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.

This work has been partially supported by NIH R01CA187780.

NOTE: Astroglial and neuronal lentivirus reporter systems were purchased as pre-packaged, concentrated lentiviral preparations. Basic knowledge of flow cytometry technique is required. Also, for a full use of this protocol the user will need access to a flow cytometer with high throughput capacity (accepts 96-well plates as sample source).
1. Lentiviral Transcriptional Reporter System
NOTE: In all flow cytometric analyses, use parental, non-transduced, cells or vector...

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+maintenance+in+glioblastoma:+a+social+network.">Heterogeneity maintenance in glioblastoma: a social network.</a> Cancer Res. 71 (12), 4055-4060 (2011).</li><li>Bao, 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=Glioma+stem+cells+promote+radioresistance+by+preferential+activation+of+the+DNA+damage+response.">Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.</a> Nature. 444 (7120), 756-760 (2006).</li><li>Sul, J., Fine, H. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+inhibition+of+glioblastoma+xenograft+initiation+and+clonogenic+growth+following+in+vivo+Notch+blockade.">Prolonged inhibition of glioblastoma xenograft initiation and clonogenic growth following in vivo Notch blockade.</a> Clin Cancer Res. 19 (12), 3224-3233 (2013).</li><li>Schreck, K. C., 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=The+Notch+target+Hes1+directly+modulates+Gli1+expression+and+Hedgehog+signaling:+a+potential+mechanism+of+therapeutic+resistance.">The Notch target Hes1 directly modulates Gli1 expression and Hedgehog signaling: a potential mechanism of therapeutic resistance.</a> Clin Cancer Res. 16 (24), 6060-6070 (2010).</li><li>Warrell, R. 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>

While most of the previous studies of GSCs focused primarily on the markers that define them, in this study we decided to take the reverse approach. We focus primarily on the differentiated progenies generated by GSCs (e.g., cells expressing astroglial and neuronal markers). Here we demonstrate the utilization of a cell-based high throughput drug screening system, which is based on human GFAP promoter-dependent expression of GFP. All the experiments were performed utilizing patient-derived neurosphere glioblastoma cell l...

Recent studies have shown that tumors contain a small population of cells, termed cancer stem cells (CSCs) or tumor-initiating cells, which are responsible for tumor progression, metastasis, and resistance to chemo- and radio-therapies 1,2. The presence of cancer stem cells and their more differentiated progenies within tumors is considered an important factor promoting intratumoral heterogeneity and thus represents a major hurdle in treating cancers3. Tumor cell hierarchy, provided by the cancer stem cell theory, has inspired the development of new strategies to treat cancers 4. One approach for targeting cancer stem cells is to identify and inhibit signaling pathways that are known to be required during embryonic development of the affected organ. Indeed, we and others have previously published multiple papers describing the ongoing requirement for the neural stem cell-relevant signaling pathways Sonic Hedgehog and Notch in glioblastoma5,6,7. This work has helped in solidifying the rationale for several GBM clinical trials. A second approach for targeting cancer stem cells is to promote their differentiation. This approach has received a lot of support due to the favorable results from preclinical and clinical studies in treating acute promyelocytic leukemia with retinoic acids (ATRA, a vitamin-A analog). Here ATRA was found to remove the maturation block and promote cancer cell differentiation8. More recently, Piccirillo and colleagues have elegantly shown that BMP-4 promotes GSC differentiation into astrocytes with significant anti-GBM effects in vitro and in vivo9.
The rationale for the current study is based on a &#34;reversed engineering&#34; approach for targeting GSCs. Given the vast heterogeneity present in GBM and with poor differentiation being one of the hallmarks of cancer, we asked if we could promote a more favorable phenotype - differentiation into an astrocyte-like state. Here, we do not have prior knowledge of the signaling pathways that maintain GSCs in a given tumor specimen but rather aim to achieve a desired phenotype (e.g. GFAP positivity).
This report describes the procedures used to establish GSC differentiation reporter-lines from the transduction of GSC-enriched cultures to GSC clonal selection. The glioblastoma neurosphere lines used were established at the laboratory of Professor Angelo Vescovi from patients with a diagnosis of primary glioblastoma at Hospital San Raffaele - Milano, Italy. These lines have been extensively studied in several publications 6,10,11,12,13,14. It is highly recommended that individuals who are interested in implementing these techniques in their laboratory determine the relevance of the reporter to cancer stem cell self-renewal capacity in the cells they plan to study (this is true for any reporter). A detailed protocol for one of the in vitro clonogenic assays accepted in the field is provided to accomplish this15,16. Finally, a detailed protocol describing the utilization of the differentiation reporter-lines in a flow-cytometry based drug screen is provided at the end. Of note, similarly to the astroglial differentiation system described here, we have successfully established and validated GSC reporter lines integrating an MAP2:GFP (neuronal differentiation) reporter. Therefore, the methodologies describe in this paper may be applied to study cellular differentiation into various cell lineages.
Some of the figures in this report can be found in a recent publication: &#34;Atracurium Besylate and other neuromuscular blocking agents promote astroglial differentiation and deplete glioblastoma stem cells18.

<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 the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

Three independent patient-derived neurospheres lines were transduced with the lentivirus astroglial reporter encoding for a green-fluorescent protein (GFP) fused in-frame with a Zeocin resistance cassette and driven by the human GFAP promoter element (Figure 1). Next individual clones were isolated by plating 0.7 cells per well in a 96 well plate (Figure 2), this was followed by flow cytometric determination of the percentage of ...

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

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

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.

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Cancer Research

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

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

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

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

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

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 of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

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 ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a 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 anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

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

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

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

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

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

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

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

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

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

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

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

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

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