WO2010050268A1 - Marqueur moléculaire de cellule souche cancéreuse - Google Patents

Marqueur moléculaire de cellule souche cancéreuse Download PDF

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WO2010050268A1
WO2010050268A1 PCT/JP2009/061154 JP2009061154W WO2010050268A1 WO 2010050268 A1 WO2010050268 A1 WO 2010050268A1 JP 2009061154 W JP2009061154 W JP 2009061154W WO 2010050268 A1 WO2010050268 A1 WO 2010050268A1
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cells
cancer
cancer stem
sox2
stem cells
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PCT/JP2009/061154
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English (en)
Japanese (ja)
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俊彦 鳥越
あかり 高橋
良彦 廣橋
昇志 佐藤
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北海道公立大学法人札幌医科大学
独立行政法人科学技術振興機構
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Publication of WO2010050268A1 publication Critical patent/WO2010050268A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention is a molecular marker for detecting cancer stem cells in a cell population to be detected, which is detected in cancer stem cells included in the detection target, but is not cancer stem cells and normal cells Relates to said molecular marker not detected in.
  • Cancer stem cells are considered to be a major cause of cancer recurrence and metastasis, and the importance of targeting cancer stem cells in cancer treatment has been pointed out.
  • cancer stem cells there is only a small proportion of cancer stem cells in the tumor tissue (Non-patent Document 4), and it is very difficult to specifically recognize and treat cancer stem cells.
  • the development of cancer stem cell detection technology and new treatment methods targeting cancer stem cells are important issues for cancer medical care.
  • molecular markers such as CD133, CD24, and CD44 are known as cancer stem cell markers (Non-Patent Documents 1 to 7), but it is said that they are effective only in a few cancer cells. In order to detect cancer stem cells in more types of cancer, it is necessary to identify new molecular markers.
  • An object of the present invention is to provide a molecular marker useful for detection of cancer stem cells.
  • the present inventors have conducted intensive research, and in at least one tissue, the expression is preferably expressed in normal cells derived from the tissue, preferably in a plurality of tissues, in the sense that it is more useful.
  • Cancer stem cells that are not seen and expressed in cancer stem cells that is, specimens used to identify cancer stem cell genes usually contain normal tissues, but are rarely expressed in normal tissues in the specimens
  • the present invention is a molecular marker for detecting a cancer stem cell in a cell population to be detected, which is detected in a cancer stem cell included in the detection target, but is a cancer cell that is not a cancer stem cell and normal Relates to said molecular marker not detected in cells.
  • the present invention also provides detection targets of heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, white blood cell, colon, stomach, bone marrow, large intestine and peripheral blood.
  • the present invention relates to the aforementioned molecular marker, which is a cell population derived from one or more cells or tissues selected from the group consisting of mononuclear cells.
  • the present invention further relates to the above-described molecular marker that is not detected in cancer cells and normal cells that are not cancer stem cells of all detection targets.
  • the molecular markers are Sox2, Smcp, Ints1, Kox12, Mdf1, FLJ13464, 667J232, Surf6, Pcdh19, Dchs2, Pcdh21, Gal3st1, Ras11b, Hes6, Znf415, Nkx2-5c, S1
  • the molecular marker is an expression product of one or more genes selected from the group consisting of Or7c1 and Dnajb8.
  • the present invention also relates to a method for determining the presence or absence of cancer stem cells in the determination target using the molecular marker as an index. Furthermore, the present invention relates to the aforementioned method, wherein the cell population comprises cancer cells that are not normal cells and / or cancer stem cells.
  • the present invention provides that the cell population is a heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, white blood cell, colon, stomach, bone marrow, large intestine and
  • the above method is derived from one or more cells or tissues selected from the group consisting of peripheral blood mononuclear cells.
  • the present invention also determines if Sox2, Smcp, Ints1, Kox12, Mdf1, FLJ13464, 667J232, Surf6, Pcdh19, Dchs2, Pcdh21, Gal3st1, Ras11b, Hes6, Znf1r, Ncx2-5, Pmcd,
  • the present invention relates to the above method, wherein it is determined that cancer stem cells are present when an expression product of one or more genes selected from the group consisting of Dnajb8 is detected.
  • the present invention further relates to the above method wherein the determination determines that there is a cancer stem cell when an expression product of the Sox2 gene is detected.
  • the present invention further relates to the above-mentioned method, wherein the gene expression product is mRNA and / or an endogenous polypeptide.
  • the present invention also relates to detecting the gene expression product is mRNA and detecting by RT-PCR.
  • the method Furthermore, the present invention relates to the aforementioned method, wherein the gene expression product is an endogenous polypeptide and comprises detection with a reagent that specifically reacts with the endogenous polypeptide.
  • the present invention relates to the aforementioned method, wherein the reagent is an antibody.
  • the present invention also relates to the aforementioned method wherein the determination is performed in vitro or in vivo.
