WO2009061837A1

WO2009061837A1 - Neoplastic stem cell system and method

Info

Publication number: WO2009061837A1
Application number: PCT/US2008/082506
Authority: WO
Inventors: Christopher Duntsch; Valery Kukekeov; Tatyana Igantova
Original assignee: Novostem Therapeutics Inc
Priority date: 2007-11-05
Filing date: 2008-11-05
Publication date: 2009-05-14

Abstract

A neoplastic stem cell population enriched for expression of the OCT4 transcription factor as well as methods for their identification, isolation and enrichment are described. The OCT4-enriched neoplastic stem cell population is further utilized for the induction and analysis of cancer in an animal. In addition, methods of preventing, abrogating, or inhibiting cancer, tumor growth, and metastasis via OCT4 inhibition are further provided.

Description

NEOPLASTIC STEM CELL SYSTEM AND METHOD

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This Application claims the benefit of United States Provisional Application Serial Number 60/996, 171 , filed November 5, 2007, which claims the benefit of United States Patent Application Serial Number 11/806,993, filed June 5, 2007, which claims the benefit of United States Provisional Application Serial Number 60/811,095, filed June 6, 2006, all of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION [002] To date, methods of analysis of neoplastic cells are neither efficient nor uniform enough for research purposes. Neoplastic stem cells isolated from tissue samples by various fractionation procedures consisted of mixed cell types. Efficient isolation of neoplastic stem cells provides a means of exploring basic mechanisms in cancer cell biology and disease. Methods for specifically and efficiently isolating and propagating a cell subpopulation to provide a large neoplastic stem cell population for in- vitro and in- vivo studies are desirable.

[003] Identification of stem cell markers in neoplastic cells provides valuable information that is useful for a variety of applications in both clinical and basic research settings. The identification and subsequent isolation of neoplastic stem cells (NSCs) from a particular tumor or metastatic lesion is useful, for example, in diagnosing a pathology and/or developing a rational therapeutic treatment that targets a developing pathology. In some instances, isolation and/or enrichment of NSCs is desirable for further in-vitro studies exploring physiological and molecular mechanisms, wherein in other instances, these cells can be used to inoculate a test animal for further studies of cancer progression or therapy. [004] NSCs can be sorted using techniques such as FACS (Fig. 10) or antibiotic selection assays that do not distinguish between sub-populations of cells based on their biological activity and/or physiological function. The assays, moreover, preclude recovery of native non- antibiotic-expressing or treated stem cells. Other methods of cellular identification and subsequent isolation and/or enrichment such as gel electrophoresis, fail to probe pure populations, suffer from contamination and/or compromise cell viability. SUMMARY OF THE INVENTION

[005] This invention relates, in one embodiment, to a method for testing a candidate compound for an ability to transform a cell to a neoplastic stem cell (NSC), comprising the steps of: (a)contacting a cell population with a candidate compound, wherein the cell population has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and (b) measuring the expression of an identifiable gene product;

[006] In another embodiment, the invention further provides a method for testing a candidate compound for an ability to inhibit OCT4 expression in a neoplastic stem cell (NSC), comprising the steps of: (a)contacting a cell population enriched for OCT4 expression with a candidate compound, wherein the cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and (b) measuring the expression of an identifiable gene product. [007] In another embodiment, the invention further provides a method for testing a candidate compound for an ability to inhibit the proliferation of a neoplastic stem cell (NSC), comprising the steps of: (a)contacting a cell population enriched for OCT4 expression with a candidate compound, wherein the cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and; (b) measuring the expression of an identifiable gene product.

[008] In another embodiment, the invention further provides a system for testing a candidate compound for an ability to inhibit a neoplastic stem cell population, comprising an isolated neoplastic stem cell population enriched for expression of OCT4, wherein isolated neoplastic stem cell population enriched for expression of OCT4 is transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product.

[009] In another embodiment, the invention further provides a system for testing a candidate compound for an ability to transform a neoplastic cell population to a neoplastic stem cell population, comprising an isolated bulk cancer cell (BCC) population, wherein isolated BCC population is transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product. [0010] In another embodiment, the invention further provides a system for testing a candidate compound for an ability to transform a non-neoplastic cell population to a neoplastic stem cell population, comprising a non-neoplastic cell population, wherein non-neoplastic cell population is transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product

[0011] In another embodiment, the invention further provides a method for testing a candidate compound for an ability to transform a neoplastic stem cell (NSC) to a bulk cancer cell (BCC) comprising the steps of: (a) contacting a cell population enriched for OCT4 expression with a candidate compound, wherein the cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and (b) measuring the expression of an identifiable gene product.

[0012] In another embodiment, the invention further provides a method for testing a candidate compound for an ability to inhibit the asymmetric division of a neoplastic stem cell (NSC) comprising the steps of: (a) contacting a cell population enriched for OCT4 expression with a candidate compound, wherein the cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and (b) measuring the expression of an identifiable gene product; [0013] In another embodiment, the invention further provides a method for testing a candidate compound for an ability to inhibit the symmetric division of a neoplastic stem cell (NSC) comprising the steps of: (a) contacting a cell population enriched for OCT4 expression with a candidate compound, wherein the cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and (b) measuring the expression of an identifiable gene product.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which: [0015] Fig. 1 presents light micrographs of serial sections of solid tumors, probed with polyclonal anti-OCT4 antibodies and control sections. Fig. IA is a cross section of a chondrosarcoma tumor; Fig. IB is a cross section of an osteosarcoma tumor; Fig. 1C is a cross section of a glioblastoma multiforme (GBM) tumor; and Fig. ID is a cross section of fetal human testis used for positive control. Arrows indicate OCT4 positive nuclei.

[0016] Fig. 2A demonstrates the results of semi-quantitive RT-PCR probing for OCT4, STAT3 and Nanog mRNA expression wherein β-tubulin and GFAP served as normalized controls in representative glioblastoma primary cultures (MT917, MT926, MT928, MT1231) and cell lines (LN18, LN229, LN428, U251). Fig. 2B demonstrates results of a Western blot analysis of OCT4, STAT3 and Nanog protein expression wherein β-tubulin and GFAP served as normalized controls in cell lines (LN18, LN229, LN319, LN428, D247, U251, U373, T98G). Blots were probed with anti-OCT4, anti-phospho-STAT3 and anti-Nanog. [0017] Fig. 3 plots the results of 2-D quantitative PCR probing OCT4 and nanog gene expression in adherent cell cultures and floating osteosarcoma-derived spheres. Substrate- attached cultures showed significantly (p<0.05) lower expression of OCT4 and Nanog. Correlation of OCT4 (X axis) and Nanog (Y axis) expression in sarcospheres is significantly (p<0.05) higher than in substrate-attached cultures.

[0018] Fig. 4 illustrates clone-forming potential of glioblastoma-derived cells suppressed by OCT4 siRNA. Fig. 4A demonstrates results of a Western blot analysis wherein suppression of exogenous OCT4 protein in a transfected cell culture was achieved by treatment with specific OCT4 siRNA comprising the DNA sequence: TTGATCCTCGGACCTGGCTAA. Fig. 4B plots the frequency of clone-formation by selected glioblastoma cells (MT317, LN-229, MT- 917). Cells were co-transfected with eGFP (Green Fluorescent Protein) and OCT4 siRNA. Experiments were performed in triplicate, bars represent standard errors. [0019] Fig. 5A is a light micrograph image (x200) of suspended mammaspheres derived from an MCF-7R breast cancer cell line and cultured in methylcellulose. Fig. 5B is a fluorescent micrograph (x200) of a mammasphere transferred from methylcellulose, attached to substratum and immunostained for OCT4 (white) and pancytokeratine (gray) expression. [0020] Fig.6 presents light micrographs (x200) of immunohistochemically stained breast cancer tumors probed for OCT4 expression. Dark punctuate staining are OCT4 positive nuclei. Fig. 6A is a cross section of ductal carcinoma tumor, and Fig. 6B is a cross section of breast cancer metastasis to brain. Arrows indicate OCT4 positive nuclei. [0021] Fig. 7 is a fluorescent microscope (x200) image of an OCT4-EGFP transfected glioblastoma cell in methyl cellulose after first division (Fig. 7A). A glioblastoma floating neurosphere (clone) of OCT-EGFP transfected cells after several rounds of divisions is shown in Fig. 7B. [0022] Fig. 8 is a fluorescent microscope image (x200) of cultured breast cancer cells (A), osteosarcoma (B), and glioblastoma multiforme cells (C) expressing EGFP through an OCT4 responsive promoter.

[0023] Fig. 9 is a fluorescent microscope image of the cultured glioblastoma cell line Ln428 (x200) (A) and osteosarcoma OS521 (xlOO) (B) expressing EGFP through Nanog responsive promoter.

[0024] Fig. 10 illustrates FACS flow isolation graphs of subpopulations of tumor cells expressing OCT4 from cultured glioblastoma cell line Ln428. M2 gate represents OCT4 positive cells. Fig. 1OA represents the initial FACS sorting for OCT4 positive cells of a mixed clonal-OCT4 cell population. Fig. 1OB represents isolated OCT4 positive cells after two passages (roughly 2 weeks) followed by FACS analysis (B) to determine their purity. This population was found to be 96.16 % pure for OCT4 protein expression.

[0025] Fig. 11 is a graph illustrating the tumor forming potential of OCT4 positive and OCT4 negative MDA MB 231 breast cancer cells transfected with OCT4-EGFR. [0026] Fig. 12 is a map illustrating DNA expression vectors Oct4hP-eGFP comprising the human Oct-4 promoter (Fig. 12A). Fig. 12B illustrates the plasmid pDsRed-Monomer-Cl Clontech en bloc (Catalog # 632466).

[0027] Fig. 13 is a microscope image (x200) of mammosphere cultures derived from an MDA-MB-435 melanoma cell line, biomarked for the presence of NSC expressing Oct-3/4. Fig. 13A is a light micrograph of suspended tumor-derived spheres cultured in methylcellulose. Fig. 13B is a fluorescent micrograph of the suspended tumor-derived spheres shown in Fig. 13A. Fig. 13C is a light micrograph of tumor spheres after attachment to the substratum. Fig. 13D is a fluorescent micrograph of the attached tumor spheres shown in Fig. 13C. [0028] Fig. 14 is a graph showing GFP expression in NSC mass cultures grown under different conditions for extended periods of time. Fig 14A is graph showing the percent of MDA-MB-435 cells that were GFP positive in either of the two media conditions at 0, 30 and 54 days as determined by FACS analysis. Fig 14B is a scatter plot showing the percent of MDA-MB-435 cells cultured in Novopro cancer stem cell media and under NSC promoting conditions.

[0029] Fig. 15 is graphs showing a measurement of GFP and RFP (Red Fluorescent Protein) fluorescence as an indirect correlation of NSC and overall stem cell viability. Fig. 15A is a graph showing measurement of GFP (NSC) and RFP (total cancer cell) expressing populations over time in NSC promoting conditions. Fig. 15B is a graph showing Ratio of GFP to RFP fluorescence for MB435 NSCs under NSC promoting conditions. The ratio is calculated as the ratio of overall Oct-4 transcription level of the population to the overall cell number. [0030] Fig. 16 is a graph showing a GFP fluorescence of MB435 NSCs grown with NSC promoting media and conditions and variable concentrations of Doxorubicin. The graph represents overall GFP fluorescence.

[0031] Fig. 17 is a graph showing RFP fluorescence of MB435 bulk cancer cells grown with NSC promoting media and conditions and variable concentrations of Doxorubicin. The graph represents overall RFP fluorescence. [0032] Fig. 18 is a graph showing RFP fluorescence of MB435 bulk cancer cells grown with NSC promoting media and conditions and variable concentrations of Doxorubicin. All sample fluorescence readings were corrected for background media auto-fluorescence by subtracting the fluorescence readings from blank wells with the appropriate amount of media. The gray lines represent overall GFP fluorescence. The black lines represent overall RFP fluorescence. [0033] Fig. 19 is a graph showing the viability of MB435 NSCs and bulk cancer cells grown with NSC promoting media conditions and variable concentrations of Doxorubicin. This graph shows a MTS Cell Proliferation Assay for the above described conditions. The gray solid line shows absorbance at 492nm for the pure NSC population. The black solid line shows absorbance at 492nm for the pure bulk cancer cell (BCC) starting population under various concentrations of Doxorubicin. This graph highlights that BCCs are much more sensitive than NSCs to the toxic effect of Doxorubicin and confirms the results of Figure 9. [0034] Fig. 20 schematically depicts the fate of NSCs daughter cells. Stage A: Fluorescent micrograph of a parental NSC assymetrically dividing to give rise to one NSC and one daughter cancer cell (a non-cancer stem cell or bulk cancer cell). Stage B: Fluorescence micrograph showing the continued asymmetric division of a NSC producing one daughter stem cell and a cluster of many BCCs. Stage C: Fluorescence micrograph showing the symmetric division of an osteosarcoma NSC producing many daughter NSCs in a sphere-like cluster. [0035] Fig. 21 schematically depicts the procedure for obtaining OCT4 enriched tumor stem cells from any tumor tissue for cancer related studies including drug discovery studies. Stage A: Preparation of Cells: 1) Surgical removal of tumor 2) Mincing and preparations to create a single cells suspension. Stage B: Stable labeling of tumor stem cells with OCT4 responsive promoter: 1) Tumor stem cell culture in a single cell suspension for expantion and selection for tumor stem cells under the appropriate conditions 2) transfection with a plasmid comprising an EGFP gene under the control of an OCT4 responsive promoter (stage C). Stage C: Creation of a highly pure tumor stem cell cultures: EGFP expressing cells are further selected via FACS and re-cultured for expansion resulting in bulk culture quantities. Stage D: Dl, tumor stem cells are further studied using rigorous cell and molecular biology techniques. D2, tumor stem cells are exposed to a vast variety of drugs Stage E: Isolated tumor stem cells are inoculated into immunodeficient mice to create xenograft tumor models followed by basic efficacy, safety (or lack of toxicity), and outcomes studies generating final drug lists. [0036] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE PRESENT INVENTION [0037] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention. [0038] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. [0039] In another embodiment, the invention comprises a neoplastic stem cell (NSC) population enriched for expression of OCT4, Nanog, STAT3 or combinations thereof. In another embodiment, NSCs represent a subpopulation of cells within a population comprising neoplastic cells, which is capable of initiating and maintaining cancer following a prolonged period of time. In another embodiment, NSCs drive the formation and growth of tumors (Fig. 11). In another embodiment, the term drive as used herein refers to guide, control, direct, initiate, go through, penetrate or combinations thereof.

