and SOX18. These SOX family genes may be used alone or in combination of two or more. Among these SOX family genes, the SOX2 gene is preferably used from the viewpoint of efficient induction into cancer stem cells.
  • the base sequence of the SOX2 gene is known (NCBI accession Number NM_003106 (human), NM_011443 (Mouse)).
  • nucleotide sequence of SOX1 gene (NCBI accession Number NM_005986 (human), NM_009233 (Mouse)), the nucleotide sequence of SOX3 gene (NCBI accession Number ⁇ NM_005634 (human), NM_009237 (Mouse)), the nucleotide sequence of SOX7 gene (NCBI accession Number NM_031439 (human), NM_011446 (Mouse)), SOX15 gene nucleotide sequence (NCBI accession Number NM_006942 (human), NM_009235 (Mouse)), SOX17 gene nucleotide sequence (NCBI accession Number NM_0022454 (human), NM_011441 (Mouse) ) And the nucleotide sequence of the SOX18 gene (NCBI accession Number NM_018419 (human), NM_009236 (Mouse)) are also known.
  • NANOG gene base sequence NCBI accession Number NM_024865 (human), NM_0280162 (Mouse)
  • LIN28 gene base sequence NCBI accession Number NM_024674 (human) , NM_145833 (Mouse)
  • nucleotide sequence of KRAS gene NCBI accession Number NM_004985, NM_033360 (human), NM_021284 (Mouse)
  • nucleotide sequence of BCL2 gene NCBI accession Number NM_000633, NM_000657 (human), NM_009741, NM_177410 (Mouse)
  • BMI1 gene base sequence NCBI accession Number NM_005180 (human), NM_007552 (Mouse)
  • ERAS gene base sequence NCBI accession Number NM_181532 (human), NM_181548
  • the tumor suppressor gene expression inhibitor is not particularly limited as long as it can suppress the expression of the cancer suppressor gene in cancer cells induced by cancer stem cells.
  • shRNA shRNA, miRNA, and Examples having an RNA interference effect such as siRNA are exemplified.
  • shRNA is preferable because it enables silencing of a target tumor suppressor gene stably in a cancer cell induced by cancer stem cells for a long period of time.
  • the tumor suppressor gene expression inhibitory factor may be directly introduced into the target cancer cells shRNA, miRNA, siRNA and the like, from the viewpoint of effectively suppressing the expression of the target cancer suppressor gene, shRNA, In order for miRNA, siRNA, and the like to be expressed in the target cancer cell, it is desirable to incorporate a DNA fragment encoding them into the vector and introduce it into the target cancer cell.
  • shRNA when shRNA is used as the tumor suppressor gene expression inhibitor, in order to enhance the silencing effect of the target tumor suppressor gene, 2 out of shRNAs that can exert an RNA interference effect on the target cancer suppressor gene. It is desirable to select and use a combination of two or more different base sequences.
  • Suppressors such as shRNA, miRNA, and siRNA are constructed by a known technique based on information on the base sequence of a tumor suppressor gene that is subject to expression suppression.
  • the base sequence of the target cancer suppressor gene is also known as shown below: CDKN2A gene base sequence (NCBI accession Number NM_000077, NM_058195, NM_058197 (human), NM_001040654, NM_009877 (Mouse)), FHIT Base sequence of the gene (NCBI accession Number NM_002012 (human), NM_010210 (Mouse)), base sequence of the TP53 gene (NCBI accession Number NM_000546, NM_001126112,26NM_001126114, NM_001126115, NM_001126116, NM_00112610011 (27), NM_0011260011 (27) )), RB1 gene base sequence (NCBI accession Number NM_000321 (human), NM_009029 (Mous
  • undifferentiation-inducing factor genes and / or tumor suppressor gene expression inhibitory factors may be used singly or in combination of two or more. Specifically, one or more of the genes of the undifferentiation inducing factor may be used, and one or more of the tumor suppressor gene expression suppressing factors may be used. Furthermore, one or more types of undifferentiation-inducing factor genes and one or more types of tumor suppressor gene expression suppressing factors may be used in combination.
  • LIN28 gene, KRAS gene, FHIT gene suppressor, PTEN gene The inhibitory factor of is mentioned.
  • the LIN gene when hepatoma cells are used as cancer cells to be induced into cancer stem cells, the LIN gene is suitable; when bile duct cancer cells are used as cancer cells to be induced into cancer stem cells, the LIN gene, KRAS gene, FHIT gene suppressors and PTEN gene suppressors are preferred; when cholangiocarcinoma cells are used as cancer cells induced in cancer stem cells, FHIT gene suppressors are preferred.
  • the gene of (1) to (3) and the gene of the undifferentiation inducer of (4) above are commonly present in mammals including humans, and those derived from any mammal can be used. It is desirable to select appropriately according to the origin of the cancer cells to be introduced. For example, when human-derived cancer cells are used, the genes (1) to (3) and the undifferentiation inducer gene (4) introduced into the cancer cells are derived from humans. It is desirable. In addition to the wild-type gene, the gene of (1) to (3) and the gene of the undifferentiation inducer of (4) are one or several (for example, 1 to 10) in the amino acid sequence of the gene product.
  • the genes (1) to (3) and the gene for the undifferentiation inducer (4) can be prepared according to conventional methods based on known sequence information.
  • cDNA of a target gene can be prepared by extracting RNA from a mammal-derived cell and cloning according to a conventional method.
  • Introduction of the above genes (1) to (3) and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor into a cancer cell can be performed by a known technique. it can.
  • a method for introducing a gene of the above (1) to (3) and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor into a cancer cell a vector Examples thereof include calcium phosphate method; lipofection method; electroporation method; microinjection method and the like. Among these, the method using a vector is preferable from the viewpoint of introduction efficiency.
  • a vector When using a vector to introduce the above genes (1) to (3), and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor into cancer cells, as vectors, viral vectors, non-viral vectors, artificial viruses and the like can be used, and viral vectors such as adenoviruses and retroviruses are preferred.
  • the above genes (1) to (3) and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor are in separate vectors. It may be integrated, or two or more genes may be integrated in one vector.
  • cancer cells into which the above genes (1) to (3) and (4) specific undifferentiation-inducing factor genes and / or specific tumor suppressor gene expression suppressors have been introduced are By culturing in a viable medium for about 7 to 35 days, the cancer cells are reprogrammed and induced into cancer stem cells that have acquired pluripotency as well as self-renewal ability.
  • the genes of the above (1) to (3), and (4) a gene of a specific undifferentiation inducer and / or a specific tumor suppressor gene are introduced
  • the genes of the above (1) to (3), and (4) a gene of a specific undifferentiation inducer and / or a specific tumor suppressor gene About 7 days after the introduction of the expression inhibitory factor, the cells were cultured in a medium containing 10% by volume of fetal bovine serum (FBS), and then (4-5 ng / ml wt% basic) in a dish coated with matrix protein. It is desirable to culture in a medium containing fibroblast growth factor (bFGF), where the matrix protein is exemplified by matrigel, fibronectin, collagen, laminin, etc. Among these, matrigel is preferred.
  • bFGF fibroblast growth factor
  • a cancer stem cell It is induced into a cancer stem cell from among the cancer cells into which the above genes (1) to (3) and (4) the gene of a specific undifferentiation inducer and / or the expression suppressor of a specific tumor suppressor gene are introduced.
  • the selection of the cells can be performed using as an index whether or not the cells have the ability to proliferate and whether or not they have the characteristics of cancer stem cells. Specifically, the selection of such cancer stem cells is carried out using the cell shape, presence / absence of specific staining for cancer stem cells, presence / absence of expression of a marker gene for cancer stem cells, etc., from among cells having proliferative ability. be able to.
  • a reporter gene construct prepared by previously binding a drug resistance gene to a cancer stem cell marker gene promoter is introduced into a cancer cell, cells that have acquired the characteristics of the cancer stem cell can grow in the presence of the drug. Therefore, cells that have acquired the characteristics of cancer stem cells can be selected using growth in the presence of a drug as an index.
  • cells induced by cancer stem cells proliferate and exhibit a circular shape when cultured in a medium coated with matrix protein in a medium containing basic fibroblast growth factor (bFGF). The above index can be used.
  • the cancer stem cell vesicle expresses a cancer stem cell marker gene (Nanog, BMI1, etc.), the presence or absence of the expression of the marker gene can also be used as the index.
  • CD24 has also been shown to be a surface marker for cancer stem cells (Eyal Sagiv et al., Targeting CD24 for Treatment of Colorectal and Pancreatic Cancer by Monoclonal Antibodies or small interfering RNA, Cancer Research, Vol. 68, pages, 2803-2812, 2008), CD24 expression can also be used as an indicator of cancer stem cell selection.
  • the characteristics of the cancer stem cells can be clarified, and further, a substance capable of inducing differentiation and controlling proliferation can be screened.
  • the test substance (candidate substance to be used for screening) is contacted with the cancer stem cells obtained above, the presence or absence of differentiation of the cancer stem cells is measured, and the test substance that has differentiated the cancer stem cells is selected By doing so, it becomes possible to screen for substances that induce differentiation of cancer stem cells.
  • a test substance or cancer in which the test substance (candidate substance to be used for screening) is brought into contact with the cancer stem cells obtained above, the degree of cancer stem cell proliferation is measured, and cancer stem cell proliferation is suppressed.
  • a test substance that promotes the proliferation of stem cells it becomes possible to screen for a substance that can suppress the proliferation of cancer stem cells or a substance that promotes the proliferation of cancer stem cells.
  • cancer stem cells are responsible for the onset, progression, and metastasis of cancer
  • the cancer stem cells obtained above can also be used for screening for cancer therapeutics and for evaluating the efficacy of cancer therapeutics.
  • a test substance candidate substance to be used for screening
  • the cancer stem cell obtained above is brought into contact with a cancer therapeutic agent, and the degree of inhibition of cancer stem cell growth is measured. It can be evaluated that the lower the degree of inhibition of cancer stem cell growth, the lower the efficacy of the therapeutic agent for cancer.
  • the growth inhibition of cancer stem cells includes not only the case of stopping the growth of cancer stem cells or attenuating the growth ability but also the case of killing cancer stem cells.
