WO2022025625A1 - Biomarker composition for detecting cancer stem cells - Google Patents

Biomarker composition for detecting cancer stem cells Download PDF

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WO2022025625A1
WO2022025625A1 PCT/KR2021/009800 KR2021009800W WO2022025625A1 WO 2022025625 A1 WO2022025625 A1 WO 2022025625A1 KR 2021009800 W KR2021009800 W KR 2021009800W WO 2022025625 A1 WO2022025625 A1 WO 2022025625A1
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cancer
cells
cancer stem
phosphatidylethanolamine
stem cells
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PCT/KR2021/009800
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French (fr)
Korean (ko)
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김재호
신민주
김대경
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부산대학교 산학협력단
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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
    • G01N33/57492—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 involving compounds localized on the membrane of tumor or cancer cells
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • 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

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  • the present invention relates to a biomarker for detecting cancer stem cells, and more particularly, a biomarker composition for detecting cancer stem cells using phosphatidylethanolamine (PE) expressed in cancer stem cells, a composition for cancer diagnosis, a cancer diagnosis kit and to a method of providing information on cancer diagnosis.
  • PE phosphatidylethanolamine
  • stem cells are essential for maintaining and regenerating the organs of the body.
  • Tumors also contain cancer stem cells, a heterogeneous population with stem cell-like properties.
  • the cancer stem cells are cells that form spheres using the self-renewal method, which is a characteristic of cancer stem cells, through floating culture, and are known to share strong cancer-forming ability and characteristics of normal stem cells compared to the original cell line.
  • cancer stem cells tend to increase as the tumor progresses to malignancy, and cancer stem cells are reported to cause cancer as well as resistance to existing cancer therapies and cause recurrence. Therefore, in order to remove cancer stem cells, it is important to recognize and target cells resistant to existing anticancer drugs.
  • cancer stem cells are known as the highest-level cells exhibiting these heterogeneous characteristics, and are closely related to anticancer drug resistance. Therefore, it is not easy to completely remove cancer stem cells despite various cell surface protein-targeted treatments. Most of the current cancer stem cell protein target biomarkers can have different expression frequencies depending on various surrounding environments, such as cell signal transduction or cell resistance acquisition. Therefore, it is important to find a cancer stem cell-specific cell surface target biomarker other than the existing protein target biomarker.
  • the present inventors completed the present invention by confirming that the expression of phosphatidylethanolamine in ovarian cancer cancer stem cells was significantly higher than that of ovarian cancer cell lines and other cell lines as a result of research to discover biomarkers for targeting cancer stem cells. did
  • an object of the present invention is to provide a biomarker composition for detecting cancer stem cells containing phosphatidylethanolamine (PE).
  • PE phosphatidylethanolamine
  • Another object of the present invention is to provide a composition for diagnosing cancer through detection of cancer stem cells, including an agent for measuring the expression of phosphaditylethanolamine, and a cancer diagnostic kit comprising the same.
  • Another object of the present invention is to provide a method for providing information on cancer diagnosis through cancer stem cell detection, comprising measuring the expression of phosphatidylethanolamine in a biological sample.
  • the present invention provides a biomarker composition for detecting cancer stem cells comprising phosphatidylethanolamine.
  • the present invention provides a composition for diagnosing cancer through cancer stem cell detection comprising an agent for measuring the expression of phosphatidylethanolamine.
  • the present invention also provides a cancer diagnosis kit through cancer stem cell detection comprising the composition for cancer diagnosis.
  • the present invention also provides a method for providing information on cancer diagnosis through cancer stem cell detection, comprising measuring the expression of phosphatidylethanolamine in a biological sample.
  • phosphatidylethanolamine a biomarker for cancer stem cell detection according to the present invention
  • FIG. 1 is a diagram showing the results of morphological analysis of ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells.
  • FIG. 2 is a diagram showing the results of confirming the expression of cancer stem cell markers in ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells through flow cytometry.
  • FIG. 3 is a diagram showing the results of analyzing the lipid expression of ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells through LC-MS.
  • FIG. 4 is a diagram showing the results of analysis of lipids with increased or decreased expression in A2780-SP cells, a cancer stem cell line, compared to A2780-AD, an ovarian cancer cell line, based on the results of FIG. 3 .
  • FIG. 5 is a diagram showing the results of comparing the expression of phosphatidylethanolamine in ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells.
  • FIG. 6 is a diagram showing the results of analyzing the lipid expression of cancer stem cell line A2780-DIF cells and cancer stem cell line A2780-SP cells that have been cancerousized through LC-MS.
  • A2780-DIF cells which are cancer stem cell lines, which have become cancerous compared to A2780-SP cells, which are cancer stem cell lines.
  • FIG. 8 is a diagram showing the results of comparing the expression of phosphatidylethanolamine in cancer stem cell line A2780-DIF cells and cancer stem cell line A2780-SP cells transformed into cancer cells.
  • EOC-SP cells which are patient-derived ovarian cancer stem cells
  • EOC-DIF cells which are patient-derived ovarian cancer stem cells, which have become cancerous.
  • FIG. 10 is a diagram showing the results of confirming the expression of cancer stem cell markers in EOC-SP cells and EOC-DIF cells.
  • FIG. 11 is a diagram showing the results of analyzing the lipid expression of EOC-SP cells and EOC-DIF cells.
  • EOC-DIF cells which are cancer stem cell lines, which have become cancerous compared to EOC-SP cells, which are patient-derived ovarian cancer cancer stem cell lines, based on the results of FIG. 11 . is the diagram shown.
  • FIG. 13 is a diagram showing the results of comparing the expression of phosphatidylethanolamine in EOC-SP cells and EOC-DIF cells.
  • FIG. 14 is a diagram showing the results of confirming whether phosphatidylethanolamine is exposed to the outside of the cell membrane in ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells through flow cytometry.
  • FIG. 15 is a diagram showing the results of confirming whether phosphatidylethanolamine is exposed to the outside of the cell membrane in ovarian cancer cell line SKOV3-AD cells and ovarian cancer stem cell line SKOV3-SP cells through flow cytometry.
  • 16 is a diagram showing the results of confirming whether phosphatidylethanolamine is exposed to the outside of the cell membrane in the patient-derived cancer stem cell lines EOC12-SP cells and EOC21-SP cells through flow cytometry.
  • 17 is a diagram showing the results of confirming the expression of phosphatidylethanolamine and external exposure to the cell membrane in human embryonic kidney cells, tonsil-derived mesenchymal stem cells, and human umbilical vein endothelial cells through flow cytometry.
  • a biomarker composition for detecting cancer stem cells comprising phosphatidylethanolamine (PE).
  • PE phosphatidylethanolamine
  • phosphatidylethanolamine is one of phospholipids, and belongs to glycerophospholipids.
  • the glycerophospholipid is a form in which two fatty acids are bonded to glycerol 3-phosphate, and phosphatidylethanolamine is a phosphoric acid group in which ethanolamine is bonded.
  • Such phosphoethanolamine is known to be involved in membrane fusion during cell division, decomposition of contractile rings, and control of membrane curvature.
  • the phosphatidylethanolamine is generally expressed in the inner membrane of the phospholipid bilayer of the cell.
  • cancer stem cell (Cancer stem cell or Tumor initiating cell) refers to a cell having the ability to generate a tumor. Cancer stem cells have characteristics similar to normal stem cells, and they generate tumors through the self-renewal and differentiation abilities of stem cells. In addition, the tumor is differentiated from other populations and causes recurrence and metastasis by generating new tumors. Therefore, the development of a specific treatment method targeting cancer stem cells can increase the survival rate of cancer patients.
  • cancer is defined as an aggressive characteristic in which cells divide and grow ignoring normal growth limits, an invasive characteristic that penetrates into surrounding tissues, and metastatic that spreads to other parts of the body ) is a generic term for diseases caused by cells with characteristics.
  • gastric cancer gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, Skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer (anal cancer), colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, small intestine cancer, endocrine cancer, Thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchogenic cancer and bone marrow cancer It is preferably at least one selected from the group consisting of (bone marrow tumor), and more preferably ovarian cancer.
  • a “biomarker” is an index that can objectively measure and evaluate changes in a living body, and can measure and evaluate an individual's pathological state, response to drugs, and the like. Accordingly, the biomarker may also be used for detection or diagnosis.
  • the phosphatidylethanolamine is preferably expressed in non-cancer cells and cancer stem cells than in cancer cells.
  • non-cancer cells may include, but are not limited to, human embryonic-derived kidney cells, tonsil-derived mesenchymal stem cells, and human umbilical vein endothelial cells.
  • phosphatidylethanolamine is expressed on the surface of cancer stem cells and is overexpressed in cancer stem cells compared to non-cancer cells and cancer cells, which is a cancer stem cell detection or cancer diagnosis bio It can be used as a marker.
  • composition for diagnosing cancer through detection of cancer stem cells and a cancer diagnosis kit comprising an agent for measuring the expression of phosphatidylethanolamine.
  • diagnosis means confirming the presence or characteristics of a pathological condition.
  • the diagnosis is meant to include not only whether or not cancer occurs, but also confirmation of prognosis, progress, and stage.
  • diagnosis means identifying the onset, prognosis, course, staging or characteristics of cancer.
  • the “agent for measuring the expression of phosphatidylethanolamine” specifically binds to phosphatidylethanolamine, a biomarker whose expression is increased in cancer stem cells, as described above, and confirms the expression level of the biomarker. It means that it can be used for detection.
  • the agent for measuring the expression of phosphatidylethanolamine is an oligopeptide that specifically binds to phosphatidylethanolamine, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, PNA (Peptide nucleic acid), an aptamer (aptamer), preferably selected from the group consisting of probes and dyes, but is not limited thereto.
  • the agent for measuring the expression of phosphatidylethanolamine may include duramycin or cinnamycin.
  • the kit for diagnosis of cancer may be a kit in various types depending on the method used, for example, the kit is quantitative analysis for flow cytometry, mass spectrometry, light absorption spectrometry, chromatography and emission spectroscopy. It may be a kit, but is not limited thereto.
  • the quantitative analysis kit may obtain a result value through a quantitative analysis device.
  • the quantitative analysis device may be at least one selected from the group consisting of flow cytometry, nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, microplate reader and mass spectrometer, but is not limited thereto.
  • the cancer diagnostic kit may include not only an agent for measuring the expression level of the phosphatidylethanolamine, but also tools and reagents commonly used in the art used for immunological analysis and quantitative analysis.
  • the tool or reagent include, but are not limited to, a suitable carrier, a labeling material capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like.
  • the labeling material is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.
  • the carrier includes a soluble carrier and an insoluble carrier
  • an example of the soluble carrier is a physiologically acceptable buffer known in the art, for example, PBS
  • examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, poly It may be acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof.
  • kit of the present invention includes the above-described composition as a component, redundant descriptions are omitted in order to avoid excessive complexity of the present specification.
  • cancer stem cells that is, cells overexpressing phosphatidylethanolamine
  • a biological sample to determine the presence of cancer stem cells, and from this, the presence of cancer as well as However, the prognosis, course, and stage can be confirmed.
  • the present invention provides a method for providing information on cancer diagnosis through cancer stem cell detection, comprising measuring the expression of phosphatidylethanolamine in a biological sample.
  • biological sample means any sample obtained from an individual in which the expression of phosphatidylethanolamine of the present invention can be detected.
  • the biological sample is any one selected from the group consisting of whole blood, serum, plasma, cells, sputum, tissue, saliva, biopsy, liquid culture, feces and urine, and is not particularly limited thereto, and the present invention It can be prepared by processing by a method commonly used in the technical field of
  • the method determines the presence or absence of cancer stem cells by checking the expression level of phosphatidylethanolamine in a biological sample, thereby providing information on diagnosis of cancer.
  • the method comprises at least one quantitative analysis device selected from the group consisting of flow cytometry, nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, microplate reader and mass spectrometer. It may further comprise the step of confirming the expression of tylethanolamine.
  • the present invention comprises the steps of (a) obtaining an assay sample from a subject; (b) detecting the presence or absence of phosphatidylethanolamine (PE) in the analysis sample of step (a); (c) diagnosing cancer when phosphatidylethanolamine is present in the analyte sample in step (b); (d) administering an effective amount of a cancer stem cell-specific anticancer agent to the individual diagnosed with cancer in step (c); provides a cancer diagnosis or treatment method comprising a.