[0040] In another embodiment, NSCs comprise properties such as longevity, self-renewal and quiescence. In another embodiment, NSCs comprise enhanced invasive capacity. In another embodiment, NSCs are multipotent, self-renewing and are able to produce proliferating sarcospheres from sarcomas, neurospheres from brain tumors or mammaspheres from breast cancers (Fig. 5). In another embodiment, NSCs are capable of keeping their self- renewal potential during 1-100 passages of in-vitro cultivation. In another embodiment, NSCs are capable of keeping their self -renewal potential during 1-90 passages of in-vitro cultivation. In another embodiment, NSCs are capable of keeping their self -renewal potential during 20-60 passages of in-vitro cultivation. In another embodiment, NSCs express genes involved in the specific functions and/or in self-renewal of NSCs, such as OCT4, Nanog, STAT3 or combinations thereof. [0041] In another embodiment, the present invention provides that these biological changes reflect the different potential fates of NSCs and BCCs when exposed to drugs and include but are not limited to apoptotic death, symmetric (Figure 20C) or asymmetric (Figure 20A) clonal production of NSCs, differentiation into progenitors and BCCs (Figure 20B), and/or reverse transition to a less differentiated status (i.e., back to or towards stem cell status). [0042] In another embodiment, the present invention provides that solid cancer represents a population of cells derived from a common founder cell, or NSC. In another embodiment, the present invention provides that tumors represent a population of cells derived from a common founder cell, or NSC. In another embodiment, the present invention provides that NSCs comprise a biological profile at the cell and at the molecular level with respect to phenotype. In another embodiment, the present invention provides that NSC phenotype is similar in many ways to that of normal stem cells. In another embodiment, the present invention provides that NSC phenotype is quite different to that of normal stem cells leading to the irregularities with respect to abnormal developmental profile. In another embodiment, the present invention provides that NSC phenotype is quite different to that of normal stem cells leading to the irregularities with respect to lack of key proliferation controls. [0043] In another embodiment, the present invention provides that NSC population comprises a mix of true or mother NSCs and the progenitor neoplastic cells derived from NSCs. In another embodiment, the present invention provides that progenitors derived from NSCs are different in key ways than mother NSCs. In another embodiment, the present invention provides that different in key ways comprise high proliferation kinetics. In another embodiment, the present invention provides that NSCs are typically present in very low percentages relative to the total cancer cell population, correlating roughly to the "hostility" of the enviroment (i.e., a natural environment such as a breast NSC in its primary breast tissue location versus a breast NSC located in a metatstatic and/or foreign location such as the brain). [0044] In another embodiment, the present invention provides that NSCs comprise about 0.001 to 1% of the parental primary cancer population. In another embodiment, the present invention provides that NSCs comprise about 0.005 to 1% of the parental primary cancer population. In another embodiment, the present invention provides that NSCs comprise about 0.01 to 0.1% of the parental primary cancer population. In another embodiment, the present invention provides that NSCs comprise about 0.05 to 0.1% of the parental primary cancer population. In another embodiment, the present invention provides that NSCs comprise about 0.005 to 0.01% of the parental primary cancer population. [0045] In another embodiment, the present invention provides that NSCs comprise about 1 to 80% of the cell population in permanent cancer cell lines parental. In another embodiment, the present invention provides that NSCs comprise about 1 to 10% of the cell population in permanent cancer cell lines parental. In another embodiment, the present invention provides that NSCs comprise about 10 to 30% of the cell population in permanent cancer cell lines parental. In another embodiment, the present invention provides that NSCs comprise about 30 to 50% of the cell population in permanent cancer cell lines parental. In another embodiment, the present invention provides that NSCs comprise about 50 to 80% of the cell population in permanent cancer cell lines parental. In another embodiment, the present invention provides that NSCs comprise about 1 to 5% of the cell population in permanent cancer cell lines parental. In another embodiment, the present invention provides that NSCs comprise about 5 to 10% of the cell population in permanent cancer cell lines parental. In another embodiment, the present invention provides that NSCs comprise about 3 to 8% of the cell population in permanent cancer cell lines parental. In another embodiment, the present invention provides that NSCs comprise about 7 to 10% of the cell population in permanent cancer cell lines parental. [0046] In another embodiment, the present invention provides that NSCs comprise about 1 to 100% of the parental metastatic cancer population cell. In another embodiment, the present invention provides that NSCs comprise about 1 to 10% of the parental metastatic cancer population cell. In another embodiment, the present invention provides that NSCs comprise about 10 to 30% of the parental metastatic cancer population cell. In another embodiment, the present invention provides that NSCs comprise about 30 to 50% of the parental metastatic cancer population cell. In another embodiment, the present invention provides that NSCs comprise about 50 to 75% of the parental metastatic cancer population cell. In another embodiment, the present invention provides that NSCs comprise about 75 to 100% of the parental metastatic cancer population cell. In another embodiment, the present invention provides that NSCs comprise about 30 to 80% of the parental metastatic cancer population cell. In another embodiment, the present invention provides that NSCs comprise about 20 to 90% of the parental metastatic cancer population cell. In another embodiment, the present invention provides that NSCs comprise about 10 to 100% of the parental metastatic cancer population cell. In another embodiment, the present invention provides that NSCs comprise about 20 to 40% of the parental metastatic cancer population cell. Bulk Cancer Cells BCCs [0047] In another embodiment, the present invention provides that BCCs comprise the majority of the cancer cell population from a primary solid tumor. In another embodiment, the present invention provides that BCCs comprise the majority of the cancer cell population from a permanent cultured cell lines derived from cancers. In another embodiment, the present invention provides that a BCC population exhibits higher proliferation kinetics. In another embodiment, the present invention provides that a BCC population exhibits increased sensitivity to chemotherapy and radiation. In another embodiment, the present invention provides that a BCC population lacks stem cell characteristics. In another embodiment, the present invention provides that a BCC population lacks OCT-4 expression. In another embodiment, the present invention provides that a BCC is any cancer cell that is not a NSC. [0048] In another embodiment, the methods for identification of neoplastic stem cells comprising the use of various identifiable fluorescent protein sequences are also employed for the identification of BCCs. In another embodiment, the present invention provides that BCCs do not comprise NSCs. In another embodiment, the methods of the present invention provide that BCCs are separated using the cell separation methods of the present invention. [0049] In another embodiment, the present invention provides that NSCs are more resistant to chemotherapy and radiotherapy than BCCs. In another embodiment, the present invention provides that NSCs express multi-drug resistance genes such as ABC transporters which make them resistant to the concentrations of anticancer drugs normally used as chemotherapeutic agents against cancer. In another embodiment, the present invention provides that these ABC transporter proteins efflux a given anti-cancer drug from the cell and do not allow these drugs to achieve the critical concentrations within the cell needed for cytotoxicity. [0050] In another embodiment, the present invention provides that NSCs are more resistant to radiation therapy than bulk cells due to their lower rates of cellular division and impaired/altered biological programs for damage response. In another embodiment, the present invention provides that NSCs are more resistant to radiation therapy than bulk cells due to their altered p53 function. In another embodiment, the present invention provides that NSCs are more resistant to radiation therapy than bulk cells due to their impaired DNA repair activity. [0051] In another embodiment, the methods of the present invention provide isolation of NSCs from cancer tissue biopsies and permanent cancer cell lines by selection of NSCs previously manipulated and biomarked to allow for detection. In another embodiment, the methods of the present invention provide stably transfecting NSCs with DNA vectors which expresses fluorescent or luminescent proteins regulated by an Oct-4 promoter (Figure 21). In another embodiment, the methods of the present invention provides separating NSCs from the total cancer cell population resulting in cultures of high purity using FACS sorting of fluorescent biomarkers. In another embodiment, the methods of the present invention provide separating NSCs from the total cancer cell population resulting in cultures of high purity using FACS sorting of those cells that express GFP (Green Fluorescent Protein) driven by an Oct4 promoter (Figure 9). Sequences

[0052] In another embodiment, the sequence of the Oct-4 CDNA of the present invention comprises the sequence: tcccttcgcaagccctcatttcaccaggcccccggcttggggcgccttccttccccatggcgggacacctggcttcggatttcgccttctcg ccc cctccaggtggtggaggtgatgggccaggggggccggagccgggctgggttgatcctcggacctggctaagcttccaaggccctcctg gagggccaggaatcgggccgggggttgggccaggctctgaggtgtgggggattcccccatgccccccgccgtatgagttctgtgggg ggatggcgtactgtgggccccaggttggagtggggctagtgccccaaggcggcttggagacctctcagcctgagggcgaagcaggag tcggggtggagagcaactccgatggggcctccccggagccctgcaccgtcacccctggtgccgtgaagctggagaaggagaagctg gagcaaaacccggaggagtcccaggacatcaaagctctgcagaaagaactcgagcaatttgccaagctcctgaagcagaagaggatc accctgggatatacacaggccgatgtggggctcaccctgggggttctatttgggaaggtattcagccaaacgaccatctgccgctttgag gctctgcagcttagcttcaagaacatgtgtaagctgcggcccttgctgcagaagtgggtggaggaagctgacaacaatgaaaatcttcag gagatatgcaaagcagaaaccctcgtgcaggcccgaaagagaaagcgaaccagtatcgagaaccgagtgagaggcaacctggagaa tttgttcctgcagtgcccgaaacccacactgcagcagatcagccacatcgcccagcagcttgggctcgagaaggatgtggtccgagtgt ggttctgtaaccggcgccagaagggcaagcgatcaagcagcgactatgcacaacgagaggattttgaggctgctgggtctcctttctcag ggggaccagtgtcctttcctctggccccagggccccattttggtaccccaggctatgggagccctcacttcactgcactgtactcctcggtc cctttccctgagggggaagcctttccccctgtctccgtcaccactctgggctctcccatgcattcaaactgaggtgcctgcccttctaggaat gggggacagggggaggggaggagctagggaaagaaaacctggagtttgtgccagggtttttgggattaagttcttcattcactaaggaa ggaattgggaacacaaagggtgggggcaggggagtttggggcaactggttggagggaaggtgaagttcaatgatgctcttgattttaatc ccacatcatgtatcacttttttcttaaataaagaagcctgggacacagtaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ . ID NO: 1). In another embodiment, the Oct-4 CDNA the present invention comprises a nucleic acid sequence homologous to SEQ. ID. NO: 1. In another embodiment, the Oct-4 CDNA sequence is a Homo sapiens Oct-4 CDNA sequence. In another embodiment, the Oct-4 CDNA sequence is from a non-human species. Each possibility represents a separate embodiment of the present invention.

[0053] In another embodiment, the sequence of the Oct-4 CDNA of the present invention comprises the sequence: gtagtcctttgttacatgcatgagtcagtgaacagggaatgggtgaatgacatttgtgggtaggttatttctagaagttaggtgggcagcttg g aaggcagaggcacttctacagactattccttggggccacacgtaggttcttgaatcccgaatggaaaggggagattgataactggtgtgttt atgttcttacaagtcttctgccttttaaaatccagtcccaggacatcaaagctctgcagaaagaactcgagcaatttgccaagctcctgaagc agaagaggatcaccctgggatatacacaggccgatgtggggctcaccctgggggttctatttgggaaggtattcagccaaacgaccatct gccgctttgaggctctgcagcttagcttcaagaacatgtgtaagctgcggcccttgctgcagaagtgggtggaggaagctgacaacaatg aaaatcttcaggagatatgcaaagcagaaaccctcgtgcaggcccgaaagagaaagcgaaccagtatcgagaaccgagtgagaggca acctggagaatttgttcctgcagtgcccgaaacccacactgcagcagatcagccacatcgcccagcagcttgggctcgagaaggatgtg gtccgagtgtggttctgtaaccggcgccagaagggcaagcgatcaagcagcgactatgcacaacgagaggattttgaggctgctgggtc tcctttctcagggggaccagtgtcctttcctctggccccagggccccattttggtaccccaggctatgggagccctcacttcactgcactgta ctcctcggtccctttccctgagggggaagcctttccccctgtctccgtcaccactctgggctctcccatgcattcaaactgaggtgcctgcc cttctaggaatgggggacagggggaggggaggagctagggaaagaaaacctggagtttgtgccagggtttttgggattaagttcttcatt cactaaggaaggaattgggaacacaaagggtgggggcaggggagtttggggcaactggttggagggaaggtgaagttcaatgatgct cttgattttaatcccacatcatgtatcacttttttcttaaataaagaagcctgggacacagtaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ. ID NO: T). In another embodiment, the Oct-4 CDNA the present invention comprises a nucleic acid sequence homologous to SEQ. ID. NO: 2. In another embodiment, the Oct-4 CDNA sequence is a Homo sapiens Oct-4 CDNA sequence. In another embodiment, the Oct-4 CDNA sequence is from a non-human species. Each possibility represents a separate embodiment of the present invention. [0054] In another embodiment, the sequence of the OCT-4 protein of the present invention comprises the sequence:

MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPG SEVWGIPPCPPPYEFCGGMA YCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGAS PEPCTVTPGA VKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRΓΓLGYTQADVGL TLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLV QARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKD VVRVWFCNRRQK GKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGE

AFPPVSVTTLGSPMHSN (SEQ. ID NO: 3). In another embodiment, the OCT-4 protein of the present invention comprises an amino acid sequence homologous to SEQ. ID. NO: 3. In another embodiment, the OCT-4 protein is a Homo sapiens OCT-4 protein. In another embodiment, the OCT-4 protein is from a non-human species. Each possibility represents a separate embodiment of the present invention.

[0055] In another embodiment, the sequence of the OCT-4 protein of the present invention comprises the sequence:

MCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLFLQCP KPT

LQQISHIAQQLGLEKD VVRVWFCNRRQKGKRSSSD YAQREDFEAAGSPFSGGPVSFP LAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSN (SEQ. ID NO: 4). In another embodiment, the OCT-4 protein of the present invention comprises an amino acid sequence homologous to SEQ. ID. NO: 4. In another embodiment, the OCT-4 protein is a Homo sapiens OCT-4 protein. In another embodiment, the OCT-4 protein is from a non- human species. Each possibility represents a separate embodiment of the present invention. [0056] In another embodiment, the sequence of the Oct-4 promoter of the present invention comprises the cacccaggggcggggccagaggtcaaggctagagggtggg (SEQ. ID NO: 5). In another embodiment, the Oct-4 promoter of the present invention comprises a nucleic acid sequence homologous to SEQ. ID. NO: 5. In another embodiment, the Oct-4 promoter sequence is a murine Oct-4 promoter sequence. In another embodiment, the Oct-4 promoter sequence is from a Homo-sapiens. In another embodiment, the Oct-4 promoter sequence is from a non- human species. Each possibility represents a separate embodiment of the present invention.

[0057] In another embodiment, the Oct-4 DNA sequence of the present invention is at least 60% homologous to anyone SEQ. ID NOs: 1-2. In another embodiment, the Oct-4 DNA sequence of the present invention is at least 70% homologous to anyone SEQ. ID NOs: 1-2. In another embodiment, the Oct-4 DNA sequence of the present invention is at least 80% homologous to anyone SEQ. ID NOs: 1-2. In another embodiment, the Oct-4 DNA sequence of the present invention is at least 90% homologous to anyone SEQ. ID NOs: 1-2. In another embodiment, the Oct-4 DNA sequence of the present invention is at least 95% homologous to anyone SEQ. ID NOs: 1-2.

[0058] In another embodiment, the Oct-4 promoter DNA sequence of the present invention is at least 60% homologous to anyone SEQ. ID NOs: 5. In another embodiment, the Oct-4 promoter DNA sequence of the present invention is at least 70% homologous to anyone SEQ. ID NOs: 5. In another embodiment, the Oct-4 promoter DNA sequence of the present invention is at least 80% homologous to anyone SEQ. ID NOs: 5. In another embodiment, the Oct-4 promoter DNA sequence of the present invention is at least 90% homologous to anyone SEQ. ID NOs: 5. In another embodiment, the Oct-4 promoter DNA sequence of the present invention is at least 95% homologous to anyone SEQ. ID NOs: 5. [0059] In another embodiment, the Oct-4 protein sequence of the present invention is at least 60% homologous to anyone SEQ. ID NOs: 3-4. In another embodiment, the Oct-4 protein sequence of the present invention is at least 70% homologous to anyone SEQ. ID NOs: 3-4. In another embodiment, the Oct-4 protein sequence of the present invention is at least 80% homologous to anyone SEQ. ID NOs: 3-4. In another embodiment, the Oct-4 protein sequence of the present invention is at least 90% homologous to anyone SEQ. ID NOs: 3-4. In another embodiment, the Oct-4 protein sequence of the present invention is at least 95% homologous to anyone SEQ. ID NOs: 3-4.

[0060] In another embodiment, the methods of the present invention provide a highly pure biomarked NSC population. In another embodiment, the methods of the present invention provides that a highly pure biomarked NSC population is studied in numerous ways by taking advantage of their fluorescent properties (Figures 7 - 9). Identification and isolation of NSCs

[0061] In another embodiment, NSCs are enriched for a stem cell marker. In another embodiment, the stem cell marker is OCT4, Nanog, STAT3 or combinations thereof. In another embodiment, the stem cell marker is a transcription factor such as OCT4. In another embodiment, OCT4 is differentially expressed in NSCs. In another embodiment, immunological methods of enriching for OCT4 expressing cells based on their affinity to surface antigens are used. In another embodiment, NSCs are enriched by an immunomagnetic based cell separation technique. In another embodiment, NSCs are enriched by the electrophoretic cell separation technique based on the electrophoretic mobility reduction via incubation with antibodies specific to surface antigen. In another embodiment, the reduction in electrophoretic mobility by incubation with surface antigen specific antibodies is performed under non-capping conditions. In another embodiment, NSCs are further enriched through fluorescence-activated cell sorter (FACS), immunomagnetic beads, or magnetic-activated cell sorter (MACS).