  • the present invention provides an antibody that specifically binds to the cancer stem cells obtained above.
  • the antibody may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
  • the antibody can be used for the purpose of detecting cancer stem cells and can also be used as a therapeutic agent for cancer.
  • Cancer stem cell inducer As described above, (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS At least one selected undifferentiation-inducing factor gene and / or at least one tumor suppressor gene expression inhibitor selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1, Cancer stem cells can be prepared by introducing into cells. Therefore, the present invention is further selected from the group consisting of (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS.
  • a cancer comprising at least one gene for an undifferentiated inducer and / or an expression suppressor for at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1
  • a stem cell inducer comprises a set of various factors used for reprogramming cancer cells to induce cancer stem cells, the gene of (1) to (3) above, and (4) specific It is desirable that the undifferentiation-inducing factor gene and / or the expression suppressor of a specific tumor suppressor gene be contained in a form that can be introduced into cancer cells.
  • Examples include vectors incorporating the genes (1) to (3) above and (4) a specific undifferentiation-inducing factor gene and / or a specific tumor suppressor gene expression suppressor.
  • the above genes (1) to (3), and (4) a specific undifferentiation-inducing factor gene and / or a specific tumor suppressor gene expression suppressor may be incorporated into different vectors.
  • two or more genes may be simultaneously incorporated into one vector.
  • the genes and vectors used for the soft cancer stem cell inducer are as described above.
  • ⁇ Experiment method> 1 ⁇ 10 6 cancer cells were inoculated into 2 ml of 10% FBS-containing DMEM medium (containing 2 ⁇ g / ml puromycin) in a 30 mm dish, and 10 ⁇ l of Lipofectamine 2000 transfection reagent and a retroviral receptor were incorporated into the expression. 3-5 ⁇ g of lentiviral vector was added and incubated at room temperature for 20 minutes. Next, after removing the supernatant and washing the cells with 10% FBS-containing DMEM medium, 2 ml of 10% FBS-containing DMEM medium was added, and 10 ⁇ l of Lipofectamine 2000 transfection reagent and the gene or DNA fragment shown in Table 1 were incorporated.
  • 10% FBS-containing DMEM medium containing 2 ⁇ g / ml puromycin
  • each gene was transfected into cancer cells by adding 5 ⁇ g each of pMXs-based retroviral vectors (4 types in total) and incubating at 37 ° C. for 24 hours.
  • the retrovirus vector used for the transfection uses a vector in which one kind of gene or DNA fragment is incorporated, and according to the combination of genes to be transfected, each cancer cell is combined with a retrovirus vector in which each gene is incorporated. Transfected.
  • Each cancer cell after transfection is cultured in DMEM medium containing 10% FBS for 7 days while changing the medium every other day.
  • DMEM medium containing 10% FBS
  • mTeSRTM1 and matrigel for BD human ES cells (StemCell Technologies Technologies Inc) was cultured while changing the medium every other day.
  • the expression level of Nanog gene was measured by RT-PCR.
  • the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was also measured by RT-PCR.
  • the primers used for this measurement are as follows.
  • the Forward Primer and Reverse Primer for the Nanog gene below are mixed with UPL (# 66) tag (Roche) and PCR reaction is performed.
  • the Forward Primer and Reverse Primer for the GAPDH gene below are UPL (# 60) tag (Roche PCR product was mixed.
  • FIGS. 1-12 show the results of measuring the expression level of NANOG gene in PLC cells (20 days after transfection) into which the combination of each gene was introduced (Example 1-6).
  • FIG. 2 shows the combination of each gene (Example As a result of measuring the expression level of NANOG gene in HuCCT-1 cells (12 days after transfection) introduced with 1, 2, and 7-12),
  • FIG. 3 shows combinations of the genes (Examples 1-3 and 7-).
  • transduced 11) (12 days after transfection) is shown.
  • the NANOG gene is specifically expressed in liver cancer cells, and its expression level is an indicator that it has been induced in cancer stem cells.
  • FIG. 4 shows the results of observing the shape of cells when the combinations of the genes of Examples 2, 5 and 6 were transfected into colon cancer cells and cultured for 20 days.
  • DLD-1 cancer cells

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Abstract

Mainly disclosed is a technique for artificially constructing a cancer stem cell from a cancer cell. A cancer cell is derived into a cancer stem cell having a self-renewal ability and acquiring an undifferentiation potency by introducing, into the cancer cell, (1) a KLF family gene, (2) an OCT family gene, (3) an SOX family gene, and (4) a gene of at least one kind of an undifferentiation-inducing factor selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1 and ERAS, and/or an expression inhibitor of at least one kind of a cancer suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN and LZTS1.

Description

癌幹細胞の製造方法Method for producing cancer stem cells
　本発明は、癌幹細胞、及びその製造方法に関する。また、本発明は、癌幹細胞を利用した各種スクリーニング方法、癌治療薬の薬効評価方法等に関する。 The present invention relates to a cancer stem cell and a method for producing the same. The present invention also relates to various screening methods using cancer stem cells, methods for evaluating the efficacy of cancer therapeutics, and the like.
　癌細胞には、自己増殖能があり、周辺組織への湿潤や離れた組織への転移が可能であるという特性がある。しかしながら、癌組織を形成している癌細胞の全てにおいて、これらの特性を備わっているのではなく、癌を発症させたり、癌を進行させる癌細胞は、癌細胞の中でもごく僅かにしか存在しない癌幹細胞であることが分かっている。癌幹細胞は、正常幹細胞と同様に未分化な表面形質を示し、自己複製能と分化能を有し、癌組織を構成する多様な分化段階にあるあらゆる癌細胞を生みだす特性を有している。即ち、癌幹細胞は、癌組織中で自己複製により自分と同じ細胞を維持しつつ、分化によって大多数の癌細胞を生み出すもとになっていると考えられている。 Cancer cells have the property of being capable of self-proliferation and being able to wet to surrounding tissues or metastasize to distant tissues. However, not all cancer cells forming a cancer tissue have these characteristics, and there are very few cancer cells that develop cancer or advance cancer. It is known to be a cancer stem cell. Cancer stem cells exhibit undifferentiated surface characteristics like normal stem cells, have self-renewal ability and differentiation ability, and have the property of producing all cancer cells in various differentiation stages constituting cancer tissue. That is, cancer stem cells are thought to be responsible for generating the majority of cancer cells by differentiation while maintaining the same cells as themselves by self-replication in cancer tissues.
　癌の制圧の目的を具現化するためには、癌幹細胞の性状を明らかにし、癌幹細胞を制圧することが重要であると考えられている。そのため、癌幹細胞は、創薬、診断薬の開発ツールとして有用であり、その標品の確立が切望されている。しかしながら、癌幹細胞は、癌組織を構成する癌細胞の中で、数％以下という寡少にしか存在しておらず、その精製作業が極めて困難であること等の理由から、医療産業上満足できる標品を確立できていないのが現状である。 In order to embody the purpose of cancer suppression, it is considered important to clarify the properties of cancer stem cells and to suppress cancer stem cells. Therefore, cancer stem cells are useful as a development tool for drug discovery and diagnostics, and the establishment of their preparations is eagerly desired. However, cancer stem cells are only a few percent or less of the cancer cells that make up cancer tissue, and the purification process is extremely difficult. The current situation is that the product has not been established.
　一方、近年、OCT3/4、KLF4、c-MYC及びSOX2をコードする各々の遺伝子を体細胞に導入することにより、体細胞を初期化して誘導多能性幹(iPS)細胞に誘導する技術が報告され、再生医薬の分野で革新的な技術が提供されている（特許文献１参照）。しかしながら、正常な体細胞から癌幹細胞を作製するための技術については、未だ報告されておらず、癌幹細胞の標品を確立できていないのが現状である。 On the other hand, in recent years, there has been a technology for reprogramming somatic cells to induce induced pluripotent stem (iPS) cells by introducing respective genes encoding OCT3 / 4, KLF4, c-MYC and SOX2 into somatic cells. Innovative technology has been reported in the field of regenerative medicine (see Patent Document 1). However, the technology for producing cancer stem cells from normal somatic cells has not yet been reported, and the standard of cancer stem cells has not been established.
　このような従来技術を背景として、医療産業を満足させる癌幹細胞の標品を製造する技術の開発が望まれている。 With the background of such conventional technology, development of a technique for producing a preparation of cancer stem cells that satisfies the medical industry is desired.
国際公開第2007/069666号パンフレットInternational Publication No. 2007/069666 Pamphlet
　本発明は、癌幹細胞を人工的に作製する技術を提供することを目的とする。更に、本発明は、人工的に作製された癌幹細胞を利用して、癌の治療方策や抗癌剤の創薬を行う各種方法を提供することを目的とする。 An object of the present invention is to provide a technique for artificially producing cancer stem cells. Furthermore, an object of the present invention is to provide various methods for cancer treatment and anticancer drug discovery using artificially produced cancer stem cells.
　本発明者等は、上記課題を解決すべく、癌組織から癌幹細胞を単離するのではなく、癌幹細胞を新たに作製するという従来にない新たな観点から、鋭意検討を行ったところ、驚くべきことに、癌細胞に対して、(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)NANOG、LIN28、KRAS、BCL2、BMI1、及びERASよりなる群から選択される少なくとも1種の未分化誘導因子の遺伝子、及び／又はCDKN2A、FHIT、TP53、RB1、PTEN、及びLZTS1よりなる群から選択される少なくとも1種の癌抑制遺伝子の発現抑制因子を導入することによって、該癌細胞を、自己複製能があり、未分化性を獲得した癌幹細胞に誘導できることを見出した。本発明は、かかる知見に基づいて、更に検討を重ねることにより完成したものである。 In order to solve the above-mentioned problems, the present inventors did not isolate a cancer stem cell from a cancer tissue, but conducted a diligent study from a new and unprecedented viewpoint of newly producing a cancer stem cell. It should be said that for cancer cells, the group consisting of (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS At least one undifferentiation-inducing factor gene selected from and / or at least one tumor suppressor gene expression inhibitor selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 Thus, the present inventors have found that the cancer cells can be induced into cancer stem cells that have self-replicating ability and have acquired undifferentiation. The present invention has been completed by further studies based on this finding.