  • PE phosphatidylethanolamine
  • the cancer stem cell-specific anticancer agent is nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib , bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscumalbum , asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tusetan, heptaplatin, methylaminolevulinic acid, ams
  • Example 1 Comparison of lipid expression of ovarian cancer cells and ovarian cancer stem cells
  • Ovarian cancer cell line A2780 cells (A2780-AD) and ovarian cancer stem cell line A2780-SP cells were prepared.
  • the ovarian cancer cell line A2780-AD was cultured at 37° C. and 5% CO 2 using RPMI1640 medium (10% FBS, 100 ⁇ g/mL Sterptomycin/Penicillin), and subcultured at regular intervals.
  • the cancer stem cell line A2780-SP cells are obtained by three-dimensional culture of the ovarian cancer cell line A2780 cells, followed by isolation of cancer stem cells.
  • the three-dimensional culture was performed in an ultra-low attachent dish using Neurobasal Plus medium (B-27 supplement (50X), 100 ⁇ g/mL Sterptomycin/Penicillin, 100 ⁇ g/mL rhFGF, 100 ⁇ g/mL rhEGF, Glutamax, 1M HEPES). was performed in The three-dimensional culture conditions are a temperature of 37° C. and 5% CO 2 conditions.
  • Morphological analysis of the prepared A2780-AD cells and A2780-SP cells was performed. Specifically, the A2780-AD cells and A2780-SP cells were cultured under each of the above conditions. The cultured cells were observed under a microscope. The morphological analysis results are shown in FIG. 1 .
  • aldehyde dehydrogenase (ALDH), a marker of cancer stem cells, was confirmed in the prepared A2780-AD cells and A2780-SP cells. Specifically, using the ALDEFOUORTM kit (STEMCELL Technologies), ALDH-expressing cells among the prepared A2780-AD cells were labeled according to the manufacturer's manual. Thereafter, cells expressing ALDH were measured using FACS AttuneNxT (ThermoFisher). A2780-SP cells were also measured for ALDH expression in the same manner. The results of measuring cells expressing cancer stem cell markers are shown in FIG. 2 .
  • A2780-SP cells which is an ovarian cancer stem cell line
  • A2780-AD ovarian cancer cell line
  • A2780-AD cells Lipid expression of the prepared A2780-AD cells and A2780-SP cells was analyzed. Specifically, adherent A2780-AD cells were treated with trypsin to collect cell pellets. In addition, A2780-SP cells, which are floating cells, were spun down after recovering the culture medium to remove the medium to collect cell pellets. MeOH 610 ⁇ l, internal standards mix 50 ⁇ l, chloroform 330 ⁇ l and Cholesteryl ester standard (1 ng/ ⁇ l) 20 ⁇ l were added to the collected cell pellet. After that, after vortexing for 30 seconds every 3 minutes, spin down and sonication were performed, and repeated 3 times. The sonicated cells were centrifuged (4°C, 1 min, 14,000 g), and the supernatant was removed to obtain a pellet.
  • sample contained in a new tube was divided into samples for non-methylation and methylation analysis, and then dried.
  • TMSD Trimethylsilyldiazomethane
  • Lipids contained in the prepared sample were analyzed through liquid chromatography mass spectrometry (LC-MS).
  • LC-MS liquid chromatography mass spectrometry
  • lipids with increased or decreased expression in A2780-SP cells were analyzed compared to A2780-AD, an ovarian cancer cell line.
  • SigmaPlot10.0 software the LC-MS raw data was plotted to analyze lipids with increased or decreased expression. The results of lipid expression analysis are shown in FIG. 4 .
  • lipids with increased expression in the ovarian cancer stem cell line A2780-SP cells compared to the ovarian cancer cell line A2780-AD are phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), mono It was confirmed that there are 93 types including glyceride (monoglyceride), and the lipids with reduced expression are 61 types including phosphatidylcholine, lysophosphatidylcholine, and diglyceride.
  • PE phosphatidylethanolamine
  • LPE lysophosphatidylethanolamine
  • the ovarian cancer stem cell line A2780-SP cells were cultured under cancer cell culture conditions to form cancer cells.
  • the cancerous ovarian cancer stem cells were named A2780-DIF.
  • Lipid expression of the A2780-DIF cells and ovarian cancer stem cells (A2780-SP) was confirmed in the same manner as in Examples 1-4.
  • a matrix was prepared based on the analysis results, and the created matrix is shown in FIG. 6 .
  • A2780-SP cells and A2780-DIF cells had different lipid expression patterns.
  • the lipid expression pattern of A2780-DIF cells was somewhat different from that of the ovarian cancer cell line A2780-AD, but had a similar tendency.
  • lipids with increased or decreased expression in A2780-DIF compared to A2780-SP cells, a cancer stem cell line, were analyzed, and the results are shown in FIG. 7 .
  • lipids with reduced expression in the ovarian cancer cell line A2780-DIF compared to the ovarian cancer stem cell line A2780-SP cells are 87 types including phosphatidylethanolamine (PE), and the lipids with increased expression are It was confirmed that phosphatidylcholine (phosphatidylcholine), lysophosphatidylcholine (lysophosphatidylcholine), diglyceride (diglyceride), such as 84 kinds.
  • PE phosphatidylethanolamine
  • Example 2 Comparison of lipid expression of patient-derived ovarian cancer cancer stem cells and cancerousized patient-derived ovarian cancer cancer stem cells
  • Ovarian cancer stem cells were isolated from the patient and named as EOC-SP.
  • the EOC-SP cells were cultured under cancer cell culture conditions to form cancer cells.
  • the ovarian cancer stem cells derived from the cancerousized patient were named EOC-DIF.
  • the prepared EOC-SP cells and EOC-DIF cells were used in the experiments described below.
  • EOC-SP cells and EOC-DIF cells were prepared EOC-SP cells and EOC-DIF cells. Specifically, the cells were each three-dimensionally cultured and then observed under a microscope. The morphological analysis results are shown in FIG. 9 .
  • EOC-SP cells and EOC-DIF cells were morphologically different.
  • the EOC-DIF cells were cancer cells, and showed a morphology similar to that of cancer cells.
  • aldehyde dehydrogenase a marker of cancer stem cells
  • EOC-SP cells and EOC-DIF cells were confirmed in EOC-SP cells and EOC-DIF cells. Specifically, using the ALDEFOUORTM kit (STEMCELL Technologies), ALDH-expressing cells among EOC-SP cells were labeled according to the manufacturer's manual. Thereafter, cells expressing ALDH were measured using FACS AttuneNxT (ThermoFisher). EOC-DIF cells were also measured for ALDH expression in the same manner. The results of measuring cells expressing cancer stem cell markers are shown in FIG. 10 .
  • EOC-SP cells and EOC-DIF cells Lipid expression of EOC-SP cells and EOC-DIF cells was analyzed. Specifically, adherent EOC-DIF cells were treated with trypsin to collect cell pellets. EOC-SP cells, which are floating cells, were spun down after recovering the culture medium to remove the medium to collect cell pellets. The collected cells were analyzed for lipid expression in the same manner as in Examples 1-4. A matrix was prepared based on the analysis results, and the created matrix is shown in FIG. 11 .
  • EOC-SP cells and EOC-DIF cells had different lipid expression patterns.
  • Lipids with increased or decreased expression in EOC-DIF cells a patient-derived ovarian cancer stem cell line that have become cancerous compared to EOC-SP cells, were analyzed, and the results are shown in FIG. 12 .
  • lipids with increased expression in EOC-SP cells compared to EOC-DIF cells are 87 types, including phosphatidylethanolamine, phosphatidylcholine, and lysophosphatidylcholine, and lipids with reduced expression are It was confirmed that 84 types including phosphatidylserine.
  • EOC-SP cells which are patient-derived ovarian cancer stem cells
  • Example 3 Analysis of exposure to phosphatidylethanolamine in ovarian cancer cancer stem cells
  • A2780-AD ovarian cancer cell line
  • A2780-SP ovarian cancer stem cell line
  • Example 3-1 Through flow cytometry, the expression of phosphatidylethanolamine in SKOV3-AD cells, another ovarian cancer cell line, and SKOV3-SP cells, a cancer stem cell line thereof, was analyzed in the same manner as in Example 3-1.
  • the ovarian cancer stem cell line SKOV3-SP cells are isolated after three-dimensional culture of the ovarian cancer cell line SKOV3-AD cells.
  • the flow cytometry results are shown in FIG. 15 .
  • the expression of phosphatidylethanolamine in the ovarian cancer stem cell line SKOV3-SP cells was significantly higher than that of the ovarian cancer cell line SKOV3-AD, and it was confirmed that the expressed phosphatidylethanolamine was exposed to the outside of the cell membrane.
  • EOC12-SP cells and EOC21-SP cells were prepared.
  • the expression of phosphatidylethanolamine in the EOC12-SP cells and the EOC21-SP cells was analyzed in the same manner as in Example 3-1.
  • the flow cytometry results are shown in FIG. 16 .
  • the patient-derived ovarian cancer stem cell lines, EOC12-SP cells and EOC21-SP cells both had high levels of phosphatidylethanolamine expression, and it was confirmed that the expressed phosphatidylethanolamine was exposed to the outside of the cell membrane.
  • TMSC, HEK293FT and HUVEC showed very little expression of phosphatidylethanolamine, and it was confirmed that the expressed phosphatidylethanolamine was exposed to the outside of the cell membrane.
  • phosphatidylethanolamine can be used as a biomarker for cancer stem cell detection, and the phosphatidylethanol of the present invention can be variously used in the fields of cancer diagnosis, prognosis prediction, and drug screening.

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Abstract

The present invention pertains to a biomarker for detecting cancer stem cells. More specifically, the present invention pertains to: a biomarker composition for detecting cancer stem cells by using phosphatidylethanolamine (PE) expressed in the cancer stem cells; a cancer diagnostic composition; a cancer diagnostic kit; and a method for providing information on cancer diagnosis. It was found that phosphatidylethanolamine, a biomarker for detecting cancer stem cells according to the present invention, is expressed at significantly higher levels in cancer stem cells than in cancer cells and other cells, and is exposed to the outside of the cell membrane when expressed. This means that cancer stem cells can be detected with higher sensitivity and accuracy when the biomarker for detecting cancer stem cells according to the present invention is used, and thus the present invention can be used in various ways in the fields of cancer diagnosis, prognosis prediction, and drug screening.

Description

암 줄기세포 검출용 바이오 마커 조성물Biomarker composition for cancer stem cell detection
본 발명은 암 줄기세포 검출용 바이오 마커에 관한 것으로, 보다 상세하게는 암 줄기세포에서 발현되는 포스파티딜에탄올아민(phosphatidylethanolamine, PE)을 이용한 암 줄기세포 검출용 바이오 마커 조성물, 암 진단용 조성물, 암 진단 키트 및 암 진단에 대한 정보 제공 방법에 관한 것이다.The present invention relates to a biomarker for detecting cancer stem cells, and more particularly, a biomarker composition for detecting cancer stem cells using phosphatidylethanolamine (PE) expressed in cancer stem cells, a composition for cancer diagnosis, a cancer diagnosis kit and to a method of providing information on cancer diagnosis.
신체의 장기를 유지하고 재생하기 위해서는 줄기세포의 존재가 필수적이다. 종양 또한 마찬가지로 줄기세포와 유사한 성질을 가지는 이질적 집단인 암 줄기세포가 존재한다. 암 줄기세포를 분리 시험관 내(in vitro) 배양하는 방법으로는 스피어 형성, 알데하이드 탈수소효소(Aldehyde dehydrogenase, ALDH) 활성, 세포 표면 표지인자 등 여러 방법이 존재한다. 상기 암 줄기세포는 부유 배양을 통하여 암 줄기세포의 특성인 자가증식법을 이용하여 스피어를 형성하는 세포로, 원 세포주에 비해 강력한 암 형성능과 정상 줄기세포의 특성을 공유한다고 알려져 있다. 또한 종양의 이질적인 특성에서 암 줄기세포는 종양이 악성으로 진행됨에 따라 증가하는 경향을 보이며, 암 줄기세포는 암의 발생은 물론 기존 암 치료법에 대한 내성을 가지며, 재발의 원인으로 보고되어 있다. 따라서 암 줄기세포를 제거하기 위해서는 기존 항암제의 내성을 갖는 세포를 인지하고 표적하는 것이 중요하다.The existence of stem cells is essential for maintaining and regenerating the organs of the body. Tumors also contain cancer stem cells, a heterogeneous population with stem cell-like properties. There are several methods for isolating and culturing cancer stem cells in vitro, such as sphere formation, aldehyde dehydrogenase (ALDH) activity, and cell surface markers. The cancer stem cells are cells that form spheres using the self-renewal method, which is a characteristic of cancer stem cells, through floating culture, and are known to share strong cancer-forming ability and characteristics of normal stem cells compared to the original cell line. In addition, due to the heterogeneous characteristics of tumors, cancer stem cells tend to increase as the tumor progresses to malignancy, and cancer stem cells are reported to cause cancer as well as resistance to existing cancer therapies and cause recurrence. Therefore, in order to remove cancer stem cells, it is important to recognize and target cells resistant to existing anticancer drugs.