[0062] In another embodiment, mixed populations of cancerous cells are grown under nonadherent cell culture conditions, wherein NSCs form spherical clusters of cells ("spheres") from which OCT4 positive NSCs can be enriched. In another embodiment, cells derived from free floating spheres express higher levels of OCT4 and Nanog mRNA than equivalent, adherent cell cultures as shown in Fig. 3. In another embodiment, the cells comprising the spheres are free floating. In another embodiment, in-vitro enrichment of NSCs from breast tumor specimens is carried out using a nonadherent mammasphere cell culture system. In another embodiment, in-vitro enrichment of NSCs from bone sarcoma tumor cells is carried out using a nonadherent sarcosphere cell culture system. In another embodiment, in-vitro enrichment of NSCs from brain tumor cells is carried out using a nonadherent neurosphere cell culture system. In another embodiment, in-vitro enrichment of NSCs from brain tumor cells is carried out using free floating spheres. [0063] In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 80% positive for OCT4 expression. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 80% positive for Nanog expression. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 80% positive for STAT3 expression. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 80% positive for the expression of OCT4, STAT 3, Nanog or combinations thereof. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 90% positive for OCT4 expression. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 90% positive for Nanog expression. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 90% positive for STAT3 expression. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 90% positive for the expression of OCT4, STAT 3, Nanog or combinations thereof. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 95% positive for OCT4 expression. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 95% positive for Nanog expression. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 95% positive for STAT3 expression. In another embodiment, the NSC-enriched subpopulation of cancerous cells is at least 95% positive for the expression of OCT4, STAT 3, Nanog or combinations thereof.

[0064] In another embodiment, the invention provides that the level of NSC-enriched subpopulation of cancerous cells is determined by FACS analysis (Fig. 10), in-situ hybridization, immunohistochemistry or a combination thereof, as described in the material and methods section.

[0065] In another embodiment, the NSC-enriched population is characterized by OCT4^hl expression. In another embodiment, OCT4^hl expression is at least twice as high as β-actin expression. In another embodiment, OCT4^hl expression is at least four times as high as β-actin expression. In another embodiment, the NSC-enriched population is further characterized by high expression of Nanog, STAT3, or combinations thereof.

[0066] In another embodiment, the expression level of OCT4, Nanog or STAT3 is determined by the mRNA transcription level. In another embodiment, the transcription levels are determined by quantitative or semi-quantitative PCR or RT-PCR methods as shown in Fig. 2A and described in the materials and methods section. In another embodiment, the expression level of OCT4, Nanog or STAT3 is determined by the protein expression level. In another embodiment, the protein expression level is determined by western blot analysis as shown in Fig. 2B and described in the materials and methods section. In another embodiment, protein expression level is determined indirectly by using a reporter gene. In another embodiment, the reporter gene comprises an EGFP construct. In another embodiment, the OCT4 expression level in an OCT4-EGFP transfected glioblastoma cell culture (Figs. 7 and 8) is determined as described in the materials and methods section. [0067] In another embodiment, the NSC subpopulation is enriched from "soft" or "hard" tumors. In another embodiment, "hard" tumors include all tumors except leukemia, lymphomas, melanomas, and multiple myeloma, which, in another embodiment, are classified as "soft." In another embodiment, the NSC subpopulation is enriched from isolated metastatic cells. In another embodiment, the NSC subpopulation is enriched from a tissue culture comprising cells derived from a tumor-derived cell line. [0068] In another embodiment, the subject invention comprises a composition comprising a population of NSCs enriched for expression of OCT4. In another embodiment, the invention comprises a population of NSCs enriched for expression of OCT4^hl. In another embodiment, the composition further comprises an appropriate environment, such as those described herein, wherein, a NSC can be induced to proliferate and generate NSC progeny. In another embodiment, the term environment in which NSC progeny are placed, refers to the combination of external or extrinsic physical and/or chemical conditions that affect and influence the growth and development of NSCs. In another embodiment, the environment can be ex- vivo or in- vivo. In another embodiment, the circulatory system (blood and lymphatic) can serve as an in-vivo environment that induces NSCs to generate progeny. In another embodiment, the environment is ex-vivo and comprises NSCs placed in cell culture medium in an incubator. [0069] In another embodiment, the environment further comprises cell culture medium comprising DMEM/F12. In another embodiment, the cell culture medium further comprises methylcellulose in a final concentration of less than 3%, more preferably, less than 1.5%. In another embodiment, the medium is supplemented with 8-20% fetal bovine serum (FBS), 30- 70% media derived from cultures of primary human foreskin fibroblasts, or a combination thereof. In another embodiment, the medium further comprises screening agents which bind OCT4. In another embodiment, the medium further comprises screening agents which interact with an OCT4 responsive element.

[0070] In another embodiment, the environment for isolating NSCs from a mixed population of cells is serum free. In another embodiment, the environment for isolating NSCs from a mixed population of cancerous cells is serum free. In another embodiment, the environment for isolating NSCs from a mixed population of cells is serum free. In another embodiment, the environment for isolating NSCs from BCCs is serum free. In another embodiment, the environment for growing NSCs in spheres is serum free. In another embodiment, BCCs do not survive in a serum free environment. In another embodiment, BCCs do not survive when grown in sarcospheres in a serum free environment. In another embodiment, non-NSCs cancer cells do not survive when grown in sarcospheres in a serum free environment.

[0071] In another embodiment, the medium is further supplemented with 5-5OnM of progesterone, 5-500μM putrescine, 2-100ng/ml recombinant EGF, 20-4OnM sodium selenit, 10-40 μg/ml transferring, 5-50 μg/ml insulin. 2-lOOng/ml recombinant FGF2 or a combination thereof. In another embodiment, the medium is supplemented with 8-20% fetal bovine serum (FBS), 30-70% media derived from cultures of primary human foreskin fibroblasts, or a combination thereof. In another embodiment, the medium comprises nucleic acids. In another embodiment, the medium comprises a plasmid DNA. In another embodiment, the plasmid DNA comprises an OCT4 responsive promoter. In another embodiment, the OCT4 responsive promoter is linked to a reporter gene (Fig. 8). In another embodiment, the OCT4 responsive promoter is linker to an antibiotic resistance gene. In another embodiment, the medium comprises siRNA. In another embodiment, the siRNA antisense encodes for anti-OCT4, anti- Nanog, anti-STAT3 or combinations thereof. In another embodiment, the anti-OCT4 siRNA inhibits clone formation (Fig. 4B) by inhibiting de-novo production of OCT4 protein (Fig. 4A).

[0072] In another embodiment, cells are plated in ultra low attachment plates. In another embodiment, the cells are kept in an incubator maintaining a temperature at 36-42°C. In another embodiment, the incubator further maintains 4-8% CO₂. In another embodiment, the incubator maintains 90-100% humidity. In another embodiment, cells are plated in a final density of 1x10²- 1x10⁶ cells/cm².

[0073] In another embodiment, NSCs of the present invention are derived from a cell line. In another embodiment, NSCs of the present invention are derived from a primary cell culture. In another embodiment, the primary cell culture comprising NSCs is derived from a tumor or cell metastasis. In another embodiment, the invention comprises tumors and cell metastasis which comprise NSCs. In another embodiment, tumors and cell metastasis are derived from but not limited to: adrenocortical carcinoma, anal cancer, bladder cancer, brain tumor, brain stem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal, pineal tumors, hypothalamic glioma, breast cancer, carcinoid tumor, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewings family of tumors (pnet), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, laryngeal cancer, leukemia, acute lymphoblastic, leukemia, oral cavity cancer, liver cancer, lung cancer, small cell, lymphoma, AIDS-related, lymphoma, central nervous system (primary), lymphoma, cutaneous T-cell, lymphoma, hodgkin's disease, non-hodgkin's disease, malignant mesothelioma, melanoma, merkel cell carcinoma, metasatic squamous carcinoma, multiple myeloma, plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, exocrine, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, salivary gland cancer, sezary syndrome, skin cancer, cutaneous T-cell lymphoma, skin cancer, kaposi's sarcoma, skin cancer, melanoma, small intestine cancer, soft tissue sarcoma, soft tissue sarcoma, testicular cancer, thymoma, malignant, thyroid cancer, urethral cancer, uterine cancer, sarcoma, unusual cancer of childhood, vaginal cancer, vulvar cancer, or wilms' tumor.

[0074] In another embodiment, the invention provides a method of identifying NSCs, comprising the steps of contacting neoplastic cells with an agent which specifically interacts with OCT4 through its employment to a cell culture comprising primary cell culture or a cell line culture. In another embodiment, NSCs subpopulation is identified in "soft or hard" tumor. In another embodiment, "Hard" tumors include all tumors except leukemia, lymphomas, melanomas, and multiple myeloma, which are classified as "soft." In another embodiment, NSCs are identified among metastatic cells.

[0075] In another embodiment, the invention provides a method of identifying NSCs, comprising the steps of contacting neoplastic cells with an agent which specifically interacts with OCT4 and identifying the cells with which the agent specifically interacted, as described herein. In another embodiment, the agent identifying OCT4 interacts with the cell membrane. In another embodiment, the agent interacts with the POU5F1 gene encoding OCT4 or a fragment thereof. In another embodiment, the agent interacts with the mRNA encoding OCT4 or a fragment thereof. In another embodiment, the agent interacts with the OCT4 protein or a fragment thereof. In another embodiment, the agent interacts with a specific post translational form of OCT4 such as, but not limited to, the phosphorylated OCT4 protein. [0076] In another embodiment, the invention provides a method of identifying NSCs using a DNA probe that specifically interacts with OCT4 mRNA in a DNA-RNA heteroduplex. In another embodiment, the method of identifying NSCs utilizes an RNA probe that specifically interacts with OCT4 mRNA in an RNA-RNA homoduplex. In another embodiment, the method of identifying NSCs utilizes a peptide nucleic acid (PNA) probe that specifically interacts with OCT4 mRNA in a PNA-RNA heteroduplex. In another embodiment, the nucleic acid probe or PNA further comprises a label which can be readily identified. In another embodiment, the methods utilize a specific probe comprising a nucleic acid that enables selective identification of OCT4 expressing cells.

[0077] In another embodiment, the invention provides a method for identification of NSCs comprising a protein that specifically interacts with OCT4 protein or a fragment thereof. In another embodiment, a monoclonal or polyclonal anti-OCT4 antibody is utilized to detect OCT4.

[0078] In another embodiment, the invention provides a method of detecting OCT4 expressing cells. In another embodiment, the detection method is direct, wherein a radioactive label is used, which in another embodiment comprises a radioactive compound such as ³²P or ¹²⁵I. In another embodiment, direct labeling comprises a fluorescent, cheniiluminescent, or gold label. In another embodiment, the detection method is indirect comprising a nucleic acid probe similar to imraunohi&tochemical probes as known to one skilled in the art, In another embodiment, probes may be labeled with hapten or biotin used to bring an enzyme which creates the detectable event (e.g., chemiluminescent, colorimetric or fluorescent) to the site of hybridization. In another embodiment, wherein amplification of the detection signal is required, a secondary labeled antibody specifically identifying the primary antibody is utilized. In another embodiment, the methods utilizing a specific probe comprising an antibody enable selective identification of OCT4 expressing cells. [0079] In another embodiment, a heterogeneous cell population for OCT4 expression is transfected with a plasmid comprising an OCT4 responsive promoter controlling the expression of an identifiable, reporting gene product. In another embodiment, the identifiable gene product comprises green fluorescent proteins such as but not limited to: GFP, Emerald, Azami Green, or ZsGreenl; blue fluorescent proteins such as but not limited to: EBFP or Sapphire; cyan fluorescent proteins such as but not limited to: Cerulean, ECFP, AmCyanl or Midoriishi-Cyan; yellow fluorescent proteins such as but not limited to: ZsYellowl, Phi YFP, Citrine, or Venus; orange fluorescent proteins such as but not limited to: Kusabira-Orange or mOrange; red fluorescent proteins such as but not limited to:, DsRed, HcRed, mPlum, mRaspberry, mTomato, mStrawberry or green-to-red fluorescent Dendra. In another embodiment, the identifiable gene product serves as a distinguishable marker between cells expressing OCT4 and cells not expressing OCT4 (Fig. 8).

[0080] In another embodiment, the invention provides a method of identifying NSCs expressing OCT4, which comprises visualizing the probed NSCs, In another embodiment, visualization of NSCs expressing OCT4 is carried out by exposing the labeled specimen to a film. In another embodiment, visualization of NSCs expressing OCT4 can be performed with a fluorescent microscope. In another embodiment, a light microscope is used for visualization of NSCs expressing OCT4, while in another embodiment, the signal is detectable using the naked eye. In another embodiment, the results of the above mentioned visualization methods can be further recorded and/or visualized on a CCD camera.

[0081] In another embodiment, the invention provides a method of identifying NSCs expressing OCT4 comprising a means of quantifying the probed NSCs, In another embodiment, quantification is assessed by a fluorometer. In another embodiment, identification and subsequent quantification of NSCs expressing OCT4 is carried out by FACS.

[0082] In another embodiment, the invention provides a method of isolating neoplastic stem cells, comprising the steps of contacting neoplastic cells with an agent which specifically interacts with OCT4. In another embodiment, a cell culture comprising primary cell culture or a cell line culture is employed.

[0083] In another embodiment, the invention provides cell separation methods which include cell isolation methods. In another embodiment, tissue dissociation techniques are utilized prior to cell separation methods. In another embodiment, enzymes such as liberase, trypsin, elastase, dispase, collagenase or combinations thereof are employed for effective tissue dissociation. In another embodiment, further trituration with a pipette tip to break apart the cell aggregates is needed.

[0084] In another embodiment, the invention provides a method of isolating neoplastic stem cells, comprising the steps of contacting neoplastic cells with an agent which specifically interacts with OCT4 and isolating the cells with which the agent specifically interacts, as described. In another embodiment, the methods described previously for identification of neoplastic stem cells, particularly the steps of contacting neoplastic cells with an agent which specifically interacts with OCT4 protein or mRNA, are also used for isolation of NSCs. [0085] In another embodiment, the invention provides a heterogeneous cell population transfected with a plasmid comprising an Oct4 responsive promoter controlling the expression of an identifiable and/or selectable gene product (Fig. 8). In another embodiment, the methods described previously for identification of neoplastic stem cells comprising the use of various identifiable fluorescent protein sequences are also employed for cell separation methods. In another embodiment, the identifiable gene product is used selectively to isolate OCT4 expressing cells resulting in a uniform OCT4 expressing NSCs.

[0086] In another embodiment, NSCs expressing OCT4 are separated in chromatography columns in which antibodies specific to OCT4 that are attached to the column bind OCT4 expressing NSCs and thereby separate them. In another embodiment, an agent that is covalently bound to magnetic particles and that specifically interacts with OCT4 is employed to retain OCT4 expressing NSCs in a magnetic field. In another embodiment, sorting of OCT4 expressing NSCs labeled with antibodies comprising a fluorescent label, through a FACS is used to separate NSCs from a heterogeneous population of cells as shown in Fig. 10. In another embodiment, the separation methods as described herein results in an isolated population of OCT4 expressing cells.

[0087] In another embodiment, the invention provides methods of enriching NSCs expressing OCT4. In another embodiment, a primary cell culture is enriched for OCT4 expressing cells. In another embodiment, the primary cell culture for which methods for enriching OCT4 expressing NSCs is employed is derived from a soft tumor, a hard tumor, or a metastatic cell population. In another embodiment, the OCT4 expressing NSC subpopulation is enriched from a tissue culture comprising cells derived from a cell line. [0088] In another embodiment, the invention provides methods of enriching OCT4 expressing NSCs which comprise transfection of a heterogeneous cell population with a plasmid comprising an Oct4 responsive promoter controlling the expression of a selectable gene product (Fig. 8). In another embodiment, the selectable gene encodes an antibiotic resistance protein. In another embodiment, the cell enrichment methods further comprise the selecting agent. In another embodiment the selecting agent is an antibiotic which selectively eradicates non-OCT4 expressing cells resulting in an enriched OCT4 expressing NSC cell population.