　即ち、本発明は、下記に掲げる態様の発明を提供する。
項１．　(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)NANOG、LIN28、KRAS、BCL2、BMI1、及びERASよりなる群から選択される少なくとも1種の未分化誘導因子の遺伝子、及び／又はCDKN2A、FHIT、TP53、RB1、PTEN、及びLZTS1よりなる群から選択される少なくとも1種の癌抑制遺伝子の発現抑制因子を、癌細胞に導入する工程を含む、癌幹細胞の製造方法。
項２．　KLFファミリー遺伝子がKLF 4遺伝子である、項１に記載の製造方法。
項３．　OCTファミリー遺伝子がOCT3/4遺伝子である、項１又は２に記載の製造方法。
項４．　SOXファミリー遺伝子がSOX2遺伝子である、項１乃至３のいずれかに記載の製造方法。
項５．　(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)NANOG、LIN28、KRAS、BCL2、BMI1、及びERASよりなる群から選択される少なくとも1種の未分化誘導因子の遺伝子、及び／又はCDKN2A、FHIT、TP53、RB1、PTEN、及びLZTS1よりなる群から選択される少なくとも1種の癌抑制遺伝子の発現抑制因子を、癌細胞に導入することにより得られる、癌幹細胞。
項６．　項５に記載の癌幹細胞に対して特異的に結合する抗体。
項７．　下記工程を含む、癌幹細胞を分化誘導させる物質のスクリーニング方法：
項５に記載の癌幹細胞に被験物質を接触させ、該癌幹細胞の分化の有無を測定する工程、及び
癌幹細胞を分化させた被験物質を、癌幹細胞を分化誘導させる物質として選択する工程。
項８．　下記工程を含む、癌幹細胞の増殖を抑制できる物質又は癌幹細胞の増殖を促進する物質のスクリーニング方法：
項５に記載の癌幹細胞に被験物質を接触させ、該癌幹細胞の増殖の程度を測定する工程、及び
癌幹細胞の増殖を抑制させた被験物質、又は癌幹細胞の増殖を促進させた被験物質を、癌幹細胞の増殖を抑制できる物質又は癌幹細胞の増殖を促進する物質として選択する工程。
項９．　下記工程を含む、癌の治療薬のスクリーニング方法：
項５に記載の癌幹細胞に被験物質を接触させ、該癌幹細胞の生育の有無を測定する工程、及び
癌幹細胞の生育を阻害した被験物質を、癌の治療薬として選択する工程。
項１０．　下記工程を含む、癌の治療薬の薬効評価方法：
項５に記載の癌幹細胞に癌の治療薬を接触させ、該癌幹細胞の生育阻害の程度をする工程、及び癌幹細胞の生育阻害の程度が大きい程、癌の治療薬の薬効が高いと判定する工程。
項１１．　(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)NANOG、LIN28、KRAS、BCL2、BMI1、及びERASよりなる群から選択される少なくとも1種の未分化誘導因子の遺伝子、及び／又はCDKN2A、FHIT、TP53、RB1、PTEN、及びLZTS1よりなる群から選択される少なくとも1種の癌抑制遺伝子の発現抑制因子を含有することを特徴とする、癌幹細胞誘導剤。 That is, this invention provides the invention of the aspect hung up below.
Item 1. (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS A cancer comprising a step of introducing into a cancer cell an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 A method for producing stem cells.
Item 2. Item 2. The production method according to Item 1, wherein the KLF family gene is a KLF4 gene.
Item 3. Item 3. The method according to Item 1 or 2, wherein the OCT family gene is an OCT3 / 4 gene.
Item 4. Item 4. The production method according to any one of Items 1 to 3, wherein the SOX family gene is a SOX2 gene.
Item 5. (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS It is obtained by introducing into a cancer cell an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1, Cancer stem cells.
Item 6. Item 6. An antibody that specifically binds to the cancer stem cell of Item 5.
Item 7. A screening method for a substance that induces differentiation of cancer stem cells, comprising the following steps:
6. A step of bringing a test substance into contact with the cancer stem cell according to Item 5 and measuring the presence or absence of differentiation of the cancer stem cell, and a step of selecting the test substance obtained by differentiating the cancer stem cell as a substance that induces differentiation of the cancer stem cell.
Item 8. A screening method for a substance that can suppress the growth of cancer stem cells or a substance that promotes the growth of cancer stem cells, comprising the following steps:
A step of contacting a test substance with the cancer stem cell according to Item 5 and measuring the degree of proliferation of the cancer stem cell, and a test substance that suppresses the growth of the cancer stem cell, or a test substance that promotes the growth of the cancer stem cell. The process of selecting as a substance which can suppress the proliferation of a cancer stem cell, or a substance which promotes the proliferation of a cancer stem cell.
Item 9. A screening method for a therapeutic agent for cancer, comprising the following steps:
6. A step of contacting a test substance with the cancer stem cell according to Item 5, measuring the presence or absence of the growth of the cancer stem cell, and a step of selecting a test substance that inhibits the growth of the cancer stem cell as a therapeutic agent for cancer.
Item 10. A method for evaluating the efficacy of a therapeutic agent for cancer, comprising the following steps:
The step of bringing a cancer therapeutic agent into contact with the cancer stem cell according to Item 5 to determine the degree of inhibition of the growth of the cancer stem cell, and the greater the degree of inhibition of the growth of the cancer stem cell, the higher the efficacy of the therapeutic agent for cancer is determined. Process.
Item 11. (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS Cancer stem cell induction characterized by comprising an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 Agent.
　本発明によれば、従来樹立できなかった癌幹細胞を人工的に作製することができる。本発明により作製される癌幹細胞は、癌の治療薬のスクリーニングや癌治療薬の薬効評価等に利用することができ、癌の治療技術の向上に寄与することが可能になる。また、本発明により作製される癌幹細胞の特性を明らかにすることにより、癌の発生、進行、転移等の機序の解明ができ、ひいては癌の根本治療に繋がる革新的な創薬及び治療法の開発に寄与できる。 According to the present invention, cancer stem cells that could not be established conventionally can be artificially produced. The cancer stem cells produced according to the present invention can be used for screening of therapeutic drugs for cancer, evaluation of the efficacy of cancer therapeutic drugs, etc., and can contribute to the improvement of cancer treatment technology. In addition, by clarifying the characteristics of cancer stem cells produced by the present invention, it is possible to elucidate the mechanisms of cancer development, progression, metastasis, etc., and thus innovative drug discovery and treatment methods that lead to the fundamental treatment of cancer Can contribute to the development of
各遺伝子の組合わせ（実施例１－６）を導入したPLC細胞について、トランスフェクション20日後のNanog遺伝子の発現量を測定した結果を示す。図中の縦軸（NANOG/GAPDH）は、GAPDH遺伝子の発現量に対するNANOG遺伝子の発現量の比率を示す。図２－３でも同様である。The results of measuring the expression level of the Nanog gene 20 days after transfection of the PLC cells into which the combination of each gene (Example 1-6) was introduced are shown. The vertical axis (NANOG / GAPDH) in the figure indicates the ratio of the NANOG gene expression level to the GAPDH gene expression level. The same applies to FIG. 2-3. 各遺伝子の組合わせ（実施例１、２及び７－１２）を導入したHuCCT細胞について、トランスフェクション12日後のNanog遺伝子の発現量を測定した結果を示す。The results of measuring the expression level of Nanog gene 12 days after transfection of HuCCT cells into which a combination of genes (Examples 1, 2 and 7-12) was introduced are shown. 各遺伝子の組合わせ（実施例３及び７－１１）を導入したDLD-1細胞について、トランスフェクション12日後のNanog遺伝子の発現量を測定した結果を示す。The results of measuring the expression level of Nanog gene 12 days after transfection of DLD-1 cells into which a combination of genes (Examples 3 and 7-11) has been introduced are shown. 各遺伝子の組合わせ（実施例２、５及び６）を大腸癌細胞にトランスフェクションし、20日間培養した際の細胞の形状を観察した結果を示す。The result of having observed the shape of the cell when the combination (Example 2, 5, and 6) of each gene was transfected to the colon cancer cell and it culture | cultivated for 20 days is shown.
１．癌幹細胞
　本発明において、「癌幹細胞」とは、自己複製能を有し、且つ未分化の状態を維持しており、分化によって癌細胞を生み出すことができる細胞を意味する。 1. Cancer Stem Cell In the present invention, the “cancer stem cell” means a cell that has self-replicating ability and maintains an undifferentiated state and can produce a cancer cell by differentiation.
　本発明の癌幹細胞の製造方法は、癌細胞に、(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)NANOG、LIN28、KRAS、BCL2、BMI1、及びERASよりなる群から選択される少なくとも1種の未分化誘導因子の遺伝子、及び／又はCDKN2A、FHIT、TP53、RB1、PTEN、及びLZTS1よりなる群から選択される少なくとも1種の癌抑制遺伝子の発現抑制因子を導入する工程を含むことを特徴とするものである。以下、本発明の製造方法について詳述する。 The method for producing cancer stem cells of the present invention comprises: (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and Expression of at least one undifferentiation inducer gene selected from the group consisting of ERAS and / or at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 The method includes a step of introducing an inhibitory factor. Hereafter, the manufacturing method of this invention is explained in full detail.
　本発明において、癌幹細胞に誘導される癌細胞としては、その種類については特に制限されず、あらゆる癌由来の癌細胞が使用できる。本発明で使用される癌細胞としては、例えば、結腸直腸癌細胞、大腸癌細胞、食道癌細胞、胃癌細胞、膵臓癌細胞、肝癌細胞、胆管癌細胞等が例示される。これらの癌細胞の中でも、癌幹細胞の効率的な誘導を行うという観点から、好ましくは、大腸癌細胞、膵臓癌細胞、肝癌細胞、胆管癌細胞、更に好ましくは大腸癌細胞が例示される。また、上記癌細胞については、癌患者から摘出したものを使用してもよく、また市販品を使用してもよい。 In the present invention, the types of cancer cells induced by cancer stem cells are not particularly limited, and cancer cells derived from any cancer can be used. Examples of cancer cells used in the present invention include colorectal cancer cells, colon cancer cells, esophageal cancer cells, gastric cancer cells, pancreatic cancer cells, liver cancer cells, bile duct cancer cells and the like. Among these cancer cells, from the viewpoint of efficiently inducing cancer stem cells, colon cancer cells, pancreatic cancer cells, liver cancer cells, cholangiocarcinoma cells, and more preferably colon cancer cells are exemplified. Moreover, about the said cancer cell, what was extracted from the cancer patient may be used, and a commercial item may be used.