현재 유방암, 간암, 대장암, 폐암, 위암 등의 분야에서 암 줄기세포를 표적하는 여러 세포 표면 단백질 바이오 마커들이 발굴 및 보고되었으나, 암 줄기세포를 표지한다고 알려진 바이오 마커들의 발현도 종양의 발병 위치, 병기, 전이, 재발에 따라 매우 다른 양상을 보인다. 이러한 암 줄기세포 표적 세포 표면 단백질 바이오 마커의 이질성을 해결하기 위하여, 암 줄기세포를 표적하는 새로운 형태의 바이오 마커 발굴 연구가 필요한 실정이다.Currently, several cell surface protein biomarkers targeting cancer stem cells have been discovered and reported in the fields of breast cancer, liver cancer, colorectal cancer, lung cancer, stomach cancer, etc., but the expression of biomarkers known to mark cancer stem cells also depends on the location of tumor onset, They show very different patterns depending on the stage, metastasis, and recurrence. In order to solve the heterogeneity of these cancer stem cell target cell surface protein biomarkers, a new type of biomarker discovery study targeting cancer stem cells is needed.
암의 경우 하나의 조직 내에서도 이질성이 매우 강하여, 항암치료 시 항암제 저항성을 가진 암 조직의 발현으로 항암치료가 실패하는 중요한 원인으로 꼽힌다. 암 줄기세포는 이러한 이질성 특징을 나타내는 가장 상위 단계의 세포로 알려져 있으며, 항암제 내성과도 밀접한 연관성을 가지고 있다. 이에 다양한 세포 표면 단백질 표적 치료에도 불구하고, 암 줄기세포를 완벽하게 제거하기는 쉽지 않다. 현재의 암 줄기세포의 단백질 표적 바이오 마커는 대부분 세포의 신호전달이나 세포의 내성 획득 등 다양한 주변 환경에 의하여 발현 빈도가 달라질 수 있다. 따라서 기존 단백질 표적 바이오 마커가 아닌 다른 형태의 암 줄기세포 특이적 세포 표면 표적 바이오 마커를 찾는 것이 중요하다.In the case of cancer, heterogeneity is very strong even within a single tissue, and the expression of cancer tissue with anticancer drug resistance during chemotherapy is considered an important cause of chemotherapy failure. Cancer stem cells are known as the highest-level cells exhibiting these heterogeneous characteristics, and are closely related to anticancer drug resistance. Therefore, it is not easy to completely remove cancer stem cells despite various cell surface protein-targeted treatments. Most of the current cancer stem cell protein target biomarkers can have different expression frequencies depending on various surrounding environments, such as cell signal transduction or cell resistance acquisition. Therefore, it is important to find a cancer stem cell-specific cell surface target biomarker other than the existing protein target biomarker.
이에 본 발명자들은 암 줄기세포를 표적하기 위한 바이오 마커를 발굴하기위하여 연구한 결과, 난소암 암 줄기세포에서 포스파티딜에탄올아민의 발현이 난소암 세포주 및 다른 세포주에 비해 현저히 높은 것을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by confirming that the expression of phosphatidylethanolamine in ovarian cancer cancer stem cells was significantly higher than that of ovarian cancer cell lines and other cell lines as a result of research to discover biomarkers for targeting cancer stem cells. did
따라서 본 발명의 목적은, 포스파티딜에탄올아민(phosphatidylethanolamine, PE)을 포함하는 암 줄기세포 검출용 바이오 마커 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a biomarker composition for detecting cancer stem cells containing phosphatidylethanolamine (PE).
본 발명의 다른 목적은, 포스파디틸에탄올아민의 발현을 측정하는 제제를 포함하는 암 줄기세포 검출을 통한 암 진단용 조성물 및 이를 포함하는 암 진단키트를 제공하는 것이다.Another object of the present invention is to provide a composition for diagnosing cancer through detection of cancer stem cells, including an agent for measuring the expression of phosphaditylethanolamine, and a cancer diagnostic kit comprising the same.
본 발명의 또 다른 목적은, 생물학적 시료에서 포스파티딜에탄올아민의 발현을 측정하는 단계를 포함하는 암 줄기세포 검출을 통한 암 진단에 대한 정보 제공 방법을 제공하는 것이다.Another object of the present invention is to provide a method for providing information on cancer diagnosis through cancer stem cell detection, comprising measuring the expression of phosphatidylethanolamine in a biological sample.
상기 목적을 달성하기 위하여, 본 발명은 포스파티딜에탄올아민을 포함하는 암 줄기세포 검출용 바이오 마커 조성물을 제공한다.In order to achieve the above object, the present invention provides a biomarker composition for detecting cancer stem cells comprising phosphatidylethanolamine.
또한 본 발명은 포스파티딜에탄올아민의 발현을 측정하는 제제를 포함하는 암 줄기세포 검출을 통한 암 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing cancer through cancer stem cell detection comprising an agent for measuring the expression of phosphatidylethanolamine.
또한 본 발명은 상기 암 진단용 조성물을 포함하는 암 줄기세포 검출을 통한 암 진단 키트를 제공한다.The present invention also provides a cancer diagnosis kit through cancer stem cell detection comprising the composition for cancer diagnosis.
또한 본 발명은 생물학적 시료에서 포스파티딜에탄올아민의 발현을 측정하는 단계를 포함하는 암 줄기세포 검출을 통한 암 진단에 대한 정보 제공 방법을 제공한다.The present invention also provides a method for providing information on cancer diagnosis through cancer stem cell detection, comprising measuring the expression of phosphatidylethanolamine in a biological sample.
본 발명에 따른 암 줄기세포 검출용 바이오 마커인 포스파티딜에탄올아민은 암세포 및 다른 세포에 비해 암 줄기세포에서 발현이 현저히 높고, 발현 시 세포막 외부로 노출된다는 것을 확인하였다. 이는 본 발명의 암 줄기세포 검출용 바이오 마커를 이용할 경우 암 줄기세포를 보다 민감하고 정확하게 검출할 수 있음을 의미하는바, 본 발명의 암의 진단, 예후 예측 및 약물 스크리닝 분야에서 다양하게 활용될 수 있다.It was confirmed that the expression of phosphatidylethanolamine, a biomarker for cancer stem cell detection according to the present invention, is significantly higher in cancer stem cells than in cancer cells and other cells, and is exposed to the outside of the cell membrane when expressed. This means that cancer stem cells can be more sensitively and accurately detected when the biomarker for cancer stem cell detection of the present invention is used. have.
도 1은 난소암 세포주 A2780-AD 세포 및 난소암 암 줄기세포주 A2780-SP 세포의 형태학적 분석 결과를 나타낸 도이다.1 is a diagram showing the results of morphological analysis of ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells.
도 2는 유세포 분석을 통해 난소암 세포주 A2780-AD 세포 및 난소암 암 줄기세포주 A2780-SP 세포의 암 줄기세포 마커 발현을 확인한 결과를 나타낸 도이다.2 is a diagram showing the results of confirming the expression of cancer stem cell markers in ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells through flow cytometry.
도 3은 LC-MS를 통해 난소암 세포주 A2780-AD 세포 및 난소암 암 줄기세포주 A2780-SP 세포의 지질 발현을 분석한 결과를 나타낸 도이다.3 is a diagram showing the results of analyzing the lipid expression of ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells through LC-MS.
도 4는 상기 도 3의 결과에 기초하여, 난소암 세포주인 A2780-AD에 비해 암 줄기세포주인 A2780-SP 세포에서 발현이 증가 또는 감소된 지질을 분석한 결과를 나타낸 도이다.4 is a diagram showing the results of analysis of lipids with increased or decreased expression in A2780-SP cells, a cancer stem cell line, compared to A2780-AD, an ovarian cancer cell line, based on the results of FIG. 3 .
도 5는 난소암 세포주 A2780-AD 세포 및 난소암 암 줄기세포주 A2780-SP 세포의 포스파티딜에탄올아민 발현을 비교한 결과를 나타낸 도이다.5 is a diagram showing the results of comparing the expression of phosphatidylethanolamine in ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells.
도 6은 LC-MS를 통해 암세포화된 암 줄기세포주 A2780-DIF 세포 및 암 줄기세포주 A2780-SP 세포의 지질 발현을 분석한 결과를 나타낸 도이다.6 is a diagram showing the results of analyzing the lipid expression of cancer stem cell line A2780-DIF cells and cancer stem cell line A2780-SP cells that have been cancerousized through LC-MS.
도 7은 암 줄기세포주인 A2780-SP 세포에 비해 암세포화된 암 줄기세포주인 A2780-DIF 세포에서 발현이 증가 또는 감소된 지질을 분석한 결과를 나타낸 도이다.7 is a diagram showing the results of analysis of lipids with increased or decreased expression in A2780-DIF cells, which are cancer stem cell lines, which have become cancerous compared to A2780-SP cells, which are cancer stem cell lines.
도 8은 암세포화된 암 줄기세포주 A2780-DIF 세포 및 암 줄기세포주 A2780-SP 세포의 포스파티딜에탄올아민 발현을 비교한 결과를 나타낸 도이다.8 is a diagram showing the results of comparing the expression of phosphatidylethanolamine in cancer stem cell line A2780-DIF cells and cancer stem cell line A2780-SP cells transformed into cancer cells.
도 9는 환자 유래 난소암 암 줄기세포인 EOC-SP 세포 및 암세포화된 환자 유래 난소암 암 줄기세포인 EOC-DIF 세포의 형태학적 분석 결과를 나타낸 도이다.9 is a diagram showing the results of morphological analysis of EOC-SP cells, which are patient-derived ovarian cancer stem cells, and EOC-DIF cells, which are patient-derived ovarian cancer stem cells, which have become cancerous.
도 10은 EOC-SP 세포 및 EOC-DIF 세포의 암 줄기세포 마커 발현을 확인한 결과를 나타낸 도이다.10 is a diagram showing the results of confirming the expression of cancer stem cell markers in EOC-SP cells and EOC-DIF cells.
도 11은 EOC-SP 세포 및 EOC-DIF 세포의 지질 발현을 분석한 결과를 나타낸 도이다.11 is a diagram showing the results of analyzing the lipid expression of EOC-SP cells and EOC-DIF cells.
도 12는 상기 도 11의 결과에 기초하여, 환자 유래 난소암 암 줄기세포주인 EOC-SP 세포에 비해 암세포화된 암 줄기세포주인 EOC-DIF 세포에서 발현이 증가 또는 감소된 지질을 분석한 결과를 나타낸 도이다.12 shows the results of analysis of lipids with increased or decreased expression in EOC-DIF cells, which are cancer stem cell lines, which have become cancerous compared to EOC-SP cells, which are patient-derived ovarian cancer cancer stem cell lines, based on the results of FIG. 11 . is the diagram shown.
도 13은 EOC-SP 세포 및 EOC-DIF 세포에서 포스파티딜에탄올아민의 발현을 비교한 결과를 나타낸 도이다.13 is a diagram showing the results of comparing the expression of phosphatidylethanolamine in EOC-SP cells and EOC-DIF cells.
도 14는 유세포 분석을 통해 난소암 세포주 A2780-AD 세포 및 난소암 암 줄기세포주 A2780-SP 세포에서 포스파티딜에탄올아민의 세포막 외부 노출 여부를 확인한 결과를 나타낸 도이다.14 is a diagram showing the results of confirming whether phosphatidylethanolamine is exposed to the outside of the cell membrane in ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells through flow cytometry.