[0089] In another embodiment, the invention provides a method of inducing cancer comprising introducing a neoplastic stem cell population enriched for expression of OCT4 to a mammal. In another embodiment, the method of inducing cancer comprises promoting cell growth that leads to cancer. In another embodiment, the method of inducing cancer comprises providing metastatic cells that induce cancer.

[0090] In another embodiment, NSCs of the invention isolated from mamaspheres, sarcospheres or neurospheres are used as cancer inducers. In another embodiment, an animal is inoculated with NSCs. In another embodiment, NSCs are injected intravenously. In another embodiment, NSCs are injected into the bone. In another embodiment, NSCs are injected into an animal intradermally, intramuscularly or intraperitoneally. In another embodiment, NSCs are injected directly to the mammary gland of a model animal. In another embodiment, inoculation comprises injection of NSCs into the fat pads of a model animal. [0091] In another embodiment, the invention provides methods of inducing cancer. In another embodiment, the methods of inducing cancer as described herein are performed in immunodeficient rodents. In another embodiment, the immunodeficient rodent is a nude mouse or rat. In another embodiment, the immunodeficient rodent is a SCID mouse. In another embodiment, the immunodeficient rodent is an NIH-III mouse.

[0092] In another embodiment, the invention provides a method of inducing tumors or metastases comprising introducing a neoplastic stem cell population enriched for expression of OCT4 to a mammal. In another embodiment, orthotopical or ectopical tumors are being induced (Fig. 11). In another embodiment, metastases take place through the lymphatic system, through the bloodstream, by spreading through body spaces, or through implantation.

[0093] In another embodiment, the invention provides a method of analyzing cancer progression and/or pathogenesis in-vivo comprising transplanting OCT4^hl neoplastic stem cells into an animal; and analyzing cancer progression and/or pathogenesis in an animal. In another embodiment, cancer comprises carcinoma, sarcoma, lymphoma, leukemia, or myeloma. [0094] In another embodiment, NSCs of the invention are labeled by transfecting OCT4^hl neoplastic stem cells with a fluorescent protein. In another embodiment, the identifiable gene product comprises various fluorescent proteins as described hereinabove. In another embodiment, the identifiable gene product comprises a luminescent protein. In another embodiment, the luminescent protein is luciferase. In another embodiment, isotopes are used for tracking the transplanted OCT4^hl neoplastic stem cells in the animal model. In another embodiment, the isotopes comprise ³²P, ¹²⁵I, ¹²⁴I, ¹²³I, ¹⁴C, ¹⁰⁹Cd, ⁵¹Cr, ⁶⁷Cu, ¹⁷⁹Ta, ¹¹¹In, ¹⁸F, or combinations thereof. In another embodiment a magnetic label is used for cell detection. [0095] In another embodiment, the transplanted labeled cells of the invention were tracked with a single-photon emission-computed tomographic (SPECT) scanner, a positron emission tomography (PET) scanner, or single photon emission commuted tomography. In another embodiment, wherein cells are labeled magnetically, MRI is used for detection. In another embodiment, a back-illuminated, cooled, charge-coupled device (CCD) camera is used for luminescent detection. In another embodiment, LED flashlights with excitation filter and an emission filter are used for detection of fluorescently labeled cells. In another embodiment, light box with fiber-optic lighting at about 490 nm and filters, placed on top of the light box, are used to image large tumors. In another embodiment, small tumors and metastases are visualized using a fluorescence dissecting microscope that incorporates a light source and filters for excitation at about 490 nm. In another embodiment, color CCD cameras as well as dual-photon lasers are used for ultra-high-resolution in-vivo imaging of fluorescent protein expression.

[0096] In another embodiment, the invention provides a method of analyzing cancer progression and/or pathogenesis in-vivo including determining cell metastasis. In another embodiment, analysis of cell metastasis comprises determination of progressive growth of cells at a site that is discontinuous from the primary tumor. In another embodiment, the site of cell metastasis analysis comprises the route of neoplastic spread. In some embodiment, cells can disperse via blood vasculature, lymphatics, within body cavities or combinations thereof. In another embodiment, cell metastasis analysis is performed in view of cell migration, dissemination, extravasation, proliferation or combinations thereof.

[0097] In another embodiment, the invention provides a method of analyzing cancer progression and/or pathogenesis in-vivo. In another embodiment, analysis of cancer progression and/or pathogenesis in-vivo comprises determining the extent of tumor progression. In another embodiment, analysis comprises the identification of the tumor (Fig. 11). In another embodiment, analysis of tumor progression is performed on the original tumor or "primary tumor". In another embodiment, analysis is performed over time depending on the type of cancer as known to one skilled in the art (Fig. 11). In another embodiment, further analysis of secondary tumors originating from metastasizing cells of the primary tumor is analyzed in-vivo. In another embodiment, the size and shape of secondary tumors are analyzed. In some embodiment, further ex-vivo analysis is performed. In another embodiment, the frequency of OCT4 expressing cells in chondrosarcoma or oteosarcoma tumors is assessed as shown in Fig. 1.

[0098] In another embodiment, the terms assessed, screened, evaluated and analyzed are used interchangeably. [0099] In another embodiment, pathological samples of metastasis or tumors are evaluated at specific points in time, as known to one skilled in the art. In another embodiment, quantitative or qualitative methods assessing tumor suppressor genes, oncogenes, apoptotic genes, signal transduction genes, receptors, transcription factors, ligands or combinations thereof comprising: PCR, western-blot, northern blot, southern blot, immunohistochemical or in situ hybridization analysis are further employed.

[00100] In another embodiment, tumor or metastatic cells are isolated from pathological samples for further analysis. In another embodiment, tumor or metastatic cells are isolated from pathological samples and grown in culture. In another embodiment, the cell proliferation potential of the primary tumor cell culture is assessed. In another embodiment, OCT4 positive cells are isolated and/or enriched from the pathological sample comprising tumor or metastatic cells according to the methods described hereinabove. In another embodiment, the OCT4 positive cells isolated from a tumor are further analyzed. In some embodiment, various agents are further employed to the tumor or metastasis primary cell culture. In another embodiment, the agent is a carcinogen. In another embodiment. The agent is a pro-apoptotic agent or a differentiating agent.

[00101] In another embodiment, the invention provides a method of assessing the effect of a carcinogen on a primary cell culture. In another embodiment, the carcinogen comprises, but is not limited to, carcinogenic substances in categories 1 through 3 of the International Agency for Research on Cancer (IARC).

[00102] In another embodiment, the invention provides a method of assessing the effect of a therapeutic agent on a primary cell culture derived from a tumor or a metastasis. In another embodiment, therapeutic agents are screened ex-vivo, on a tumor- or metastasis-derived primary cell culture. In another embodiment, the therapeutic agents comprise interferons, interleukins, colony-stimulating, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, steroid hormones or combinations thereof. In another embodiment, the therapeutic agent is a chemotherapy agent. In another embodiment, the chemotherapy agent is non-specific and hence may kill a cancerous cell during any phase of the cell-cycle. In another embodiment, the chemotherapy agent is specific and is thus able to kill a cancerous cell during a specific phase of the cell-cycle.

[00103] In another embodiment, the present invention provides that NSCs are responsible for metastasis to systemic organs. In another embodiment, the present invention provides that NSCs are required for metastasis to systemic organs. In another embodiment, the present invention provides that NSCs are responsible for recurrent cancer growth in the primary location after attempts at treatment (i.e., surgery, radiation, and chemotherapy). In another embodiment, the methods of the present invention provide a platform which allows for the identification of drugs that target those NSCs with metastatic potential. System for screening compounds [00104] In another embodiment, the methods of the present invention provide that NSC populations are expanded into large volume mass cultures for extended periods of time without losing their desired pure NSC phenotype (Figures 14). [00105] In another embodiment, the present invention provides that methods for identification, isolation and growth of NSCs are further utilized to create cancer cell models and methods for detection that can be used to test both conventional and novel drugs and compounds for anti-NSC and anti-cancer effects (Figures 16-18). [00106] In another embodiment, the present invention provides that several desired effects of anti-NSC compounds can be monitored via the use of the methods of the present invention. In another embodiment, the desired effect of anti-NSC comprises killing NSCs. In another embodiment, the desired effect of anti-NSC comprises differentiating NSCs into progeny which are more chemosensitive. In another embodiment, the desired effect of anti-NSC comprises differentiating NSCs into progeny which are more radiosensitive thereby allowing conventional drugs to be effective.

[00107] In another embodiment, a candidate compound kills NSCs. In another embodiment, a candidate compound causes NSCs to mature and thus lose their "sternness" and NSCs markers. In another embodiment, the effect of a candidate compound on a NSC population is measured by FACS. In another embodiment, compounds which inhibit the proliferation of NSCs, cause NSCs to mature, or kill NSCs will cause a decline in FACS counting of fluorescence markers compared to control NSCs treated with PBS. In another embodiment, FACS counting of cells comprising NSC markers correlates to the number of NSCs. [00108] In another embodiment, compounds identified by the present invention induce NSCs to mature (loose sternness). In another embodiment, compounds identified by the present invention induce NSCs to differentiate. In another embodiment, compounds identified by the present invention induce NSCs to transform into mature cancer cells or BCCs. In another embodiment, compounds identified by the present invention remove cancer stem cells. In another embodiment, compounds identified by the present invention create cancer cells that are more susceptible to chemotherapy and/or radiation.

[00109] In another embodiment, compounds identified by the present invention provide insight into transforming mechanisms in situ. In another embodiment, compounds identified by the present invention provide insight into the molecular mechanisms that are activated and whose action result in the phenotypic changes. In another embodiment, compounds of the present invention provide clues to the true origins of cancer (ie, vis-a-vis the understanding of the origin of a NSCs). In another embodiment, such a breakthrough could lead to major advances in immunomodulatory approaches, preventive medicine, diagnostic assays, and even drugs that prevent the transforming/initiation steps. [00110] In another embodiment, asymmetric division is NSC division in which an identical clone is created, while at the same time non-NSC progeny are given off which go on to rapidly divide and grow into the tumor body. In another embodiment, this is the typically situation of a growing tumor in which the NSCs are not under any particular survival pressure. [00111] In another embodiment, symmetric division is NSC division in which NSCs only clone themselves, and do so in a highly proliferative manner relative to their typical growth kinetics (very slow). In another embodiment, a NSC is under significant survival pressure and overcome such pressure or responds to a challenge in which plasticity is key. [00112] In another embodiment, a compound of the present invention induces apoptosis in NSCs. In another embodiment, a compound of the present invention induces cell cycle arrest in NSCs. In another embodiment, a compound of the present invention induces a NSC to convert to a BCC.

[00113] In another embodiment, the present invention provides that a candidate or a test compound of the present invention is screened in a tissue culture well comprising an isolated population of NSCs. In another embodiment, the present invention provides that a candidate or a test compound of the present invention is screened in a tissue culture well comprising an enriched population of NSCs. In another embodiment, the present invention provides that a candidate or a test compound of the present invention is screened in a tissue culture well comprising an isolated or enriched population of NSCs incubated in a cell culture medium which promotes NSCs.

[00114] In another embodiment, the present invention provides methods for testing compounds which inhibit NSCs by converting NSCs to BCCs. In another embodiment, the present invention provides methods for testing compounds which inhibit NSCs by differentiating NSCs. In another embodiment, the present invention provides methods for testing compounds which inhibit NSCs by differentiating NSCs to non-cancerous cells.

[00115] In another embodiment, the present invention provides methods for testing compounds which cause BCCs to convert to NSCs. In another embodiment, the present invention provides methods for testing compounds which cause matured cells to convert to NSCs. In another embodiment, the present invention provides methods for testing compounds which cause differentiated cells to convert to NSCs. In another embodiment, the present invention provides that any matured or differentiated cell culture can be used for testing compounds which cause cells to be transformed to NSCs. In another embodiment, the methods of the present invention provide an additional category to test carcinogens according to their ability to transform various cell types to NSCs.

[00116] In another embodiment, the present invention provides that the dynamics of the NSC population and its biological response to environmental changes and toxic challenges can be mathematically modeled allowing for efficient real-time analysis in large-scale drug discovery approaches.

[00117] In another embodiment, the present invention provides that the mathematical model comprises parameters that can be detected by the present invention drug discovery platform. In another embodiment, the present invention provides that a mathematical model enables to calculate the probability that a stem cell will die-P₀. In another embodiment, the present invention provides that a mathematical model enables to calculate the probability that stem cell will divide asymmetrically and produce one daughter stem cell (NSC) and one progenitor cell (BCC)-P_a (Fig. 20A). In another embodiment, the present invention provides that a mathematical model enables to calculate the probability that stem cell will divide symmetrically and produce two daughter stem cells-P_s (Figure 20C). In another embodiment, the present invention provides that a mathematical model enables to calculate the probability that stem cell will transit to a progenitor (bulk) state-P_t (Fig. 20B). In another embodiment, the present invention provides that a mathematical model enables to calculate the probability that a bulk cell will die-Pbo. In another embodiment, the present invention provides that a mathematical model enables to calculate the probability that bulk cell will divide symmetrically and produce two progenitor cells-P_bs. In another embodiment, the present invention provides that a mathematical model enables to calculate the probability that bulk cell will transit to the more mature state and stop proliferation (senescence)-Pbt. In another embodiment, the present invention provides that a mathematical model enables to calculate the probability that bulk cell will reverse to stem cell state-Pb_r.

[00118] In another embodiment, the invention provides a method for testing a candidate compound for an ability to inhibit OCT4 expression in a neoplastic stem cell (NSC), comprising the steps of: contacting a cell population enriched for OCT4 expression with a candidate compound, wherein the cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and measuring the expression of an identifiable gene product; thereby testing a candidate compound for an ability to inhibit OCT4 expression in a neoplastic stem cell. In another embodiment, the methods of the present invention provide that the cell population enriched for OCT4 expression is characterized by OCT4^hl expression. [00119] In another embodiment, the invention provides a method for testing a candidate compound for an ability to inhibit the proliferation of a neoplastic stem cell (NSC), comprising the steps of: contacting a cell population enriched for OCT4 expression with a candidate compound, wherein the cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and; measuring the expression of an identifiable gene product; thereby testing a candidate compound for an ability to inhibit the proliferation of a NSC. In another embodiment, the cell population enriched for OCT4 expression is characterized by OCT4^hl expression.

[00120] In another embodiment, the invention provides a method for testing a candidate compound for an ability to transform a BCC to a neoplastic stem cell (NSC), comprising the steps of: contacting a cell population with a candidate compound. In another embodiment, the cell population has been transfected with an expression vector comprising a gene encoding an identifiable gene product under control of a cancer stem cell responsive promoter. In another embodiment, the cell population has been transfected with an expression vector comprising a gene encoding an identifiable gene product under control of an Oct4 responsive promoter. In another embodiment, the invention provides that a cell population has been enriched by selecting cells that express the identifiable gene product. In another embodiment, the invention provides that a cell population has been enriched by selecting cells that express the identifiable gene product at high levels.

