Name: flow cytometry-based drug screening system for the identification of small molecules that promote cellular differentiation of glioblastoma stem cells
Uploaded: 2018-01-10T14:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells at JoVE.

Este trabalho foi parcialmente apoiado pela NIH R01CA187780.

Nota: Sistemas de repórter de lentivirus neuronal e Ralevic foram comprados como preparações de Lentivirus pré-embalados, concentradas. Conhecimento básico da técnica de citometria de fluxo é necessário. Também, para um uso completo do presente protocolo o usuário terá acesso a um citômetro de fluxo com capacidade de produção elevada (aceita placas de 96 poços como fonte de exemplo).
1. Lentivirus repórter Transcriptional sistema
Nota: Em todas as aná...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+gliomas:+new+translational+therapies.">Malignant gliomas: new translational therapies.</a> Mt Sinai J Med. 77 (6), 655-666 (2010).</li><li>Bar, E. E., Chaudhry, A., Farah, M. H., Eberhart, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+promotes+medulloblastoma+survival+via+Bc/II.">Hedgehog signaling promotes medulloblastoma survival via Bc/II.</a> Am J Pathol. 170 (1), 347-355 (2007).</li><li>Chu, Q., Orr, B. A., Semenkow, S., Bar, E. E., Eberhart, C. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+therapy+of+acute+promyelocytic+leukemia+with+tretinoin+(all-trans-retinoic+acid).">Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid).</a> N Engl J Med. 324 (20), 1385-1393 (1991).</li><li>Piccirillo, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+proteins+inhibit+the+tumorigenic+potential+of+human+brain+tumour-initiating+cells.">Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.</a> Nature. 444 (7120), 761-765 (2006).</li><li>Bar, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclopamine-mediated+hedgehog+pathway+inhibition+depletes+stem-like+cancer+cells+in+glioblastoma.">Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma.</a> Stem Cells. 25 (10), 2524-2533 (2007).</li><li>Bar, E. E., Lin, A., Mahairaki, V., Matsui, W., Eberhart, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+increases+the+expression+of+stem-cell+markers+and+promotes+clonogenicity+in+glioblastoma+neurospheres.">Hypoxia increases the expression of stem-cell markers and promotes clonogenicity in glioblastoma neurospheres.</a> Am J Pathol. 177 (3), 1491-1502 (2010).</li><li>Kahlert, U. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133/CD15+defines+distinct+cell+subpopulations+with+differential+in+vitro+clonogenic+activity+and+stem+cell-related+gene+expression+profile+in+in+vitro+propagated+glioblastoma+multiforme-derived+cell+line+with+a+PNET-like+component.">CD133/CD15 defines distinct cell subpopulations with differential in vitro clonogenic activity and stem cell-related gene expression profile in in vitro propagated glioblastoma multiforme-derived cell line with a PNET-like component.</a> Folia Neuropathol. 50 (4), 357-368 (2012).</li><li>Lim, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+monocarboxylate+transporter-4+depletes+stem-like+glioblastoma+cells+and+inhibits+HIF+transcriptional+response+in+a+lactate-independent+manner.">Inhibition of monocarboxylate transporter-4 depletes stem-like glioblastoma cells and inhibits HIF transcriptional response in a lactate-independent manner.</a> Oncogene. 33 (35), 4433-4441 (2014).</li><li>Kahlert, U. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZEB1+Promotes+Invasion+in+Human+Fetal+Neural+Stem+Cells+and+Hypoxic+Glioma+Neurospheres.">ZEB1 Promotes Invasion in Human Fetal Neural Stem Cells and Hypoxic Glioma Neurospheres.</a> Brain Pathol. 25 (6), 724-732 (2014).</li><li>Hu, Y., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELDA:+extreme+limiting+dilution+analysis+for+comparing+depleted+and+enriched+populations+in+stem+cell+and+other+assays.">ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays.</a> J Immunol Methods. 347 (1-2), 70-78 (2009).</li><li>Meyer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell-derived+clonal+analysis+of+human+glioblastoma+links+functional+and+genomic+heterogeneity.">Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity.</a> Proc Natl Acad Sci U S A. 112 (3), 851-856 (2015).</li><li>Galli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64 (19), 7011-7021 (2004).</li><li>Spina, R., Voss, D. M., Asnaghi, L., Sloan, A., Bar, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atracurium+Besylate+and+other+neuromuscular+blocking+agents+promote+astroglial+differentiation+and+deplete+glioblastoma+stem+cells.">Atracurium Besylate and other neuromuscular blocking agents promote astroglial differentiation and deplete glioblastoma stem cells.</a> Oncotarget. 7 (1), 459-472 (2016).</li></ol>

Enquanto a maioria dos estudos anteriores de GSCs centradas-se principalmente os marcadores que os definem, neste estudo decidimos tomar a abordagem inversa. Focamos principalmente em progênies diferenciadas gerados pelo GSCs (por exemplo, células expressando marcadores neuronais e Ralevic). Aqui vamos demonstrar a utilização de uma sistema, que é baseado na expressão humana do promotor-dependente GFAP de GFP de despistagem de drogas baseada em célula alto throughput. Todos os experimentos foram realizados utiliza...