도 15는 유세포 분석을 통해 난소암 세포주 SKOV3-AD 세포 및 난소암 암 줄기세포주 SKOV3-SP 세포에서 포스파티딜에탄올아민의 세포막 외부 노출 여부를 확인한 결과를 나타낸 도이다.15 is a diagram showing the results of confirming whether phosphatidylethanolamine is exposed to the outside of the cell membrane in ovarian cancer cell line SKOV3-AD cells and ovarian cancer stem cell line SKOV3-SP cells through flow cytometry.
도 16은 유세포 분석을 통해 환자 유래 암 줄기세포주 EOC12-SP 세포 및 EOC21-SP 세포에서 포스파티딜에탄올아민의 세포막 외부 노출 여부를 확인한 결과를 나타낸 도이다.16 is a diagram showing the results of confirming whether phosphatidylethanolamine is exposed to the outside of the cell membrane in the patient-derived cancer stem cell lines EOC12-SP cells and EOC21-SP cells through flow cytometry.
도 17은 유세포 분석을 통해 인간 배아 유래 신장세포, 편도 유래 중간엽 줄기세포 및 인간 탯줄 정맥내피세포에서 포스파티딜에탄올아민의 발현 및 세포막 외부 노출 여부를 확인한 결과를 나타낸 도이다.17 is a diagram showing the results of confirming the expression of phosphatidylethanolamine and external exposure to the cell membrane in human embryonic kidney cells, tonsil-derived mesenchymal stem cells, and human umbilical vein endothelial cells through flow cytometry.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 양태에 따르면, 본 발명은 포스파티딜에탄올아민(phosphatidylethanolamine, PE)을 포함하는 암 줄기세포 검출용 바이오 마커 조성물을 제공한다.According to an aspect of the present invention, there is provided a biomarker composition for detecting cancer stem cells comprising phosphatidylethanolamine (PE).
본 발명에 있어서, “포스파티딜에탄올아민”은 인지질(phospholipid)의 하나로, 글리세로인지질(glycerophospholipid)에 속한다. 상기 글리세로인지질은 글리세롤 3인산(glycerol 3-phosphate)에 지방산 2개가 결합한 형태인데, 포스파티딜에탄올아민은 인산기에 에탄올아민(ethanolamine)이 결합된 것이다. 이와 같은 포스포에탄올아민은 세포 분열 동안 막의 융합, 수축환의 분해 및 막의 곡률 조절에 관여한다고 알려져 있다.In the present invention, “phosphatidylethanolamine” is one of phospholipids, and belongs to glycerophospholipids. The glycerophospholipid is a form in which two fatty acids are bonded to glycerol 3-phosphate, and phosphatidylethanolamine is a phosphoric acid group in which ethanolamine is bonded. Such phosphoethanolamine is known to be involved in membrane fusion during cell division, decomposition of contractile rings, and control of membrane curvature.
본 발명의 구체예에서, 상기 포스파티딜에탄올아민은 일반적으로 세포의 인지질 이중층 내막에서 발현되는 것이 바람직하다.In an embodiment of the present invention, it is preferred that the phosphatidylethanolamine is generally expressed in the inner membrane of the phospholipid bilayer of the cell.
본 발명에 있어서, “암 줄기세포(Cancer stem cell 또는 Tumor initiating cell)”는 종양을 생성할 수 있는 능력을 가지는 세포를 의미한다. 암 줄기세포는 정상적인 줄기세포와 유사한 특징을 가지며, 줄기세포의 특성인 자기재생능 및 분화능을 통해 종양을 발생시킨다. 또한 종양에서 다른 집단과 구별되어 새로운 종양을 발생시킴으로써 재발 및 전이의 원인이 된다. 그러므로 암 줄기세포를 타겟으로 하여 특이적 치료방법의 발달은 암환자의 생존율을 증가시킬 수 있다.In the present invention, "cancer stem cell (Cancer stem cell or Tumor initiating cell)" refers to a cell having the ability to generate a tumor. Cancer stem cells have characteristics similar to normal stem cells, and they generate tumors through the self-renewal and differentiation abilities of stem cells. In addition, the tumor is differentiated from other populations and causes recurrence and metastasis by generating new tumors. Therefore, the development of a specific treatment method targeting cancer stem cells can increase the survival rate of cancer patients.
본 발명에 있어서, “암”은 세포가 정상적인 성장 한계를 무시하고 분열 및 성장하는 공격적(aggressive) 특성, 주위 조직에 침투하는 침투적(invasive) 특성, 및 체내의 다른 부위로 퍼지는 전이적(metastatic) 특성을 갖는 세포에 의한 질병을 총칭하는 의미이다. In the present invention, “cancer” is defined as an aggressive characteristic in which cells divide and grow ignoring normal growth limits, an invasive characteristic that penetrates into surrounding tissues, and metastatic that spreads to other parts of the body ) is a generic term for diseases caused by cells with characteristics.
본 발명의 구체예에서, 위암(gastric cancer), 유방암(breast cancer), 폐암(lung cancer), 간암(liver cancer), 혈액암(blood cancer), 뼈암(bone cancer), 췌장암(pancreatic cancer), 피부암(skin cancer), 머리 또는 목암(head or neck cancer), 피부 또는 안구 흑색종(cutaneous or intraocular melanoma), 자궁육종(uterine sarcoma), 난소암(ovarian cancer), 직장암(rectal cancer), 항문암(anal cancer), 대장암(colon cancer), 난관암(fallopian tube carcinoma), 자궁내막암(endometrial carcinoma), 자궁경부암(cervical cancer), 소장암(small intestine cancer), 내분비암(endocrine cancer), 갑상선암(thyroid cancer), 부갑상선암(parathyroid cancer), 신장암(adrenal cancer), 연조직종양(soft tissue tumor), 요도암(urethral cancer), 전립선암(prostate cancer), 기관지암(bronchogenic cancer) 및 골수암(bone marrow tumor)으로 이루어진 군에서 선택된 1 종 이상인 것이 바람직하며, 난소암인 것이 더 바람직하다.In an embodiment of the present invention, gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, Skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer (anal cancer), colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, small intestine cancer, endocrine cancer, Thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchogenic cancer and bone marrow cancer It is preferably at least one selected from the group consisting of (bone marrow tumor), and more preferably ovarian cancer.
본 발명에 있어서, “바이오 마커”는 생체 내 변화를 객관적으로 측정 및 평가할 수 있는 지표로, 개체의 병리적인 상태, 약물에 대한 반응도 등을 측정·평가할 수 있다. 이에, 바이오 마커는 검출 또는 진단 용도로도 쓰일 수 있다.In the present invention, a “biomarker” is an index that can objectively measure and evaluate changes in a living body, and can measure and evaluate an individual's pathological state, response to drugs, and the like. Accordingly, the biomarker may also be used for detection or diagnosis.
본 발명의 구체예에서, 상기 포스파티딜에탄올아민은 암이 아닌 세포 및 암세포보다 암 줄기세포에서 발현이 높은 것이 바람직하다. 상기 암이 아닌 세포의 예로는 인간 배아 유래 신장세포, 편도 유래 중간엽 줄기세포 및 인간 탯줄정맥내피세포일 수 있으며, 이에 제한되지 않는다.In an embodiment of the present invention, the phosphatidylethanolamine is preferably expressed in non-cancer cells and cancer stem cells than in cancer cells. Examples of the non-cancer cells may include, but are not limited to, human embryonic-derived kidney cells, tonsil-derived mesenchymal stem cells, and human umbilical vein endothelial cells.
본 발명의 일 실시예에서, 포스파티딜에탄올아민이 암 줄기세포의 표면에서 발현되며, 암이 아닌 세포 및 암세포에 비해 암 줄기세포에서 과발현됨을 확인하였는바, 이는 암 줄기세포의 검출용 또는 암 진단용 바이오마커로 활용될 수 있다.In one embodiment of the present invention, it was confirmed that phosphatidylethanolamine is expressed on the surface of cancer stem cells and is overexpressed in cancer stem cells compared to non-cancer cells and cancer cells, which is a cancer stem cell detection or cancer diagnosis bio It can be used as a marker.
본 발명의 다른 양태에 따르면, 본 발명은 포스파티딜에탄올아민의 발현을 측정하는 제제를 포함하는 암 줄기세포 검출을 통한 암 진단용 조성물 및 암 진단 키트를 제공한다.According to another aspect of the present invention, there is provided a composition for diagnosing cancer through detection of cancer stem cells and a cancer diagnosis kit comprising an agent for measuring the expression of phosphatidylethanolamine.
본 발명에 있어서, “진단”은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 상기 진단은 암의 발병 여부뿐만 아니라 예후, 경과, 병기 등을 확인하는 것을 모두 포함하는 의미이다. 본 발명의 목적상, 진단은 암의 발병, 예후, 경과, 병기 상태 또는 특징을 확인하는 것을 의미한다.In the present invention, “diagnosis” means confirming the presence or characteristics of a pathological condition. The diagnosis is meant to include not only whether or not cancer occurs, but also confirmation of prognosis, progress, and stage. For the purposes of the present invention, diagnosis means identifying the onset, prognosis, course, staging or characteristics of cancer.
본 발명에 있어서, “포스파티딜에탄올아민의 발현을 측정하는 제제”는 상기와 같이 암 줄기세포에서 발현이 증가하는 바이오 마커인 포스파티딜에탄올아민에 특이적으로 결합하여, 이의 발현 수준을 확인함으로써 바이오 마커의 검출에 사용될 수 있는 것을 의미한다.In the present invention, the “agent for measuring the expression of phosphatidylethanolamine” specifically binds to phosphatidylethanolamine, a biomarker whose expression is increased in cancer stem cells, as described above, and confirms the expression level of the biomarker. It means that it can be used for detection.
본 발명의 구체예에서, 상기 포스파티딜에탄올아민의 발현을 측정하는 제제는 포스파티딜에탄올아민에 특이적으로 결합하는 올리고펩타이드, 모노클로날 항체, 폴리클로날 항체, 키메릭(chimeric) 항체, 리간드, PNA(Peptide nucleic acid), 앱타머(aptamer), 프로브 및 염료로 이루어진 군에서 선택되는 것이 바람직하나, 이에 제한되지 않는다.In an embodiment of the present invention, the agent for measuring the expression of phosphatidylethanolamine is an oligopeptide that specifically binds to phosphatidylethanolamine, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, PNA (Peptide nucleic acid), an aptamer (aptamer), preferably selected from the group consisting of probes and dyes, but is not limited thereto.
본 발명의 바람직한 구체예에서, 포스파티딜에탄올아민의 발현을 측정하기 위한 제제는 듀라마이신(duramycin) 또는 신나마이신(cinnamycin)을 포함할 수 있다.In a preferred embodiment of the present invention, the agent for measuring the expression of phosphatidylethanolamine may include duramycin or cinnamycin.
본 발명의 구체예에서, 암 진단용 키트는 사용하는 방법에 따라 다양한 형태의 키트일 수 있으며, 예를 들어 상기 키트는 유세포분석법, 질량분석법, 광흡수분석법, 크로마토그래피 및 발광분광분석법을 위한 정량 분석 키트일 수 있으며, 이에 제한되지 않는다. 또한 상기 정량 분석 키트는 정량 분석 장치를 통해 결과 값을 획득할 수 있다. 상기 정량 분석 장치는 유세포분석기, 핵자기공명분광기, 크로마토그래피, 자외선분광기, 적외선분광기, 형광분광기, 마이크로플레이트리더 및 질량분석기로 이루어진 군에서 선택된 1 이상일 수 있으나, 이에 제한되지 않는다.In an embodiment of the present invention, the kit for diagnosis of cancer may be a kit in various types depending on the method used, for example, the kit is quantitative analysis for flow cytometry, mass spectrometry, light absorption spectrometry, chromatography and emission spectroscopy. It may be a kit, but is not limited thereto. In addition, the quantitative analysis kit may obtain a result value through a quantitative analysis device. The quantitative analysis device may be at least one selected from the group consisting of flow cytometry, nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, microplate reader and mass spectrometer, but is not limited thereto.