　また、上記癌細胞は、癌幹細胞の使用目的等に応じて、ヒト、マウス、ラット、ハムスター、ウサギ、ネコ、イヌ、ヒツジ、ブタ、ウシ、ヤギ、サル等の哺乳動物由来のものから適宜選択されるが、作製された癌幹細胞をヒトの癌に対する創薬ツールや診断薬の開発ツールとして使用する場合には、ヒト由来のものが好適である。また、ヒト由来の体細胞を使用する場合、胎児、幼児、小児、及び成人のいずれに由来するものであってもよい。 The cancer cells are appropriately selected from those derived from mammals such as humans, mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, etc., depending on the intended use of cancer stem cells. However, when the prepared cancer stem cells are used as a drug discovery tool for human cancers or a development tool for diagnostic agents, those derived from humans are preferable. Moreover, when using human-derived somatic cells, they may be derived from any of fetuses, infants, children, and adults.
　本発明では、癌細胞を癌幹細胞に再プログラミングさせる因子として、(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子を使用する。このように、上記(1)～(3)の遺伝子と共に、(4)の未分化誘導因子の遺伝子及び／又は癌抑制遺伝子の発現抑制因子を使用することによって、癌細胞を癌幹細胞に誘導することが可能になる。 In the present invention, as factors for reprogramming cancer cells into cancer stem cells, (1) KLF family genes, (2) OCT family genes, (3) SOX family genes, and (4) specific undifferentiation inducer genes and Use of a specific tumor suppressor gene suppression factor. As described above, by using the gene of (4) undifferentiation-inducing factor and / or tumor suppressor gene expression inhibitor in addition to the genes of (1) to (3) above, cancer cells are induced into cancer stem cells. It becomes possible.
　KLFファミリー遺伝子としては、KLF1、KLF2、KLF4、及びKLF5等が挙げられる。これらのKLFファミリー遺伝子は、1種単独で使用してもよく、また2種以上を組み合わせて使用してもよい。これらのKLFファミリー遺伝子の中でも、癌幹細胞への効率的な誘導という観点から、KLF4遺伝子が好適に使用される。KLF4遺伝子の塩基配列は公知（NCBI accession Number NM_010637（human）、NM_004235（Mouse））である。また、KLF1遺伝子の塩基配列（NCBI accession Number NM_006563（human）、NM_010635（Mouse））、KLF2遺伝子の塩基配列（NCBI accession Number NM_016270（human）、NM_008452（Mouse））、及びKLF5遺伝子の塩基配列（NCBI accession Number NM_001730（human）、NM_009769（Mouse））についても公知である。なお、本明細書において、NCBIとは、米国立生物工学情報センター（National Center for Biotechnology Information）の略である。 Examples of KLF family genes include KLF1, KLF2, KLF4, and KLF5. These KLF family genes may be used alone or in combination of two or more. Among these KLF family genes, the KLF4 gene is preferably used from the viewpoint of efficient induction into cancer stem cells. The base sequence of the KLF4 gene is known (NCBI accession Number NM_010637 (human), NM_004235 (Mouse)). In addition, the nucleotide sequence of the KLF1 gene (NCBI accession Number NM_006563 (human), NM_010635 (Mouse)), the nucleotide sequence of the KLF2 gene (NCBI accession Number NM_016270 (human), NM_008452 (Mouse)), and the nucleotide sequence of the KLF5 gene (NCBI accession Number NM_001730 (human), NM_009769 (Mouse)) is also known. In the present specification, NCBI is an abbreviation for National Center for Biotechnology Information.
　OCTファミリー遺伝子としては、OCT3/4、OCT1A、及びOCT6等が挙げられる。これらのOCTファミリー遺伝子は、1種単独で使用してもよく、また2種以上を組み合わせて使用してもよい。これらのOCTファミリー遺伝子の中でも、本癌幹細胞への効率的な誘導という観点から、OCT3/4遺伝子が好適に使用される。OCT3/4の塩基配列は、公知（NCBI accession Number NM_002701（human）、NM_013633（Mouse））である。また、OCT1A遺伝子の塩基配列（NCBI accession Number NM_002697（human）、NM_198934（Mouse））、OCT6遺伝子の塩基配列（NCBI accession Number NM_002699（human）、NM_011141（Mouse））についても公知である。 OCT family genes include OCT3 / 4, OCT1A, and OCT6. These OCT family genes may be used alone or in combination of two or more. Among these OCT family genes, the OCT3 / 4 gene is preferably used from the viewpoint of efficient induction into the cancer stem cells. The base sequence of OCT3 / 4 is known (NCBI accession Number NM_002701 (human), NM_013633 (Mouse)). The base sequence of the OCT1A gene (NCBI accession Number NM_002697 (human), NM_198934 (Mouse)) and the base sequence of the OCT6 gene (NCBI accession Number NM_002699 (human), NM_011141 (Mouse)) are also known.
　SOXファミリー遺伝子としては、SOX1、SOX2、SOX3、SOX7、SOX15、SOX17、及びSOX18が挙げられる。これらのSOXファミリー遺伝子は、1種単独で使用してもよく、また2種以上を組み合わせて使用してもよい。これらのSOXファミリー遺伝子の中でも、癌幹細胞への効率的な誘導という観点から、SOX2遺伝子が好適に使用される。SOX2遺伝子の塩基配列は、公知（NCBI accession Number NM_003106（human）、NM_011443（Mouse））である。また、SOX1遺伝子の塩基配列（NCBI accession Number NM_005986（human）、NM_009233（Mouse））、SOX3遺伝子の塩基配列（NCBI accession Number NM_005634（human）、NM_009237（Mouse））、SOX7遺伝子の塩基配列（NCBI accession Number NM_031439（human）、NM_011446（Mouse））、SOX15遺伝子の塩基配列（NCBI accession Number NM_006942（human）、NM_009235（Mouse））、SOX17遺伝子の塩基配列（NCBI accession Number NM_0022454（human）、NM_011441（Mouse））、SOX18遺伝子の塩基配列（NCBI accession Number NM_018419（human）、NM_009236（Mouse））についても公知である。 SOX family genes include SOX1, SOX2, SOX3, SOX7, SOX15, SOX17, and SOX18. These SOX family genes may be used alone or in combination of two or more. Among these SOX family genes, the SOX2 gene is preferably used from the viewpoint of efficient induction into cancer stem cells. The base sequence of the SOX2 gene is known (NCBI accession Number NM_003106 (human), NM_011443 (Mouse)). In addition, the nucleotide sequence of SOX1 gene (NCBI accession Number NM_005986 (human), NM_009233 (Mouse)), the nucleotide sequence of SOX3 gene (NCBI accession Number 、 NM_005634 (human), NM_009237 (Mouse)), the nucleotide sequence of SOX7 gene (NCBI accession Number NM_031439 (human), NM_011446 (Mouse)), SOX15 gene nucleotide sequence (NCBI accession Number NM_006942 (human), NM_009235 (Mouse)), SOX17 gene nucleotide sequence (NCBI accession Number NM_0022454 (human), NM_011441 (Mouse) ) And the nucleotide sequence of the SOX18 gene (NCBI accession Number NM_018419 (human), NM_009236 (Mouse)) are also known.
　また、上記未分化誘導因子の遺伝子の塩基配列については公知である。これらの遺伝子の具体的な配列情報は、次の通りである：NANOG遺伝子の塩基配列（NCBI accession Number NM_024865（human）、NM_0280162（Mouse））、LIN28遺伝子の塩基配列（NCBI accession Number NM_024674（human）、NM_145833（Mouse））、KRAS遺伝子の塩基配列（NCBI accession Number NM_004985, NM_033360（human）、NM_021284（Mouse））、BCL2遺伝子の塩基配列（NCBI accession Number NM_000633, NM_000657（human）、NM_009741, NM_177410（Mouse））、BMI1遺伝子の塩基配列（NCBI accession Number NM_005180（human）、NM_007552（Mouse））、ERAS遺伝子の塩基配列（NCBI accession Number NM_181532（human）、NM_181548（Mouse））。 Further, the base sequence of the gene of the undifferentiation inducer is known. Specific sequence information of these genes is as follows: NANOG gene base sequence (NCBI accession Number NM_024865 (human), NM_0280162 (Mouse)), LIN28 gene base sequence (NCBI accession Number NM_024674 (human) , NM_145833 (Mouse)), nucleotide sequence of KRAS gene (NCBI accession Number NM_004985, NM_033360 (human), NM_021284 (Mouse)), nucleotide sequence of BCL2 gene (NCBI accession Number NM_000633, NM_000657 (human), NM_009741, NM_177410 (Mouse) )), BMI1 gene base sequence (NCBI accession Number NM_005180 (human), NM_007552 (Mouse)), ERAS gene base sequence (NCBI accession Number NM_181532 (human), NM_181548 (Mouse)).