  • the present invention further includes i) a step of measuring a detection amount A of the molecular marker before administering a candidate compound to a cell population, ii) administering the candidate compound to a cell population; iii) measuring the detected amount B of the molecular marker measured in i) after administering the candidate compound to the cell population, and iv) comparing A and B, and if A is significantly greater than B, the candidate Determining that the compound is a cancer drug candidate,
  • the present invention relates to a method for screening cancer therapeutic agents, including
  • the present invention further relates to a kit for determining the presence or absence of cancer stem cells, comprising the reagent for detecting the molecular marker.
  • the reagent for detection is Sox2, Smcp, Ints1, Kox12, Mdf1, FLJ13464, 667J232, Surf6, Pcdh19, Dchs2, Pcdh21, Gal3st1, Ras11b, Hes6, Znf415, t
  • a probe and / or primer having a base sequence complementary to the gene for detecting mRNA which is an expression product of one or more genes selected from the group consisting of Scgb3a1, Or7c1 and Dnajb8, About the kit.
  • the present invention relates to the kit, wherein the reagent for detection is a probe and / or primer having a base sequence complementary to the gene for detecting mRNA that is an expression product of the Sox2 gene.
  • the reagent for detection is Sox2, Smcp, Ints1, Kox12, Mdf1, FLJ13464, 667J232, Surf6, Pcdh19, Dchs2, Pcdh21, Gal3st1, Ras11b, Hes6, Znf415, Ncx5,
  • the present invention relates to the above kit, which is an antibody for detecting a polypeptide that is an expression product of one or more genes selected from the group consisting of Pnmt, Scgb3a1, Or7c1 and Dnajb8.
  • the present invention also relates to the aforementioned kit, wherein the reagent for detection is an antibody for detecting a polypeptide that is an expression product of the Sox2 gene.
  • the present invention further relates to a method for determining cancer using the kit.
  • the present invention is also a polypeptide that can be used as an antigen for inhibiting the function of cancer stem cells or killing cancer stem cells, and is Sox2, Smcp, Ints1, Kox12, Mdf1, FLJ13464, 667J232, Surf6.
  • polypeptide is a polypeptide in which several amino acids are deleted, substituted or added.
  • the present invention also includes Sox2, Smcp, Ints1, Kox12, Mdf1, FLJ13464, 667J232, Surf6, Pcdh19, Dchs2, Pcdh21, Gal3st1, Ras11b, Hes6, Znf415, Nkx2-5, Pamci, G7
  • the present invention relates to an antibody that specifically reacts with an epitope derived from the expression product of one or more genes selected from the group.
  • the present invention relates to a pharmaceutical composition comprising at least one of the aforementioned polypeptides and / or the aforementioned antibodies.
  • this invention relates to the said pharmaceutical composition whose pharmaceutical composition is a cancer therapeutic agent.
  • the present invention also relates to a method for inhibiting the function of cancer stem cells or killing cancer stem cells using the polypeptide as an antigen.
  • the present invention further relates to the above method using the above antibody.
  • the present invention further provides i) a step of producing a target polypeptide fragmented into peptides of appropriate length, ii) culturing together the fragmented peptide obtained in i), the antigen-presenting cell and the T cell sensitized with the target polypeptide and / or the fragmented peptide obtained in i), iii) measuring the degree of activation of the T cells obtained in ii), iv) selecting a fragmented peptide that activated T cells in iii), A method for screening a peptide presented as an antigen on a major histocompatibility antigen, wherein the polypeptide of interest is the aforementioned polypeptide.
  • the present invention provides Sox2, Smcp, Ints1, Kox12, Mdf1, FLJ13464, 667J232, Surf6, Pcdh19, Dchs2, Pcdh21, Gal3st1, Ras11b, Hes6, Znf415, Nkx2-5a, mk1-5g, , A nucleic acid used to suppress the expression of a gene selected from the group consisting of Or7c1 and Dnajb8. Furthermore, the present invention relates to the nucleic acid as described above, wherein the nucleic acid is DNA and / or RNA. Furthermore, the present invention relates to a pharmaceutical composition comprising at least one of the above nucleic acids. The present invention also relates to the aforementioned pharmaceutical composition, wherein the pharmaceutical composition is a cancer therapeutic agent. The present invention further relates to a method for inhibiting the function of cancer stem cells using the nucleic acid.
  • the molecular marker provided by the present invention can discriminate between cancer stem cells contained in cancer cells and cancer cells that are not cancer stem cells. Furthermore, since the discrimination can be performed by detecting a single molecular marker, cancer stem cells can be discriminated extremely easily. Moreover, since the molecular marker of the present invention can be used in common for a plurality of cancer stem cells such as lung cancer cells, breast cancer cells, and colon cancer cells, it is extremely effective as a molecular marker for general-purpose cancer stem cells. . Furthermore, since the molecular marker of the present invention is not detected at all or is hardly detected in normal cells, it is useful for discriminating cancer stem cells and further for discriminating tumor tissues containing cancer stem cells. In addition, the molecular marker of the present invention is highly related to tumorigenicity, and cancer stem cell immunotherapy, molecular target therapy, gene transfer therapy, drugs that can be used for cancer therapy, peptides targeting them It is also useful for screening.