상기 암 진단용 키트에는 상기 포스파티딜에탄올아민의 발현 수준을 측정하는 제제 뿐만 아니라, 면역학적 분석 및 정량 분석에 사용되는 당 분야에서 일반적으로 사용되는 도구, 시약 등이 포함될 수 있다. 상기 도구 또는 시약의 일 예로, 적합한 담체, 검출 가능한 신호를 생성할 수 있는 표지 물질, 발색단(chromophores), 용해제, 세정제, 완충제, 안정화제 등이 포함되나 이에 제한되지 않는다. 표지물질이 효소인 경우에는 효소 활성을 측정할 수 있는 기질 및 반응 정지제를 포함할 수 있다. 담체는 가용성 담체, 불용성 담체가 있고, 가용성 담체의 일 예로 당 분야에서 공지된 생리학적으로 허용되는 완충액, 예를 들어 PBS가 있고, 불용성 담체의 예로는 폴리스틸렌, 폴리에틸렌, 폴리프로필렌, 폴리에스테르, 폴리아크릴로니트릴, 불소 수지, 가교 덱스트란, 폴리사카라이드, 라텍스에 금속을 도금한 자성 미립자와 같은 고분자, 기타 종이, 유리, 금속, 아가로오스 및 이들의 조합일 수 있다.The cancer diagnostic kit may include not only an agent for measuring the expression level of the phosphatidylethanolamine, but also tools and reagents commonly used in the art used for immunological analysis and quantitative analysis. Examples of the tool or reagent include, but are not limited to, a suitable carrier, a labeling material capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling material is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. The carrier includes a soluble carrier and an insoluble carrier, and an example of the soluble carrier is a physiologically acceptable buffer known in the art, for example, PBS, and examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, poly It may be acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof.
본 발명의 키트는 상술한 조성물을 구성으로 포함하므로, 중복된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.Since the kit of the present invention includes the above-described composition as a component, redundant descriptions are omitted in order to avoid excessive complexity of the present specification.
본 발명의 암 진단용 조성물 및 암 진단 키트를 이용하여, 생물학적 시료에서 암 줄기세포, 즉, 포스파티딜에탄올아민이 과발현되는 세포를 검출하여 암 줄기세포의 존재 여부를 확인하고, 이로부터 암의 존재 여부 뿐만 아니라 예후, 경과, 병기 등을 확인할 수 있다.By using the cancer diagnosis composition and cancer diagnosis kit of the present invention, cancer stem cells, that is, cells overexpressing phosphatidylethanolamine, are detected in a biological sample to determine the presence of cancer stem cells, and from this, the presence of cancer as well as However, the prognosis, course, and stage can be confirmed.
본 발명의 또 다른 양태에 따르면, 본 발명은 생물학적 시료에서 포스파티딜에탄올아민의 발현을 측정하는 단계를 포함하는 암 줄기세포 검출을 통한 암 진단에 대한 정보 제공 방법을 제공한다.According to another aspect of the present invention, the present invention provides a method for providing information on cancer diagnosis through cancer stem cell detection, comprising measuring the expression of phosphatidylethanolamine in a biological sample.
본 발명에 있어서, “생물학적 시료”는 본 발명의 포스파티딜에탄올아민의 발현이 검출될 수 있는 개체로부터 얻어지는 모든 시료를 의미한다. 상기 생물학적 시료 는 전혈, 혈청, 혈장, 세포, 객담, 조직, 타액(saliva), 생검(biopsy), 액체 배양물, 분변 및 소변으로 이루어진 군에서 선택된 어느 하나이며, 특별히 이에 제한되지 않고, 본 발명의 기술 분야에서 통상적으로 사용되는 방법으로 처리하여 준비될 수 있다.In the present invention, "biological sample" means any sample obtained from an individual in which the expression of phosphatidylethanolamine of the present invention can be detected. The biological sample is any one selected from the group consisting of whole blood, serum, plasma, cells, sputum, tissue, saliva, biopsy, liquid culture, feces and urine, and is not particularly limited thereto, and the present invention It can be prepared by processing by a method commonly used in the technical field of
본 발명의 구체예에서, 상기 방법은 생물학적 시료에서 포스파티딜에탄올아민의 발현 정도를 확인함으로써 암 줄기세포의 존재 여부를 판단하며, 이를 통해 암의 진단에 대한 정보를 제공할 수 있다.In an embodiment of the present invention, the method determines the presence or absence of cancer stem cells by checking the expression level of phosphatidylethanolamine in a biological sample, thereby providing information on diagnosis of cancer.
본 발명의 구체예에서, 상기 방법은 유세포분석기, 핵자기공명분광기, 크로마토그래피, 자외선분광기, 적외선분광기, 형광분광기, 마이크로플레이트리더 및 질량분석기로 이루어진 군에서 선택된 1 이상의 정량 분석 장치를 통해 포스파디틸에탄올아민의 발현을 확인하는 단계를 더 포함할 수 있다.In an embodiment of the present invention, the method comprises at least one quantitative analysis device selected from the group consisting of flow cytometry, nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, microplate reader and mass spectrometer. It may further comprise the step of confirming the expression of tylethanolamine.
본 발명의 또 다른 양태에 따르면, 본 발명은 (a) 개체로부터 분석 시료를 얻는 단계; (b) 단계 (a)의 분석 시료에서 포스파티딜에탄올아민(phosphatidylethanolamine, PE)의 존재여부를 검출하는 단계; (c) 단계 (b)에서 포스파티딜에탄올아민이 분석 시료에 존재할 경우 암으로 진단하는 단계; (d) 단계 (c)에서 암으로 진단된 개체에 유효량의 암 줄기세포 특이적 항암제를 투여하는 단계;를 포함하는 암 진단 또는 치료방법을 제공한다.According to another aspect of the present invention, the present invention comprises the steps of (a) obtaining an assay sample from a subject; (b) detecting the presence or absence of phosphatidylethanolamine (PE) in the analysis sample of step (a); (c) diagnosing cancer when phosphatidylethanolamine is present in the analyte sample in step (b); (d) administering an effective amount of a cancer stem cell-specific anticancer agent to the individual diagnosed with cancer in step (c); provides a cancer diagnosis or treatment method comprising a.
본 발명의 구체예에서, 상기 암 줄기세포 특이적 항암제는 나이트로젠 머스타드, 이마티닙, 옥살리플라틴, 리툭시맙, 엘로티닙, 네라티닙, 라파티닙, 제피티닙, 반데타닙, 니로티닙, 세마사닙, 보수티닙, 악시티닙, 세디라닙, 레스타우르티닙, 트라스투주맙, 게피티니브, 보르테조밉, 수니티닙, 카보플라틴, 소라페닙, 베바시주맙, 시스플라틴, 세툭시맙, 비스쿰알붐, 아스파라기나제, 트레티노인, 하이드록시카바마이드, 다사티닙, 에스트라머스틴, 겜투주맵오조가마이신, 이브리투모맙튜세탄, 헵타플라틴, 메칠아미노레불린산, 암사크린, 알렘투주맙, 프로카르바진, 알프로스타딜, 질산홀뮴 키토산, 젬시타빈, 독시플루리딘, 페메트렉세드, 테가푸르, 카페시타빈, 기메라신, 오테라실, 아자시티딘, 메토트렉세이트, 우라실, 시타라빈, 플루오로우라실, 플루다가빈, 에노시타빈, 플루타미드, 데시타빈, 카페시타빈, 머캅토푸린, 티오구아닌, 클라드리빈, 카르모퍼, 랄티트렉세드, 도세탁셀, 파클리탁셀, 이리노테칸, 벨로테칸, 토포테칸, 비노렐빈, 에토포시드, 빈크리스틴, 빈블라스틴, 테니포시드, 독소루비신, 이다루비신, 에피루비신, 미톡산트론, 미토마이신, 블레로마이신, 다우노루비신, 닥티노마이신, 피라루비신, 아클라루비신, 페프로마이신, 템시롤리무스, 테모졸로마이드, 부설판, 이포스파미드, 사이클로포스파미드, 멜파란, 알트레트민, 다카바진, 치오테파, 니무스틴, 클로람부실, 미토락톨, 레우코보린, 트레토닌, 엑스메스탄, 아미노글루테시미드, 아나그렐리드, 나벨빈, 파드라졸, 타목시펜, 토레미펜, 테스토락톤, 아나스트로졸, 레트로졸, 보로졸, 비칼루타미드, 로무스틴, 보리노스텟, 엔티노스텟, 5FU 및 카르무스틴으로 이루어진 군에서 선택된 1종 이상인 것이 바람직하나, 이에 제한되지 않는다.In an embodiment of the present invention, the cancer stem cell-specific anticancer agent is nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib , bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscumalbum , asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tusetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, pro Carbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxyfluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, Fluorouracil, fludabine, enocitabine, flutamide, decitabine, capecitabine, mercaptopurine, thioguanine, cladribine, carmopher, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, Topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, Pyrarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melparan, altretmine, dacarbazine, thiotepa, nimustine, clo Rambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutethimide, anagrelide, nabelbine, fadrazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, At least one selected from the group consisting of vorozol, bicalutamide, lomustine, vorinostat, entinostat, 5FU and carmustine is preferable, but is not limited thereto.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 난소암 세포 및 난소암 암 줄기세포의 지질 발현 비교Example 1. Comparison of lipid expression of ovarian cancer cells and ovarian cancer stem cells
1-1. 세포 준비1-1. cell preparation
난소암 세포주 A2780 세포(A2780-AD)와 난소암 암 줄기세포주 A2780-SP 세포를 준비하였다. 난소암 세포주 A2780-AD은 RPMI1640 배지(10% FBS, 100 μg/mL Sterptomycin/Penicillin)를 이용하여 37℃ 및 5% CO2 조건에서 배양하였고, 일정 기간마다 계대 배양하였다. 상기 암 줄기세포주 A2780-SP 세포는 난소암 세포주인 A2780 세포를 3차원 배양한 후 암 줄기세포를 분리한 것이다. 상기 3차원 배양은 Neurobasal Plus 배지(B-27 supplement (50X), 100 μg/mL Sterptomycin/Penicillin, 100 μg/mL rhFGF, 100 μg/mL rhEGF, Glutamax, 1M HEPES)를 이용하여 Ultra-low attachent dish에서 수행하였다. 상기 3차원 배양조건은 온도 37℃ 및 5% CO2 조건이다.Ovarian cancer cell line A2780 cells (A2780-AD) and ovarian cancer stem cell line A2780-SP cells were prepared. The ovarian cancer cell line A2780-AD was cultured at 37° C. and 5% CO 2 using RPMI1640 medium (10% FBS, 100 μg/mL Sterptomycin/Penicillin), and subcultured at regular intervals. The cancer stem cell line A2780-SP cells are obtained by three-dimensional culture of the ovarian cancer cell line A2780 cells, followed by isolation of cancer stem cells. The three-dimensional culture was performed in an ultra-low attachent dish using Neurobasal Plus medium (B-27 supplement (50X), 100 μg/mL Sterptomycin/Penicillin, 100 μg/mL rhFGF, 100 μg/mL rhEGF, Glutamax, 1M HEPES). was performed in The three-dimensional culture conditions are a temperature of 37° C. and 5% CO 2 conditions.
1-2. 형태학적 분석1-2. Morphological analysis
준비된 A2780-AD 세포 및 A2780-SP 세포의 형태학적 분석을 수행하였다. 구체적으로, 상기 A2780-AD 세포 및 A2780-SP 세포를 각각의 상기 조건으로 배양하였다. 배양된 세포를 현미경으로 관찰하였다. 형태학적 분석 결과는 도 1에 나타내었다.Morphological analysis of the prepared A2780-AD cells and A2780-SP cells was performed. Specifically, the A2780-AD cells and A2780-SP cells were cultured under each of the above conditions. The cultured cells were observed under a microscope. The morphological analysis results are shown in FIG. 1 .
도 1에 나타낸 바와 같이, 난소암 세포주인 A2780-AD 세포 및 난소암 암줄기세포주인 A2780-SP 세포는 형태학적으로 차이가 있는 것을 확인하였다.As shown in Figure 1, it was confirmed that the morphological difference between the ovarian cancer cell line A2780-AD cells and the ovarian cancer stem cell line A2780-SP cells.