　また、上記癌抑制遺伝子の発現抑制因子は、癌幹細胞に誘導される癌細胞内で上記癌抑制遺伝子の発現を抑制できるものであれば、特に制限されないが、その一例として、shRNA、miRNA、及びsiRNA等のRNA干渉効果を有するものが例示される。これらの中でも、shRNAは、癌幹細胞に誘導される癌細胞内で長期間安定に標的癌抑制遺伝子のサイレンシングを可能にするので、好適である。また、上記癌抑制遺伝子の発現抑制因子は、shRNA、miRNA、及びsiRNA等をそのまま標的癌細胞に導入してもよいが、標的癌抑制遺伝子の発現抑制を効果的に行うという観点から、shRNA、miRNA、及びsiRNA等が標的癌細胞内で発現するように、これらをコードしているDNA断片をベクターに組み込んで標的癌細胞に導入することが望ましい。また、上記癌抑制遺伝子の発現抑制因子として、shRNAを使用する場合、標的癌抑制遺伝子のサイレンシング効果を高めるために、標的癌抑制遺伝子に対してRNA干渉効果を奏し得るshRNAの中から、２種以上の異なる塩基配列のものを選択して併用することが望ましい。 In addition, the tumor suppressor gene expression inhibitor is not particularly limited as long as it can suppress the expression of the cancer suppressor gene in cancer cells induced by cancer stem cells.For example, shRNA, miRNA, and Examples having an RNA interference effect such as siRNA are exemplified. Among these, shRNA is preferable because it enables silencing of a target tumor suppressor gene stably in a cancer cell induced by cancer stem cells for a long period of time. In addition, the tumor suppressor gene expression inhibitory factor may be directly introduced into the target cancer cells shRNA, miRNA, siRNA and the like, from the viewpoint of effectively suppressing the expression of the target cancer suppressor gene, shRNA, In order for miRNA, siRNA, and the like to be expressed in the target cancer cell, it is desirable to incorporate a DNA fragment encoding them into the vector and introduce it into the target cancer cell. In addition, when shRNA is used as the tumor suppressor gene expression inhibitor, in order to enhance the silencing effect of the target tumor suppressor gene, 2 out of shRNAs that can exert an RNA interference effect on the target cancer suppressor gene. It is desirable to select and use a combination of two or more different base sequences.
　shRNA、miRNA、及びsiRNA等の抑制因子は、発現抑制の対象となる癌抑制遺伝子の塩基配列の情報に基づいて、公知の手法で構築される。なお、上記の標的癌抑制遺伝子の塩基配列についても、次に示す通り、公知である：CDKN2A遺伝子の塩基配列（NCBI accession Number NM_000077, NM_058195, NM_058197（human）、NM_001040654, NM_009877（Mouse））、FHIT遺伝子の塩基配列（NCBI accession Number NM_002012（human）、NM_010210（Mouse））、TP53遺伝子の塩基配列（NCBI accession Number NM_000546, NM_001126112, NM_001126113, NM_001126114, NM_001126115, NM_001126116, NM_001126117（human）、NM_001127233, NM_011640（Mouse））、RB1遺伝子の塩基配列（NCBI accession Number NM_000321（human）、NM_009029（Mouse））、PTEN遺伝子の塩基配列（NCBI accession Number NM_000314（human）、NM_008960（Mouse））、及びLZTS1遺伝子の塩基配列（NCBI accession Number NM_021020（human）、NM_199364（Mouse））。 Suppressors such as shRNA, miRNA, and siRNA are constructed by a known technique based on information on the base sequence of a tumor suppressor gene that is subject to expression suppression. The base sequence of the target cancer suppressor gene is also known as shown below: CDKN2A gene base sequence (NCBI accession Number NM_000077, NM_058195, NM_058197 (human), NM_001040654, NM_009877 (Mouse)), FHIT Base sequence of the gene (NCBI accession Number NM_002012 (human), NM_010210 (Mouse)), base sequence of the TP53 gene (NCBI accession Number NM_000546, NM_001126112,26NM_001126114, NM_001126115, NM_001126116, NM_00112610011 (27), NM_0011260011 (27) )), RB1 gene base sequence (NCBI accession Number NM_000321 (human), NM_009029 (Mouse)), PTEN gene base sequence (NCBI accession Number NM_000314 (human), NM_008960 (Mouse)), and LZTS1 gene base sequence ( NCBI accession Number NM_021020 (human), NM_199364 (Mouse)).
　これらの未分化誘導因子の遺伝子及び／又は癌抑制遺伝子の発現抑制因子は、１種単独で使用してもよく、また２種以上を組み合わせて使用してもよい。具体的には、未分化誘導因子の遺伝子の内から１種又は２種以上を使用してもよく、また癌抑制遺伝子の発現抑制因子の内から１種又は２種以上を使用してもよく、更には未分化誘導因子の遺伝子の１種以上と癌抑制遺伝子の発現抑制因子の１種以上とを組み合わせて使用してもよい。これらの未分化誘導因子の遺伝子及び／又は癌抑制遺伝子の発現抑制因子の中でも、癌幹細胞への効率的な誘導という観点から、好ましくは、LIN28遺伝子、KRAS遺伝子、FHIT遺伝子の抑制因子、PTEN遺伝子の抑制因子が挙げられる。とりわけ、癌幹細胞に誘導する癌細胞として肝癌細胞を使用する場合には、LIN遺伝子が好適であり；癌幹細胞に誘導する癌細胞として胆管癌細胞を使用する場合には、LIN遺伝子、KRAS遺伝子、FHIT遺伝子の抑制因子、PTEN遺伝子の抑制因子が好適であり；癌幹細胞に誘導する癌細胞として胆管癌細胞を使用する場合には、FHIT遺伝子の抑制因子が好適である。 These undifferentiation-inducing factor genes and / or tumor suppressor gene expression inhibitory factors may be used singly or in combination of two or more. Specifically, one or more of the genes of the undifferentiation inducing factor may be used, and one or more of the tumor suppressor gene expression suppressing factors may be used. Furthermore, one or more types of undifferentiation-inducing factor genes and one or more types of tumor suppressor gene expression suppressing factors may be used in combination. Among these undifferentiation inducer genes and / or tumor suppressor gene expression suppressors, from the viewpoint of efficient induction into cancer stem cells, preferably, LIN28 gene, KRAS gene, FHIT gene suppressor, PTEN gene The inhibitory factor of is mentioned. In particular, when hepatoma cells are used as cancer cells to be induced into cancer stem cells, the LIN gene is suitable; when bile duct cancer cells are used as cancer cells to be induced into cancer stem cells, the LIN gene, KRAS gene, FHIT gene suppressors and PTEN gene suppressors are preferred; when cholangiocarcinoma cells are used as cancer cells induced in cancer stem cells, FHIT gene suppressors are preferred.
　上記(1)～(3)の遺伝子及び上記(4)の未分化誘導因子の遺伝子は、ヒトを含む哺乳動物において共通して存在しており、任意の哺乳動物由来のものを使用できるが、導入する癌細胞の由来に応じて適宜選択することが望ましい。例えば、癌細胞としてヒト由来のものを使用する場合であれば、癌細胞に導入される上記(1)～(3)の遺伝子及び上記(4)の未分化誘導因子の遺伝子はヒト由来であることが望ましい。また、上記(1)～(3)の遺伝子及び上記(4)の未分化誘導因子の遺伝子は、野生型遺伝子以外に、その遺伝子産物のアミノ酸配列における１若しくは数個（例えば１～１０個、好ましくは１～６個、更に好ましくは１～４個、より好ましくは１～３個、特に好ましくは１又は２個）のアミノ酸が置換、欠失、及び／又は挿入されており、且つ、野生型の遺伝子産物と同等の機能を有する変異遺伝子産物をコードしている変異遺伝子であってもよい。 The gene of (1) to (3) and the gene of the undifferentiation inducer of (4) above are commonly present in mammals including humans, and those derived from any mammal can be used. It is desirable to select appropriately according to the origin of the cancer cells to be introduced. For example, when human-derived cancer cells are used, the genes (1) to (3) and the undifferentiation inducer gene (4) introduced into the cancer cells are derived from humans. It is desirable. In addition to the wild-type gene, the gene of (1) to (3) and the gene of the undifferentiation inducer of (4) are one or several (for example, 1 to 10) in the amino acid sequence of the gene product. Preferably 1 to 6, more preferably 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2 amino acids are substituted, deleted and / or inserted, and wild It may be a mutated gene encoding a mutated gene product having a function equivalent to that of the type gene product.
　本発明において、上記(1)～(3)の遺伝子及び上記(4)の未分化誘導因子の遺伝子は、公知の配列情報に基づいて、常法に従って調製することができる。例えば、哺乳動物由来の細胞からRNAを抽出し、常法に従ってクローニングすることにより、目的とする遺伝子のcDNAを調製することができる。 In the present invention, the genes (1) to (3) and the gene for the undifferentiation inducer (4) can be prepared according to conventional methods based on known sequence information. For example, cDNA of a target gene can be prepared by extracting RNA from a mammal-derived cell and cloning according to a conventional method.
　上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子の癌細胞への導入は、公知の手法によって行うことができる。具体的には、上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子を癌細胞へ導入する方法として、ベクターを使用する方法；リン酸カルシウム法；リポフェクション法；エレクトロポレーション法；マイクロインジェクション法等が例示される。これらの中でも、導入効率の点から、ベクターを使用する方法が好ましい。ベクターを使用して上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子を癌細胞に導入する場合には、ベクターとして、ウイルスベクター、非ウイルスベクター、人工ウイルス等を用いることができるが、アデノウイルス及びレトロウイルス等のウイルスベクターが好適である。なお、ベクターを使用する場合、上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子は、各々別のベクターに組み込まれていてもよく、１つのベクターに２種以上の遺伝子が組み込まれていてもよい。 Introduction of the above genes (1) to (3) and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor into a cancer cell can be performed by a known technique. it can. Specifically, as a method for introducing a gene of the above (1) to (3) and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor into a cancer cell, a vector Examples thereof include calcium phosphate method; lipofection method; electroporation method; microinjection method and the like. Among these, the method using a vector is preferable from the viewpoint of introduction efficiency. When using a vector to introduce the above genes (1) to (3), and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor into cancer cells, As vectors, viral vectors, non-viral vectors, artificial viruses and the like can be used, and viral vectors such as adenoviruses and retroviruses are preferred. When a vector is used, the above genes (1) to (3) and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor are in separate vectors. It may be integrated, or two or more genes may be integrated in one vector.
　また、ベクターを利用して上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子を、癌細胞に導入する場合、まず、癌細胞を、レトロウイルスレセプターを発現するレンチウイルスベクターで形質転換した後に、上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子を各々組み込んだレトロウイルスベクターで形質転換する方法が好適である。このような方法を採用することによって、遺伝子の導入効率を高め、目的の癌幹細胞の回収率を高めることができる。 In addition, when a vector is used to introduce the above genes (1) to (3) and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor into cancer cells First, after transforming a cancer cell with a lentiviral vector that expresses a retroviral receptor, the above genes (1) to (3), and (4) a gene of a specific undifferentiation inducer and / or a specific A method of transforming with a retroviral vector each incorporating a tumor suppressor gene expression suppressor is preferred. By adopting such a method, it is possible to increase the efficiency of gene introduction and increase the recovery rate of the target cancer stem cells.
