We thank The National Natural Science Foundation of China (81672962), the Jiangsu Provincial Innovation Team Program Foundation, and the Joint Key Project Foundation of Southeast University and Nanjing Medical University for their support.

GBM specimen was acquired from a patient during a routine operation after obtaining fully informed consent by human research ethics committee of The First Affiliated Hospital of Nanjing Medical University.
1. Isolation and culture of patient-derived GSCs
<ol>
	<li>Place fresh surgically resected glioblastoma tissue in a 15 mL centrifuge tube filled with sterile PBS and store the tissue on ice until further operation.</li>
	<li>Mince the GBM tissue into approximately 0.5 to 1 mm diameter piec...

<ol>
	<li>Lathia, J. D., Mack, S. C., Mulkearns-Hubert, E. E., Valentim, C. L., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+in+glioblastoma.">Cancer stem cells in glioblastoma.</a> Genes &amp; Development. 29 (12), 1203-1217 (2015).</li><li>Binello, E., Germano, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+glioma+stem+cells:+a+novel+framework+for+brain+tumors.">Targeting glioma stem cells: a novel framework for brain tumors.</a> Cancer Science. 102 (11), 1958-1966 (2011).</li><li>Mathews Griner, L. A., 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=High-throughput+combinatorial+screening+identifies+drugs+that+cooperate+with+ibrutinib+to+kill+activated+B-cell-like+diffuse+large+B-cell+lymphoma+cells.">High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (6), 2349-2354 (2014).</li><li>Di Veroli, G. Y., 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=Combenefit:+an+interactive+platform+for+the+analysis+and+visualization+of+drug+combinations.">Combenefit: an interactive platform for the analysis and visualization of drug combinations.</a> Bioinformatics. 32 (18), 2866-2868 (2016).</li><li>Shi, Y., 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=Ibrutinib+inactivates+BMX-STAT3+in+glioma+stem+cells+to+impair+malignant+growth+and+radioresistance.">Ibrutinib inactivates BMX-STAT3 in glioma stem cells to impair malignant growth and radioresistance.</a> Science Translational Medicine. 10 (443), 1-13 (2018).</li><li>Tan, X., 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=Systematic+identification+of+synergistic+drug+pairs+targeting+HIV.">Systematic identification of synergistic drug pairs targeting HIV.</a> Nature Biotechnology. 30 (11), 1125-1130 (2012).</li><li>Jansen, V. 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=Kinome-wide+RNA+interference+screen+reveals+a+role+for+PDK1+in+acquired+resistance+to+CDK4/6+inhibition+in+ER-positive+breast+cancer.">Kinome-wide RNA interference screen reveals a role for PDK1 in acquired resistance to CDK4/6 inhibition in ER-positive breast cancer.</a> Cancer Research. 77 (9), 2488-2499 (2017).</li><li>Malyutina, A., 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=Drug+combination+sensitivity+scoring+facilitates+the+discovery+of+synergistic+and+efficacious+drug+combinations+in+cancer.">Drug combination sensitivity scoring facilitates the discovery of synergistic and efficacious drug combinations in cancer.</a> PLoS Computational Biology. 15 (5), 1006752 (2019).</li><li>He, L., 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=Methods+for+High-throughput+drug+combination+screening+and+synergy+scoring.">Methods for High-throughput drug combination screening and synergy scoring.</a> Cancer Systems Biology. 1711, 351-398 (2018).</li><li>Chen, 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=Targeting+the+synthetic+vulnerability+of+PTEN-deficient+glioblastoma+cells+with+MCL1+inhibitors.">Targeting the synthetic vulnerability of PTEN-deficient glioblastoma cells with MCL1 inhibitors.</a> Molecular Cancer Therapeutics. 19 (10), 2001-2011 (2020).</li></ol>

In the present study, a protocol that can be applied to identify potential combination therapy for GBM using patient-derived GSCs was described. Unlike the standard synergy/additivity metric model such as Loewe, BLISS, or HSA methods, a simple and quick workflow was used that does not require a drug pair to be combined at multiple concentrations in a full factorial manner as the traditional methods. In this workflow, SI (sensitivity index) which is originated from a study to evaluate the sensitizing effect of siRNAs in c...