[00121] In another embodiment, the invention provides culturing a cell population under NSC promoting conditions. In another embodiment, the invention provides culturing a cell population under NSC suppressing conditions. In another embodiment, the invention provides culturing a cell population under BCC suppressing conditions. In another embodiment, the invention provides culturing a cell population under BCC promoting conditions. In another embodiment, the invention provides culturing a cell population under NSC promoting and BCC suppressing conditions. In another embodiment, the invention provides culturing a cell population under NSC suppressing and BCC promoting conditions. In another embodiment, the invention provides that the cell population expressing the identifiable gene product is characterized by OCT4hi expression. [00122] In another embodiment, the invention provides that the step of measuring the expression of an identifiable gene product comprises measuring the level of expression of an identifiable gene product in a single cell. In another embodiment, the invention provides that the step of measuring the expression of an identifiable gene product comprises measuring the level of expression of an identifiable gene product in a small group of cells. In another embodiment, the invention provides that changes in the level of expression of a single cell or small group of cells are relative to other cells in the culture. In another embodiment, the invention provides that a change of a function of already present equals change in signal intensity. [00123] In another embodiment, the invention provides a method for testing a candidate compound for an ability to transform a NSC to a BCC comprising the steps of: (a) contacting a cell population with a candidate compound, wherein the cell population has been transfected with an expression vector comprising a gene encoding an identifiable gene product under control of an Oct4 responsive promoter, and wherein the cell population has been enriched by selecting cells that express said identifiable gene product at high levels and culturing said cell population under NSC promoting and BCC suppressing conditions, and wherein said cell population expressing the identifiable gene product is characterized by OCT4hi expression; and [00124] In another embodiment, the invention provides a method for testing a candidate compound for an ability to inhibit the asymmetric division of a cancer stem cell (CSC), comprising the steps of: (a) contacting a cell population with a candidate compound, wherein said cell population has been transfected with an expression vector comprising a gene encoding an identifiable gene product under control of an Oct4 responsive promoter. In another embodiment, the cell population has been enriched by selecting cells that express an identifiable gene product. In another embodiment, the invention provides culturing cell population under NSC promoting and BCC suppressing conditions or vice versa. In another embodiment, the invention provides that the cell population expressing the identifiable gene product is characterized by OCT4M expression. [00125] In another embodiment, the invention provides a method for testing a candidate compound for an ability to inhibit the symmetric division of a cancer stem cell (CSC), comprising the steps of: (a) contacting a cell population with a candidate compound, wherein said cell population has been transfected with an expression vector comprising a gene encoding an identifiable gene product under control of an Oct4 responsive promoter. In another embodiment, the cell population has been enriched by selecting cells that express an identifiable gene product. In another embodiment, the invention provides culturing cell population under NSC promoting and BCC suppressing conditions or vice versa. In another embodiment, the invention provides that the cell population expressing the identifiable gene product is characterized by OCT4M expression. [00126]

[00127] In another embodiment, the invention provides that the terms cancerous stem cells and neoplastic stem cells are used interchangeably. [00128] In another embodiment, the invention further provides a method for testing a candidate compound for an ability to arrest the cell cycle, prevent cell division, or otherwise prohibit growth (cytostatic), or cause cell death (cytotoxic), comprising the steps of: (a) contacting a cell population with a candidate compound, wherein a cell population has been transfected with an expression vector comprising a gene encoding an identifiable gene product under control of an Oct4 responsive promoter. In another embodiment, the invention provides that the cell population has been enriched by selecting cells that express an identifiable gene product. In another embodiment, the invention provides that the cell population has been enriched by selecting cells that express an identifiable gene product at high levels. In another embodiment, the invention provides culturing a cell population under CSC promoting and BCC suppressing conditions or vice versa. [00129] In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 1-10% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 7-13% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 5-20% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 12-24% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 18-25% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 20-35% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 28-35% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 30-50% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 35-45% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 45-60% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 55-65% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 55-70% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 65-80% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 70-85% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 75-98% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 80-90% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 85-100% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 90-100% of NSCs in a NSC population. In another embodiment, the invention provides that a candidate compound inhibits the proliferation of 95-100% of NSCs in a NSC population.

[00130] In another embodiment, the methods of the present invention provide that the proliferation rate of NSCs is measured by FACS. In another embodiment, FACS measures the amount of DNA in a NSC. In another embodiment, the methods of the present invention provide that the proliferation rate of NSCs is measured by XTT. In another embodiment, the methods of the present invention provide that the proliferation rate of NSCs is measured by MTT. In another embodiment, the methods of the present invention provide that the proliferation rate of NSCs is measured by any relevant means known to one of skill in the art. [00131] In another embodiment, the invention provides a method for testing a candidate compound for an ability to transform cells to NSCs, comprising the steps of contacting a cell population with a candidate compound, wherein the cell population has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and measuring the expression of an identifiable gene product. In another embodiment, the invention provides a method for testing a candidate compound for an ability to transform non-neoplastic cells to NSCs, comprising the steps of contacting a cell population with a candidate compound, wherein the cell population has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and measuring the expression of an identifiable gene product. In another embodiment, the invention provides a method for testing a candidate compound for an ability to transform differentiated cells to NSCs, comprising the steps of contacting a cell population with a candidate compound, wherein the cell population has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and measuring the expression of an identifiable gene product. In another embodiment, the invention provides a method for testing a candidate compound for an ability to transform mammal non-neoplastic dividing cells to NSCs, comprising the steps of contacting a cell population with a candidate compound, wherein the cell population has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and measuring the expression of an identifiable gene product.

[00132] In another embodiment, the invention provides that the candidate compound transforms 1-10% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 5-15% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 10- 20% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 15-25% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 20-30% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 25-40% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 30-50% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 40-60% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 50-60% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 60-80% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 60-70% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 70-80% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 80-100% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 80-90% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 90-95% of the non-neoplastic cells to NSCs. In another embodiment, the invention provides that the candidate compound transforms 95-100% of the non-neoplastic cells to NSCs. [00133] In another embodiment, the invention provides that the candidate compound is a protein. In another embodiment, the invention provides that the candidate compound is a mutated protein. In another embodiment, the invention provides that the candidate compound is an oncogenic protein.

[00134] In another embodiment, the invention provides that the candidate compound is a nucleic acid. In another embodiment, the invention provides that the candidate compound is a viral nucleic acid. In another embodiment, the invention provides that the candidate compound is a bacterial nucleic acid. In another embodiment, the invention provides that the candidate compound is a mammalian nucleic acid. In another embodiment, the invention provides that the candidate compound is a mutated nucleic acid.

[00135] In another embodiment, the invention provides that the candidate compound is an organic compound. In another embodiment, the invention provides that the candidate compound is an organo-metallic compound. In another embodiment, the invention provides that the candidate compound is an inorganic compound.

[00136] In another embodiment, the invention provides the measurement of the expression of an identifiable gene product. In another embodiment, the invention provides that measuring the expression of an identifiable gene product comprises measuring the number of cells expressing the identifiable gene product. In another embodiment, a candidate compound inhibits NSCs and thus inhibits OCT4 expression and consequently reduces the number of cells expressing an identifiable gene product controlled by OCT4 promoter. In another embodiment, a candidate compound induces NSCs and thus promotes OCT4 expression and consequently increases the number of cells expressing an identifiable gene product controlled by OCT4 promoter. In another embodiment, a candidate compound induces OCT4 expression in BCCs and consequently increases the number of cells expressing an identifiable gene product controlled by OCT4 promoter. In another embodiment, a candidate compound that induces OCT4 expression in BCCs induces the transformation of BCCs to NSCs. In another embodiment, a candidate compound induces OCT4 expression in non-cancerous cell population and consequently increases the number of cells expressing an identifiable gene product controlled by OCT4 promoter. In another embodiment, a candidate compound that induces OCT4 expression in non-cancerous cell population induces stem cell neoplastic transformation. In another embodiment, a candidate compound that induces OCT4 expression in non-cancerous cell population is a carcinogen.

[00137] In another embodiment, the invention provides the measurement of the expression of an identifiable gene product. In another embodiment, the invention provides that measuring the expression of an identifiable gene product comprises measuring the overall identifiable gene product controlled by OCT4 expression. In another embodiment, a candidate compound inhibits NSCs and thus inhibits OCT4 expression and consequently reduces the overall identifiable product in a cell population compared to a control comprising the same population treated with an inert compound. In another embodiment, a candidate compound induces NSCs and thus promotes OCT4 expression and consequently increases the overall identifiable gene product controlled by OCT4 promoter. In another embodiment, a candidate compound induces OCT4 expression in BCCs and consequently increases the overall identifiable gene product controlled by OCT4 promoter. In another embodiment, a candidate compound that induces OCT4 expression in BCCs induces the transformation of BCCs to NSCs. In another embodiment, a candidate compound induces OCT4 expression in non-cancerous cell population and consequently increases the overall identifiable gene product controlled by OCT4 promoter. In another embodiment, a candidate compound that induces OCT4 expression in non-cancerous cell population induces stem cell neoplastic transformation. In another embodiment, a candidate compound that induces OCT4 expression in non-cancerous cell population is a carcinogen.

[00138] In another embodiment, the present invention provides means of measuring the number of cells expressing an identifiable gene product controlled by OCT4 promoter. In another embodiment, the present invention provides that a FACS is used for measuring the number of cells expressing an identifiable gene product controlled by OCT4 promoter. In another embodiment, a FACS is further used to separate the cells induced for expression of an identifiable gene product controlled by OCT4 promoter. In another embodiment, the OCT4 positive enriched cell population can be further studied and manipulated by means known to one of skill in the art. In another embodiment, the OCT4 positive enriched cell population can be further propagated by the methods of the present invention. [00139] In another embodiment, the present invention provides that a microscope is used for measuring the number of cells expressing an identifiable gene product controlled by OCT4 promoter. In another embodiment, the present invention provides that a confocal microscope is used for measuring the number of cells expressing an identifiable gene product controlled by OCT4 promoter. In another embodiment, the present invention provides that a fluorescent microscope is used for measuring the number of cells expressing an identifiable gene product controlled by OCT4 promoter. In another embodiment, the present invention provides that an electron microscope is used for measuring the number of cells expressing an identifiable gene product controlled by OCT4 promoter. In another embodiment, the present invention further provides a CCD camera attached to a microscope of the present invention. In another embodiment, the present invention further provides that a CCD camera is further attached to a computer comprising a software assessing the number of cells expressing an identifiable gene controlled by OCT4 promoter (positive) and the number of cells that are not expressing an identifiable gene controlled by OCT4 promoter (negative). In another embodiment, a system of the present invention comprise a CCD camera attached to a computer comprising a software assessing the number of cells expressing an identifiable gene controlled by OCT4 promoter (positive) and the number of cells that are not expressing an identifiable gene controlled by OCT4 promoter (negative). [00140] In another embodiment, the system of the present invention enables the identification of compounds inducing the expression of an identifiable gene controlled by OCT4 promoter. In another embodiment, the system of the present invention enables the identification of compounds inhibiting the expression of an identifiable gene controlled by OCT4 promoter. [00141] In another embodiment, the present invention provides means of measuring the overall amount of an identifiable gene product controlled by OCT4 promoter in a sample comprising the cells of the present invention and a candidate compound of the present invention. In another embodiment, the present invention provides means of measuring the overall amount of an identifiable gene product controlled by OCT4 promoter in a sample comprising the cells of the present invention and an inert compound of the present invention. In another embodiment, the present invention provides that a fluorometer is used for measuring the overall amount of an identifiable gene product controlled by OCT4 promoter in a sample comprising the cells of the present invention. In another embodiment, the present invention provides that a luminescence reader is used for measuring the overall amount of an identifiable gene product controlled by OCT4 promoter in a sample comprising the cells of the present invention.

[00142] In another embodiment, the present invention provides that a microscope is used for measuring the overall amount of an identifiable gene product controlled by OCT4 promoter in a sample comprising the cells of the present invention. In another embodiment, the present invention provides that a confocal microscope is used for measuring the overall amount of an identifiable gene product controlled by OCT4 promoter in a sample comprising the cells of the present invention. In another embodiment, the present invention provides that a fluorescent microscope is used for measuring the overall amount of an identifiable gene product controlled by OCT4 promoter in a sample comprising the cells of the present invention. In another embodiment, the present invention provides that an electron microscope is used for measuring the overall amount of an identifiable gene product controlled by OCT4 promoter in a sample comprising the cells of the present invention. In another embodiment, the present invention further provides a CCD camera attached to a microscope of the present invention. In another embodiment, the present invention further provides that a CCD camera is further attached to a computer comprising a software assessing the overall amount of an identifiable gene product controlled by OCT4 promoter in a sample comprising the cells of the present invention. [00143] In another embodiment, the present invention provides a system for testing a candidate compound for an ability to inhibit a neoplastic stem cell population, comprising an isolated neoplastic stem cell population enriched for expression of OCT4, wherein the isolated neoplastic stem cell population enriched for expression of OCT4 is transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product. In another embodiment, the present invention provides a system for testing a candidate compound for an ability to transform a neoplastic cell population to a neoplastic stem cell population, comprising an isolated BCC population wherein isolated BCC population is transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product. In another embodiment, the present invention provides a system for testing a candidate compound for an ability to transform a nonneoplastic cell population to a neoplastic stem cell population, comprising a non-neoplastic cell population wherein non-neoplastic cell population is transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product

[00144] In another embodiment, the system of the present invention comprises a bank of candidate compounds. In another embodiment, the system of the present invention comprises a bank of control compounds. In another embodiment, the system of the present invention comprises a bank of control-inert compounds. In another embodiment, the system of the present invention comprises a fluorescent microscope, a FACS, a fluorometer, a luminescence reader, or any combination thereof. In another embodiment, the system of the present invention comprises any means, for identifying or quantifying an identifiable gene of the present invention, as known to one of skill in the art.

[00145] In another embodiment, a system of the present invention comprise a CCD camera attached to a computer comprising a software assessing the overall amount of an identifiable gene product controlled by OCT4 promoter in a sample comprising the cells of the present invention. In another embodiment, a sample of the present invention further comprises a candidate compound. In another embodiment, a sample of the present invention further comprises an inert compound. In another embodiment, a sample comprising an inert compound is used as a control sample. [00146] In another embodiment, the luminescence or fluorescence signal is detected by a microscopic imaging system comprising algorithmic imaging software. In another embodiment, the algorithmic imaging software monitors the amount of labeled cells in a given specimen. In another embodiment, the algorithmic imaging software monitors the relative amount of labeled cells. [00147] In another embodiment, the method and system of the present invention comprise using cells cultured in miniaturized format. In another embodiment, cells cultured in miniaturized format of the present invention comprise multi-well plates. In another embodiment, a multi-well plate of the present invention comprises 96 wells. In another embodiment, a multi-well plate of the present invention comprises 384 wells (example X). In another embodiment, a multi-well plate of the present invention comprises 1536 wells. In another embodiment, a multi-well plate of the present invention comprises from 2-5000 wells. In another embodiment, a multi-well plate of the present invention comprises from 20-3000 wells. In another embodiment, a multi-well plate of the present invention comprises from 96- 2000 wells. [00148] In another embodiment, the invention provides a heterogeneous cell population transfected with a plasmid comprising a CMV responsive promoter controlling the expression of an identifiable and/or selectable gene product (Example 9, Fig. 17). In another embodiment, the methods described previously for identification of neoplastic stem cells comprising the use of various identifiable fluorescent protein sequences are also employed for the identification of BCCs. In another embodiment, the present invention provides bulk cancer cells (BCCs) do not comprise NSCs. In another embodiment, the methods of the present invention provide that BCCs are separated using the cell separation methods of the present invention. [00149] In another embodiment, the invention provides a heterogeneous cell population transfected with a plasmid comprising a general, non-specific responsive promoter controlling the expression of an identifiable and/or selectable gene product which differs from the identifiable and/or selectable gene product directed by the Oct4 responsive promoter. (Example 9, Fig. 19). In another embodiment, the invention provides a heterogeneous cell population transfected with at least two plasmids wherein one plasmid comprises a CMV responsive promoter controlling the expression of a first identifiable and/or selectable gene product and a second plasmid comprising Oct4 responsive promoter controlling the expression of a second identifiable and/or selectable gene product. In another embodiment, the invention provides a heterogeneous cell population transfected with at least two plasmids wherein one plasmid comprises a CMV responsive promoter controlling the expression of RFP (Red Fluorescent Protein) and a second plasmid comprising Oct4 responsive promoter controlling the expression of an identifiable and/or selectable gene product. [00150] In another embodiment, the methods of the present invention provide that NSCs are transfected with both plasmids, wherein one plasmid comprises a CMV responsive promoter controlling the expression of a first identifiable and/or selectable gene product and a second plasmid comprising Oct4 responsive promoter controlling the expression of a second identifiable and/or selectable gene product, express both identifiable gene products. In another embodiment, an isolated population of NSCs transfected with both plasmids is used for testing candidate compounds that inhibit NSCs by converting NSCs to BCCs. In another embodiment, the methods of the present invention provide that compounds which convert NSCs to BCCs will inhibit the expression of an identifiable and/or selectable gene directed by Oct4 responsive promoter. In another embodiment, the present invention provides that the effect of a compound converting NSCs to BCCs correlates to percentage of cells which do not express an identifiable and/or selectable gene directed by Oct4 responsive promoter. In another embodiment, the present invention provides that the effect of compounds which increase the percentage of BCCs in a NSC enriched populations is measures by FACS. In another embodiment, compounds that cause NSCs to convert to BCCs, cause NSCs to loose their "sternness". [00151] In another embodiment, the methods of the present invention provide that BCCs are transfected with both plasmids, wherein one plasmid comprises a CMV responsive promoter controlling the expression of a first identifiable and/or selectable gene product and a second plasmid comprising Oct4 responsive promoter controlling the expression of a second identifiable and/or selectable gene product. In another embodiment, the present invention provides that BCCs express only the first plasmid comprising a CMV responsive promoter controlling the expression of a first identifiable and/or selectable gene product. In another embodiment, the methods of the present invention provide that compounds which convert BCCs to NSCs will induce the expression of the identifiable and/or selectable gene directed by Oct4 responsive promoter. In another embodiment, the present invention provides that the effect of a compound converting BCCs to NSCs correlates to the percentage of cells which express an identifiable and/or selectable gene directed by Oct4 responsive promoter. In another embodiment, the present invention provides that the effect of compounds which increase the percentage of NSCs in a BCC enriched or isolated population is measured by FACS. In another embodiment, compounds that cause BCCs to convert to NSCs, cause BCC to gain "sternness".

