Cancer stem cells and uses thereof

Abstract

Disclosed are enriched preparations of neuroblastoma tumor initiating cells (NB TICs). The NB TICs are capable of self-renewal, initiating neuroblastoma tumor growth in vivo and are capable of being passaged in high frequency. These NB TICs have chromosomal abnormalities and are capable of giving rise to secondary tumor spheres. Methods are also disclosed for preparing the enriched preparations of NB TICs, such as from neuroblastoma tumor tissue and metastasized bone marrow. Also disclosed are methods of screening candidate substances to identify therapeutic agents for the treatment of neuroblastoma. Methods are also provided for screening a sample for neuroblastoma, as well as for screening a sample to identify the stage of neuroblastoma present. Kits are also provided for selecting appropriate anti-neuroblastoma compounds for a patient, and utilize isolated compositions of the patients' neuroblastoma tumor initiating cells. In this manner, a customized medicinal profile for the patient may be devised.

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       5 . A method for identifying compounds having anti-neuroblastoma tumor initiating cell activity comprising:
 preparing an enriched preparation of neuroblastoma tumor cells from a patient having neuroblastoma to provide an enriched test cell dissociated sample of cells;   depositing a number of cells from the enriched test cell dissociated sample of cells into a desired number of wells of the multi-well assay plate to provide test cell wells and two or more control wells;   adding a volume of a potential anti-neuroblastoma compound to each of said test cell wells to provide a loaded multi-well assay plate;   adding a cell proliferation indicator agent to each well of said loaded multi-well assay plate and incubating said loaded multi-well assay plate;   comparing indicator agent intensity in each well of said test cell wells to said control wells; and   selecting potential anti-neuroblastoma compounds that elicit a cell proliferation indicator agent intensity that is two (2) fold or less intense than the indicator agent intensity observed in a control cell well.   
   
   
       6 . The method of  claim 5  wherein the cell proliferation indicator agent is a cell viability dye. 
   
   
       7 . The method of  claim 6  wherein the cell viability dye is Alamar Blue or another cell viability dye. 
   
   
       8 . A method for selecting compounds having anti-neuroblastoma tumor-initiating cell activity comprising:
 preparing a culture of cells enriched for neuroblastoma tumor initiating cells to provide an enriched test cell culture;   providing a culture of cells other than neuroblastoma tumor-initiating cells to provide a control cell culture;   providing a first test volume of a compound from a compound library of interest to a sample of the test cell culture and a second test volume of the compound of interest to a sample of the control cell culture from the compound library of interest;   assessing cell activity in the test cell culture sample and in the control cell culture sample; and   selecting a compound that reduces the activity of the cells in the test cell culture sample and that does not reduce the activity of the cells in the control cell culture sample.   
   
   
       9 . The method of  claim 8  wherein an indicator dye of cell viability is added to each well. 
   
   
       10 . The method of  claim 9  wherein the indicator dye of cell viability is Alamar blue. 
   
   
       11 . The method of  claim 8  further comprising one or more wells having a known anti-neuroblastoma tumor initiating cell therapeutic agent or known anti-neuroblastoma therapeutic agent. 
   
   
       12 . The method of clam  11  wherein the known anti-neuroblastoma tumor initiating cell therapeutic agent is ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, or vincristine sulfate. 
   
   
       13 . A kit for selecting an anti-neuroblastoma tumor initiating cell preparation for a patient, said kit comprising:
 an assay plate that includes a plurality of wells, each well of said assay plate being suitable for containing an anti-neuroblastoma tumor initiating cell pharmacologically active agent and a volume of cells;   a volume of a panel of individual anti-neuroblastoma tumor initiating cell pharmacologically active agents, wherein 2 or more of said wells each contain a volume of each anti-neuroblastoma tumor initiating cell pharmacologically active agent.   
   
   
       14 . The kit of  claim 13  further defined as comprising a 96-well assay plate. 
   
   
       15 . The kit of  claim 13  wherein the panel of individual anti-neuroblastoma tumor initiating cell pharmacologically active agents comprises a volume of one or more of each of the compounds listed below:
 2.3-Dimethoxy-1.4-naphthoquinone,   Aklavine Hydrochloride,   Amodiaquin dihydrochloride dehydrate;   Amsacrine Hydrochloride;   Azaguanine-8;   beta-peltatin;   Camptothecine (S.+);   CGP-74514A hydrochloride;   Chelerythrine chloride;   Cholestan-3beta.5alpha.6beta-Triol;   Ciclopirox Olamine;   Clofazimine;   Colchicine;   Convallatoxin;   Crassin Acetate;   Crinamine;   Dequalinium analog. C-14 linker;   Dequalinium dichloride;   Digitoxin;   Digoxigenin;   Dihydrogambogic acid;   Dihydroouabain;   Erysolin;   Gambogic acid;   Mechlorethamine;   Meclizine hydrochloride;   MG 624;   Mitoxanthrone Hydrochloride;   Ouabain;   Oxybendazole;   Oxybendazole;   Paclitaxel;   Parthenolide;   Patulin;   Periplocymarin;   Peruvoside;   Primaquine diphosphate;   Quinacrine dihydrochloride;   Sanguinarine chloride; or   Tomatine.   
   
