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-transduced (non-fluorescent) cells for establishing baseline fluorescence. Also, note that in all steps where mechanical trituration is called for, be gentle. Harsh trituration can kill a significant number of the fragile GSCs and influence flow cytometry results.
<ol>
	<li>Plate 1x106 cells in 2 mL of complete neural stem cell growth medium in a 6-multiwell plate.</li>
	<li>Transduce cells by adding lentivirus reporter at a multiplicity of infection (moi) equal to 5.</li>
	<li>Add 2 &#181;L of polybrene for a final concentration of 8 &#181;g/mL.</li>
	<li>Incubate the cells at 37 &#176;C and 5% CO2 overnight.</li>
	<li>The next day, replace growth medium to remove unbound virus. Place the plate back in the incubator at 37 &#176;C and 5% CO2 and incubate for 24 h.</li>
	<li>Harvest 0.5 mL of the total volume; spin down cells at 360 x g for 5 min at room temperature in a 15- mL conical tube and remove supernatant by aspiration. Add 0.5 mL of fresh neural stem cell growth medium to the remaining cells and return the plate to the incubator for continued expansion.</li>
	<li>Add 200 &#181;L of dissociation reagent and incubate for 5 min at 37 &#176;C.</li>
	<li>Dissociate cells by gently pipetting up and down. 
	NOTE: Harsh trituration may result in significant killing of GSCs, complete dissociation is usually achieved after 20 - 30 times.</li>
	<li>To minimize cell attachment or aggregation, add 800 &#181;L of Hank&#39;s Balanced Salt Solution (HBSS) for a final volume of 1 mL.</li>
	<li>Transfer 200 &#181;L of each cell suspension to a well of a 96-multiwell plate.</li>
	<li>For this procedure, use a benchtop flow cytometer with 96-well plate capability equipped with a blue laser for excitation and with the capability of detecting green fluorescent protein. Perform flow cytometric analysis with at least 10,000 viable cells for each acquisition.</li>
	<li>Determine the percentage of GFP-positive cells by flow cytometry.</li>
</ol>
2. Subclone Selection, Expansion, and Validation
<ol>
	<li>Plate cells in 100 &#181;L of neural stem cell medium at a density of 0.7 cells per well of a 96-multiwell plate.</li>
	<li>Culture clones for 11 days at 37 &#176;C and 5% CO2. This step is highly cell type dependent and will likely require an adjustment based on the cell line used. As a general rule, a sphere diameter &#8805; 100 &#181;m is a good indication of the presence of clonogenic GSCs in the neurospheres.</li>
	<li>Using a fluorescent microscope equipped with a FITC filter, mark the wells that contain a single neurosphere where ~1-5% of the cells are GFP-positive. 
	NOTE: Determining the exact percentage of GFP-positive cells is not too critical at this point. Each of the subclones will later be evaluated by flow cytometry. This step is performed to reduce the total number of clones to be analyzed with emphasis on neurospheres that originate by undifferentiated, GFAP-negative, GSC.</li>
	<li>Expand selected reporter clones until there are a sufficient number of cells for analysis by flow cytometry. A sub-confluent well of a 6-multiwell plate containing &#8804; 1.5x105 cells/mL should provide a sufficient number of cells. 
	NOTE: A detailed procedure for the isolation and expansion of GSCs is available 17.</li>
</ol>
3. Determination of GFAP:GFP Expression by Flow Cytometry
<ol>
	<li>Harvest an aliquot of cells (0.5 mL) from each reporter clone and non-transduced controls to determine the exact percentage of GFP-positive cells. Considering reporter clones that contain ~1 - 5% GFP-positive cells.</li>
	<li>Spin cells at 360 x g for 5 min at room temperature and remove supernatant by aspiration.</li>
	<li>To each pellet, add 200 &#181;L of cell dissociation reagent and incubate the tubes in a water bath set to 37 &#176;C.</li>
	<li>Triturate cells gently to achieve a single cell suspension (usually between 20 to 30 times).</li>
	<li>To minimize cell attachment or aggregation, add 800 &#181;L HBSS to a final volume of 1 mL.</li>
	<li>Transfer 200 &#181;L per well of a 96-multiwell plate from each cell suspension.</li>
	<li>For this procedure, use a benchtop flow cytometer with 96-well plate capability. Perform flow cytometric analysis with at least 10,000 viable cells for each acquisition.</li>
</ol>
4. ELDA Self-Renewal Assay to Assess Clonogenic Capacity
NOTE: For controls, both parental, non-transduced, as well as GFP-expressing lentivirus-transduced cells, should be used to determine the relative clonogenic potential of the GSC differentiation reporter lines to the original GSC cultures from which they were derived.
<ol>
	<li>Dissociate differentiation-reporter subclones into single-cell suspensions as described above.</li>
	<li>Plate cells in 96-multiwell plates in 100 &#181;L of complete neural stem cell growth media at cell densities ranging between 5 and 500 cells per well.</li>
	<li>Incubate cells for 9 to 11 days at 37 &#176;C and 5% CO2.</li>
	<li>Score positive wells by direct visualization of neurospheres under a light microscope. A well should be considered &#34;positive&#34; if it contains at least a single large neurosphere.</li>
	<li>Plug the data: total wells analyzed and the number of positive wells using the ELDA online interface available at&#160;http://bioinf.wehi.edu.au/software/elda/index.html.</li>
</ol>
5. Drug Library Dilution Preparation
<ol>
	<li>Remove library plates from storage at -80 &#176;C, cover with aluminum foil (protect light-sensitive compounds). Thaw for approximately 30 min to an hour at room temperature.</li>
	<li>Use a 12-channel multichannel pipettor to dilute the library compounds to 0.2 mM in complete neural stem cell medium. Dilute DMSO to 10% in growth medium 
	NOTE: The final concentration of DMSO should be 0.1% when added to cells. A higher concentration of DMSO in the culture may result in toxicity. For this reason, it is recommended to test sensitivity to DMSO in the cell line used, before treatment.</li>
	<li>Use DMSO as vehicle control to treat cells found in the left and right columns of each plate (Columns 1 and 12; Wells A through H).</li>
	<li>Cover the diluted library plates with aluminum foil and return the original library plates to the -80 &#176;C freezer for long-term storage.</li>
</ol>
6. Drug Screen
NOTE: DMSO-treated cells should be used to set the baseline fluorescence and adjust gating.
<ol>
	<li>Plate 5x103 cells in a 96-multiwell plate in 99 &#181;L of complete neural stem cell growth medium.</li>
	<li>Using a 12-channel multichannel pipettor, treat cells with diluted library compounds at a final concentration of 2 &#181;M (1 &#181;L of 0.2 mM drug into 99 &#181;L of cell suspension) or with DMSO (control). Incubate the plates for 72 h at 37 &#176;C and 5% CO2.</li>
	<li>Using a 12-channel multichannel pipettor add 150 &#181;L of cell dissociation reagent to each well and incubate for 20 min at 37 &#176;C.</li>
	<li>Dissociate cells by gently triturating with a 12-channel multichannel pipettor until single cell suspension is achieved (usually between 20 to 30 times). The amount of time that is required to dissociate neurospheres completely will vary between GSC lines. While the recommended cell dissociation reagent (see materials) is safe and incubation for up to 45 minutes had no effect on viability in multiple GSC lines tested, verify for each GSC line to be used in screening. 
	NOTE: The final volume in each well should be roughly 250 &#181;L (100 &#181;L of cell suspension + 150 &#181;L of cell dissociation reagent).</li>
	<li>Determine the percentage of cells expressing the GFAP:GFP reporter by flow cytometry. Use standard gating strategy. First, plot forward and side scatters to get a general sense of cell size and viability. Then place a gate on the viable single-cell population. This is the population for which green fluorescence (eGFP) will be determined. Any compound that results in an increase in the percentage of GFP-positive cells by over three standard deviations over control (DMSO-treated) is considered as a positive &#34;hit.&#34;&#160;This threshold should be adjusted according to the specific application and the desired stringency. 
	NOTE: For the high throughput screen is recommended using a single GSC reporter subclone. Following hit identification, each hit should be validated against additional reporter subclones from the same GSC line. To increase the confidence for true hits is also recommend testing compounds against reporter subclones isolated from different GSC neurospheres lines.</li>
</ol>