Glioblastoma (GBM) is the most common and aggressive type of primary brain tumor. Currently, the overall survival of GBM patients who received maximal treatment (a combination of surgery, chemotherapy, and radiotherapy) is still shorter than 15 months; so novel and effective therapies for GBM are urgently required.
The presence of glioma stem cells (GSCs) in GBM constitutes a considerable challenge for the conventional treatment as these stem-like cells play pivot roles in the maintenance of tumor microenvironment, drug resistance, and tumor recurrence1. Therefore, targeting GSCs could be a promising strategy for GBM treatment2. Nevertheless, a major drawback for the drug efficacy in GBM is its heterogenetic nature, including but not limited to the difference in genetic mutations, mixed subtypes, epigenetic regulation, and tumor microenvironment which makes them very refractory for treatment. After many failed clinical trials, scientists and clinical researchers realized that single-agent targeted therapy is probably incapable of fully controlling the progression of highly heterogeneous cancers such as GBM. Whereas, carefully selected drug combinations have been approved for their effectiveness by synergistically enhancing the effect of each other, thus providing a promising solution for GBM treatment.
Although there are many ways to evaluate the drug-drug interactions of a drug combination, such as the CI (Combination Index), HSA (Highest Single Agent), and Bliss values, etc.3,4, these calculation methods are usually based on multiple concentration combinations. Indeed, these methods can provide affirmative assessment of drug-drug interaction but can be very laborious if they are applied in high-throughput screening. To simplify the process, a screening workflow for rapidly identifying the potential drug combinations that inhibit the growth of GSCs originated from surgical biopsies of patient GBM was developed. A sensitivity Index (SI) that reflects the difference of the expected combined effect and the observed combined effect was introduced into this method to quantify the synergizing effect of each drug, so the potential candidates can be easily identified by the SI ranking. Meanwhile, this protocol demonstrates an example screen to identify the potential candidate(s) that can synergize the anti-glioma effect with temozolomide, the first-line chemotherapy for GBM treatment, among 20 small molecular inhibitors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>50X</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Gibco</td>
			<td>PHG0313</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>Gibco</td>
			<td>PHG0263</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>Gluta Max</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td>P: 10,000 units/ml&#160;&#160;&#160;&#160; S:&#160; 10,000 ug/ml</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>2088876</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td colspan="4">Table 1. The&#160;formulation&#160;of GSC&#160;complete culture medium.&#160;&#160;</td>
		</tr>
		<tr>
			<td>ABT-737</td>
			<td>MCE</td>
			<td></td>
			<td>Selective and BH3 mimetic&#160;Bcl-2,&#160;Bcl-xL&#160;and&#160;Bcl-w&#160;inhibitor</td>
		</tr>
		<tr>
			<td>Adavosertib (MK-1775)</td>
			<td>MCE</td>
			<td></td>
			<td>Wee1&#160;inhibitor</td>
		</tr>
		<tr>
			<td>Axitinib</td>
			<td>MCE</td>
			<td></td>
			<td>Multi-targeted tyrosine kinase inhibitor</td>
		</tr>
		<tr>
			<td>AZD5991</td>
			<td>MCE</td>
			<td></td>
			<td>Mcl-1&#160;inhibitor</td>
		</tr>
		<tr>
			<td>A 83-01</td>
			<td>MCE</td>
			<td></td>
			<td>Potent inhibitor of TGF-&#946; type I receptor&#160;ALK5 kinase</td>
		</tr>
		<tr>
			<td>CGP57380</td>
			<td>Selleck</td>
			<td></td>
			<td>Potent&#160;MNK1&#160;inhibitor</td>
		</tr>
		<tr>
			<td>Dactolisib (BEZ235)</td>
			<td>Selleck</td>
			<td></td>
			<td>Dual ATP-competitive&#160;PI3K&#160;and&#160;mTOR&#160;inhibitor</td>
		</tr>
		<tr>
			<td>Dasatinib</td>
			<td>MCE</td>
			<td></td>
			<td>Dual&#160;Bcr-Abl and Src family tyrosine kinase&#160;inhibitor</td>
		</tr>
		<tr>
			<td>Erlotinib</td>
			<td>MCE</td>
			<td></td>
			<td>EGFR&#160;tyrosine kinase inhibitor</td>
		</tr>
		<tr>
			<td>Gefitinib</td>
			<td>MCE</td>
			<td></td>
			<td>EGFR tyrosine kinase&#160;inhibitor</td>
		</tr>
		<tr>
			<td>Linifanib</td>
			<td>MCE</td>
			<td></td>
			<td>Multi-target inhibitor of&#160;VEGFR&#160;and&#160;PDGFR&#160;family</td>
		</tr>
		<tr>
			<td>Masitinib</td>
			<td>MCE</td>
			<td></td>
			<td>Inhibitor of&#160;c-Kit</td>
		</tr>
		<tr>
			<td>ML141</td>
			<td>Selleck</td>
			<td></td>
			<td>Non-competitive inhibitor of&#160;Cdc42 GTPase&#160;</td>
		</tr>
		<tr>
			<td>OSI-930</td>
			<td>MCE</td>
			<td></td>
			<td>Multi-target inhibitor of Kit, KDR and CSF-1R&#160;</td>
		</tr>
		<tr>
			<td>Palbociclib</td>
			<td>MCE</td>
			<td></td>
			<td>Selective&#160;CDK4&#160;and&#160;CDK6&#160;inhibitor</td>
		</tr>
		<tr>
			<td>SB 202190</td>
			<td>MCE</td>
			<td></td>
			<td>Selective&#160;p38 MAP kinase&#160;inhibitor</td>
		</tr>
		<tr>
			<td>Sepantronium bromide (YM-155)</td>
			<td>MCE</td>
			<td></td>
			<td>Survivin&#160;inhibitor</td>
		</tr>
		<tr>
			<td>TCS 359</td>
			<td>Selleck</td>
			<td></td>
			<td>Potent FLT3 inhibitor</td>
		</tr>
		<tr>
			<td>UMI-77</td>
			<td>MCE</td>
			<td></td>
			<td>Selective&#160;Mcl-1&#160;inhibitor</td>
		</tr>
		<tr>
			<td>4-Hydroxytamoxifen(Afimoxifene)</td>
			<td>Selleck</td>
			<td></td>
			<td>Selective&#160;estrogen receptor (ER)&#160;modulator</td>
		</tr>
		<tr>
			<td colspan="4">Table 2. The information of 20 targeted agents used in the test screen. All of these are target selective small molecular inhibitors. The provider, name, and targets were given in the table.</td>
		</tr>
	</tbody>
</table>