  • FIG. 1 is a diagram showing SP analysis results in a lung cancer cell line. The part surrounded by a line is each SP fraction, and% indicates the ratio of the SP fraction cells to the total cells.
  • FIG. 2 is a diagram showing the results of a tumorigenicity experiment in NOD / SCID mice. a) A photograph of a mouse 8 weeks after transplantation. b) is a table showing the mean value of tumor diameter ⁇ standard deviation of all mice transplanted with LHK2 (lung cancer cells), the number of mice engrafted with tumor / the total number of transplanted mice.
  • FIG. 3a shows the expression of Sox2 in the SP fraction and MP fraction of a cancer cell cell line, in which human adult normal cells and b).
  • FIG. 4a) shows the expression of Smcp and FLJ13464 in the SP fraction and the MP fraction of a cancer cell line, in which human adult normal cells and b).
  • FIG. 5 is a diagram showing a list of the expression of the investigated genes in each cell.
  • FIG. 6 is a diagram showing the expression of Sox2 in other cancer cell lines.
  • FIG. 7 is a view showing the expression of Smcp in another cancer cell line. The results are shown with the PCR cycle set to 35 cycles and 40 cycles, respectively.
  • Caki-1, ACHN, SMKTR-1 to 4 are renal cell carcinoma cell lines
  • Sq-1 is a lung cancer squamous cell carcinoma cell line
  • 1-87, A549, and LHK2 are lung cancer glands.
  • LC817 is a cell line for small cell lung cancer
  • 86-2 and Lu99 are cell lines for large cell lung cancer
  • HPC3 is a cell line for pancreatic cancer
  • LCC ( Mouse) is a mouse lung cancer cell.
  • FIG. 8a) shows the expression of Ints1 in the SP and MP fractions of normal cells and cancer cell lines SW480, KM12, LHK2, and MCF7.
  • b) is a diagram showing expression of Ints1 in a cancer cell line.
  • FIG. 9 shows the results of immunohistochemical staining in lung cancer tissue.
  • Case 1 and Case 2 are stained images of lung squamous cell carcinoma tissues derived from different cancer patients. The blue part represents the nucleus stained by hematoxylin staining, and the brown part is the SOX2 antigen protein stained with anti-SOX2 antibody.
  • FIG. 10 shows the results of immunohistochemical staining in breast cancer tissue. Case 1 is a cytoplasmic pattern, Case 2 is a stained image of a nuclear pattern, CK5 is a stained image stained with an anti-CK5 antibody, and SOX2 is a stained image stained with an anti-SOX2 antibody.
  • FIG. 11 shows the results of immunohistochemical staining in a) the brain, b) the lung, c) the pancreas, and d) the stomach.
  • the blue part represents the nucleus stained by hematoxylin staining, and the brown part is the SOX2 antigen protein stained with anti-SOX2 antibody.
  • the part indicated by the black arrow indicates cells slightly stained with the SOX2 antibody.
  • FIG. 12 is a graph observing changes in tumorigenic ability due to forced expression of SOX2.
  • the photograph on the left is a photograph of a mouse 9 weeks after tumor implantation.
  • the graph on the right is a graph showing the average value of the tumor size over 9 weeks in the group transplanted with 10000 cells.
  • black squares represent LHK2 cells forcibly expressing SOX2, and black diamonds represent mock-transfected LHK2 cells.
  • FIG. 13 is a graph showing patient survival rates in SOX2-positive breast cancer patients and SOX2-negative breast cancer patients.
  • FIG. 14a) shows the results of examining the expression level of the SOX2 gene in the cells into which the sh-SOX2 gene was introduced and in the cells into which the sh-EGFP was introduced by the RT-PCR method.
  • b) is a graph showing the cell viability for each cisplatin concentration when each cell was cultured in a cisplatin-containing medium for 24 hours.
  • 15a) is a schematic diagram of a plasmid used for SMCP gene transfer and a schematic diagram of a transplanted mouse
  • b) is a graph showing transplantation of LHK2 cells introduced with an SMCP expression gene and mock-transfected LHK2 cells. It is a photograph of a mouse after a week.
  • c) is a graph in which the size of a tumor in each week was measured after transplanting 1000 and 10,000 LHK2 cells into which a SMCP gene had been introduced and mock-transfected LHK2 cells, respectively.
  • black diamonds represent LHK2 cells forcibly expressing the SMCP gene
  • black squares represent mock-transfected LHK2 cells.
  • 16a shows the expression level of the SMCP gene in cells into which siRNAs against two types of SMCP were introduced, and cells into which "invitrogen Stealth RNAi TM siRNA Negative Control Hi GC Catalog No. 12935-400" was introduced as a negative control. It is the result investigated by RT-PCR method.