Estudos recentes têm mostrado que os tumores contêm uma pequena população de células, denominadas células-tronco câncer (CSCs) ou células de tumor-iniciando, que são responsáveis pela progressão do tumor, metástase e resistência à quimioterapia e radio-terapias 1, 2. a presença de câncer células-tronco e suas progênies mais diferenciados dentro de tumores é considerada fator importante promover a heterogeneidade intratumoral e, portanto, representa um grande obstáculo no tratamento de câncer3. Hierarquia de células de tumor, fornecida pela teoria de células-tronco do câncer, inspirou o desenvolvimento de novas estratégias para tratar cancros 4. Uma abordagem para o direcionamento de câncer células-tronco é identificar e inibir vias de sinalização que são conhecidas por serem necessárias durante o desenvolvimento embrionário do órgão afetado. Na verdade, nós e os outros anteriormente publicaram vários artigos descrevendo a exigência em curso para as células-tronco-relevantes sinalização vias neurais Sonic Hedgehog e entalhe em glioblastoma5,6,7. Este trabalho tem ajudado no solidificando a justificativa para vários ensaios clínicos de GBM. Uma segunda abordagem para o direcionamento de câncer células-tronco é promover a sua diferenciação. Esta abordagem tem recebido muito apoio devido os resultados favoráveis de estudos pré-clínicos e clínicos no tratamento da leucemia promielocítica aguda com ácidos retinoico (ATRA, um análogo da vitamina A). Aqui ATRA foi encontrado para remover o bloco de maturação e promover câncer de diferenciação celular8. Mais recentemente, Piccirillo e colegas elegantemente mostraram que BMP-4 promove a diferenciação de GSC em astrócitos com anti-GBM significativos efeitos in vitro e in vivo9.
A justificativa para o estudo atual é baseada em uma abordagem de "engenharia invertida" para segmentação GSCs. Dada a vasta heterogeneidade presente em GBM e com pobre diferenciação, sendo uma das marcas registradas do câncer, pedimos que se nós poderia promover um fenótipo mais favorável - diferenciação em um estado de astrocyte. Aqui, não temos conhecimento prévio dos caminhos sinalização que manter GSCs em uma amostra de determinado tumor mas prefiro visam alcançar um fenótipo desejado (por exemplo, positividade GFAP).
Este relatório descreve os procedimentos utilizados para estabelecer o GSC diferenciação repórter-linhas da transdução de culturas GSC-enriquecido a Seleção clonal de GSC. Estabeleceram-se as linhas de neurosphere de glioblastoma usadas no laboratório do Professor Angelo Vescovi de pacientes com diagnóstico de glioblastoma primário no Hospital San Raffaele - Milano, Itália. Estas linhas têm sido muito estudadas em várias publicações 6,10,11,12,13,14. É altamente recomendado que os indivíduos que estão interessados em implementar estas técnicas em seu laboratório determinam a relevância do repórter, a capacidade de auto-renovação de células-tronco de câncer nas células eles planejam estudar (isto é verdadeiro para qualquer repórter). Um protocolo detalhado para um dos ensaios clonogenic em vitro aceitados no campo é fornecido para realizar este15,16. Finalmente, um protocolo detalhado descrevendo a utilização das linhas de repórter de diferenciação em um teste de drogas baseada em citometria de fluxo é fornecido no final. Da nota, da mesma forma para o sistema de diferenciação de Ralevic descrito aqui, nós temos com sucesso estabelecido e validado linhas de repórter GSC integrando um repórter (diferenciação neuronal) de MAP2:GFP. Portanto, as metodologias descrevem neste livro pode ser aplicado para estudar a diferenciação celular em várias linhagens de células.
Algumas das figuras neste relatório podem ser encontradas em uma recente publicação: "Atracurium Besylate e outro agentes de bloqueio neuromuscular promover a diferenciação de Ralevic e empobrecem o glioblastoma as células-tronco18.