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 XG387 cells formed neurospheres in the culture medium described in the Table 1 in an ultra-low attachment 6-well culture plate or a non-coated plate5 (Figure 1A). First, a test was performed to check whether the bio-luminescence intensity from XG387-Luc cells was proportional to the cell number. As shown in Figure 1B, the bio-luminescence intensity increased proportionally to the cell density and resulted in a linear ...

Watch this Scientific Journal Video about A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells at JoVE.com

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 ...

Glioma stem Cells are sub-population of cells in the glioma, which are usually resistant to chemo and radiotherapy. Here we described the method for rapid identification of combination therapies, targeting glioma stem cells, instead of differentiated glioma cells. Compared with other methods, which may be a laborious and complicated.This protocol is a relatively simple and rapid, to identify potential useful drug combinations. Begin by collecting the glioma stem cells or GSCs from the culture medium, and centrifuging them at 70 times G for three minutes at room temperature. After removing the supernatant, digest the cells with accutase for four minutes at 37 degrees Celsius.Using a 200 microliter tip, pipette the cells repeatedly to dissociate, and resuspend the cell pellet. Add the GSCs at a density of 200, 000 cells in one milliliter of culture medium, into each well of a 12 well culture plate, and incubate them overnight. Oh the next day, add 30 microliters of luciferase-eGFP virus supernatant into each well of the plate.Centrifuge the cells at 1000 times G for two hours, at 25 degrees Celsius and incubate them overnight. On the following day, replace the medium in the wells by collecting the GSCs, centrifuging them, removing the supernatant, resuspending them in fresh medium, and replating them. Culture the cells for another 48 hours.Observe the plate under a fluorescent microscope and confirm the appearance of GFP-positive cells. Sort and collect the GSCs with a high fluorescence using a flow cell sorter and culture them further. Coat, 96 well optical bottom plates with an extracellular matrix mixture, such as matrigel, and incubate them for one hour at 37 degrees Celsius.Remove the excess mixture and gently rinse the plates once with PBS, then seed XG387-Luc cells at a density of 1000 cells in 100 microliters of culture medium, and to each well of the coated plates and culture them overnight. On the following day, observe the cells under a microscope to confirm their attachment to the plate. Next prepare a 200 micromolar temozolomide solution and two micromolar solutions of the targeted agents, in culture medium.For combination drug screening, remove the blank medium and add the medium containing either 200 micromolar temozolomide or two micromolar targeted agent, or a combination of both, into each well for three technical replicates per treatment. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for three days. Take images of the cellular bioluminescence in the plate using the IVIS spectrum imaging system.Using the built-in software, create multiple circular areas of the region of interest and quantify the cellular bioluminescence. To identify potential candidates that could enhance the antiglioblastoma effect of temozolomide, 20 target selective small molecule inhibitors were analyzed through drug combination screening. The sensitive index values of 13 targeted agents were above zero and five of them were above 0.1.The sensitive index of the top two candidate drugs UMI-77 and A 83-01, were higher than 0.25, suggesting their potential to synergize with temozolomide. The combined treatment of temozolomide and UMI-77 was evaluated using an anti-proliferative assay in a six by six dose titration matrix. The combination index values were less than one.And the high single agent values were greater than zero for most of the combinations of temozolomide and UMI-77 at different concentrations. Suggesting an overall synergistic interaction of temozolomide and UMI-77 in both XG387 and XG328 GSCs. This protocol can also be applied in finding drug combination using adherent cancer cells by simply removing the plate coating step.

a-rapid-screening-workflow-to-identify-potential-combination-therapy

Research

JoVE Journal

Cancer Research

2.7K 次观看.  Nanjing Medical University. 胶质瘤干细胞是胶质瘤细胞的亚种群，通常对化疗和放射治疗有抵抗力。在这里，我们描述了快速识别组合疗法的方法，针对胶质瘤干细胞，而不是分化胶质瘤细胞。与其他方法相比，这可能是一个费力和复杂的。此协议是一个相对简单和快速的，以识别潜在的有用的药物组合。首先从培养介质中收集胶质瘤干细胞或GCS，并在室温下以70倍G离心三分钟。去除超高分子后?...