  • b) is a graph showing changes in the number of cells when each cell is cultured under the same conditions. In the graph, black squares represent negative control LHK2 cells, black triangles represent LHK2 cells into which SMCP1 has been introduced, and black circles represent LHK2 cells into which SMCP3 has been introduced.
  • FIG. 17 shows transplantation of 200000 cells transfected with 2 types of SMCP cells and cells introduced with “Invitrogen Stealth RNAi TM siRNA Negative Control Hi GC Catalog No. 12935-400” as a negative control into NOD / SCID mice. It is the graph which measured the magnitude
  • black squares represent negative control LHK2 cells
  • black triangles represent LHK2 cells into which SMCP1 has been introduced
  • the molecular marker of the present invention is a molecular marker for detecting cancer stem cells in a cell population to be detected, and is detected in cancer stem cells contained in the detection target, but is not cancer stem cells. And not detected in normal cells. Therefore, it is typically used to detect cancer stem cells from a cancer cell population. Furthermore, for example, when a cell population to be detected is collected, normal cells may be mixed into a cancer cell population that is a mixture of cancer stem cells, cancer cells that are not cancer stem cells, and normal cells. Even in such a case, cancer stem cells can be accurately detected.
  • the “cancer stem cell” in the present invention is a cell having the properties of a stem cell among cancer cells.
  • Stem cells refer to cells that maintain their differentiation potential even after cell division.
  • cancer stem cells are stained with Hoechst fluorescent dye (Hoechst 33342) and detected using a UV laser (wavelength of about 350 nm) as excitation light using flow cytometry, they are concentrated in the Side Population (SP) fraction.
  • the SP fraction refers to a fraction that is not stained by discharging the dye out of the cell via an ABC transporter or the like with respect to the Main Population (MP) fraction that is stained with Hoechst fluorescent dye.
  • the “normal cell” in the present invention refers to a cell having a normal function in the activity of a living body or tissue. Normal cells may include somatic stem cells, but are preferably mature cells.
  • the molecular marker of the present invention is preferably detected in common to cancer stem cells of a plurality of cancer types, and can be used as a single marker in a plurality of cancer types.
  • cells such as heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, leukocyte, colon, stomach, bone marrow, colon and peripheral blood mononuclear cells or Since it is commonly expressed in cancer stem cells of multiple cancer types derived from tissues, it can be detected.
  • the molecular marker of the present invention is not detected in cancer cells and normal cells that are not cancer stem cells.
  • cancer stem cells Preferably, for example, heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, white blood cell, colon, stomach, bone marrow, large intestine and peripheral blood mononuclear cells, etc.
  • the molecular marker of the present invention can be expressed or detected in cancer stem cells but cannot be expressed or detected in normal cells, cancer stem cells can be detected in a cell population arbitrarily collected in cells or tissues containing cancer stem cells. It becomes possible.
  • a general-purpose molecular marker it is preferable that it can be used as a single marker in a plurality of cancer types, and typically, heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas , Spleen, thymus, prostate, testis, ovary, small intestine, leukocytes, colon, stomach, bone marrow, large intestine and peripheral blood are not detected in normal cells in at least two or more cells, preferably at least Not detected in more than two. Most preferably it is not detected in normal cells in all cells or tissues.
  • gene expression refers to a series of biological reactions starting from transcription of the gene
  • expression product refers to a series of biological reactions such as mRNA and endogenous polypeptide.
  • “expressed” means that an expression product can be confirmed by methods known to those skilled in the art, such as RT-PCR, in-situ hybridization, immunoassay, chromatography, and the like. Further, “not detected” means that an expression product cannot be confirmed by the method for confirming an expression product.
  • the expression product of the gene detected in the present invention is preferably an expression product of a gene having a known sequence, but may be a homologue thereof.
  • the expression product of a gene having the following mRNA or cDNA sequence is preferable.
  • NM_001080453 Kox12 Gene accession no. NM_152907 Mdf1: Gene accession no. NM_005586 FLJ13464: Gene accession no. AK023526 667J232: Gene accession no. AL83332 Surf6: Gene accession no. NM_006753 Pcdh19: Gene accession no. NM_001105243 Dchs2: Gene accession no. NM_017639 Pcdh21: Gene accession no. NM_033100 Gal3st1: Gene accession no. NM_004861 Rasllb: Gene accession no. NM_023940 Hes6: Gene accession no. NM_018645 Znf415: Gene accession no.
  • NM_018355 Nkx2-5 Gene accession no. NM_004387 Pamci: Gene accession no. NM_005447 Pnmt: Gene accession no. NM_002686 Scgb3a1: Gene accession no. NM_052863 Or7c1: Gene accession no. NM_198944 Dnajb8: Gene accession no.