1-3. 암 줄기세포 마커 발현 확인1-3. Confirmation of cancer stem cell marker expression
준비된 A2780-AD 세포 및 A2780-SP 세포에서 암 줄기세포의 마커인 알데하이드 탈수소효소(Aldehyde dehydrogenase, ALDH)의 발현을 확인하였다. 구체적으로, ALDEFOUOR™ 키트(STEMCELL Technologies)를 사용하여, 준비된 A2780-AD 세포 중 ALDH를 발현하는 세포를 제조사의 매뉴얼에 따라 표지하였다. 그 후 FACS AttuneNxT(ThermoFisher)를 이용하여 ALDH를 발현하는 세포를 측정하였다. A2780-SP 세포도 동일한 방법으로 ALDH의 발현을 측정하였다. 암 줄기세포 마커를 발현하는 세포를 측정한 결과는 도 2에 나타내었다.The expression of aldehyde dehydrogenase (ALDH), a marker of cancer stem cells, was confirmed in the prepared A2780-AD cells and A2780-SP cells. Specifically, using the ALDEFOUOR™ kit (STEMCELL Technologies), ALDH-expressing cells among the prepared A2780-AD cells were labeled according to the manufacturer's manual. Thereafter, cells expressing ALDH were measured using FACS AttuneNxT (ThermoFisher). A2780-SP cells were also measured for ALDH expression in the same manner. The results of measuring cells expressing cancer stem cell markers are shown in FIG. 2 .
도 2에 나타낸 바와 같이, 난소암 암 줄기세포주인 A2780-SP 세포는 ALDH의 발현이 난소암 세포주(A2780-AD)에 비해 현저히 높은 것을 확인하였다. As shown in FIG. 2 , it was confirmed that the expression of ALDH in A2780-SP cells, which is an ovarian cancer stem cell line, was significantly higher than that in the ovarian cancer cell line (A2780-AD).
1-4. 난소암 세포 및 난소암 암 줄기세포의 지질 발현 분석1-4. Lipid Expression Analysis of Ovarian Cancer Cells and Ovarian Cancer Stem Cells
준비된 A2780-AD 세포 및 A2780-SP 세포의 지질 발현을 분석하였다. 구체적으로, 부착세포인 A2780-AD 세포는 트립신을 처리하여 세포 펠릿을 수집하였다. 또한 부유세포인 A2780-SP 세포는 배양액을 회수한 후 스핀다운(spin down)하여 배지를 제거하여 세포 펠릿을 수집하였다. 수집된 세포 펠릿에 MeOH 610 μl, Internal standards mix 50 μl, 클로로폼(Chloroform) 330 μl 및 Cholesteryl ester standard(1 ng/μl) 20 μl을 첨가하였다. 그 후 3분 마다 30초씩 볼텍싱(vortexing)한 후 스핀다운(spin down) 및 초음파 처리하였으며, 3회 반복하였다. 초음파 처리된 세포를 원심분리(4℃, 1분, 14,000 g)하였고, 상층액을 제거하여 펠릿을 얻었다.Lipid expression of the prepared A2780-AD cells and A2780-SP cells was analyzed. Specifically, adherent A2780-AD cells were treated with trypsin to collect cell pellets. In addition, A2780-SP cells, which are floating cells, were spun down after recovering the culture medium to remove the medium to collect cell pellets. MeOH 610 μl, internal standards mix 50 μl, chloroform 330 μl and Cholesteryl ester standard (1 ng/μl) 20 μl were added to the collected cell pellet. After that, after vortexing for 30 seconds every 3 minutes, spin down and sonication were performed, and repeated 3 times. The sonicated cells were centrifuged (4°C, 1 min, 14,000 g), and the supernatant was removed to obtain a pellet.
수득된 펠릿에 MeOH 496 μl, CH3Cl 248 μl을 첨가하고, 30% HCl 6 μl을 첨가하였다. 그 후 5분마다 30초씩 볼텍싱한 후 스핀다운하였으며, 이 과정을 3회 반복하였다. 그 후 CH3Cl 250 μl 및 0.1N HCl 450 μl을 첨가한 후 1분 동안 볼텍싱한 후 원심분리(4℃, 1분, 6,500 g)하여 층분리를 확인하였다. 아랫층을 파이펫으로 취하였고, 새 튜브에 옮겼다.496 μl of MeOH, 248 μl of CH 3 Cl were added to the obtained pellet, and 6 μl of 30% HCl was added. Then, after vortexing for 30 seconds every 5 minutes, spin down was performed, and this process was repeated 3 times. Then, after adding 250 μl of CH 3 Cl and 450 μl of 0.1N HCl, vortexing was performed for 1 minute, followed by centrifugation (4° C., 1 minute, 6,500 g) to confirm layer separation. The lower layer was pipetted and transferred to a new tube.
새 튜브에 담긴 시료를 비-메틸화(non-methylation) 및 메틸화(methylation) 분석용 시료로 나눈 후 건조시켰다. 비-메틸화 분석용 시료는 용액(A:B=2:1) 50 μl에 희석하여, 메틸화 분석용 시료는 MeOH 50 μl에 희석하여 준비된 샘플을 -80℃에 보관하였다.The sample contained in a new tube was divided into samples for non-methylation and methylation analysis, and then dried. A sample for non-methylation analysis was diluted in 50 μl of a solution (A:B=2:1), and a sample for methylation analysis was diluted in 50 μl of MeOH, and the prepared sample was stored at -80°C.
준비된 샘플 및 TMSD(Trimethylsilyldiazomethane) 시약을 1:1(v/v) 비율로 혼합한 후 35 내지 40℃에서 15분 동안 반응시켰다. 반응 후 아세트산을 전체 부피의 5% 정도 첨가한 후 용액이 투명해질 때까지 한 방울씩 첨가하였다. 투명해진 용액을 분석을 위한 시료로 사용하였다.The prepared sample and TMSD (Trimethylsilyldiazomethane) reagent were mixed at a 1:1 (v/v) ratio and reacted at 35 to 40° C. for 15 minutes. After the reaction, about 5% of the total volume of acetic acid was added and then added dropwise until the solution became transparent. The clear solution was used as a sample for analysis.
준비된 시료에 포함된 지질을 LC-MS(liquid chromatography mass spectrometry)를 통해 분석하였다. 분석 결과에 기초하여 매트릭스를 작성하였으며, 작성된 매트릭스는 도 3에 나타내었다.Lipids contained in the prepared sample were analyzed through liquid chromatography mass spectrometry (LC-MS). A matrix was prepared based on the analysis results, and the created matrix is shown in FIG. 3 .
도 3에 나타낸 바와 같이, A2780-AD 세포 및 A2780-SP 세포는 지질 발현 패턴이 상이한 것을 확인하였다. As shown in FIG. 3 , it was confirmed that A2780-AD cells and A2780-SP cells had different lipid expression patterns.
또한 난소암 세포주인 A2780-AD에 비해 암 줄기세포주인 A2780-SP 세포에서 발현이 증가 또는 감소된 지질을 분석하였다. 구체적으로, SigmaPlot10.0 소프트웨어를 이용하여, 상기 LC-MS 로우 데이터를 도식화하여 발현이 증가 또는 감소된 지질을 분석하였다. 지질 발현 분석 결과는 도 4에 나타내었다.In addition, lipids with increased or decreased expression in A2780-SP cells, a cancer stem cell line, were analyzed compared to A2780-AD, an ovarian cancer cell line. Specifically, using SigmaPlot10.0 software, the LC-MS raw data was plotted to analyze lipids with increased or decreased expression. The results of lipid expression analysis are shown in FIG. 4 .
도 4에 나타낸 바와 같이, 난소암 세포주 A2780-AD에 비해 난소암 줄기세포주 A2780-SP 세포에서 발현이 증가된 지질은 포스파티딜에탄올아민(phosphatidylethanolamine, PE), 리소포스파티딜에탄올아민(lysophosphatidylethanolamine, LPE), 모노글리세라이드(monoglyceride)를 포함한 93종이며, 발현이 감소된 지질은 포스파티딜콜린(phosphatidylcholine), 리소포스파티딜콜린(lysophosphatidylcholine), 다이글리세라이드(diglyceride)를 포함한 61종인 것을 확인하였다.As shown in Figure 4, lipids with increased expression in the ovarian cancer stem cell line A2780-SP cells compared to the ovarian cancer cell line A2780-AD are phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), mono It was confirmed that there are 93 types including glyceride (monoglyceride), and the lipids with reduced expression are 61 types including phosphatidylcholine, lysophosphatidylcholine, and diglyceride.
특히, 난소암 세포주 A2780-AD에 비해 난소암 줄기세포주 A2780-SP 세포에서 발현이 현저히 증가된 포스파티딜에탄올아민의 발현을 비교하였으며, 그 결과는 도 5에 나타내었다.In particular, the expression of phosphatidylethanolamine, which was significantly increased in the ovarian cancer stem cell line A2780-SP cells compared to the ovarian cancer cell line A2780-AD, was compared, and the results are shown in FIG. 5 .
도 5에 나타낸 바와 같이, 난소암 줄기세포주 A2780-SP 세포는 포스파티딜에탄올아민의 발현이 난소암 세포주 A2780-AD보다 3배 이상 증가한 것을 확인하였다.As shown in FIG. 5 , it was confirmed that the expression of phosphatidylethanolamine in the ovarian cancer stem cell line A2780-SP cells increased more than 3-fold compared to the ovarian cancer cell line A2780-AD.
1-5. 암세포화된 암 줄기세포의 지질 발현 분석1-5. Lipid Expression Analysis of Cancerized Cancer Stem Cells
암세포화된 암 줄기세포의 지질 발현을 분석하기 위하여, 난소암 암 줄기세포주 A2780-SP 세포를 암세포 배양 조건으로 배양하여 암세포화시켰다. 상기 암세포화된 난소암 암 줄기세포는 A2780-DIF로 명명하였다. 상기 A2780-DIF 세포 및 난소암 암 줄기세포(A2780-SP)의 지질 발현은 상기 실시예 1-4와 동일한 방법으로 확인하였다. 분석 결과에 기초하여 매트릭스를 작성하였으며, 작성된 매트릭스는 도 6에 나타내었다.In order to analyze the lipid expression of cancer stem cells that have become cancerous, the ovarian cancer stem cell line A2780-SP cells were cultured under cancer cell culture conditions to form cancer cells. The cancerous ovarian cancer stem cells were named A2780-DIF. Lipid expression of the A2780-DIF cells and ovarian cancer stem cells (A2780-SP) was confirmed in the same manner as in Examples 1-4. A matrix was prepared based on the analysis results, and the created matrix is shown in FIG. 6 .
도 6에 나타낸 바와 같이, A2780-SP 세포 및 A2780-DIF 세포는 지질 발현 패턴이 상이한 것을 확인하였다. 또한 A2780-DIF 세포의 지질 발현 패턴은 난소암 세포주 A2780-AD와 일부 차이가 있으나, 유사한 경향을 가지는 것을 확인하였다.As shown in FIG. 6 , it was confirmed that A2780-SP cells and A2780-DIF cells had different lipid expression patterns. In addition, it was confirmed that the lipid expression pattern of A2780-DIF cells was somewhat different from that of the ovarian cancer cell line A2780-AD, but had a similar tendency.
또한 암 줄기세포주인 A2780-SP 세포에 비해 A2780-DIF에서 발현이 증가 또는 감소된 지질을 분석하였으며, 그 결과는 도 7에 나타내었다.In addition, lipids with increased or decreased expression in A2780-DIF compared to A2780-SP cells, a cancer stem cell line, were analyzed, and the results are shown in FIG. 7 .
도 7에 나타낸 바와 같이, 난소암 줄기세포주 A2780-SP 세포에 비해 난소암 세포주 A2780-DIF에서 발현이 감소된 지질은 포스파티딜에탄올아민(phosphatidylethanolamine, PE)을 포함한 87종이며, 발현이 증가된 지질은 포스파티딜콜린(phosphatidylcholine), 리소포스파티딜콜린(lysophosphatidylcholine), 다이글리세라이드(diglyceride) 등 84종인 것을 확인하였다.As shown in Figure 7, lipids with reduced expression in the ovarian cancer cell line A2780-DIF compared to the ovarian cancer stem cell line A2780-SP cells are 87 types including phosphatidylethanolamine (PE), and the lipids with increased expression are It was confirmed that phosphatidylcholine (phosphatidylcholine), lysophosphatidylcholine (lysophosphatidylcholine), diglyceride (diglyceride), such as 84 kinds.