<table>
	<tbody>
		<tr>
			<td>ESGRO Complete Accutase</td>
			<td>EMD Millipore</td>
			<td>SF006</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (Hank&#39;s Balanced Salt Solution)</td>
			<td>Sigma Aldrich</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GFAP Differentiation Reporter (pGreenZeo, Virus)</td>
			<td>SBI (System Biosciences)</td>
			<td>SR10015VA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml sterile disposable reagent reservoirs</td>
			<td>Corning</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolite T25 cm&#178; Flask Vented</td>
			<td>Thermo Fisher Scientific</td>
			<td>130189</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolite T75 cm&#178; Flask Vented</td>
			<td>Thermo Fisher Scientific</td>
			<td>130190</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Centrifuge tubes</td>
			<td>Celltreat</td>
			<td>229411</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml Microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovation Multi Channel Pipette, 12 Channel, 0.5 - 20uL</td>
			<td>VistaLab Technologies</td>
			<td>1060-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovation Multi Channel Pipette, 12 Channel, 5-250uL</td>
			<td>VistaLab Technologies</td>
			<td>1060-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi 12-channel pipette tips 25 &#956;l</td>
			<td>VistaLab Technologies</td>
			<td>4060-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi 12-channel pipette tips 250 &#956;l</td>
			<td>VistaLab Technologies</td>
			<td>4060-9025</td>
			<td></td>
		</tr>
		<tr>
			<td>Guava easyCyte 5HT Benchtop Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>0500-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH Clinical Collections 1 and 2 small molecule libraries</td>
			<td>Evotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For the preparation of neural stem cell media (500 mL)</td>
			<td></td>
			<td></td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>GoldBio.com</td>
			<td>A-421-250</td>
			<td>0.20%</td>
		</tr>
		<tr>
			<td>DMEM/F12 10X</td>
			<td>Corning</td>
			<td>90-091-PB</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>H3149</td>
			<td>0.0002%</td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Sigma Aldrich</td>
			<td>H4036</td>
			<td>5.4 mM</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin- Selenium (ITS -G) (100X)</td>
			<td>Life Technologies</td>
			<td>41400-045</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S-5761</td>
			<td>14.5 mM</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL) 100X</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma Aldrich</td>
			<td>P8783</td>
			<td>16 nM</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma Aldrich</td>
			<td>P5780</td>
			<td>4.8 &#181;M</td>
		</tr>
		<tr>
			<td>Basic FGF (FGF2), Human</td>
			<td>GoldBio</td>
			<td>1140-02-50</td>
			<td>10 ng/ml</td>
		</tr>
		<tr>
			<td>EGF, Human</td>
			<td>GoldBio</td>
			<td>1150-04-100</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>Bottle-Top Filter, 150ml, 33mm, 0.22um, Pes, S, Ind</td>
			<td>Corning</td>
			<td>431160</td>
			<td>Use to filter sterlize media</td>
		</tr>
	</tbody>
</table>

flow cytometry-based drug screening system for the identification of small molecules that promote cellular differentiation of glioblastoma stem cells

Glioblastoma (GBM) é o tumor cerebral primário mais comum e mais letal em adultos, causando cerca de 14.000 mortes a cada ano só nos EUA. Sobrevida média após o diagnóstico é menor que 15 meses com quimioterapia máxima de temozolomide, radiação e ressecção cirúrgica. Os desafios inerentes ao desenvolvimento de tratamentos mais eficazes de GBM tornaram-se cada vez mais claros e incluem sua invasividade inflexível, sua resistência aos tratamentos padrão, sua complexidade genética e molecular adaptabilidade e subpopulações de GBM células com semelhanças fenotípicas de células-tronco normais, doravante conhecido como glioblastoma as células-tronco (GSCs). Porque GSCs são necessários para o crescimento do tumor e progressão, terapia baseada em diferenciação representa uma modalidade de tratamento viável para estas Neoplasias incuráveis.
O seguinte protocolo descreve uma coleção de procedimentos para estabelecer um alto throughput plataforma vista a identificação de pequenas moléculas que promovem a diferenciação de GSC Ralevic de triagem. No núcleo do sistema é uma proteína ácida fibrilar glial (GFAP) repórter-construção de diferenciação. O protocolo contém os seguintes procedimentos gerais: (1) estabelecer linhas de repórter GSC diferenciação; (2) testar/validar a relevância do repórter, a capacidade de autorenovação / clonogenic GSC; e triagem de drogas de alta capacidade-citometria de fluxo com base (3).
A plataforma de triagem fornece uma abordagem simples e barata para identificar pequenas moléculas que promovem a diferenciação de GSCs. Além disso, a utilização de bibliotecas de drogas FDA-approved possui o potencial para a identificação dos agentes que pode ser reaproveitado mais rapidamente. Também, terapias que promovem a diferenciação de células-tronco de câncer são esperadas para trabalhar em sinergia com a atual terapias "padrão de cuidados" que foram mostradas para atacar e eliminar as células do câncer diferenciado principalmente mais.

