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.

Dit werk werd gedeeltelijk ondersteund door de NIH R01CA187780.

Opmerking: Astroglial en neuronale lentivirus verslaggever systemen werden aangekocht als voorverpakte, geconcentreerde lentivirale preparaten. Basiskennis van stroom cytometry techniek is vereist. Voor een optimaal gebruik van dit protocol moet de gebruiker ook toegang tot een cytometer van de stroom met hoge doorvoersnelheid capaciteit (accepteert 96-wells-platen als monsterbron).
1. lentivirale transcriptionele verslaggever systeem
Opmerking: In alle stroom cytomet...

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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>

Terwijl de meeste van de eerdere studies van GSCs was vooral gericht op de markeringen die ze bepalen, in dit onderzoek besloten hebben we om de omgekeerde aanpak. We concentreren ons vooral op de gedifferentieerde nakomelingschap gegenereerd door GSCs (bijvoorbeeld cellen uiten van astroglial en neuronale stiften). Hier tonen we het gebruik van een hoge-doorvoer-cel-gebaseerde drug screening systeem, dat gebaseerd is op menselijke GFAP promotor-afhankelijke expressie van GFP. Alle experimenten werden uitgevoerd met behu...

Recente studies hebben aangetoond dat tumoren een kleine populatie bevatten van cellen, cellen van de stam van kanker (CSCs) of inleiding van de tumor cellen, genoemd die belast zijn met progressie van de tumor, uitzaaiingen en weerstand tegen chemo - en radio-therapieën 1, 2. de aanwezigheid van cellen van de stam van kanker en hun meer gedifferentieerde nakomelingschap binnen tumoren wordt beschouwd als een belangrijke factor die bevordering van intratumoral heterogeniteit en vormt aldus een grote hindernis in het behandelen van kankers3. Tumor cel hiërarchie, geboden door de theorie van de stamcel kanker, heeft de ontwikkeling van nieuwe strategieën voor de behandeling van kankers 4geïnspireerd. Een aanpak voor het richten van de cellen van de stam van kanker is het identificeren en remmen signaalroutes waarvan bekend is dat tijdens de embryonale ontwikkeling van het betrokken orgaan verplicht worden. Inderdaad, wij en anderen eerder hebben gepubliceerd meerdere documenten beschrijven de voortdurende eis van de neurale stamcellen-relevante signaalroutes Sonic Hedgehog en Notch in glioblastoma5,6,7. Dit werk heeft geholpen bij het stollen van de motivering van de verschillende GBM klinische proeven. Een tweede aanpak voor het richten van de cellen van de stam van kanker is het bevorderen van hun differentiatie. Deze aanpak heeft ontvangen veel steun als gevolg van de gunstige resultaten van preklinische en klinische studies in het behandelen van acute promyelocytic leukemie met retinoic zuren (ATRA, een vitamine-A-analoog). Hier bleek ATRA te verwijderen van het blok van rijping en kanker cel differentiatie8te bevorderen. Meer recent, Piccirillo en collega's elegant is gebleken dat BMP-4 GSC differentiatie in astrocyten met belangrijke anti-GBM effecten in vitro en in vivo9 bevordert.
De reden voor de huidige studie is gebaseerd op een "omgekeerde engineering" aanpak voor het richten van de GSCs. Gezien de grote heterogeniteit aanwezig in GBM en met arme differentiatie wordt een van de kenmerken van de kanker, vroegen we als we een meer gunstige fenotype - differentiatie in een Astrocyt-achtige toestand kunnen bevorderen. Wij hebben hier geen voorkennis van de signaalroutes die handhaven GSCs een gegeven tumor specimen maar veeleer streven naar een gewenste fenotype (bijvoorbeeld GFAP positiviteit).
Dit verslag beschrijft de procedures die zijn gebruikt om de GSC differentiatie verslaggever-lijnen van de transductie van GSC-verrijkt culturen aan GSC kloonselectie. Het glioblastoma neurosphere lijnen gebruikt werden opgericht aan het laboratorium van Professor Angelo Vescovi van patiënten met een diagnose van primaire glioblastoma bij ziekenhuis San Raffaele - Milano, Italië. Deze lijnen zijn uitvoerig bestudeerd in verschillende publicaties 6,10,11,12,13,14. Het is sterk aanbevolen dat personen die geïnteresseerd zijn in het implementeren van deze technieken in hun laboratorium bepalen de relevantie van de verslaggever voor Kanker stamcel zelf-vernieuwing capaciteit in de cellen die zij van plan om te studeren (dit geldt voor elke verslaggever). Een gedetailleerd protocol voor één van de in vitro clonogenic tests geaccepteerd in het veld wordt geleverd aan het bereiken van deze15,16. Ten slotte, een gedetailleerd protocol met een beschrijving van het gebruik van de differentiatie verslaggever-lijnen in een flow-cytometry gebaseerd drug scherm wordt geleverd aan het eind. Van de nota, hebben ook aan het astroglial differentiatie systeem hier beschreven, we met succes vastgesteld en gevalideerd GSC verslaggever lijnen integratie van een MAP2:GFP (neuronale differentiatie) verslaggever. Daarom de methoden beschrijven in dit papier kan worden toegepast om te bestuderen van celdifferentiatie in verschillende geslachten van de cel.
Enkele van de cijfers in dit verslag kan worden gevonden in een recente publicatie: "Atracurium Besylate en andere neuromusculaire blokkering agenten bevorderen astroglial differentiatie en afbreken van glioblastoma stamcellen18.