  • the determination target in the present invention is preferably human, human-derived tissue and / or human-derived cell, but non-human animals (for example, rodents such as mice, rats, guinea pigs, hamsters, primates such as chimpanzees, It may be an artiodactyl such as a cow, a goat or a sheep, an ostracod such as a horse, a rabbit, a dog, a cat, etc.), a tissue derived from the animal, and / or a cell derived from the animal.
  • the determination is made when there is a cancer stem cell when the molecular marker of the present invention is detected, and the determination target may include normal cells and / or cancer cells that are not cancer stem cells.
  • Sox2 preferably Sox2, Smcp, Ints1, Kox12, Mdf1, FLJ13464, 667J232, Surf6, Pcdh19, Dchs2, Pcdh21, Gal3st1, Ras11b, Hes6, Znf415, -5, Pamci, Pnmt, Scgb3a1, Or7c1, and Dnajb8, most preferably Sox2 expression products are detected and determined.
  • the molecular marker of the present invention is mRNA
  • a reagent that specifically binds to mRNA such as a probe or primer is used for the detection.
  • detection is preferably performed by RT-PCR.
  • a reagent that specifically binds to a peptide such as an antibody or a ligand is used for the detection.
  • a polyclonal antibody and / or a monoclonal antibody can be used as the antibody.
  • the determination may be an in vivo or in vitro determination.
  • in vitro determination refers to determination of tissue or cells collected from a living body after growth in an in vitro environment such as a culture solution.
  • in vivo determination means determination directly in vivo, or determination immediately after immobilization of tissue or cells from a living body or after immobilization.
  • the collection of tissue or cells is not limited to this, but is performed by, for example, incision, cell aspiration, blood collection, urine collection, or the like.
  • the determination may be performed using in vivo detection methods known to those skilled in the art.
  • the determination in vivo may be performed using a known detection method such as blood test, in situ hybridization, in situ PCR, immunohistochemical staining, and the like.
  • the determination may be performed using in vitro detection methods known to those skilled in the art, such as, but not limited to, immunohistochemical staining and RT-PCR.
  • the number of cycles is preferably 30 to 35 cycles. Determination in tumor tissue after tissue culture is also included in in vitro determination. Since the molecular marker of the present invention is specifically expressed in cancer stem cells, the detected amount is presumed to be correlated with the number of cancer stem cells. Therefore, if the detection amount of the molecular marker of the present invention decreases before and after administration of the candidate compound to the subject, it is considered that the number of cancer stem cells has decreased.
  • the present invention also includes a kit containing a reagent for detecting the molecular marker.
  • the kit contains reagents that specifically detect mRNA, such as probes and primers for detecting mRNA, and specifically detects ligands, antibodies, and other polypeptides to detect polypeptides. Reagents to be included.
  • the kit can include at least one or more of the reagents.
  • the kit may contain additional reagents suitable for the mode of use, such as a reaction buffer and a reaction accelerator.
  • the gene sequence is preferably Sox2, Smcp, Ints1, Kox12, Mdf1, FLJ13464, 667J232, Surf6, Pcdh19, Dchs2, Pcdh21, Gal3st1, Ras11b, Hes11, Reagents for detecting the gene product of Znf415, Nkx2-5, Pamci, Pnmt, Scgb3a1, Or7c1, or Dnajb8, most preferably Sox2, are included.
  • the “probe and / or primer having a base sequence complementary to a gene” in the present invention is DNA or RNA having a complementary sequence so as to specifically bind to a partial sequence of the gene sequence.
  • polypeptide that functions as a molecular marker of the present invention can be used as an antigen for inhibiting or killing the function of cancer stem cells.
  • These polypeptides only need to have a functional site such as an antibody recognition site or a protein binding site, and may have a length of, for example, about 9 to 11 amino acids.
  • the functional site may appear after some modification or deformation It is only necessary to have this functional site structure.
  • These polypeptides are preferably Sox2, Smcp, Ints1, Kox12, Mdf1, FLJ13464, 667J232, Surf6, Pcdh19, Dchs2, Pcdh21, Gal3st1, Ras11b, Hes6, Znf415, Nkx2-5nmc, And an expression product of a gene selected from the group consisting of Dnajb8.
  • Methods for killing cancer stem cells using these polypeptides as antigens are not limited to these, but include, for example, a method based on CTL induction, a method of causing an immune reaction using a polypeptide-specific antibody, and a binding with a polypeptide.
  • a method based on CTL induction a method of causing an immune reaction using a polypeptide-specific antibody, and a binding with a polypeptide.
  • the epitope derived from the expression product of the gene of the present invention is bound to an antibody that specifically reacts with the epitope
  • the immune reaction is activated using the bound antibody as a signal. Since the epitope is a molecular marker that is specifically expressed in cancer stem cells or a part of the structure thereof, the specific antibody binds to the epitope of the immune cascade that specifically recognizes cancer stem cells. It is strongly suggested that activation can occur.