[00152] In another embodiment, the present invention provides a biotechnology platform for testing conventional anti-cancer drugs. In another embodiment, the present invention provides a biotechnology platform for testing novel compounds for anti-cancer activity against NSCs. In another embodiment, the present invention provides a biotechnology platform which allows for the testing of NSCs derived from a given heterogeneous cancer cell population. In another embodiment, the present invention provides that NSCs comprise a rare subset of cancer cells. [00153] In another embodiment, the present invention provides that NSCs of the present invention are key target in cancer drug discovery platform of the present invention. In another embodiment, the present invention provides that NSCs are the determinants of tumor initiation. In another embodiment, the present invention provides that NSCs are the determinants of tumor growth. In another embodiment, the present invention provides that NSCs are the determinants of metastatic spread. In another embodiment, the present invention provides that the majority of cancer cells are unable create similar biological events in a cancer life cycle. In another embodiment, the present invention provides that BCCs, are unable create similar biological events in a cancer life cycle.

[00154] In another embodiment, the present invention provides that heterogeneous cancer cell populations derived from clinical tumor specimens (whether primary or metastatic) or from permanent tumor cell lines can be manipulated to allow for the isolation and propagation of their respective cancer stem cell populations. In another embodiment, the present invention provides methods for the identification, sorting and stable maintenance in culture subsets of NSCs based on their ability to maintain the expression of fluorescent (or luminescent) proteins driven by the promoter of the Oct3/4 transcription factor. In another embodiment, the present invention provides that Oct3/4 transcription factor in concert with SOX-2, Nanog and STAT3, are the regulators of normal stem cell phenotype in the context of embryonic development including the process of self -renewal. [00155] In another embodiment, the present invention provides that in the context of cancer, NSCs do not have the appropriate proliferation controls allowing the process of self -renewal to go unchecked resulting in dysplastic tissue mass at site of proliferation. In another embodiment, the methods of the present invention provides the use of biomarkers that are regulated in parallel to the molecular machinery mentioned above. In another embodiment, these regulated biomarkers monitor the "sternness" of a given cancer cell. . In another embodiment, these regulated biomarkers are used for a multitude of sensitive cellular/molecular assays and for their identification, isolation, and propagation en-masse. [00156] In another embodiment, the present invention allows for the monitoring of the relative viability and "sternness" of the NSC population, as well as the absolute cell number of the above described cancer cell populations when exposed to a given anti-cancer drug or a novel compound. In another embodiment, the present invention provides methods for qualitatively and quantitatively monitoring of all dynamic biological shifts (with respect to phenotype) occurring in cancer cell subsets when exposed various test drugs or compounds. [00157] In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 0.1 μM- 100 mM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 0.1 - 4 μM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 0.1-0.5 μM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 1-2 μM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 2-3 μM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 3-5 μM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 5-10 μM.

[00158] In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 10-100 μM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 100 μM-1 mM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 1-5 mM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 5-15 mM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 15-30 mM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 30-50 mM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 50-75 mM. In another embodiment, the candidate compound screened according to the methods of the present invention is applied in a concentration of 75-100 mM.

[00159] In another embodiment, a candidate compound kills NSCs. In another embodiment, a candidate compound causes NSCs to mature and thus lose their "sternness" and NSCs markers. In another embodiment, the effect of a candidate compound on a NSC population is measured by FACS. In another embodiment, compounds which inhibit the proliferation of NSCs, cause NSCs to mature, or kill NSCs will cause a decline in FACS counting of fluorescence markers compared to control NSCs treated with PBS. In another embodiment, FACS counting of cells comprising NSC markers correlates to the number of NSCs. [00160] In another embodiment, candidate compounds that kill cause senescence, or mature NSCs are considered as NSCs inhibitors. In another embodiment, candidate compounds that cause NSCs inhibition identified by the methods of the present invention can treat breast cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat prostate cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat leukemia. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat chronic myeloid leukemia. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat skin cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat colorectal cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat premalignant polyps. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat adenocarcinoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat mammary adenocarcinoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat lymphoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat mantle cell lymphoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat a carcinoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat oral squamous cell carcinoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat pancreatic tumors. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat bladder cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat lung cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat gastrointestinal tumors. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat head tumors. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat neck tumors. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat Hodgkin's lymphoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat neuroblastoma. [00161] In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat adrenocortical carcinoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat anal cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat bladder cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat brain tumor. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat brain stem glioma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat cerebellar astrocytoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat cerebral astrocytoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat ependymoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat medulloblastoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat supratentorial primitive neuroectodermal. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat pineal tumors. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat hypothalamic glioma.

[00162] In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat breast cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat carcinoid tumor. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat sarcoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat cervical cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat colon cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat endometrial cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat esophageal cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat extrahepatic bile duct cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat ewings family of tumors (pnet). In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat extracranial germ cell tumor. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat eye cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat intraocular melanoma. [00163] In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat gallbladder cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat gastric cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat germ cell tumor. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat extragonadal. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat gestational trophoblastic tumor. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat hypopharyngeal cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat islet cell carcinoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat laryngeal cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat leukemia. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat acute lymphoblastic. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat liver cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat lung cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat small cell lymphoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat AIDS-related lymphoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat central nervous system (primary) lymphoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat cutaneous T-cell lymphoma.

[00164] In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat hodgkin's disease. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat non-hodgkin's disease. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat malignant mesothelioma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat melanoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat merkel cell carcinoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat metasatic squamous carcinoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat multiple myeloma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat plasma cell neoplasms. [00165] In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat mycosis fungoides. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat myelodysplastic syndrome. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat myeloproliferative disorders. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat nasopharyngeal cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat neuroblastoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat oropharyngeal cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat osteosarcoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat ovarian epithelial cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat ovarian germ cell tumor. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat ovarian low malignant potential tumor. [00166] In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat pancreatic cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat pancreatic cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat islet cell carcinoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat paranasal sinus and nasal cavity cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat parathyroid cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat penile cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat pheochromocytoma cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat pituitary cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat plasma cell neoplasm. [00167] In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat rhabdomyosarcoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat renal cell cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat salivary gland cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat sezary syndrome. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat cutaneous T-cell lymphoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat kaposi's sarcoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat melanoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat small intestine cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat soft tissue sarcoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat testicular cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat thymoma. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat thyroid cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat urethral cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat uterine cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat unusual cancer of childhood. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat vaginal cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat vulvar cancer. In another embodiment, NSCs inhibitors identified by the methods of the present invention can treat wilms' tumor. [00168] In another embodiment, candidate compounds of the present invention are screened on NSC culture obtained from a primary cell culture derived from a tumor. In another embodiment, candidate compounds of the present invention are screened on NSC culture obtained from a cancer patient suffering from one of the cancers listed hereinabove. In another embodiment, the cells obtained from a tumor or a cancer patient is further transfected with a plasmid of the invention comprising a NSC marker.

[00169] In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 1-48 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 1-3 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 3-5 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 5-7 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 7-9 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 4-6 hours after administration.

[00170] In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 9-12 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 12-15 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 15-18 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 18-21 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 21-24 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 24-30 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 30-36 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 36- 42 hours after administration. In another embodiment, candidate compound that act as NSCs inhibitors inhibit NSCs within 42-48 hours after administration. [00171] In another embodiment, the invention provides a method of evaluating the effect of photodynamic therapy (PDT) on tumor derived primary cell culture. In another embodiment, the effect of radiation therapy or radiofrequency ablation alone or in combination with any other form of a therapeutic agent on tumor primary cell culture is further assessed. In another embodiment, the effect of chemoembolization on tumor derived primary cell culture is analyzed. In another embodiment, the effect of local hyperthermia on tumor derived primary cell culture is analyzed. In some embodiment, the in-vivo effect of various agents and conditions is desired.

[00172] In another embodiment, the invention provides a method wherein an agent of interest is further administered in-vivo to an animal that has been transplanted with OCT4 expressing NSCs. In another embodiment, the OCT4 expressing NSCs express OCT4^hl. In another embodiment, administration of an agent is according to procedures known to one skilled in the art. In another embodiment, single or multiple administrations of an agent or agents are required, as known to one skilled in the art. In another embodiment, the agent or agents are administered over a period of days to weeks or over a period of months to years, depending on cancer progression and/or regression, as known to one skilled in the art. In another embodiment, the agent is a carcinogen which in another embodiment is a carcinogenic substance in categories 1 through 3 of the International Agency for Research on Cancer (IARC). In another embodiment, the agent is a therapeutic agent. [00173] In another embodiment, the invention provides a means of exploring the effects of a therapeutic agent on cancer progression (Fig. 12). In another embodiment, the effects of a therapeutic agent on cell metastasis potential are evaluated. In another embodiment, the effects of a therapeutic agent on a soft tumor are evaluated. In another embodiment, the effects of a therapeutic agent on a hard tumor are evaluated. In another embodiment, the effect of a therapeutic agent on primary and/or secondary tumor growth is evaluated.

[00174] In another embodiment, the therapeutic agent or agents administered in-vivo to an animal transplanted with OCT4 or OCT4^hl neoplastic stem cells (Fig. 12) comprise: interferons, interleukins, colony-stimulating, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, steroid hormones or combinations thereof. In another embodiment, the therapeutic agent is a chemotherapy agent. In another embodiment, the chemotherapy agent is non-specific and therefore has the potential to kill a cancerous cell during any phase of the cell-cycle. In another embodiment, the chemotherapy agent is specific and thus is able to kill cancerous cells during a specific cell cycle phase. [00175] In another embodiment, the in-vivo effect of PDT on an animal transplanted with OCT4^hl neoplastic stem cells is evaluated. In another embodiment, the in-vivo effects of radiation therapy or radiofrequency ablation alone or in combination with any other form of a therapeutic agent in-vivo are further assessed. In another embodiment, the in-vivo effects of chemoembolization or local hyperthermia, on cancer progression and/or regression are evaluated.

[00176] In another embodiment, the in-vivo effect of biological therapy on an animal transplanted with OCT4^hl neoplastic stem cells derived from tumor or primary cell culture is analyzed. In another embodiment, biological therapies comprise immunotherapy. In another embodiment, immunotherapy comprises the use of a vaccine comprising immunogenic fragments derived from Nanog, STAT3, OCT4, or combinations thereof, as described hereinabove. In another embodiment, the effect of nonspecific immunomodulating agent or agents is assessed. In another embodiment, the nonspecific immunomodulating agent is bacillus Calmette-Guerin (BCG) or levamisole.

[00177] In another embodiment, OCT4 modifiers are screened in-vivo for cancer progression or regression in an animal transplanted with OCT4^hl neoplastic stem cells (Fig. 12). In another embodiment, OCT4 monoclonal antibodies are screened in-vivo. In another embodiment, intrabodies specific to an OCT4 protein are screened in-vivo. In another embodiment, PNAs, aptamers, or antisense siRNA are further evaluated in-vivo as shown in Fig. 4.

[00178] In another embodiment, the invention provides a method of preventing, abrogating, or inhibiting cancer, tumor growth, cell metastasis or combinations thereof comprising the step of contacting neoplastic cells with an agent that inhibits OCT4 expression or function. In another embodiment, OCT4 is inhibited transiently. In other embodiments, OCT4 is inhibited constitutively.

[00179] In another embodiment, the invention provides a method of inhibiting OCT4 comprising targeting OCT4 expression at the DNA level and thus inhibiting or abrogating OCT4 transcription. In another embodiment, inhibition of OCT4 at the DNA level is accomplished via the formation of DNA triple- stranded structures. In another embodiment, the triple helix inhibition complex, is designed as an OCT4 gene-specific oligonucleotide and thus inhibits OCT4 transcription.

[00180] In another embodiment, the invention provides a method of inhibiting OCT4 comprising targeting OCT4 expression at the RNA level and thus inhibiting OCT4 expression. In another embodiment, RNA is mRNA. In another embodiment, antisense based therapeutics are used to inhibit OCT4. In another embodiment, synthetic oligonucleotides are designed to be complementary in sequence to a specific OCT4 mRNA sequence and thus inhibit OCT4 expression. [00181] In another embodiment, the invention provides peptide nucleic acids (PNAs) that are artificially constructed to hybridize to an OCT4 mRNA sequence and thus inhibit OCT4 expression. In another embodiment, the binding agent is a specifically engineered ribozyme, which cleaves OCT4 mRNA transcripts and subsequently inhibits OCT4 expression. [00182] In another embodiment, the method of inhibiting OCT4 function comprises targeting OCT4 protein. In another embodiment, inhibition of OCT4 function is achieved through the specific binding of an antibody to an OCT4 protein and thus inhibiting or abrogating OCT4 protein binding to an OCT4 responsive DNA element. In another embodiment, intrabody or antibodies raised subsequent to OCT4 immunotherapy, inhibit or abrogate OCT4 function. [00183] In another embodiment, the invention provides an intrabody specific to OCT4 protein. In another embodiment, intrabodies comprise a single chain of a coupled variable domain of the heavy chain to the variable domain of the light chain through a peptide linker and are used to interfere with the binding of the OCT4 protein to an OCT4 DNA responsive element. In another embodiment, intrabodies are directed to the cell nucleus where they inhibit or abrogate binding of an OCT4 protein to an OCT4 DNA responsive element. In another embodiment, the intrabodies target the OCT4 protein DNA binding domain on the OCT4 protein and hence, inhibit or abrogate OCT4 protein binding to an OCT4 DNA responsive element. [00184] In another embodiment, the invention provides a vaccine comprising an OCT4 peptide. In another embodiment, the OCT4 peptide elevates OCT4 specific antibodies. In another embodiment, the peptide consists of the full length OCT4 gene. In another embodiment, the peptide is a mutated form of OCT4. In another embodiment, a 4-18 amino acid long OCT4 peptide is used. In another embodiment, the vaccine comprises OCT4 peptides of uniform length and sequence. In another embodiment, the vaccine comprises a mixture of OCT4 peptides that differ in both length and sequence.

[00185] In another embodiment, oligonucleotide aptamers are used to bind specific OCT4 protein sequence and thus inhibit or abrogate OCT4 protein binding to an OCT4 DNA responsive element. [00186] In another embodiment, the invention provides a mutated OCT4 protein. In another embodiment, the mutated OCT4 protein is used to block the transcription of downstream OCT4 responsive genes. In another embodiment, the mutated OCT4 protein used to inhibit or abrogate OCT4 responsive gene expression and the wild type OCT4 protein have similar affinities to the OCT4 DNA responsive element. In another embodiment, the mutated OCT4 protein used to inhibit or abrogate OCT4 responsive genes expression have a higher affinity to the OCT4 DNA responsive element compared to the wild type OCT4 protein.