Três linhas independentes neurospheres paciente-derivados foram transfectadas com o repórter Ralevic de lentivirus codificação para uma proteína verde fluorescente (GFP) fundida em-frame com uma gaveta de resistência Zeocin e conduzidas pelo elemento humano para a promotora GFAP ( Figura 1). Próximo clones individuais foram isolados por chapeamento 0,7 células por poço em um prato bem 96 (Figura 2), este foi seguido por ...

Watch this Scientific Journal Video about Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells at JoVE.

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Drogas baseada em citometria de fluxo sistema de triagem para a identificação de pequenas moléculas que promovem a diferenciação celular de células-tronco Glioblastoma

Um protocolo de triagem eficiente é apresentado para a identificação de pequenas moléculas que promovem a diferenciação de Ralevic em células-tronco glioblastoma (GSCs). O ensaio baseia-se em um repórter de diferenciação de células-tronco no qual a expressão da GFP reforçada (eGFP) é conduzida pelo promotor GFAP humano.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

The overall goal of this procedure is to screen small molecule libraries for agents that promote astroglial differentiation in glioblastoma stem cells. This method can help answer key questions in the cancer stem cell field, such as what are the signaling pathways that are required for maintaining glioblastoma stem cells in the undifferentiated state. Proper execution of this technique allows for the straightforward identification of small molecules that promote glioblastoma stem cell differentiation.The implication of this technique extend toward therapy for Ehlers-Danlos glioblastoma because reducing the percentage of glioblastoma stem cells may decrease the likelihood of recurrence. This method can also be applied to setting differentiation into other cell lineages using other reporter constructs. Our report is the first to establish a high throughput approach to screen for agents that force glioma stem cells to differentiate.Working with lentivirus particles in cancer cells derived from patients can be extremely hazardous. Therefore standard procedure for working with bloodborne pathogens should always be followed. To begin, seed one million glioblastoma stem cells or GSCs in two milliliters of complete neural stem cell growth medium in a six-well plate.Next, transduce the cells with the lentivirus reporter at a multiplicity of infection equal to five. To the cell and virus mixture, add two microliters of polybrene such that the final concentration is eight micrograms per milliliter, and incubate the cells at 37 degrees Celsius and 5%CO2 overnight. After incubation, replace the growth medium to remove unbound virus.And centrifuge cells at 360 times G for five minutes. Then carefully aspirate medium and plate cells back in a six-well plate. And continue to incubate the cells for 24 hours.Harvest 0.5 milliliters of the total culture volume. Pellet cells by centrifugation at 360 times G for five minutes at room temperature. Resuspend cells in 0.5 milliliters fresh neural stem cell growth medium.Place the plate back in the incubator to allow for expansion for at least 72 hours. Add 200 microliters of dissociation reagent and incubate for five minutes in a water bath set to 37 degrees Celsius. Dissociate cells by pippetting up and down gently.To the dissociated cells add 800 microliters of HBSS to ensure minimal cell attachment or aggregation. Next, transfer 200 microliters of each cell suspension into wells of a 96-multiwell plate. Determine the percentage of cells expressing the green fluorescent protein by flow cytometry.Record at least 10, 000 viable cells in each acquisition. In all flow cytometric analysis use parental non-transduced cells or vector-transduced nonfluorescent cells to establish baseline fluorescence. To select and expand individual subclones seed cells at a density of 0.7 cells per well in 100 microliters of neural stem cell medium in a 96-well plate.Maintain these clones in culture for 11 days. Look out for neurospheres with a diameter greater than 100 microns. We have previously established this cutoff size to retrospectively determine if the neuorsphere was originated by a self-renewing glioblastoma stem cell.This cutoff size may need optimization when working with different glioblastoma cell models. Observe the cells under a fluorescence microscope equipped with a FITC filter and mark wells that contain a single neurosphere in which approximately one 1%to 5%of the cells are GFP-positive. After marking, expand individual clones until a sufficient number of cells is available for flow cytometry analysis.Assuming the reporter clones contain 1%to 5%of GFP-positive cells, harvest 0.5 milliliters from each clone along with an equal quantity of non-transduced control cells to determine the exact percentage of GFP-positive cells. After pelleting cells at 360 times G for five minutes at room temperature, aspirate the supernatant. Add 200 microliters of dissociation reagent to each pellet and incubate the tubes in a water bath set to 37 degrees Celsius.After incubation, triturate the cells about 20 to 30 times to achieve a single cell suspension. Then add 