   
       16 . The kit of  claim 15  wherein the panel of individual anti-neuroblastoma tumor initiating cell pharmacologically active agents comprises a volume of ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, or vincristine sulfate. 
   
   
       17 . The kit of  claim 13  wherein the kit further comprises an instructional manual. 
   
   
       18 . A kit for screening a patient of interest to identify an appropriate anti-tumor cell agent, said kit comprising:
 an assay plate comprising a plurality of wells suitable for containing a volume of one of each compound listed below:   2.3-Dimethoxy-1.4-naphthoquinone,   Aklavine Hydrochloride,   Amodiaquin dihydrochloride dehydrate;   Amsacrine Hydrochloride;   Azaguanine-8;   beta-peltatin;   Camptothecine (S.+);   CGP-74514A hydrochloride;   Chelerythrine chloride;   Cholestan-3beta.5alpha.6beta-Triol;   Ciclopirox Olamine;   Clofazimine;   Colchicine;   Convallatoxin;   Crassin Acetate;   Crinamine;   Dequalinium analog. C-14 linker;   Dequalinium dichloride;   Digitoxin;   Digoxigenin;   Dihydrogambogic acid;   Dihydroouabain;   Erysolin;   Gambogic acid;   Mechlorethamine;   Meclizine hydrochloride;   MG 624;   Mitoxanthrone Hydrochloride;   Ouabain;   Oxybendazole;   Oxybendazole;   Paclitaxel;   Parthenolide;   Patulin;   Periplocymarin;   Peruvoside;   Primaquine diphosphate;   Quinacrine dihydrochloride;   Sanguinarine chloride; or   Tomatine, and   wherein one or more assay plate wells is a positive control well that contains ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, or vincristine sulfate; and   an instructional sheet describing a method by which an enriched preparation of neuroblastoma tumor initiating cells may be isolated from the patient of interest.   
   
   
       19 . The kit of  claim 18  further comprising positive control compound wells that are absent an anti-tumor cell agent. 
   
   
       20 . The kit of  claim 18  further comprising a cell viability indicator dye. 
   
   
       21 . A kit for screening a patient of interest to identify an appropriate anti-tumor initiating cell active agent for tumor-initiating cells of leukemia, melanoma, breast cancer, brain cancer, or colon cancer, said kit comprising:
 an assay plate comprising a plurality of wells suitable for containing an anti-tumor initiating cell compound, wherein each well contains a volume of one of each compound listed below:   2.3-Dimethoxy-1,4-naphtho quinone,   Aklavine Hydrochloride,   Amodiaquin dihydrochloride dehydrate;   Amsacrine Hydrochloride;   Azaguanine-8;   beta-peltatin;   Camptothecine (S.+);   CGP-74514A hydrochloride;   Chelerythrine chloride;   Cholestan-3beta.5alpha.6beta-Triol;   Ciclopirox Olamine;   Clofazimine;   Colchicine;   Convallatoxin;   Crassin Acetate;   Crinamine;   Dequalinium analog. C-14 linker;   Dequalinium dichloride;   Digitoxin;   Digoxigenin;   Dihydrogambogic acid;   Dihydroouabain;   Erysolin;   Gambogic acid;   Mechlorethamine;   Meclizine hydrochloride;   MG 624;   Mitoxanthrone Hydrochloride;   Ouabain;   Oxybendazole;   Oxybendazole;   Paclitaxel;   Parthenolide;   Patulin;   Periplocymarin;   Peruvoside;   Primaquine diphosphate;   Quinacrine dihydrochloride;   Sanguinarine chloride; or   Tomatine, and   wherein one or more assay plate wells is a positive control well that contains ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, or vincristine sulfate; and   an instructional sheet describing a method by which an enriched preparation of tumor initiating cells may be isolated from the patient of interest.   
   
   
       22 . The kit of  claim 21  further comprising positive control compound wells that are absent an anti-tumor initiating cell active agent. 
   
   
       23 . The kit of  claim 21  further comprising a cell viability indicator dye.