　斯くして上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子が導入された癌細胞は、該癌細胞が生育可能な培地で7～35日間程度培養することによって、癌細胞がリプログラミングされ、自己複製能と共に多分化能が獲得された癌幹細胞に誘導される。上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子が導入された癌細胞の培養に使用される培地については、該癌細胞が生育可能であることを限度として特に制限されないが、上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子の導入後7日程度は、10容量％のウシ胎仔血清（FBS）を含む培地で培養し、その後は、マトリクスタンパク質でコーティングしたディッシュにおいて、（4～5ng/ml重量％の塩基性繊維芽細胞増殖因子（bFGF）を含む培地で培養することが望ましい。ここで、マトリクスタンパク質としては、マトリゲル、フィブロネクチン、コラーゲン、ラミニン等が例示され、これらの中でもマトリゲルが好適である。 Thus, cancer cells into which the above genes (1) to (3) and (4) specific undifferentiation-inducing factor genes and / or specific tumor suppressor gene expression suppressors have been introduced are By culturing in a viable medium for about 7 to 35 days, the cancer cells are reprogrammed and induced into cancer stem cells that have acquired pluripotency as well as self-renewal ability. Regarding the medium used for culturing cancer cells into which the gene of (1) to (3) above and (4) the gene of a specific undifferentiation inducer and / or the expression suppressor of a specific tumor suppressor gene are introduced Although not particularly limited as long as the cancer cells can grow, the genes of the above (1) to (3), and (4) a gene of a specific undifferentiation inducer and / or a specific tumor suppressor gene About 7 days after the introduction of the expression inhibitory factor, the cells were cultured in a medium containing 10% by volume of fetal bovine serum (FBS), and then (4-5 ng / ml wt% basic) in a dish coated with matrix protein. It is desirable to culture in a medium containing fibroblast growth factor (bFGF), where the matrix protein is exemplified by matrigel, fibronectin, collagen, laminin, etc. Among these, matrigel is preferred.
　上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子が導入された癌細胞の中から、癌幹細胞に誘導された細胞の選択は、細胞の増殖能の有無、及び癌幹細胞の特性を有するか否かを指標として行うことができる。このような癌幹細胞の選択は、具体的には、増殖能を有する細胞の中から、細胞の形状、癌幹細胞に対する特異的染色の有無、癌幹細胞のマーカー遺伝子の発現の有無等を指標として行うことができる。癌細胞に、予め癌幹細胞マーカー遺伝子のプロモーターに薬剤耐性遺伝子を結合して作ったレポーター遺伝子コンストラクトを導入した場合には、癌幹細胞の特性を獲得した細胞は該薬剤の存在下で生育可能になるので、薬剤存在下での生育を指標として、癌幹細胞の特性を獲得した細胞を選択することもできる。また、癌幹細胞に誘導された細胞は、マトリクスタンパク質でコーティングしたディッシュにおいて、塩基性繊維芽細胞増殖因子（bFGF）を含む培地で培養すると、増殖すると共に、円形の形状を呈するので、かかる形状を上記指標とすることができる。更に、癌幹細胞胞は、癌幹細胞マーカー遺伝子（Nanog、BMI1等）を発現しているので、当該マーカー遺伝子の発現の有無を上記指標とすることもできる。また、CD24は、癌幹細胞の表面マーカーであることも明らかにされており（Eyal Sagiv et al., Targeting CD24 for Treatment of Colorectal and Pancreatic Cancer by Monoclonal Antibodies or small interfering RNA, Cancer Research, Vol. 68, pages, 2803-2812, 2008）、CD24の発現を癌幹細胞の選択の指標として使用することもできる。 It is induced into a cancer stem cell from among the cancer cells into which the above genes (1) to (3) and (4) the gene of a specific undifferentiation inducer and / or the expression suppressor of a specific tumor suppressor gene are introduced. The selection of the cells can be performed using as an index whether or not the cells have the ability to proliferate and whether or not they have the characteristics of cancer stem cells. Specifically, the selection of such cancer stem cells is carried out using the cell shape, presence / absence of specific staining for cancer stem cells, presence / absence of expression of a marker gene for cancer stem cells, etc., from among cells having proliferative ability. be able to. When a reporter gene construct prepared by previously binding a drug resistance gene to a cancer stem cell marker gene promoter is introduced into a cancer cell, cells that have acquired the characteristics of the cancer stem cell can grow in the presence of the drug. Therefore, cells that have acquired the characteristics of cancer stem cells can be selected using growth in the presence of a drug as an index. In addition, cells induced by cancer stem cells proliferate and exhibit a circular shape when cultured in a medium coated with matrix protein in a medium containing basic fibroblast growth factor (bFGF). The above index can be used. Furthermore, since the cancer stem cell vesicle expresses a cancer stem cell marker gene (Nanog, BMI1, etc.), the presence or absence of the expression of the marker gene can also be used as the index. CD24 has also been shown to be a surface marker for cancer stem cells (Eyal Sagiv et al., Targeting CD24 for Treatment of Colorectal and Pancreatic Cancer by Monoclonal Antibodies or small interfering RNA, Cancer Research, Vol. 68, pages, 2803-2812, 2008), CD24 expression can also be used as an indicator of cancer stem cell selection.
　斯くして得られる癌幹細胞を利用することによって、癌幹細胞の特性を明らかにすることができ、更にはその分化誘導や増殖制御を行うことができる物質をスクリーニングすることが可能になる。具体的には、上記で得られる癌幹細胞に対して、被験物質（スクリーニングに供される候補物質）を接触させ、癌幹細胞の分化の有無を測定し、癌幹細胞を分化させた被験物質を選択することによって、癌幹細胞を分化誘導させる物質をスクリーニングすることが可能になる。また、上記で得られる癌幹細胞に対して、被験物質（スクリーニングに供される候補物質）を接触させ、癌幹細胞の増殖の程度を測定し、癌幹細胞の増殖を抑制させた被験物質、又は癌幹細胞の増殖を促進させた被験物質を選択することによって、癌幹細胞の増殖を抑制できる物質又は癌幹細胞の増殖を促進する物質をスクリーニングすることが可能になる。 By using the cancer stem cells thus obtained, the characteristics of the cancer stem cells can be clarified, and further, a substance capable of inducing differentiation and controlling proliferation can be screened. Specifically, the test substance (candidate substance to be used for screening) is contacted with the cancer stem cells obtained above, the presence or absence of differentiation of the cancer stem cells is measured, and the test substance that has differentiated the cancer stem cells is selected By doing so, it becomes possible to screen for substances that induce differentiation of cancer stem cells. Further, a test substance or cancer in which the test substance (candidate substance to be used for screening) is brought into contact with the cancer stem cells obtained above, the degree of cancer stem cell proliferation is measured, and cancer stem cell proliferation is suppressed. By selecting a test substance that promotes the proliferation of stem cells, it becomes possible to screen for a substance that can suppress the proliferation of cancer stem cells or a substance that promotes the proliferation of cancer stem cells.
　また、癌幹細胞は、癌の発症、進行、転移を担っているので、上記で得られる癌幹細胞は、癌の治療薬のスクリーニングや癌の治療薬の薬効評価に使用することもできる。具体的には、上記で得られる癌幹細胞に対して、被験物質（スクリーニングに供される候補物質）を接触させ、癌幹細胞の生育の有無を測定し、癌幹細胞の生育を阻害した被験物質を選択することによって、癌の治療薬をスクリーニングすることが可能になる。また、上記で得られる癌幹細胞に対して、癌の治療薬を接触させ、癌幹細胞の生育阻害の程度を測定することによって、癌幹細胞の生育阻害の程度が大きい程、癌の治療薬の薬効は高く、癌幹細胞の生育阻害の程度が小さい程、癌の治療薬の薬効は低いと評価することができる。なお、本明細書において、癌幹細胞の生育阻害とは、癌幹細胞の生育を止める又は生育能を減弱化させる場合のみならず、癌幹細胞を死滅させる場合も包含する。 In addition, since cancer stem cells are responsible for the onset, progression, and metastasis of cancer, the cancer stem cells obtained above can also be used for screening for cancer therapeutics and for evaluating the efficacy of cancer therapeutics. Specifically, a test substance (candidate substance to be used for screening) is brought into contact with the cancer stem cells obtained above, the presence or absence of growth of cancer stem cells is measured, and a test substance that inhibits the growth of cancer stem cells is obtained. Selection makes it possible to screen for cancer therapeutics. In addition, the cancer stem cell obtained above is brought into contact with a cancer therapeutic agent, and the degree of inhibition of cancer stem cell growth is measured. It can be evaluated that the lower the degree of inhibition of cancer stem cell growth, the lower the efficacy of the therapeutic agent for cancer. In this specification, the growth inhibition of cancer stem cells includes not only the case of stopping the growth of cancer stem cells or attenuating the growth ability but also the case of killing cancer stem cells.
　更に、本発明は、上記で得られる癌幹細胞に対して特異的に結合する抗体を提供する。該抗体は、モノクローナル抗体、ポリクローナル抗体のいずれであってもよいが、好ましくはモノクローナル抗体である。該抗体は、癌幹細胞を検出する目的で使用でき、また癌の治療薬としても使用し得る。 Furthermore, the present invention provides an antibody that specifically binds to the cancer stem cells obtained above. The antibody may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. The antibody can be used for the purpose of detecting cancer stem cells and can also be used as a therapeutic agent for cancer.