  • the antibody used for the said method and the pharmaceutical composition containing polypeptide and / or an antibody are also included by this invention.
  • the pharmaceutical composition include, but are not limited to, cancer vaccines and anticancer agents.
  • these compositions can appropriately contain, for example, a drug having an antitumor action, an adjuvant, a pharmaceutically acceptable carrier and the like as necessary. .
  • fragments of the polypeptide that is the molecular marker of the present invention there are those that are presented as an antigen by a protein called a major histocompatibility antigen (MHC molecule).
  • MHC molecule major histocompatibility antigen
  • CTLs Cytotoxic T cells
  • HLA-A24 HLA-A24
  • polypeptide of the present invention can be applied to a method for specifically treating cancer stem cells. Since the molecular marker of the present invention is specifically expressed in cancer stem cells, it is suggested that some of the polypeptides are presented as antigens by MHC molecules. Screening a polypeptide (epitope peptide) presented as an antigen on this MHC molecule is useful in the treatment of cancer. Therefore, such a screening method is also included in the present invention. A normal epitope mapping technique can be applied to the screening.
  • the target polypeptide and / or fragmented peptide are cultured together to sense T cells. Make. Thereafter, the sensitized T cells, antigen presenting cells and fragmented peptides are cultured together to re-stimulate the T cells. Thereafter, the degree of activation of T cells is measured, peptides having a high degree of activation are selected, and the epitope sequence is determined. At this time, the degree of activation can be appropriately selected from methods well known to those skilled in the art, such as measurement of cytotoxic activity and cytokine production.
  • an antigenic peptide restricted by a specific HLA type is selected.
  • the DNA that functions as the molecular marker of the present invention is DNA that is specifically expressed in cancer stem cells, and it is considered that the functions of cancer stem cells can be suppressed by suppressing the expression. Therefore, a nucleic acid for suppressing the expression of DNA that functions as the molecular marker of the present invention is also included in the present invention. Examples of methods for suppressing expression include, but are not limited to, RNAi and repressor expression. Thus, nucleic acids for suppressing DNA expression include, but are not limited to, siRNA, shRNA, shRNA, and the like.
  • the nucleic acid may have any length as long as it has a sufficient number of bases to suppress the expression of DNA.
  • the nucleic acid may be a nucleic acid analog in addition to DNA and RNA, but is preferably DNA and / or RNA from the viewpoint of versatility. This suggests the possibility of specifically and / or efficiently attacking cancer stem cells by suppressing the expression of DNA that functions as the molecular marker of the present invention. That is, it is suggested that the above nucleic acid can be applied to gene transfer therapy. Accordingly, a pharmaceutical composition containing the nucleic acid of the present invention is also encompassed by the present invention.
  • said pharmaceutical composition is not limited to this, For example, it can be used as cancer therapeutic agents, such as an anticancer agent, a metastasis inhibitor, and a cancer vaccine.
  • these pharmaceutical compositions can appropriately contain, for example, a drug having an antitumor action, an adjuvant, and a pharmaceutically acceptable carrier as necessary.
  • a method for suppressing the expression of DNA functioning as the molecular marker of the present invention in cancer stem cells using the nucleic acid of the present invention is also encompassed by the present invention.
  • the following experimental examples explain the present invention more specifically, and do not limit the scope of the present invention. Those skilled in the art having ordinary knowledge and techniques can make various modifications to the embodiments shown in the following experimental examples without departing from the spirit of the present invention. include.
  • 5% FCS + DMEM was added to adjust the cell concentration to 1 ⁇ 10 6 cells / ml, and 1 ml was collected in a falcon tube for verapamil (+).
  • Verapamil (+) and the remaining cells were incubated in a water bath at 37 ° C. for 10 minutes. After the incubation, a verapamil solution is added so that the final concentration of verapamil is 50 to 75 ⁇ M for verapamil (+), and then the final concentration of Hoechst 33342 is 2.5 ⁇ M for verapamil (+) and verapamil ( ⁇ ).
  • the Hoechst 33342 solution was added to a concentration of ⁇ 5.0 ⁇ M.
  • the SP fraction When confirmed, the SP fraction is gated, and verapamil (+) cells are flowed to see the SP fraction cells. I confirmed that it disappeared. If it disappeared, it was determined that the cells of the fraction were SP fraction cells, and the cells of the fraction were isolated. The isolated cells were centrifuged at 1500 rpm for 15 minutes at 4 ° C., the supernatant was removed, and then suspended in 100 to 200 ⁇ l of 1 ⁇ PBS, and the number of cells was counted. As a result, SP fraction cells were detected in adenocarcinoma A549, LHK2, and small cell carcinoma Lc817. The result is shown in FIG.
  • mRNA extracted from the SP fraction was amplified using the RNA amplification kit obtained from Sigma Genosys, and further amplified using the mRNA labeling kit obtained from Sigma Genosys. Cy5 was labeled on the extracted one, Cy3 was labeled on the extracted MP fraction, Cy3 was labeled on the SP fraction after exchanging the dye, and Cy5 was labeled on the extracted MP fraction.