800 microliters of HBSS to ensure minimal cell attachment or aggregation. Now transfer 200 microliters of each cell suspension into wells of a 96-multiwell plate.Finally, perform flow cytometry, recording at least 10, 000 viable cells in each acquisition. To assess the clonogenic capacity prepare single cell suspensions of differentiation reporter subclones by triturating the cells about 20 to 30 times. Then plate these cells in 100 microliters of complete neural stem cell growth medium at a density of about five to 500 cells per well in 96-multiwell plates.Maintain these cells in culture for nine to 11 days. Look for neurospheres under a light microscope. A well containing at least one single large neurosphere is considered positive.Considering parental non-transduced cells and GFP-positive lentivirus-transduced cells as controls input the total wells analyzed and the number of positive cells on the ELDA online interface. To begin screening, plate 5000 cells in 99 microliters of complete neural stem cell growth medium in a 96-well plate. Then with a 12-channel pipettor treat cells with two micromolar final concentration of diluted library compounds or with the DMSO control.Incubate the cells for 72 hours. Now add 150 microliters of cell dissociation reagent using a 12-channel pipettor in each well and incubate at 37 degrees Celsius for 20 minutes. Then gently triturate the cells in each well about 20 to 30 times using a 12-channel pipettor until a single cell suspension is achieved.Now perform flow cytometry and determine the percentage of cells expressing the GFAP:GFP reporter. First determine the cell size and viability from the forward and side scatters and gate the viable cell population. Then determine the GFP-positive cells within the viable cell population.An increase of percentage of GFP-positive cells by three standard deviations over controls is considered to be a positive hit which may be adjusted to accommodate user preference of stringency. Representative images of HSR-GBM1 GSC subclones expressing low and high levels of GFAP:GFP are shown. The subclones positive for GFAP:GFP expression were selected by flow cytometry for the patient-derived neurosphere line.Here, low GFAP:GFP indicates that less than 5%of the cells in the clone are GFP-positive, while high GFAP:GFP indicates that greater than 75%of the cells in the clone are GFP-positive. These results show that in vitro self-renewal capacity of GFAP-low subclones is higher than their GFAP-high counterparts as determined by ELDA. Therefore GH subclones are more differentiated than GL subclones.Finally, drug libraries were screened to identify agents capable to induce astroglial differentiation in GSCs. 12 drugs were found to significantly increase the percentage of cells expressing the GFAP:GFP reporter. Once mastered, this technique can be done in a few weeks if it is performed properly.Following this procedure other methods like alamarBlue assay, clonogenic assay in semisolid media, flank or intracranial xenograph injection can be performed in order to answer additional questions like the effect of selective drugs on cellular proliferation and clonogenicity in vitro and tumorigenicity in vivo. After watching this video, you should have a good understanding of how to identify compounds capable of inducing astroglial differentiation in glioblastoma stem cells.

Research

JoVE Journal

Cancer Research

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Imagem latente da viver-pilha ensaios de migração de células-tronco do estudo Glioblastoma cerebral Tumor e invasão

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Pesquisa do câncer

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Fluxo Cytometric deteção da recém-formada mama câncer células-tronco células após a reversão de apoptose

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

Spheroids 3D derivados do paciente fisiológico para a seleção antineoplástica da droga para alvejar pilhas de haste do cancro

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Diferenciação das células Epiteliais do Peito do Camundongo HC11 e EpH4

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Identificação de reguladores de fatores de transcrição usando triagem de média-duração de bibliotecas arrayed e um repórter baseado em dupla-luciferase

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Triagem de drogas de xenoenxertos tumorais derivados do paciente primário em zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Co-cultura de células-tronco semelhantes ao Glioblastoma em neurônios padronizados para estudar migração e interações celulares

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

In vivo Triagem de imunogenicidade de vesículas extracelulares derivadas do tumor por citometria de fluxo de células T esplênicas

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Triagem de biblioteca shRNA agrupada para identificar fatores que modulam um fenótipo de resistência a drogas

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Detecção simultânea baseada em imagens e flutometria de marcadores de senescência fluorescentes múltiplas em células cancerígenas senescentes induzidas por terapia

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...