２．癌幹細胞誘導剤
　前述するように、(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)NANOG、LIN28、KRAS、BCL2、BMI1、及びERASよりなる群から選択される少なくとも1種の未分化誘導因子の遺伝子、及び／又はCDKN2A、FHIT、TP53、RB1、PTEN、及びLZTS1よりなる群から選択される少なくとも1種の癌抑制遺伝子の発現抑制因子を、癌細胞に導入することにより、癌幹細胞を調製することができる。従って、本発明は、更に、(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)NANOG、LIN28、KRAS、BCL2、BMI1、及びERASよりなる群から選択される少なくとも1種の未分化誘導因子の遺伝子、及び／又はCDKN2A、FHIT、TP53、RB1、PTEN、及びLZTS1よりなる群から選択される少なくとも1種の癌抑制遺伝子の発現抑制因子を含む、癌幹細胞誘導剤を提供する。該癌幹細胞誘導剤は、癌細胞をリプログラミングして癌幹細胞を誘導するために使用される各種因子のセットを含むものであり、上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子が癌細胞に導入可能な形態で含まれていることが望ましい。上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子が癌細胞に導入可能な形態として、具体的には、上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子が組み込まれたベクターが例示される。ここで、上記(1)～(3)の遺伝子、並びに(4)特定の未分化誘導因子の遺伝子及び／又は特定の癌抑制遺伝子の発現抑制因子は、各々別のベクターに組み込まれていてもよく、１つのベクターに２種以上の遺伝子が同時に組み込まれていてもよい。 2. Cancer stem cell inducer As described above, (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS At least one selected undifferentiation-inducing factor gene and / or at least one tumor suppressor gene expression inhibitor selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1, Cancer stem cells can be prepared by introducing into cells. Therefore, the present invention is further selected from the group consisting of (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS. A cancer comprising at least one gene for an undifferentiated inducer and / or an expression suppressor for at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 A stem cell inducer is provided. The cancer stem cell inducer comprises a set of various factors used for reprogramming cancer cells to induce cancer stem cells, the gene of (1) to (3) above, and (4) specific It is desirable that the undifferentiation-inducing factor gene and / or the expression suppressor of a specific tumor suppressor gene be contained in a form that can be introduced into cancer cells. As a form in which the gene of (1) to (3) above and (4) the gene of a specific undifferentiation inducer and / or the expression suppressor of a specific tumor suppressor gene can be introduced into cancer cells, specifically, Examples include vectors incorporating the genes (1) to (3) above and (4) a specific undifferentiation-inducing factor gene and / or a specific tumor suppressor gene expression suppressor. Here, the above genes (1) to (3), and (4) a specific undifferentiation-inducing factor gene and / or a specific tumor suppressor gene expression suppressor may be incorporated into different vectors. In addition, two or more genes may be simultaneously incorporated into one vector.
　該軟癌幹細胞誘導剤に使用される遺伝子、ベクターの種類等については、前述の通りである。 The genes and vectors used for the soft cancer stem cell inducer are as described above.
　以下に、実施例等に基づいて本発明を詳細に説明するが、本発明はこれらによって限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples and the like, but the present invention is not limited thereto.
実施例
　ヒト肝癌細胞（PLC）、ヒト胆管癌細胞（HuCCT-1）、及びヒト大腸癌細胞（DLD-1、John Hopkins大学（米国）のB. Vogelsteinより供与）をリプログラミングして、癌幹細胞に誘導する実験を行った。 Examples Reprogramming human hepatoma cells (PLC), human cholangiocarcinoma cells (HuCCT-1), and human colon cancer cells (DLD-1, provided by B. Vogelstein, John Hopkins University, USA), cancer stem cells Experiments to induce
＜実験方法＞
　まず、30mmディッシュに1×106個の各癌細胞を2mlの10%FBS含有DMEM培地（2μg/ml puromycin含有）中に接種し、Lipofectamine 2000トランスフェクション試薬10μlとレトロウイルスレセプターを発現に組み込んだレンチウイルスベクター3-5μgを添加して、室温で20分インキュベートした。次いで、上清を除去し、10%FBS含有DMEM培地で細胞を洗浄した後に、2mlの10%FBS含有DMEM培地を入れ、更にLipofectamine 2000トランスフェクション試薬10μlと表１に示す遺伝子又はDNA断片を組み込んだpMXsベースのレトロウイルスベクター（計4種）各5μgを添加して、37℃で24時間インキュベートすることにより、各遺伝子を癌細胞にトランスフェクトした。なお、トランスフェクションに使用したレトロウイルスベクターは１種の遺伝子又はDNA断片を組み込んだものを使用し、トランスフェクトする遺伝子の組合せに応じて、各遺伝子を組み込んだレトロウイルスベクターを組み合わせて各癌細胞にトランスフェクトした。 <Experiment method>
First, 1 × 10 6 cancer cells were inoculated into 2 ml of 10% FBS-containing DMEM medium (containing 2 μg / ml puromycin) in a 30 mm dish, and 10 μl of Lipofectamine 2000 transfection reagent and a retroviral receptor were incorporated into the expression. 3-5 μg of lentiviral vector was added and incubated at room temperature for 20 minutes. Next, after removing the supernatant and washing the cells with 10% FBS-containing DMEM medium, 2 ml of 10% FBS-containing DMEM medium was added, and 10 μl of Lipofectamine 2000 transfection reagent and the gene or DNA fragment shown in Table 1 were incorporated. Each gene was transfected into cancer cells by adding 5 μg each of pMXs-based retroviral vectors (4 types in total) and incubating at 37 ° C. for 24 hours. In addition, the retrovirus vector used for the transfection uses a vector in which one kind of gene or DNA fragment is incorporated, and according to the combination of genes to be transfected, each cancer cell is combined with a retrovirus vector in which each gene is incorporated. Transfected.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　トランスフェクション後の各癌細胞を10%FBS含有DMEM培地で一日おきに培地を交換しながら７日間培養し、トランスフェクションから７日目以降はmTeSRTM1とBDヒトES細胞用マトリゲル（StemCell Technologies Inc）を用いて一日おきに培地を交換しながら培養を行った。 Each cancer cell after transfection is cultured in DMEM medium containing 10% FBS for 7 days while changing the medium every other day. After 7 days from transfection, mTeSRTM1 and matrigel for BD human ES cells (StemCell Technologies Technologies Inc) Was cultured while changing the medium every other day.
　斯くして培養された細胞の癌幹細胞への誘導を確認するために、Nanog遺伝子の発現量をRT-PCRにより測定した。また、コントロールとして、グリセルアルデヒド3-リン酸デヒドロゲナーゼ（GAPDH）遺伝子の発現量についてもRT-PCRにより測定した。本測定に使用したプライマーは以下の通りである。なお、下記Nanog遺伝子に対するForward Primer及びReverse PrimerはUPL（#66）タグ(Roche社製)を混合してPCR反応を行い、下記GAPDH遺伝子に対するForward Primer及びReverse PrimerはUPL（#60）タグ(Roche社製)を混合してPCR反応を行った。
Forward Primer for Nanog：5'-ATG CCT CAC ACG GAG ACT GT-3'（配列番号１）
Reverse Primer for Nanog：5'-AGG GCT GTC CTG AAT AAG CA-3'（配列番号２）
Forward Primer for GAPDH：5'-AGC CAC ATC GCT CAG ACA C-3'（配列番号３）
Reverse Primer for GAPDH：5'-GCC CAA TAC GAC CAA ATC C-3'（配列番号４）。 In order to confirm the induction of the thus cultured cells into cancer stem cells, the expression level of Nanog gene was measured by RT-PCR. As a control, the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was also measured by RT-PCR. The primers used for this measurement are as follows. In addition, the Forward Primer and Reverse Primer for the Nanog gene below are mixed with UPL (# 66) tag (Roche) and PCR reaction is performed. The Forward Primer and Reverse Primer for the GAPDH gene below are UPL (# 60) tag (Roche PCR product was mixed.
Forward Primer for Nanog: 5'-ATG CCT CAC ACG GAG ACT GT-3 '(SEQ ID NO: 1)
Reverse Primer for Nanog: 5'-AGG GCT GTC CTG AAT AAG CA-3 '(SEQ ID NO: 2)
Forward Primer for GAPDH: 5'-AGC CAC ATC GCT CAG ACA C-3 '(SEQ ID NO: 3)
Reverse Primer for GAPDH: 5′-GCC CAA TAC GAC CAA ATC C-3 ′ (SEQ ID NO: 4).
＜実験結果＞
　実施例１－１２のいずれの組合せでも、PLC、HuCCT、及びDLD-1において、Nanog発現量の明らかな増加が認められた。得られた結果の代表例を図１～３に示す。図１には各遺伝子の組合わせ（実施例１－６）を導入したPLC細胞（トランスフェクション20日後）のNANOG遺伝子の発現量を測定した結果、図２には各遺伝子の組合わせ（実施例１、２及び７－１２）を導入したHuCCT-1細胞（トランスフェクション12日後）のNANOG遺伝子の発現量を測定した結果、図３には各遺伝子の組合わせ（実施例１－３及び７－１１）を導入したDLD-1細胞（トランスフェクション12日後）のNANOG遺伝子の発現量を測定した結果を示す。なお、NANOG遺伝子は肝癌細胞で特異的に発現しており、その発現量は、癌幹細胞に誘導されたことの指標とされている。 <Experimental result>
In any combination of Examples 1-12, a clear increase in Nanog expression was observed in PLC, HuCCT, and DLD-1. Representative examples of the results obtained are shown in FIGS. FIG. 1 shows the results of measuring the expression level of NANOG gene in PLC cells (20 days after transfection) into which the combination of each gene was introduced (Example 1-6). FIG. 2 shows the combination of each gene (Example As a result of measuring the expression level of NANOG gene in HuCCT-1 cells (12 days after transfection) introduced with 1, 2, and 7-12), FIG. 3 shows combinations of the genes (Examples 1-3 and 7-). The result of having measured the expression level of the NANOG gene of DLD-1 cell which introduce | transduced 11) (12 days after transfection) is shown. The NANOG gene is specifically expressed in liver cancer cells, and its expression level is an indicator that it has been induced in cancer stem cells.