  • Hybridization was performed as described above, and mRNA expression was analyzed.
  • Table 1 shows a list of primers used in RT-PCR in the experimental examples of primers used in RT-PCR.
  • b) Expression of Sox2 in Normal Cells In order to confirm the usefulness of Sox2 confirmed in Experimental Example 3c), the expression was examined in human adult normal cells.
  • a human adult normal tissue-derived mRNA panel was obtained from Clontech and RT-PCR was performed using it.
  • mRNA panel is derived from adult normal cells and tissues of heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, large intestine, peripheral blood mononuclear cells Contains mRNA.
  • cDNA was synthesized from mRNA using SuperScript TM III reverse transcriptase (Invitrogen) according to the protocol of the kit.
  • the synthesized cDNA was amplified by RT-PCR using forward and reverse primers (see Table 1).
  • G3PDH cDNA was amplified in the same manner.
  • the PCR conditions are shown in Table 2.
  • the amplified product was subjected to electrophoresis at 100 V for 25 minutes using a 1.5% agarose gel. The results are shown in Fig. 3a).
  • Sox2 is a marker that does not recognize normal cells in the tissue, and suggests the possibility of treating tumor cells specifically by using Sox2.
  • all strains were strongly expressed in the SP fraction cells, and almost no expression was observed in the MP fraction cells.
  • Sox2 can be used as a marker specific to cells of the SP fraction, that is, cancer stem cells.
  • Experimental Example 5 Expression of Smcp and FLJ13464 Regarding Smcp and FLJ13464 confirmed in Experimental Example 3c), the expression was examined in human adult normal tissues and other cancer cells in order to confirm the usefulness.
  • Human adult normal tissues obtained from Clontech include humans including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, white blood cell, colon, stomach, bone marrow A normal tissue mRNA panel was used, and lung cancer cell line LHK2, colon cancer cell line SW480, KM12LM, breast cancer cell line MCF7, and malignant fibrous histiocytoma cell line MFH03 were used as cancer cells.
  • CDNA was synthesized from mRNA of each cell and tissue in the same manner as in Experimental Example 4b).
  • the synthesized cDNA was amplified using the Smcp forward primer and reverse primer (see Table 1), and the FLJ13464 forward primer and reverse primer (see Table 1) under the PCR conditions shown in Table 2. did.
  • G3PDH cDNA was amplified in the same manner as described above.
  • the amplified product was subjected to electrophoresis at 100 V for 25 minutes using a 1.5% agarose gel. The results are shown in FIG.
  • Experimental Example 7 Expression in other cancer cell cell lines of Sox2
  • 14 types of lung cancer cell line, renal cells were In the same manner as in Experimental Example 4, the expression of Sox2 was confirmed in 3 types of cancer cell lines, 1 type of prostate cancer cell line, and HeLa cells of the uterine cancer cell line. Fetal brain cells were used as a positive control, and double distilled water was used as a negative control. The results are shown in FIG. Discussion Sox2 expression was confirmed in all cancer cell lines examined . This indicates that Sox2 functions as a cancer stem cell molecular marker for a much wider variety of cancer cell cell lines than other currently known cancer stem cell molecular markers.
  • Experimental Example 8 Expression in other cancer cells of Smcp Similar to Experimental Example 5, it was examined in detail whether Smcp was expressed in other cancer cell lines. The expression of Smcp was confirmed in the same manner as in Experimental Example 5 in 6 types of renal cell cancer cell lines, 7 types of lung cancer cell cell lines, and 1 type of prostate cancer cell cell line as cancer cell cell lines. Testicular cells were used as a positive control, and mouse lung cancer cells were used as a negative control. FIG. 7 shows the results of the PCR performed with 35 and 40 cycles. Discussion Smcp expression was confirmed in all cancer cell lines examined.
  • Ints1 was not confirmed in normal cells except that very little expression was observed in the pancreas and spleen. This result indicates that Ints1 is a marker that does not recognize normal cells in tissues in almost all tissues, depending on the sensitivity of detection in tissues where expression is not observed in normal cells, or even in the pancreas and spleen seen. Suggests the possibility of treatment specific to tumor cells. Similarly to the results of Experimental Example 4, SW480, KM12, LHK2, and MCF7 are also expressed almost exclusively in SP cells, suggesting that they are useful as markers for cancer stem cells. In other cancer cell lines, expression was confirmed in most cancer cell lines except for a part.
  • the section was immersed in a mixed solution of hydrogen peroxide solution and DAB substrate (Simple Stain MAX-PO, manufactured by Nichirei Co., Ltd.) to cause a color reaction for 1 to 2 minutes. Thereafter, the sections were washed with running water for 1 minute, and then stained with hematoxylin for 1-2 minutes. The results are shown in FIG. Discussion
  • SOX2 antigen protein is stained brown. Some of the cells were stained dark brown, and the cells corresponding to the advanced tumor were stained lightly in a band shape. This result shows that it is possible to clarify the form, number and position of cancer stem cells in tissues in lung cancer by immunostaining with anti-SOX2 antibody.