또한 A2780-DIF 세포 및 A2780-SP 세포의 포스파티딜에탄올아민 발현을 비교하였으며, 그 결과는 도 8에 나타내었다.In addition, the expression of phosphatidylethanolamine in A2780-DIF cells and A2780-SP cells was compared, and the results are shown in FIG. 8 .
도 8에 나타낸 바와 같이, A2780-DIF 세포는 A2780-SP에 비해 포스파티딜에탄올아민의 발현이 감소한 것을 확인하였다.As shown in FIG. 8 , it was confirmed that the expression of phosphatidylethanolamine was reduced in A2780-DIF cells compared to A2780-SP.
실시예 2. 환자 유래 난소암 암 줄기세포 및 암세포화된 환자 유래 난소암 암 줄기세포의 지질 발현 비교Example 2. Comparison of lipid expression of patient-derived ovarian cancer cancer stem cells and cancerousized patient-derived ovarian cancer cancer stem cells
환자로부터 난소암 암 줄기세포를 분리하였으며, EOC-SP로 명명하였다. 상기 EOC-SP 세포를 암세포 배양 조건으로 배양하여 암세포화시켰다. 상기 암세포화된 환자 유래 난소암 암 줄기세포는 EOC-DIF로 명명하였다.Ovarian cancer stem cells were isolated from the patient and named as EOC-SP. The EOC-SP cells were cultured under cancer cell culture conditions to form cancer cells. The ovarian cancer stem cells derived from the cancerousized patient were named EOC-DIF.
준비된 EOC-SP 세포 및 EOC-DIF 세포는 후술되는 실험에 이용되었다.The prepared EOC-SP cells and EOC-DIF cells were used in the experiments described below.
2-1. 형태학적 분석2-1. Morphological analysis
준비된 EOC-SP 세포 및 EOC-DIF 세포의 형태학적 분석을 수행하였다. 구체적으로, 상기 세포들을 각각 3차원 배양한 후 현미경으로 관찰하였다. 형태학적 분석 결과는 도 9에 나타내었다.Morphological analysis of the prepared EOC-SP cells and EOC-DIF cells was performed. Specifically, the cells were each three-dimensionally cultured and then observed under a microscope. The morphological analysis results are shown in FIG. 9 .
도 9에 나타낸 바와 같이, EOC-SP 세포 및 EOC-DIF 세포는 형태학적으로 차이가 있는 것을 확인하였다. 특히, EOC-DIF 세포는 암세포화된 것인바, 암세포와 유사한 형태를 보였다.As shown in FIG. 9 , it was confirmed that EOC-SP cells and EOC-DIF cells were morphologically different. In particular, the EOC-DIF cells were cancer cells, and showed a morphology similar to that of cancer cells.
2-2. 암 줄기세포 마커 발현 확인2-2. Confirmation of cancer stem cell marker expression
EOC-SP 세포 및 EOC-DIF 세포에서 암 줄기세포의 마커인 알데하이드 탈수소효소의 발현을 확인하였다. 구체적으로, ALDEFOUOR™ 키트(STEMCELL Technologies)를 사용하여, EOC-SP 세포 중 ALDH를 발현하는 세포를 제조사의 매뉴얼에 따라 표지하였다. 그 후 FACS AttuneNxT(ThermoFisher)를 이용하여 ALDH를 발현하는 세포를 측정하였다. EOC-DIF 세포도 동일한 방법으로 ALDH의 발현을 측정하였다. 암 줄기세포 마커를 발현하는 세포를 측정한 결과는 도 10에 나타내었다.Expression of aldehyde dehydrogenase, a marker of cancer stem cells, was confirmed in EOC-SP cells and EOC-DIF cells. Specifically, using the ALDEFOUOR™ kit (STEMCELL Technologies), ALDH-expressing cells among EOC-SP cells were labeled according to the manufacturer's manual. Thereafter, cells expressing ALDH were measured using FACS AttuneNxT (ThermoFisher). EOC-DIF cells were also measured for ALDH expression in the same manner. The results of measuring cells expressing cancer stem cell markers are shown in FIG. 10 .
도 10에 나타낸 바와 같이, 환자 유래 난소암 암 줄기세포주인 EOC-SP 세포는 ALDH의 발현이 EOC-DIF 세포에 비해 현저히 높은 것을 확인하였다.As shown in FIG. 10 , it was confirmed that the expression of ALDH in EOC-SP cells, a patient-derived ovarian cancer stem cell line, was significantly higher than in EOC-DIF cells.
2-3. EOC-SP 세포 및 EOC-DIF 세포의 지질 발현 분석2-3. Lipid Expression Analysis of EOC-SP Cells and EOC-DIF Cells
EOC-SP 세포 및 EOC-DIF 세포의 지질 발현을 분석하였다. 구체적으로, 부착세포인 EOC-DIF 세포는 트립신을 처리하여 세포 펠릿을 수집하였다. 부유세포인 EOC-SP 세포는 배양액을 회수한 후 스핀다운(spin down)하여 배지를 제거하여 세포 펠릿을 수집하였다. 수집된 세포는 실시예 1-4의 방법과 같은 방법으로 지질 발현을 분석하였다. 분석 결과에 기초하여 매트릭스를 작성하였으며, 작성된 매트릭스는 도 11에 나타내었다.Lipid expression of EOC-SP cells and EOC-DIF cells was analyzed. Specifically, adherent EOC-DIF cells were treated with trypsin to collect cell pellets. EOC-SP cells, which are floating cells, were spun down after recovering the culture medium to remove the medium to collect cell pellets. The collected cells were analyzed for lipid expression in the same manner as in Examples 1-4. A matrix was prepared based on the analysis results, and the created matrix is shown in FIG. 11 .
도 11에 나타낸 바와 같이, EOC-SP 세포 및 EOC-DIF 세포는 지질 발현 패턴이 상이한 것을 확인하였다.11 , it was confirmed that EOC-SP cells and EOC-DIF cells had different lipid expression patterns.
EOC-SP 세포에 비해 암세포화 된 환자 유래 난소암 암 줄기세포주인 EOC-DIF 세포에서 발현이 증가 또는 감소된 지질을 분석하였으며, 그 결과는 도 12에 나타내었다.Lipids with increased or decreased expression in EOC-DIF cells, a patient-derived ovarian cancer stem cell line that have become cancerous compared to EOC-SP cells, were analyzed, and the results are shown in FIG. 12 .
도 12에 나타낸 바와 같이, EOC-DIF 세포에 비해 EOC-SP 세포에서 발현이 증가된 지질은 포스파티딜에탄올아민, 포스파티딜콜린 (phosphatidylcholine), 리소포스파티딜콜린(lysophosphatidylcholine)을 포함한 87종이며, 발현이 감소 된 지질은 포스파티딜세린(phosphatidylserine)을 포함한 84종인 것을 확인하였다.As shown in FIG. 12, lipids with increased expression in EOC-SP cells compared to EOC-DIF cells are 87 types, including phosphatidylethanolamine, phosphatidylcholine, and lysophosphatidylcholine, and lipids with reduced expression are It was confirmed that 84 types including phosphatidylserine.
EOC-SP 세포 및 EOC-DIF 세포의 포스파티딜에탄올아민의 발현을 비교하였으며, 그 결과는 도 13에 나타내었다.The expression of phosphatidylethanolamine in EOC-SP cells and EOC-DIF cells was compared, and the results are shown in FIG. 13 .
도 13에 나타낸 바와 같이, 환자 유래 난소암 암 줄기세포인 EOC-SP 세포는 포스파티딜에탄올아민의 발현이 EOC-DIF 세포에 비해 2.47배 높은 것을 확인하였다.As shown in FIG. 13 , it was confirmed that the expression of phosphatidylethanolamine in EOC-SP cells, which are patient-derived ovarian cancer stem cells, was 2.47 times higher than in EOC-DIF cells.
실시예 3. 난소암 암 줄기세포에서 포스파티딜에탄올아민의 노출 여부 분석Example 3. Analysis of exposure to phosphatidylethanolamine in ovarian cancer cancer stem cells
3-1. 난소암 세포주 A2780-AD 세포 및 난소암 암 줄기세포주 A2780-SP 세포의 비교3-1. Comparison of ovarian cancer cell line A2780-AD cells and ovarian cancer stem cell line A2780-SP cells
유세포 분석을 통해 난소암 세포주(A2780-AD) 및 난소암 암 줄기세포주(A2780-SP)에서 포스파티딜에탄올아민의 노출 여부를 확인하였다. 구체적으로, 포스파티틸에탄올아민에 특이적으로 결합하는 Duramycin-Cy5 Conjugate 형광 염료와 A2780-AD 세포를 함께 배양한 후 유세포 분석을 수행하였다. 유세포 분석 결과에서 형광은 포스파티딜에탄올아민이 세포막 외부로 노출된 것임을 의미한다. A2780-SP 세포도 위와 동일한 방법으로 유세포 분석을 수행하였다. 유세포 분석 결과는 도 14에 나타내었다.Through flow cytometry, it was confirmed whether phosphatidylethanolamine was exposed in the ovarian cancer cell line (A2780-AD) and the ovarian cancer cancer stem cell line (A2780-SP). Specifically, A2780-AD cells were co-cultured with Duramycin-Cy5 Conjugate fluorescent dye that specifically binds to phosphatethylethanolamine, and then flow cytometry was performed. Fluorescence in flow cytometry results means that phosphatidylethanolamine is exposed to the outside of the cell membrane. A2780-SP cells were also subjected to flow cytometry analysis in the same manner as above. The flow cytometry results are shown in FIG. 14 .
도 14에 나타낸 바와 같이, 난소암 암 줄기세포주 A2780-SP 세포는 포스파티딜에탄올아민의 발현이 A2780-AD 세포에 비해 높았고, 발현된 포스파티딜에탄올아민은 세포막 외부로 노출된다는 것을 확인하였다.As shown in FIG. 14 , it was confirmed that the expression of phosphatidylethanolamine was higher in the ovarian cancer stem cell line A2780-SP cells than in A2780-AD cells, and the expressed phosphatidylethanolamine was exposed to the outside of the cell membrane.
3-2. 난소암 세포주 SKOV3-AD 세포 및 난소암 암 줄기세포주 SKOV3-SP 세포의 비교3-2. Comparison of ovarian cancer cell line SKOV3-AD cells and ovarian cancer stem cell line SKOV3-SP cells
유세포 분석을 통해, 다른 난소암 세포주인 SKOV3-AD 세포 및 이의 암 줄기세포주인 SKOV3-SP 세포에서 포스파티딜에탄올아민의 발현을 실시예 3-1과 동일한 방법으로 분석하였다. 상기 난소암 암 줄기세포주 SKOV3-SP 세포는 난소암 세포주 SKOV3-AD 세포를 3차원 배양한 후 분리한 것이다. 유세포 분석 결과는 도 15에 나타내었다. Through flow cytometry, the expression of phosphatidylethanolamine in SKOV3-AD cells, another ovarian cancer cell line, and SKOV3-SP cells, a cancer stem cell line thereof, was analyzed in the same manner as in Example 3-1. The ovarian cancer stem cell line SKOV3-SP cells are isolated after three-dimensional culture of the ovarian cancer cell line SKOV3-AD cells. The flow cytometry results are shown in FIG. 15 .
도 15에 나타낸 바와 같이, 난소암 암 줄기세포주 SKOV3-SP 세포는 포스파티딜에탄올아민의 발현이 난소암 세포주 SKOV3-AD에 비해 현저히 높았고, 발현된 포스파티딜에탄올아민은 세포막 외부로 노출된다는 것을 확인하였다.As shown in FIG. 15 , the expression of phosphatidylethanolamine in the ovarian cancer stem cell line SKOV3-SP cells was significantly higher than that of the ovarian cancer cell line SKOV3-AD, and it was confirmed that the expressed phosphatidylethanolamine was exposed to the outside of the cell membrane.