　図１～３から明らかなように、実施例１－１２のいずれの遺伝子の組合せを導入した細胞でも、Nanog発現量の著しい増加が認められた。とりわけ、PLCに対しては実施例１の遺伝子の組合せ、HuCCTに対しては実施例１、２、８及び１１の遺伝子の組合せ、並びにDLD-1に対しては実施例８の遺伝子の組合せが、癌細胞への誘導に特に有効であることが明らかとなった。一方、KLF4、OCT3/4、及びSOX2と共に、c-Mycを導入した細胞（比較例１）では、Nanogの発現量の増加は殆ど認められなかった。つまり、比較例１の結果から、体細胞を誘導多能性幹（iPS）細胞を創出可能な再プログラム因子（KLF4、OCT3/4、SOX2、及びc-Mycの組み合わせ）を使用しても、癌細胞を癌幹細胞に誘導できなかった。 As is clear from FIGS. 1 to 3, a significant increase in the expression level of Nanog was observed in cells into which any combination of genes of Example 1-12 was introduced. In particular, the combination of genes of Example 1 for PLC, the combination of genes of Examples 1, 2, 8, and 11 for HuCCT, and the combination of genes of Example 8 for DLD-1. It was proved to be particularly effective for induction into cancer cells. On the other hand, in the cells into which c-Myc was introduced together with KLF4, OCT3 / 4, and SOX2 (Comparative Example 1), almost no increase in the expression level of Nanog was observed. In other words, from the results of Comparative Example 1, using a reprogramming factor (a combination of KLF4, OCT3 / 4, SOX2, and c-Myc) that can create somatic cells induced pluripotent stem (iPS) cells, Cancer cells could not be induced into cancer stem cells.
　また、実施例１－１２のいずれの遺伝子の組合せを導入した細胞でも、トランスフェクション20日後には、球状の形状を示しており、癌細胞が未分化状態の形態に変化していることも確認された。代表例として、図４に、実施例２、５及び６の各遺伝子の組合せを大腸癌細胞にトランスフェクションし、20日間培養した際の細胞の形状を観察した結果を示す。 In addition, the cells into which any of the gene combinations of Example 1-12 were introduced showed a spherical shape 20 days after transfection, and it was confirmed that the cancer cells had changed to an undifferentiated state. It was done. As a representative example, FIG. 4 shows the results of observing the shape of cells when the combinations of the genes of Examples 2, 5 and 6 were transfected into colon cancer cells and cultured for 20 days.
　更に、実施例１－１２の遺伝子の組合せを導入した細胞のいずれでも、自己増殖能を有していることが確認された。 Furthermore, it was confirmed that any of the cells into which the gene combination of Example 1-12 was introduced had self-proliferating ability.
　以上のNanog発現量、細胞の形状、及び細胞の自己増殖能の結果から、KLF4、OCT3/4、及びSOX2と共に、Nanog、LIN28、BCL2、BMI1、ERAS、KRAS、CDKN2A、FHIT、TP53、RB1、PTEN、又はLZTS1を癌細胞に導入することによって、癌細胞がリプログラミングされて癌幹細胞に誘導されることが分かった。 From the results of the above Nanog expression level, cell shape, and cell self-proliferation ability, together with KLF4, OCT3 / 4, and SOX2, Nanog, LIN28, BCL2, BMI1, ERAS, KRAS, CDKN2A, FHIT, TP53, RB1, It was found that by introducing PTEN or LZTS1 into cancer cells, the cancer cells were reprogrammed and induced into cancer stem cells.
比較例
　KLF4遺伝子、OCT3/4遺伝子、及びSOX2遺伝子と共に、SNAI1遺伝子を組み合わせた４つの遺伝子を、上記と同様の手法で、癌細胞（PLC、HuCCT-1、DLD-1）に導入したところ、細胞の形状から明らかに癌幹細胞に誘導されていないと判断されたため、Nanog発現量の測定は行わなかった。 Comparative Example When four genes combining SNAI1 gene together with KLF4 gene, OCT3 / 4 gene, and SOX2 gene were introduced into cancer cells (PLC, HuCCT-1, DLD-1) by the same method as above, Since it was judged from the cell shape that it was clearly not induced in cancer stem cells, the expression level of Nanog was not measured.
　また、KLF4遺伝子、OCT3/4遺伝子、SOX2遺伝子、C-MYC遺伝子、NANOG遺伝子、LIN28遺伝子、KRAS遺伝子、BCL2遺伝子、BMI1遺伝子、ERAS遺伝子、CDKN2A遺伝子、FHIT遺伝子、TP53遺伝子、RB1遺伝子、PTEN遺伝子、又はLZTS1遺伝子の各１遺伝子を上記と同様の手法で、癌細胞（DLD-1）に導入したところ、Nanogの発現は認められなかった（いずれの場合も、NANOG/GPDHは０であった）。 In addition, KLF4 gene, OCT3 / 4 gene, SOX2 gene, C-MYC gene, NANOG gene, LIN28 gene, KRAS gene, BCL2 gene, BMI1 gene, ERAS gene, CDKN2A gene, FHIT gene, TP53 gene, RB1 gene, PTEN gene Alternatively, when one gene of each of the LZTS1 genes was introduced into cancer cells (DLD-1) in the same manner as described above, no Nanog expression was observed (NANOG / GPDH was 0 in either case) ).
　以上の結果から、KLF4、OCT3/4、及びSOX2と共に、特定の遺伝子を選択し、これらを組み合わせて癌細胞に導入した場合に限り、癌幹細胞への誘導が実現できることが明らかとなった。 From the above results, it has been clarified that induction into cancer stem cells can be realized only when specific genes are selected together with KLF4, OCT3 / 4, and SOX2, and these are combined and introduced into cancer cells.

Claims (11)

  1. 　(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)NANOG、LIN28、KRAS、BCL2、BMI1、及びERASよりなる群から選択される少なくとも1種の未分化誘導因子の遺伝子、及び／又はCDKN2A、FHIT、TP53、RB1、PTEN、及びLZTS1よりなる群から選択される少なくとも1種の癌抑制遺伝子の発現抑制因子を、癌細胞に導入する工程を含む、癌幹細胞の製造方法。 (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS A cancer comprising a step of introducing into a cancer cell an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 A method for producing stem cells.
  2. 　KLFファミリー遺伝子がKLF 4遺伝子である、請求項１に記載の製造方法。 The production method according to claim 1, wherein the KLF family gene is a KLF-4 gene.
  3. 　OCTファミリー遺伝子がOCT3/4遺伝子である、請求項１に記載の製造方法。 The production method according to claim 1, wherein the OCT family gene is an OCT3 / 4 gene.
  4. 　SOXファミリー遺伝子がSOX2遺伝子である、請求項１に記載の製造方法。 The production method according to claim 1, wherein the SOX family gene is a SOX2 gene.
  5. 　(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)NANOG、LIN28、KRAS、BCL2、BMI1、及びERASよりなる群から選択される少なくとも1種の未分化誘導因子の遺伝子、及び／又はCDKN2A、FHIT、TP53、RB1、PTEN、及びLZTS1よりなる群から選択される少なくとも1種の癌抑制遺伝子の発現抑制因子を、癌細胞に導入することにより得られる、癌幹細胞。 (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS It is obtained by introducing into a cancer cell an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1, Cancer stem cells.
  6. 　請求項５に記載の癌幹細胞に対して特異的に結合する抗体。 An antibody that specifically binds to the cancer stem cell according to claim 5.
  7. 　下記工程を含む、癌幹細胞を分化誘導させる物質のスクリーニング方法：
    請求項５に記載の癌幹細胞に被験物質を接触させ、該癌幹細胞の分化の有無を測定する工程、及び
    癌幹細胞を分化させた被験物質を、癌幹細胞を分化誘導させる物質として選択する工程。     A screening method for a substance that induces differentiation of cancer stem cells, comprising the following steps:
    6. A step of contacting a test substance with the cancer stem cell according to claim 5 and measuring the presence or absence of differentiation of the cancer stem cell, and a step of selecting a test substance obtained by differentiating the cancer stem cell as a substance that induces differentiation of the cancer stem cell.
  8. 　下記工程を含む、癌幹細胞の増殖を抑制できる物質又は癌幹細胞の増殖を促進する物質のスクリーニング方法：
    請求項５に記載の癌幹細胞に被験物質を接触させ、該癌幹細胞の増殖の程度を測定する工程、及び
    癌幹細胞の増殖を抑制させた被験物質、又は癌幹細胞の増殖を促進させた被験物質を、癌幹細胞の増殖を抑制できる物質又は癌幹細胞の増殖を促進する物質として選択する工程。     A screening method for a substance that can suppress the growth of cancer stem cells or a substance that promotes the growth of cancer stem cells, comprising the following steps:
    6. A step of contacting a test substance with the cancer stem cell according to claim 5 and measuring the degree of proliferation of the cancer stem cell; and a test substance in which the proliferation of the cancer stem cell is suppressed; or a test substance that promotes the proliferation of the cancer stem cell Is selected as a substance that can suppress the growth of cancer stem cells or a substance that promotes the growth of cancer stem cells.
  9. 　下記工程を含む、癌の治療薬のスクリーニング方法：
    請求項５に記載の癌幹細胞に被験物質を接触させ、該癌幹細胞の生育の有無を測定する工程、及び
    癌幹細胞の生育を阻害した被験物質を、癌の治療薬として選択する工程。     A screening method for a therapeutic agent for cancer, comprising the following steps:
    6. A step of bringing a test substance into contact with the cancer stem cells according to claim 5 and measuring the presence or absence of growth of the cancer stem cells; and a step of selecting a test substance that inhibits the growth of cancer stem cells as a therapeutic agent for cancer.
  10. 　下記工程を含む、癌の治療薬の薬効評価方法：
    請求項５に記載の癌幹細胞に癌の治療薬を接触させ、該癌幹細胞の生育阻害の程度をする工程、及び
    癌幹細胞の生育阻害の程度が大きい程、癌の治療薬の薬効が高いと判定する工程。     A method for evaluating the efficacy of a therapeutic agent for cancer, comprising the following steps:
    The step of bringing a cancer therapeutic agent into contact with the cancer stem cell according to claim 5 to inhibit the growth of the cancer stem cell, and the greater the inhibition of the growth of the cancer stem cell, the higher the efficacy of the cancer therapeutic agent. A step of determining.
  11. 　(1)KLFファミリー遺伝子、(2)OCTファミリー遺伝子、(3)SOXファミリー遺伝子、並びに(4)NANOG、LIN28、KRAS、BCL2、BMI1、及びERASよりなる群から選択される少なくとも1種の未分化誘導因子の遺伝子、及び／又はCDKN2A、FHIT、TP53、RB1、PTEN、及びLZTS1よりなる群から選択される少なくとも1種の癌抑制遺伝子の発現抑制因子を含有することを特徴とする、癌幹細胞誘導剤。 (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS Cancer stem cell induction characterized by comprising an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 Agent.
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