  • Experimental Example 11 Immunohistochemical staining of breast cancer tissue using anti-SOX2 antibody Similar to Experimental Example 10, immunohistochemical staining was performed on Basaloid type breast cancer tissue. As a comparative example, staining of CK (cytokeratin) 5 which is a marker of basal cells was also performed. The results are shown in FIG. SOX2 antigen protein was also stained brown and observed in breast cancer tissue. This indicates that SOX2 can clarify the form, number and location of cancer stem cells even in breast cancer. Basaloid type breast cancer generally has a poor prognosis and is recognized as having a high metastasis rate, suggesting that SOX2 may be involved in this.
  • CK cytokeratin
  • Experimental Example 12 Immunohistochemical staining of normal tissue using anti-SOX2 antibody Immunohistological staining was performed on normal tissues of the brain, lung, stomach, and pancreas in the same manner as Experimental Example 10. The results are shown in FIG. A very dense image of the nucleus was observed in a small part of the stomach, but expression was not observed in other tissues. This indicates that SOX2 is hardly expressed in normal tissues.
  • the LHK2 cell line was transfected with sh-SOX2 plasmid and sh-EGFP plasmid as a negative control, and cultured in DMEM medium containing cisplatin at concentrations of 0, 10, 30, and 100 ⁇ M, respectively, at 37 ° C. for 24 hours. Thereafter, cell viability was measured by the MTT method. The results are shown in FIG. Discussion In cells into which sh-SOX2 was introduced, the expression of Sox2 gene was remarkably suppressed. Furthermore, the survival rate of the cells in which the expression of the Sox2 gene was suppressed was significantly lower in the culture in the cisplatin-containing medium than in the cells that were not suppressed.
  • SMCP expression suppression si-RNA used was Stealth Select RNAi TM siRNA (Catalog # 1299003) (invitrogen).
  • SMCP1 refers to that of OligoID HSS142897
  • SMCP3 refers to that of OligoID HSS142899.
  • Stealth RNA TM siRNA Negative Control Hi GC 12935-400 was used. 100 pmol of Si-RNA and 4 ⁇ l of Lipofectamine RNAiMAX were mixed, cultured in DMEM culture medium at 37 ° C.
  • the molecular marker used in the discrimination method provided by the present invention is usually not expressed in normal adult cells, and is expressed in many cancer stem cells. By specifically recognizing only tumor cells, diagnosis with much higher accuracy than conventional cancer diagnostic techniques is possible. In particular, since cancer stem cells are specifically recognized, it can be expected to contribute widely to the medical industry, such as the development of cancer immunotherapy, molecular target therapy, and gene transfer therapy.

Abstract

La présente invention concerne un marqueur moléculaire permettant la détection d’une cellule souche cancéreuse dans une population cellulaire  d’intérêt, qui peut être détecté dans une cellule souche cancéreuse continue dans la population cellulaire d’intérêt mais qui ne peut pas être détecté dans une cellule normale ou dans une cellule cancéreuse  qui n’est pas une cellule souche cancéreuse. L’invention concerne également un procédé permettant de déterminer la présence ou l’absence d’une cellule souche cancéreuse dans une population cellulaire d’intérêt par l’utilisation du marqueur moléculaire comme indicateur; un kit permettant de déterminer la présence ou l’absence d’une cellule souche cancéreuse, qui comprend au moins un réactif pour détecter le marqueur moléculaire; un polypeptide codé par le marqueur moléculaire; un anticorps qui peut reconnaître un épitope d’un produit d’expression d’un gène dérivé du marqueur moléculaire; et un acide nucléique capable d’inhiber l’expression du marqueur moléculaire.
PCT/JP2009/061154 2008-10-27 2009-06-12 Marqueur moléculaire de cellule souche cancéreuse WO2010050268A1 (fr)

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WO2015050259A1 (fr) 2013-10-03 2015-04-09 大日本住友製薬株式会社 Peptide d'antigène tumoral
WO2017115798A1 (fr) * 2015-12-28 2017-07-06 北海道公立大学法人 札幌医科大学 Peptide antigénique tumoral
US10765729B2 (en) 2014-12-09 2020-09-08 Sapporo Medical University Tumor antigen peptide

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WO2015050259A1 (fr) 2013-10-03 2015-04-09 大日本住友製薬株式会社 Peptide d'antigène tumoral
US10765729B2 (en) 2014-12-09 2020-09-08 Sapporo Medical University Tumor antigen peptide
WO2017115798A1 (fr) * 2015-12-28 2017-07-06 北海道公立大学法人 札幌医科大学 Peptide antigénique tumoral

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