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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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The authors have nothing to disclose.

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&#039;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&amp;sup2; Flask Vented</td><td>Thermo Fisher Scientific</td><td>130189</td><td></td></tr><tr><td>Biolite T75 cm&amp;sup2; 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 &amp;mu;l</td><td>VistaLab Technologies</td><td>4060-1002</td><td></td></tr><tr><td>Multi 12-channel pipette tips 250 &amp;mu;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For the preparation of neural stem cell media (500 mL)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Final concentration&lt;/strong&gt;</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 &amp;micro;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 ...

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8.3K Views.  Case Western Reserve University School of Medicine. 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.

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

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Medicine

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

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 ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Immunology and Infection

LEGO: A Human GBM Organoid Model Revealing Lipid Dysregulation and Drug Targets

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

Here, we outline a comprehensive protocol for generating and utilizing laboratory-engineered glioblastoma organoids (LEGO) to investigate genotype-phenotype dependencies and screen potential drugs for glioblastoma treatment.

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and ...

Cellular Bioluminescence Based Assay: A High Throughput Method for Combinatorial Drug Screening

Source: Hu, Z. et al. <a href="https://www.jove.com/v/62312/a-rapid-screening-workflow-to-identify-potential-combination-therapy">A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells.</a>&#160;J. Vis. Exp.&#160;(2021)
In this video, we describe a method highlighting the utility of bioluminescence assay for sensitive and rapid screening of drugs. These drugs can be tested alone or in combination to assess the synergism in their mode of action.

Source: Hu, Z. et al. A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells.&#160;J. Vis. ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vitro studies

Luciferase-tagged Glioma Stem Cell Model: A Lentivirus-mediated Method to Prepare Luciferase-tagged Glioma Stem Cells for In Vitro Therapeutic Studies

Source: Hu, Z. et al. <a href="https://www.jove.com/v/62312/a-rapid-screening-workflow-to-identify-potential-combination-therapy">A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells.</a> J. Vis. Exp. (2021)
This video demonstrates the establishment of luciferase-tagged glioma stem cells via lentivirus-mediated gene delivery. Luciferase acts as a reporter gene and helps facilitate rapid drug screening experiments.

Source: Hu, Z. et al. A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells. J. Vis. Exp. ...

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

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