3-3. 환자 유래 난소암 암 줄기세포주 EOC12-SP 및 EOC21SP 세포의 비교3-3. Comparison of patient-derived ovarian cancer cancer stem cell lines EOC12-SP and EOC21SP cells
환자 유래 난소암 암 줄기세포주 EOC12-SP 세포 및 EOC21-SP 세포를 준비하였다. 상기 EOC12-SP 세포 및 EOC21-SP 세포에서 포스파티딜에탄올아민의 발현을 실시예 3-1과 동일한 방법으로 분석하였다. 유세포 분석 결과는 도 16에 나타내었다.Patient-derived ovarian cancer cancer stem cell lines EOC12-SP cells and EOC21-SP cells were prepared. The expression of phosphatidylethanolamine in the EOC12-SP cells and the EOC21-SP cells was analyzed in the same manner as in Example 3-1. The flow cytometry results are shown in FIG. 16 .
도 16에 나타낸 바와 같이, 환자 유래 난소암 암 줄기세포주인 EOC12-SP 세포 및 EOC21-SP 세포는 모두 포스파티딜에탄올아민의 발현 수준이 높으며, 발현된 포스파티딜에탄올아민은 세포막 외부로 노출된다는 것을 확인하였다.As shown in Figure 16, the patient-derived ovarian cancer stem cell lines, EOC12-SP cells and EOC21-SP cells, both had high levels of phosphatidylethanolamine expression, and it was confirmed that the expressed phosphatidylethanolamine was exposed to the outside of the cell membrane.
3-4. 인간 배아 유래 신장세포, 편도 유래 중간엽 줄기세포 및 인간 탯줄 정맥내피세포의 비교3-4. Comparison of human embryonic kidney cells, tonsil-derived mesenchymal stem cells, and human umbilical vein endothelial cells
상기 실시예 3-1 내지 3-3의 난소암 암 줄기세포와 비교하기 위하여, 인간 배아 유래 신장세포(HEK293FT), 편도 유래 중간엽 줄기세포(Tonsil-derived mesenchymal stem cell, TMSC) 및 인간 탯줄 정맥내피세포(Human umbilical vein endothelial cell, HUVEC)에서 유세포 분석을 통해 포스파티딜에탄올아민의 발현을 확인하였다. 상기 유세포 분석은 실시예 3-1과 동일한 방법으로 수행하였으며, 그 결과는 도 17에 나타내었다.In order to compare with the ovarian cancer stem cells of Examples 3-1 to 3-3, human embryonic kidney cells (HEK293FT), tonsil-derived mesenchymal stem cells (Tonsil-derived mesenchymal stem cells, TMSC) and human umbilical vein The expression of phosphatidylethanolamine was confirmed by flow cytometry in human umbilical vein endothelial cells (HUVEC). The flow cytometry was performed in the same manner as in Example 3-1, and the results are shown in FIG. 17 .
도 17에 나타낸 바와 같이, TMSC, HEK293FT 및 HUVEC는 포스파티딜에탄올아민의 발현이 매우 적으며, 발현된 포스파티딜에탄올아민은 세포막 외부에 노출된다는 것을 확인하였다.As shown in FIG. 17, TMSC, HEK293FT and HUVEC showed very little expression of phosphatidylethanolamine, and it was confirmed that the expressed phosphatidylethanolamine was exposed to the outside of the cell membrane.
종합적으로 본 발명자들은 난소암 암 줄기세포에서 포스파티딜에탄올아민의 발현이 난소암 세포주 및 다른 세포주에 비해 현저히 높은 것을 확인하였다. 또한 상기 난소암 암 줄기세포는 발현된 포스파티딜에탄올아민이 세포막 외부로 노출된다는 것을 추가 실험을 통해 확인하였다. 이는 포스파티딜에탄올아민이 암 줄기세포 검출용 바이오 마커로 이용될 수 있음을 의미하는바, 본 발명의 포스파티딜에탄올은 암의 진단, 예후 예측 및 약물 스크리닝 분야에서 다양하게 활용될 수 있다.Overall, the present inventors confirmed that the expression of phosphatidylethanolamine in ovarian cancer stem cells was significantly higher than in ovarian cancer cell lines and other cell lines. In addition, it was confirmed through additional experiments that the ovarian cancer stem cells are exposed to the outside of the cell membrane expressed phosphatidylethanolamine. This means that phosphatidylethanolamine can be used as a biomarker for cancer stem cell detection, and the phosphatidylethanol of the present invention can be variously used in the fields of cancer diagnosis, prognosis prediction, and drug screening.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다. Above, specific parts of the present invention have been described in detail, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (10)

  1. 포스파티딜에탄올아민(phosphatidylethanolamine, PE)을 포함하는 암 줄기세포 검출용 바이오 마커 조성물.A biomarker composition for detecting cancer stem cells comprising phosphatidylethanolamine (PE).
  2. 제1항에 있어서,According to claim 1,
    상기 포스파티딜에탄올아민은 세포의 표면에서 발현되는 것인, 암 줄기세포 검출용 바이오 마커 조성물.The phosphatidylethanolamine is expressed on the surface of the cell, a biomarker composition for detecting cancer stem cells.
  3. 제1항에 있어서,According to claim 1,
    상기 암은 위암(gastric cancer), 유방암(breast cancer), 폐암(lung cancer), 간암(liver cancer), 혈액암(blood cancer), 뼈암(bone cancer), 췌장암(pancreatic cancer), 피부암(skin cancer), 머리 또는 목암(head or neck cancer), 피부 또는 안구 흑색종(cutaneous or intraocular melanoma), 자궁육종(uterine sarcoma), 난소암(ovarian cancer), 직장암(rectal cancer), 항문암(anal cancer), 대장암(colon cancer), 난관암(fallopian tube carcinoma), 자궁내막암(endometrial carcinoma), 자궁경부암(cervical cancer), 소장암(small intestine cancer), 내분비암(endocrine cancer), 갑상선암(thyroid cancer), 부갑상선암(parathyroid cancer), 신장암(adrenal cancer), 연조직종양(soft tissue tumor), 요도암(urethral cancer), 전립선암(prostate cancer), 기관지암(bronchogenic cancer) 및 골수암(bone marrow tumor)으로 이루어진 군에서 선택된 1 종 이상인, 암 줄기세포 검출용 바이오 마커 조성물.The cancer is gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer ), head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer , colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer ), parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchogenic cancer and bone marrow tumor ) at least one selected from the group consisting of, a biomarker composition for detecting cancer stem cells.
  4. 제1항에 있어서,According to claim 1,
    상기 포스파티딜에탄올아민은 암세포보다 암 줄기세포에서 발현이 높은 것인, 암 줄기세포 검출용 바이오 마커 조성물.The phosphatidylethanolamine is a biomarker composition for detecting cancer stem cells, wherein the expression is higher in cancer stem cells than in cancer cells.
  5. 포스파티딜에탄올아민(phosphatidylethanolamine, PE)의 발현을 측정하는 제제를 포함하는, 암 줄기세포 검출을 통한 암 진단용 조성물.A composition for diagnosing cancer through cancer stem cell detection, comprising an agent for measuring the expression of phosphatidylethanolamine (PE).
  6. 제5항에 있어서,6. The method of claim 5,
    상기 포스파티딜에탄올아민의 발현을 측정하는 제제는 포스파티딜에탄올아민에 특이적으로 결합하는 올리고펩타이드, 모노클로날 항체, 폴리클로날 항체, 키메릭(chimeric) 항체, 리간드, PNA(Peptide nucleic acid), 앱타머(aptamer), 프로브 및 염료로 이루어진 군에서 선택되는 것인, 암 줄기세포 검출을 통한 암 진단용 조성물.The agent for measuring the expression of the phosphatidylethanolamine is an oligopeptide, monoclonal antibody, polyclonal antibody, chimeric antibody, ligand, PNA (Peptide nucleic acid), app that specifically binds to phosphatidylethanolamine. A composition for diagnosing cancer through detection of cancer stem cells, which is selected from the group consisting of tamers, probes and dyes.
  7. 제5항 또는 제6항의 조성물을 포함하는 암 줄기세포 검출을 통한 암 진단 키트.A cancer diagnosis kit through cancer stem cell detection comprising the composition of claim 5 or 6.
  8. 생물학적 시료에서 포스파티딜에탄올아민(phosphatidylethanolamine, PE)의 발현을 측정하는 단계를 포함하는, 암 줄기세포 검출을 통한 암 진단에 대한 정보 제공 방법.A method for providing information on cancer diagnosis through cancer stem cell detection, comprising measuring the expression of phosphatidylethanolamine (PE) in a biological sample.
  9. (a) 개체로부터 분석 시료를 얻는 단계;(a) obtaining an assay sample from the subject;
    (b) 단계 (a)의 분석 시료에서 포스파티딜에탄올아민(phosphatidylethanolamine, PE)의 존재여부를 검출하는 단계;(b) detecting the presence or absence of phosphatidylethanolamine (PE) in the analysis sample of step (a);
    (c) 단계 (b)에서 포스파티딜에탄올아민이 분석 시료에 존재할 경우 암으로 진단하는 단계;(c) diagnosing cancer when phosphatidylethanolamine is present in the analyte sample in step (b);
    (d) 단계 (c)에서 암으로 진단된 개체에 유효량의 암 줄기세포 특이적 항암제를 투여하는 단계;를 포함하는 암 진단 또는 치료방법.(d) administering an effective amount of a cancer stem cell-specific anticancer agent to the individual diagnosed with cancer in step (c); cancer diagnosis or treatment method comprising a.
  10. 제9항에 있어서,10. The method of claim 9,
    상기 암 줄기세포 특이적 항암제는 나이트로젠 머스타드, 이마티닙, 옥살리플라틴, 리툭시맙, 엘로티닙, 네라티닙, 라파티닙, 제피티닙, 반데타닙, 니로티닙, 세마사닙, 보수티닙, 악시티닙, 세디라닙, 레스타우르티닙, 트라스투주맙, 게피티니브, 보르테조밉, 수니티닙, 카보플라틴, 소라페닙, 베바시주맙, 시스플라틴, 세툭시맙, 비스쿰알붐, 아스파라기나제, 트레티노인, 하이드록시카바마이드, 다사티닙, 에스트라머스틴, 겜투주맵오조가마이신, 이브리투모맙튜세탄, 헵타플라틴, 메칠아미노레불린산, 암사크린, 알렘투주맙, 프로카르바진, 알프로스타딜, 질산홀뮴 키토산, 젬시타빈, 독시플루리딘, 페메트렉세드, 테가푸르, 카페시타빈, 기메라신, 오테라실, 아자시티딘, 메토트렉세이트, 우라실, 시타라빈, 플루오로우라실, 플루다가빈, 에노시타빈, 플루타미드, 데시타빈, 카페시타빈, 머캅토푸린, 티오구아닌, 클라드리빈, 카르모퍼, 랄티트렉세드, 도세탁셀, 파클리탁셀, 이리노테칸, 벨로테칸, 토포테칸, 비노렐빈, 에토포시드, 빈크리스틴, 빈블라스틴, 테니포시드, 독소루비신, 이다루비신, 에피루비신, 미톡산트론, 미토마이신, 블레로마이신, 다우노루비신, 닥티노마이신, 피라루비신, 아클라루비신, 페프로마이신, 템시롤리무스, 테모졸로마이드, 부설판, 이포스파미드, 사이클로포스파미드, 멜파란, 알트레트민, 다카바진, 치오테파, 니무스틴, 클로람부실, 미토락톨, 레우코보린, 트레토닌, 엑스메스탄, 아미노글루테시미드, 아나그렐리드, 나벨빈, 파드라졸, 타목시펜, 토레미펜, 테스토락톤, 아나스트로졸, 레트로졸, 보로졸, 비칼루타미드, 로무스틴, 보리노스텟, 엔티노스텟, 5FU 및 카르무스틴으로 이루어진 군에서 선택된 1종 이상인, 암 진단 또는 치료방법.The cancer stem cell-specific anticancer agent is nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib , cediranib, restautinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscumalbum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tuccetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil , holmium nitrate chitosan, gemcitabine, doxyfluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine , enocitabine, flutamide, decitabine, capecitabine, mercaptopurine, thioguanine, cladribine, carmofer, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, eto Forsyd, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pyrarubicin, aclarubicin Cin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melparan, altretmine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, re Ucovorin, tretonin, exemestane, aminoglutethimide, anagrelide, nabelbine, fadrazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, Lomustine, vorinostat, entinostat, 5FU and at least one selected from the group consisting of carmustine, cancer diagnosis or treatment method.
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