<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) is de meest voorkomende en meest dodelijke primaire hersentumor bij volwassenen, waardoor ongeveer 14.000 doden elk jaar in de VS alleen. Mediane overleving na diagnose is minder dan 15 maanden met maximale chirurgische resectie, straling en temozolomide chemotherapie. De uitdagingen die inherent zijn aan de ontwikkeling van effectievere behandelingen GBM steeds duidelijk geworden, en omvatten de onverzettelijke invasiviteit, haar weerstand tegen standaard behandelingen zijn genetische complexiteit en moleculaire aanpassingsvermogen en subpopulaties van GBM cellen met fenotypische overeenkomsten met normale cellen van de stam, hierna te noemen glioblastoma stamcellen (GSCs). Omdat GSCs vereist voor tumorgroei en progressie zijn, vertegenwoordigt differentiatie gebaseerde therapie een levensvatbare behandeling modaliteit voor deze ongeneeslijke gezwellen.
Het volgende protocol beschrijft een verzameling van procedures om een hoge throughput screening platform gericht op de identificatie van kleine moleculen ter bevordering van de GSC astroglial differentiatie. De kern van het systeem is een gliale fibrillary zuur eiwit (GFAP) differentiatie verslaggever-construct. Het protocol bevat de volgende algemene procedures: (1) tot oprichting van de GSC differentiatie verslaggever lijnen; (2) testen/valideren de relevantie van de verslaggever voor GSC zelf-vernieuwing/clonogenic capaciteit; en (3) hoge capaciteit stroom-cytometry gebaseerd drug screening.
De screening-platform biedt een eenvoudige en goedkope aanpak ter identificatie van de kleine moleculen ter bevordering van de differentiatie van de GSCs. Gebruik van bibliotheken van FDA-goedgekeurde geneesmiddelen houdt bovendien de mogelijkheden voor de identificatie van agenten die sneller andere doelen kan worden gebruikt. Ook worden therapieën die bevordering van Kanker stamcel differentiatie geacht te werken synergetisch met huidige "standaard van zorg" therapieën die te richten en te elimineren vooral meer gedifferentieerde kankercellen is gebleken.

Drie onafhankelijke patiënt afkomstige neurospheres lijnen werden getransduceerde met de lentivirus astroglial verslaggever codering voor een groen fluorescent proteïne (GFP) gesmolten in-frame met een Zeocin weerstand cassette en gedreven door het menselijke element van GFAP promotor ( Figuur 1). Volgende individuele klonen werden geïsoleerd door plating 0.7 cellen per putje in een 96 goed bord (Figuur 2), dit werd gevolgd do...

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

Stroom Cytometry gebaseerde Drug Screening systeem voor de identificatie van kleine moleculen die bevordering van celdifferentiatie van Glioblastoma stamcellen

Een efficiënte screening-protocol wordt gepresenteerd voor de identificatie van kleine moleculen ter bevordering van astroglial differentiatie in glioblastoma stamcellen (GSCs). De bepaling is gebaseerd op een stamcel differentiatie verslaggever waarbij de expressie van de verbeterde GFP (eGFP) wordt gedreven door de menselijke promotor van het algemeen kaderakkoord voor vrede.

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

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Generatie van High-Throughput driedimensionale Tumor spheroïden voor Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

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

Live-cel Imaging tests studie Glioblastoma Brain Tumor stamcel migratie en invasie

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

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

Stroom cytometrische detectie van nieuwgevormde stamcel-achtige borstkankercellen na Apoptosis omkering

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

Fysiologisch patiënt afgeleide 3D-Spheroids voor anti-neoplastische geneesmiddelen screening om kanker stamcellen te targeten

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

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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 ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Oprichting en karakterisering van de neuro-endocriene tumor Spheroids van de kleine darm

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

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

Identificatie van Transcription Factor Regulators met behulp van Medium-Throughput Screening van arrayed bibliotheken en een Dual-Luciferase-Based Reporter

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

Drug Screening van primaire patiënt afgeleid tumor Xenografts bij zebravissen

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-cultuur van Glioblastoom Stam-achtige cellen op patroonneuronen om migratie en cellulaire interacties te bestuderen

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 Immunogeniciteitsscreening van tumor-afgeleide extracellulaire blaasjes door flowcytometrie van milt-T-cellen

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