Materials and Methods

Immunohistochemical identification of OCT4, STAT3 or Nanog positive cells in tissue [00187] Immunohistochemical staining for OCT4, STAT3 or Nanog histological analyses of tumor biopsies from human and from mice were preformed as follows: Formalin fixed paraffin embedded tissue sections (5μm) were sequentially deparaffinized, rehydrated and blocked for endogenous peroxidase activity following a 95° C degree, 25 minute antigen retrieval in Trilogy unmasking solution (Cell Marque, Hot Springs AR). Slides were biotin blocked, serum blocked and immunostained using a goat ABC Elite Kit (Vector Labs, Burlingame, CA) Antibodies to OCT 3/4, STAT3 and Nanog (R&D Systems, Minneapolis, MN) were applied at 1:50 dilution for one hour at room temperature. Positive staining was detected with DAB (3,3'-Diaminobenzidene) and light green SF yellowish or hematoxylin (Sigma, St. Louis, MO) was used as counterstain. Alternatively, primary breast cancer foci and the brain and lung were harvested for sectioning by cryostat. Tissues were cut on a cryostat at 16μm to generate sets that are in the axial plane (breast and lung) and coronal plane (brain). Hematoxylin-eosin (H&E) staining was performed on one set, and immunohistochemistry on a second set. The immunohistochemistry was performed using the following procedures. The frozen breast, lung, and brain sections were (1) incubated in 2% non-fat milk and 0.3% Triton-X in PBS for 1 hour; (2) incubated in OCT4, STAT3 or Nanog antibodies in 3% donkey serum and 0.1% Triton-X overnight at room temperature; (3) washed with PBS for 3 times; (4) incubated with secondary antibody for 4 hours in dark at room temperature; (5) washed with PBS for 3 times; and (6) dehydrated through graded ethanol, cleared with xylene, and coverslipped with DPX mounting medium (44581, Fluka Biochemika). Immunoreactivity was visualized with a Bio- Rad confocal microscope and images collected on a computer for later analysis.

Immunocvtochemical identification of OCT4, STAT3 or Nanog positive cell culture [00188] Immunohistochemical staining for identification of OCT4, STAT3 or Nanog positive cells in primary culture of tumor biopsies from human and mice was carried out by mincing the specimens into small particles in DMEM/F12 medium digested with 300 U/ml Collagenase Type II ( Gibco BRL Invitrogen Corporation, Grand Island, NY, USA) for 3-6 hours and passed through a 70 μm Cell Strainer (Becton Dickinson Lab Ware, Franklin Lakes, NJ USA) to prepare single-cell suspension. Cells were then drained of all medium rinsed with PBS, suspended in culture medium and plated. After cells attachment fixation solution containing fresh 4% formaldehyde solution with 0.1% Triton X-IOO was applied. Cells were blocked for endogenous peroxidase activity following biotin blocked, serum blocked and immunostained using a goat ABC Elite Kit (Vector Labs, Burlingame, CA) Antibodies to OCT 3/4, STAT3 and Nanog (R&D Systems, Minneapolis, MN) were applied at 1:200 for 30 minutes at room temperature. Positive staining was detected with DAB (3,3'- Diaminobenzidene) and hematoxylin (Sigma, St. Louis, MO) was used as the counterstain.

The OCT4-EGFP and Nanog- EGFP constructs

[00189] The OCT4-EGFP and Nanog- EGFP constructs were engineered using strategies and techniques previously described (Gerrard et al., 2005). A plasmid containing the EGFP reporter (pEGFPl, BD Biosciences) and the selectable marker G418 under the control of the OCT4 and Nanog promoter was used. The promoter fragment of human OCT4 spans from base -3917 to base +55 of the OCT4 gene (hOCT4pr, from 67539 to 71490 in the human DNA sequence), and contains two appropriate regulatory elements which drove developmentally specific EGFP expression. The promoter fragment of human Nanog spans from base -132 to base +300 (from base 697969 to base 701269 in the human genomic DNA sequence of Chromosome 12).

Expression vectors used to create biomarked cells

Oct4hP-eGFP plasmid was constructed using the human Oct-4 promoter (Oct4hP, from 67539 to 71490 in human DNA sequence with accession number AP000509)_that was amplified by polymerase chain reaction with primers Oct4hP-F (5'-TT CCC ATG TCA AGT AAG TGG GGT GG-3') and Oct4hP-R (5'-CGA GAA GGC AAA ATC TGA AGC CAG G-3') using human genomic DNA (Promega G3041) as a template. The fragment was cloned into a TOPO vector (Invitrogen) and the fidelity of the DNA sequence was confirmed with bi-directional DNA sequencing. Oct4hP was then cloned into the expression vector pEGFPl (Clontech Cat # 6086-1, Genbank Accession # U55761) by insertion into the HindIH and BamHl sites upstream of eGFP (Figure 12A).

Modification of the neurosphere culture system for isolation and analyses of NSCs from glioblastoma, bone sarcomas, and breast cancer

[00190] The neurosphere culture system proposed by Weiss and Raynolds (1992) was modified by inhibiting the potential of cells for substrate attachment by exploiting pleiotropical growth factors - EGF, FGF2 and insulin in semi-solid methylcellulose (MC). In this experiment NSCs were transfected with EGFP reporter plasmid having the EFGP and G418 genes under the human promoter of OCT4 as shown in Fig. 7.

Lesions and foci dissections

[00191] The lesions and foci were dissected from their respective tissue locations using a Leica MZl 6FA dissecting microscope with a GFP3 filter (for fluorescence capability) and Q- imaging Retiga EXi monochrome digital camera with RGB filter for in-vitro studies and molecular analysis. This tumor material was further studied by using different assays.

RNA isolation and target cDNA amplification

[00192] Total RNA was isolated using the RNeasy Mini Kit (and treated with RNAase-Free DNase Set (Qiagen Sciences, MD, USA), by using DNAase treatment. A Superscript II RNase H⁺ Reverse Transcriptase first-strand synthesis system (InVitrogen Life Technologies, Carlsbad, CA) was used to synthesize the cDNA, from 1.5 μg of total RNA by priming with Oligo(dT)₁₂-i₈ (Invitrogen Life Technologies, Carlsbad, CA). The target cDNA was amplified by using Platinum TaqDNA Polymerase (InVitrogen Life Technologies, Carlsbad, CA) and 37 cycles of PCR. Primers for human beta tubulin IH amplified 1356-1497 bp product transcribed from the non-translated 3' UTR-region of the Hbeta4 gene as described in (Kavallaris et al., 1997). The primers for human OCT3/4 (Accession # Zl 1898), Nanog (NM 024865), Stat3 (NM 139276), Gata-4 (NM 002052), -6 (NM 005257), AFP (NM 001134), Runx 1 (NM 001754), were originally generated by using the Oligo5.1 program. All primers are provided in Table 1. Table 1. Primers used for gene expression analysis by RT PCR

Western blot analysis

5 [00193] Cells were dissolved in lysing buffer containing 50 mM Tris-HCl, 150 mM NaCl, ImM EDTA, 1% NP40, 0.1% SDS, l%Na-deoxycholate, ImM Na-vanadate, and protease inhibitors: 5 μg/ml pepstatin, ImM phenylmethylsulphonylfluoride, 10 μg/ml leupeptin, ImM NaF ( Sigma Chemical Co., St. Louis, MO) for at least 1 hour on ice. After centrifugation (12,000 g for 10 min at 4°C), the protein concentration of the supernatant was measured by

10 BCA Protein Assay kit (Pierce, Rockford, IL) using Benchmark Microplate Reader (Bio Rad Laboratories, Hercules, CA USA). Lysates were mixed (1:1) with Laemmli Buffer (Sigma Chemical Co., St. Louis, MO). 15 μg of protein was loaded per lane of 8-16% or 10-20% Tris- HCl Ready Gels (Bio Rad Laboratories, Hercules, CA) and separated by electrophoresis. The nitrocellulose membranes (Sigma Chemical Co., St. Louis, MO) with transferred proteins was

15 blocked as the manufacture recommended and incubated over night with shaking at 4°C with the corresponding primary antibodies against: STAT3 (R&D Systems), phospho- Tyr705 Stat3 (Cell Signaling Technology, IL), beta-IH tubulin (BAbCO, Berkeley,CA), beta-actin (Sigma Chemical Co., St. Louis, MO), alpha fetoprotein (Santa Cruz Biotech, CA), OCT-3/4 (Santa

Cruz Biotech., CA, Nanog from (R&D systems), diluted in solution containing 5% bovine albumin, (Sigma Chemical Co., St. Louis, MO) in Tris-Buffered Saline (TBS) and 0.1% Tween-20 (Bio-Rad Laboratories, Hercules, CA USA). After washing in TBS with 0.1% Tween-20, the blots were incubated with secondary peroxidase-conjugated goat antibodies to mouse or rabbit IgG (Cell Signaling Technology, IL) or rabbit antibodies to goat IgG (Jackson Immuno Research Laboratories, West Grove, PA). Immunoreactive bands were detected by ECL+ Western Blotting Detection Reagents (Amersham Biosciences,UK) for 60 seconds or more and exposed to X-ray films.

Pre and post transfection Cell culture media

[00194] Cells were cultured in DMEM/F12 medium supplemented with 10% (volume/volume) of characterized fetal bovine serum (FBS) (HyClone, Logan, Utah USAUSA) at 37°C, 7.0% CO₂. All cells were tranfected by electroporation with a Gibco Cell Porator pulser. The cell suspension was supplemented with 10 μg of plasmid DNA, and transfected according to protocols known to one skilled in the art (Fig. HC). After electroporation, a whole suspension was plated at a density of 60,000 cells/2ml/well in DMEM/F12 with 0.8% of MC, supplemented with progesterone (2OnM), putrescine (100 μM), sodium selenite (3OnM), transferrin (25 μg/ml), insulin (20 μg/ml) (Sigma Chemical Co., St. Louis, MO USA) and the growth factors EGF (lOng/ml) and recombinant FGF2 (lOng/ml).

Selection of transfected EGFP- mammasphere clones

[00195] To select transfected EGFP- mammasphere clones, G418 was added after 3 days of culturing (200mg/ml). In plates with G418, only green EGFP-positive clones were generated and collected for further manipulations. The generated EGFP- positive mammasphere were used for establishing EGFP- subpopulation with stable integration of EGFP. The green mammasphere expressing EGFP under the control of the Oct4 promoter grew as un-attached suspended mammasphere (Fig. 7B). EGFP cells were isolated directly under fluorescent microscope (Fig. HD).

Example 1:

The frequency of OCT4 expressing cells in tumors

[00196] In order to better explore the frequency of OCT4 expressing cells in tumors, immunohistochemical analysis was performed on serial sections derived from: osteosarcoma tumor, glioblastoma tumor, and ductal carcinoma. The results in Fig. 1 indicate that OCT4 positive nuclei are present in osteosarcoma and glioblastoma tumors; furthermore, the results in Fig. 6A-B indicate that OCT4 positive nuclei are also present in ductal carcinoma and breast cancer metastasis to the brain, respectively. Although, OCT4-positive cells were observed in both ductal carcinoma and breast cancer metastasis, more frequent OCT4 positive nuclei were indicated in breast cancer metastasis to the brain (Fig. 6B), compared to primary breast cancer embodied in ductal carcinoma (Fig. 6A). Example 2:

OCT4, Nanog and STAT3 are expressed in concert in glioblastomas clinical specimens and cell lines

[00197] In order to determine whether OCT4, Nanog and STAT3 are co-expressed in glioblastomas clinical specimens and cell lines LNl 8, LN229, LN428 and U251 semi- quantitative RT-PCR analysis followed by western blot analysis was performed. The results as indicated in Fig 2A show a moderate to high mRNA expression of OCT4, Nanog and STAT3. The protein expression levels were in correlation with the mRNA expression levels as shown in Fig. 2B, wherein, moderate to high protein expression levels of OCT4, Nanog and STAT3 are presented. Example 3:

OCT4 expression in tumor cells grown attached or unattached to a substrate [00198] In order to test the impact of cell-substrate attachment on OCT4 expression in tumor cells, bone sarcoma and mammary tumor cells were grown attached to a substrate or unattached as sarcospheres or mammasphere, respectively. The results as shown in Fig. 3 indicate that hi OCT4 and Nanog expression is dependent on cell attachment and thus, tumor cells grown unattached in sarcospheres and mammasphere highly expresses OCT4 and Nanog, in contrast to their suppression in substrate-attached tumor cells wherein the expression of Nanog and OCT4 is relatively low.

Example 4: OCT4 role in clone generation from glioma-derived tumor stem cells cultured in a neurosphere system

[00199] To test the functional role of OCT4 gene in maintenance of self -renewal in a model culture system, siRNA silencing of POU5F1/OCT4 gene was assessed (Fig. 4). Toward this end, tumor cells derived from three representatives of glioblastoma cell types, including two cell lines and a primary tumor culture isolated from a patient, were co-transfected with EGFP and OCT4 siRNA, and plated in a neurosphere culture system. In a set of parallel control experiments, cells were co-transfected with EGFP and scrambled control siRNA. Clones transfected with EGFP and siRNAs became visible on the 7th day after plating (Fig. 4A). The proportion of clones with detectable EGFP, formed by cells transfected with OCT4-siRNA, fell by more than 60% when compared to controls (Figure 4B). These findings demonstrated that down regulation of endogenous OCT4 by siRNA in plated tumor-derived cells under growth constraining conditions of a neurosphere culture system results in a significant decrease of clone-formation by tumor-derived EGFP-containing cells.

Example 5: Tagging MDA MB 231 breast cancer cell line with EGFP to asses NSCs involvement in orthotopic tumor formation

[00200] The potential of neoplastic stem cells derived from breast cancer cell line-MDA MB 231 stably expressing EGFP under Oct4 promoter in nude mice was assessed through NSCs involvement in orthotopic tumor formation in nude mice. This assay is based on the presumption that EGFP expression correlates with endogenous OCT4 gene expression. OCT4 positive and OCT4 negative MDA MB 231 breast cancer cells transfected with OCT4-EGFR construct and sorted by FACS were inoculated into fat pad of twelve nude mice which were grouped in four groups each group comprising 3 animal: Group 1 was inoculated with 5,000 OCT4 positive cells/animal, Group 2 was inoculated with 50,000 OCT4 positive cells/animal, Group 3 was inoculated with 500,000 OCT4 positive cells/animal, Group 4 was inoculated with 500,000 OCT4 negative cells/animal. The graph in Fig. 11 shows that animals inoculated with OCT4-positive cells began developing tumors on the 37^th day post inoculation; however, animals inoculated with OCT4-negative cells began developing tumors on the 50^th day. The results also indicate that the isolated EGFP-positive cells expressed OCT4, EGFP, CD44, AC 133 and ES cell marker SSEA4 as shown in Table 2.

Table 2. FACS analysis for stem cell markers in MDA-MB 231 breast cancer cells and control stem cells positive (results are given in percentage)

Example 6 Fluorescence biomarker driven by the Oct3/4 promoter is strong and specific for cancer stem cells

[00201] Mammosphere cultures were derived from an MDA-MB-435 melanoma cell line and biomarked for the presence of cancer stem cells expressing Oct-3/4. MDA-MB-435 cells were stably transfected with Oct4hP-eGFP and CMV-mRFP. The cells were then FACS sorted for GFP expressing cells to create a highly pure cancer stem cell population for further studies. Fluorescent micrograph of suspended tumor-derived spheres shown in Figure 13B demonstrated that the fluorescence biomarker driven by the Oct3/4 promoter is strong and specific for cancer stem cells. Furthermore, fluorescent micrograph of the attached tumor spheres shown (Figure 13D) exhibited a similar phenomenon. These findings demonstrate that fluorescence driven by the Oct3/4 promoter is strong and specific for cancer stem cells biomarked for study as therapeutic targets in various systems.

Example 7

Fluorescence biomarker driven by the Oct3/4 promoter is strong, specific, and stable over time in for cancer stem cells

[00202] Analysis of GFP expression in NSC mass cultures grown under different conditions for extended periods of time was preformed. MDA-MB-435 cells were stably transfected with Oct4hP-eGFP and CMV-mRFP. Cells were then FACS sorted for GFP expressing cells to create a highly pure cancer stem cell population. These cells were continuously cultured in large quantities for extended periods of time in Novopro cancer stem cell media or standard DFlO Media. DFlO Media is the standard media for BCC culture conditions and comprises Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). The data presented in Figure 14A demonstrated that fluorescence driven from the human Oct3/4 promoter is strong, specific, and stable for at least 54 days in NSCs cultured in these conditions. Figure 14B further shows the high percent of MDA-MB-435 cells cultured in Novopro cancer stem cell media and under NSC promoting conditions that were GFP positive (M2 gate) at 30 days. Thus, large quantities of NSCs can be cultured for extended periods of time without loss of purity (with respect to the percentage of true NSCs relative to BCCs or the desired NSC phenotype of a given cell.

Example 8

NSCs viability relative to the total cancer cell population [00203] GFP and RFP fluorescence were measured as an indirect correlation of NSC and overall stem cell viability. MB435 cells were stably transfected with Oct4hP-eGFP and CMV- mRFP and FACS sorted for GFP expressing cells to create a highly pure NSC population. These cells were plated on a 96- well plate at a cell density of 5000 cells/well and grown for six days in Novopro cancer stem cell media. Fluorescence was measured each day on a FLx800 Multi-Detection Microplate Reader (BioTek). For GFP fluorescence measurements, a 485nm (20nm window) excitation filter and a 528nm (20nm window) emission filter were used with a sensitivity setting of 80. For RFP fluorescence measurements, a 540nm (35nm window) excitation and 620nm (40nm window) emission filter were used with a sensitivity setting of 60. All sample fluorescence readings were corrected for background media auto-fluorescence by subtracting the fluorescence readings from blank wells with the appropriate amount of media. GFP (NSC) and RFP (total cancer cell) were measured for the expressing populations over time in NSC promoting conditions (Figure 15A). Figure 15B represents the ratio of overall Oct-4 transcription level of the population to the overall cell number. The gray line in Figure 15A represents overall GFP fluorescence which correlated with the expression of Oct-4 and thus the NSC population.

[00204] The black line in Figure 15 A represents overall RFP fluorescence and is population proportional to the overall cell number.

[00205] The results as demonstrated in Figure 15 demonstrate the ability of NSC viability to be detected relative to the total cancer cell population.

Example 9

NSCs and BCC viability after exposure to toxic agents

[00206] GFP fluorescence of MB435 NSCs grown with NSC promoting media and conditions and variable concentrations of Doxorubicin was measured. MB435 cells were stably transfected with Oct4hP-eGFP and CMV-mRFP and FACS sorted for GFP expressing cells to create a highly pure NSC population. These cells were plated on 96-well plates at a cell density of 5000 cells/well and grown for six days in Novopro cancer stem cell media (patent pending) and either 0.1 μM and 1.0 μM Doxorubicin. Fluorescence was measured each day on a FLx800 Multi-Detection Microplate Reader (BioTek). For GFP fluorescence measurements, a 485nm (20nm window) excitation filter and a 528nm (20nm window) emission filter were used with a sensitivity setting of 80. All sample fluorescence readings were corrected for background media auto-fluorescence by subtracting the fluorescence readings from blank wells with the appropriate amount of media. As shown in the Figures 16 and 18, NSCs were resistant to the cytotoxic effects Doxorubicin even at the supraphysiologic concentration of 1.0 μM.

[00207] RFP fluorescence of MB435 BCCs grown with NSC promoting media and conditions and variable concentrations of Doxorubicin was measured. MB435 cells were stably transfected with Oct4hP-eGFP and CMV-mRFP and FACS sorted for RFP expressing cells to create a highly pure BCC (non-cancer stem cell) population. These cells were then plated on 96-well plates at a cell density of 5000 cells/well and grown for six days in Novopro cancer stem cell media and either 0.1 μM and 1.0 μM Doxorubicin. Fluorescence was measured each day on a FLx800 Multi-Detection Microplate Reader (BioTek). For RFP fluorescence measurements, a 545nm (20nm window) excitation filter and a 620nm (20nm window) emission filter were used with a sensitivity setting of 60. All sample fluorescence readings were corrected for background media auto-fluorescence by subtracting the fluorescence readings from blank wells with the appropriate amount of media. [00208] MB435 cells were stably transfected with Oct4hP-eGFP and CMV-mRFP and FACS sorted separately for GFP and RFP expressing cells to create a highly pure NSC and BCC (non-cancer stem cell) populations respectively. These cells were plated on 96-well plates at a cell density of 5000 cells/well and grown for six days in Novopro cancer stem cell media and escalating doses of Doxorubicin. [00209] The lines in Figure 16 represent overall GFP fluorescence which correlated with the expression of Oct-4 and thus the NSC population. The results as demonstrated in Figure 16 show the ability of the methods of the present invention to detect NSC viability relative to the total cancer cell population after exposure to toxic agents.

[00210] The black lines in Figure 17 represent overall RFP fluorescence which correlated with the overall BCC number. The results as demonstrated in Figure 17 show that BCCs were sensitive to the cytotoxic effects Doxorubicin. Furthermore, the graph in Figure 17 demonstrates the ability to detect BCC viability after exposure to toxic agents such as Doxorubicin.

[00211] The results as demonstrated in Figure 18 re-confirm that, NSCs were resistant to the cytotoxic effects of Doxorubicin even at the supraphysiologic concentration of 1.0 μM. The gray lines represent overall GFP fluorescence which correlated with the expression of Oct-4 and thus the NSC population. The black lines represent overall RFP fluorescence which correlated with the overall BCC number. The results represented in Figure 18 demonstrated the ability to detect NSC viability relative to the total cancer stem cell population in the same culture system, in realtime, after exposure to toxic agents. Further, this graph highlights that BCCs are much more sensitive to the toxic effects of Doxorubicin than NSCs.

[00212] Figure 19 shows a graph representing an MTS Cell Proliferation Assay (CellTiter96 AQ One Solution Cell Proliferation Assay, Promega Cat # G3580) for the above described conditions. All absorbance at 492nm is specified by the assay manufacturer as linearly proportional to cell number. The gray solid line shows absorbance at 492nm for the pure NSC population. The black solid line shows absorbance at 492nm for the pure BCC starting population under various concentrations of Doxorubicin. The graph depicted in Figure 19 highlighted that BCCs are much more sensitive than NSCs to the toxic effect of Doxorubicin and thus confirmed the results presented in Figure 18.

Claims

CLAIMSWhat is claimed is:

1. A method for testing a candidate compound for an ability to transform a cell to a neoplastic stem cell (NSC), comprising the steps of:

(a) contacting a cell population with said candidate compound, wherein said cell population has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and

(b) measuring the expression of said identifiable gene product; thereby testing a candidate compound for an ability to transform a cell to a neoplastic stem cell.

2. The method of claim 1, wherein said candidate compound is a protein.

3. The method of claim 1, wherein said candidate compound is a nucleic acid.

4. The method of claim 1, wherein said candidate compound is an organic compound.

5. The method of claim 1, wherein said candidate compound is an inorganic compound.

6. The method of claim 1, wherein said measuring the expression of said identifiable gene product further comprises measuring the number of cells expressing said identifiable gene product.

7. The method of claim 6, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescence-activated cell sorter (FACS).

8. The method of claim 6, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescent microscope.

9. The method of claim 1, wherein said measuring the expression of said identifiable gene product further comprises the use of a fluorometer.

10. The method of claim 1, wherein said measuring the expression of said identifiable gene product further comprises the use of a luminescence reader.

11. A method for testing a candidate compound for an ability to inhibit OCT4 expression in a neoplastic stem cell (NSC), comprising the steps of:

(a) contacting a cell population enriched for OCT4 expression with said candidate compound, wherein said cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and

(b) measuring the expression of said identifiable gene product; thereby testing a candidate compound for an ability to inhibit OCT4 expression in a neoplastic stem cell.

12. The method of claim 11, wherein said cell population enriched for OCT4 expression is characterized by OCT4^hl expression.

13. The method of claim 11, wherein said candidate compound is a protein.

14. The method of claim 11, wherein said candidate compound is a nucleic acid.

15. The method of claim 11, wherein said candidate compound is an organic compound.

16. The method of claim 1, wherein said candidate compound is an inorganic compound.

17. The method of claim 11, wherein said measuring the expression of said identifiable gene product further comprises measuring the number of cells expressing said identifiable gene product.

18. The method of claim 17, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescence-activated cell sorter (FACS).

19. The method of claim 17, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescent microscope.

20. The method of claim 11, wherein said measuring the expression of said identifiable gene product further comprises the use of a fluorometer.

21. The method of claim 11, wherein said measuring the expression of said identifiable gene product further comprises the use of a luminescence reader.

22. A method for testing a candidate compound for an ability to inhibit the proliferation of a neoplastic stem cell (NSC), comprising the steps of:

(a) contacting a cell population enriched for OCT4 expression with said candidate compound, wherein said cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and;

(b) measuring the expression of said identifiable gene product; thereby testing a candidate compound for an ability to inhibit the proliferation of a NSC.

23. The method of claim 22, wherein said cell population enriched for OCT4 expression is characterized by OCT4^hl expression.

24. The method of claim 22, wherein said candidate compound is a protein.

25. The method of claim 22, wherein said candidate compound is a nucleic acid.

26. The method of claim 22, wherein said candidate compound is an organic compound.

27. The method of claim 1, wherein said candidate compound is an inorganic compound.

28. The method of claim 22, wherein said measuring the expression of said identifiable gene product further comprises measuring the number of cells expressing said identifiable gene product.

29. The method of claim 28, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescence-activated cell sorter (FACS).

30. The method of claim 28, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescent microscope.

31. The method of claim 22, wherein said measuring the expression of said identifiable gene product further comprises the use of a fluorometer.

32. The method of claim 22, wherein said measuring the expression of said identifiable gene product further comprises the use of a luminescence reader.

33. A system for testing a candidate compound for an ability to inhibit a neoplastic stem cell population, comprising an isolated neoplastic stem cell population enriched for expression of OCT4, said isolated neoplastic stem cell population enriched for expression of OCT4 is transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product.

34. The system of claim 33, wherein said cell population enriched for OCT4 expression is characterized by OCT4^hl expression.

35. The system of claim 33, wherein said candidate compound is a protein.

36. The system of claim 33, wherein said candidate compound is a nucleic acid.

37. The system of claim 33, wherein said candidate compound is an organic compound.

38. The system of claim 33, wherein said candidate compound is an inorganic compound.

39. The system of claim 33, further comprising a fluorescent microscope measuring the number of cells expressing said identifiable gene product.

40. The system of claim 33, further comprising a fluorescence-activated cell sorter (FACS) measuring the number of cells expressing said identifiable gene product.

41. The system of claim 33, further comprising a fhiorometer measuring the expression of said identifiable gene product in said isolated neoplastic stem cell population.

42. The system of claim 33, further comprising a luminescence reader measuring the expression of said identifiable gene product in said isolated neoplastic stem cell population.

43. A system for testing a candidate compound for an ability to transform a neoplastic cell population to a neoplastic stem cell population, comprising an isolated bulk cancer cell (BCC) population said isolated BCC population is transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product.

44. The system of claim 43, wherein said candidate compound is a protein.

45. The system of claim 43, wherein said candidate compound is a nucleic acid.

46. The system of claim 43, wherein said candidate compound is an organic compound.

47. The system of claim 43, wherein said candidate compound is an organic compound.

48. The system of claim 43, further comprising a fluorescent microscope measuring the number of cells expressing said identifiable gene product.

49. The system of claim 43, further comprising a fluorescence-activated cell sorter (FACS) measuring the number of cells expressing said identifiable gene product.

50. The system of claim 43, further comprising a fluorometer measuring the expression of said identifiable gene product in said isolated neoplastic stem cell population.

51. The system of claim 43, further comprising a luminescence reader measuring the expression of said identifiable gene product in said isolated neoplastic stem cell population.

52. A system for testing a candidate compound for an ability to transform a nonneoplastic cell population to a neoplastic stem cell population, comprising a nonneoplastic cell population said non-neoplastic cell population is transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product

53. The system of claim 52, wherein said candidate compound is a protein.

54. The system of claim 52, wherein said candidate compound is a nucleic acid.

55. The system of claim 52, wherein said candidate compound is an organic compound.

56. The system of claim 52, wherein said candidate compound is an organic compound.

57. The system of claim 52, further comprising a fluorescent microscope measuring the number of cells expressing said identifiable gene product.

58. The system of claim 52, further comprising a fluorescence-activated cell sorter (FACS) measuring the number of cells expressing said identifiable gene product.

59. The system of claim 52, further comprising a fluorometer measuring the expression of said identifiable gene product in said isolated neoplastic stem cell population.

60. The system of claim 52, further comprising a luminescence reader measuring the expression of said identifiable gene product in said isolated neoplastic stem cell population.

61. A method for testing a candidate compound for an ability to transform a neoplastic stem cell (NSC) to a bulk cancer cell (BCC) comprising the steps of: (a) contacting a cell population enriched for OCT4 expression with said candidate compound, wherein said cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and (b) measuring the expression of said identifiable gene product; thereby testing a candidate compound for an ability to inhibit OCT4 expression in a neoplastic stem cell.

62. The method of claim 61, wherein said NSC expresses OCT4.

63. The method of claim 61, wherein said candidate compound is a protein.

64. The method of claim 61, wherein said candidate compound is a nucleic acid.

65. The method of claim 61, wherein said candidate compound is an organic compound.

66. The method of claim 61, wherein said candidate compound is an inorganic compound.

67. The method of claim 61, wherein said measuring the expression of said identifiable gene product further comprises measuring the number of cells expressing said identifiable gene product.

68. The method of claim 67, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescence-activated cell sorter (FACS).

69. The method of claim 67, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescent microscope.

70. The method of claim 61, wherein said measuring the expression of said identifiable gene product further comprises the use of a fluorometer.

71. The method of claim 61, wherein said measuring the expression of said identifiable gene product further comprises the use of a luminescence reader.

72. A method for testing a candidate compound for an ability to inhibit the asymmetric division of a neoplastic stem cell (NSC) comprising the steps of: (a) contacting a cell population enriched for OCT4 expression with said candidate compound, wherein said cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and (b) measuring the expression of said identifiable gene product; thereby testing a candidate compound for an ability to inhibit OCT4 expression in a neoplastic stem cell.

73. The method of claim 72, wherein said candidate compound is a protein.

74. The method of claim 72, wherein said candidate compound is a nucleic acid.

75. The method of claim 72, wherein said candidate compound is an organic compound.

76. The method of claim 72, wherein said candidate compound is an inorganic compound.

77. The method of claim 72, wherein said measuring the expression of said identifiable gene product further comprises measuring the number of cells expressing said identifiable gene product.

78. The method of claim 77, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescence-activated cell sorter (FACS).

79. The method of claim 77, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescent microscope.

80. The method of claim 72, wherein said measuring the expression of said identifiable gene product further comprises the use of a fluorometer.

81. The method of claim 72, wherein said measuring the expression of said identifiable gene product further comprises the use of a luminescence reader.

82. A method for testing a candidate compound for an ability to inhibit the symmetric division of a neoplastic stem cell (NSC) comprising the steps of: (a) contacting a cell population enriched for OCT4 expression with said candidate compound, wherein said cell population enriched for OCT4 expression has been transfected with an expression vector comprising an Oct4 responsive promoter controlling the expression of an identifiable gene product; and (b) measuring the expression of said identifiable gene product; thereby testing a candidate compound for an ability to inhibit OCT4 expression in a neoplastic stem cell.

83. The method of claim 82, wherein said candidate compound is a protein.

84. The method of claim 82, wherein said candidate compound is a nucleic acid.

85. The method of claim 82, wherein said candidate compound is an organic compound.

86. The method of claim 82, wherein said candidate compound is an inorganic compound.

87. The method of claim 82, wherein said measuring the expression of said identifiable gene product further comprises measuring the number of cells expressing said identifiable gene product.

88. The method of claim 87, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescence-activated cell sorter (FACS).

89. The method of claim 87, wherein said measuring the number of cells expressing said identifiable gene product comprises the use of a fluorescent microscope.

90. The method of claim 82, wherein said measuring the expression of said identifiable gene product further comprises the use of a fluorometer.

91. The method of claim 82, wherein said measuring the expression of said identifiable gene product further comprises the use of a luminescence reader.