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 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.
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.
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
2. Preparing luciferase-tagged GSCs
3. Bio-luminescence based measurement of cell viability
4. Temozolomide treatment and combination screening
5. Combination treatment of temozolomide and UMI-77 in XG387-Luc and XG328-Luc cell lines
6. Data analysis
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 correction between them (Pearson r = 0.9872; p < 0.0001; Figure 1C). Since the bio-luminescence of luciferase tagged cells is easy and quick to measure, this provides a simple method to measure the density of viable GSCs. Next, the anti-proliferative activity of temozolomide was assessed. As shown in Figure 1D, 400 µM temozolomide caused approximately 20% proliferation inhibition of XG387-Luc cells, suggesting it is useful but its anti-GBM effect can be further improved. The concentration of 200 µM was selected for the combination screening.
To give an example, 20 target-selective small molecule inhibitors was utilized for the drug combination screening to identify the potential candidate(s) that enhances the anti-GBM effect of temozolomide. As a result, the sensitive index (SI) values of 13 targeted agents were above 0, and 5 of them were above 0.1 (Figure 2B,C). Especially, the SI of the top two candidate drugs UMI-77 and A 83-01 were higher than 0.25, suggesting their potential to synergize with temozolomide.
To validate the above finding, the classical synergy models of HSA and Bliss3,4,6 were applied to determine the combined effect of temozolomide and UMI-77 in GSCs. In addition, XG328-another patient-derived GSC model established early-was used to perform the same evaluation. Anti-proliferative assay of the combined treatment of temozolomide and UMI-77 was performed in a six-by-six dose titration matrix. The results were analyzed to acquire the HSA and Bliss values which are readouts for synergistic inhibition and depict the difference between the expected inhibition and the observed inhibition. As shown in Figure 3B-D, the combination index (CI) values <1 and the high single agent (HSA) values >0 for most of the combinations of temozolomide and UMI-77 at different concentrations, suggests an overall synergistic interaction of temozolomide and UMI-77 in both XG387 and XG328 GSCs.
Figure 1: GBM patient-derived GSCs XG387. (A) Neurosphere formation. (B) Bio-luminescence generation of luciferase tagged GSCs. (C) Bio-luminescence generated by XG387-Luc cells was proportional to the cell density. (D) Temozolomide treatment of XG387. Each treatment was performed in triplicate with two independent experiments. The data are expressed as the mean ± SD. Please click here to view a larger version of this figure.
Figure 2: Drug combination screening using GSCs. (A) The formula to calculate the SI (sensitivity index) of 20 targeted agents with temozolomide (TMZ) in the combination screen. (B) The distribution of SI values of 20 targeted agents. Red dots: the top five candidate drugs. (C) Information of the top five candidate targeted agents. Please click here to view a larger version of this figure.
Figure 3: Combination treatment of temozolomide (TMZ) and UMI-77 in XG387-Luc and XG328-Luc cell lines. (A) Single and combinatorial titration of temozolomide and UMI-77 in a proliferation assay in XG387-Luc and XG328-Luc cell lines. (B) Isobologram and combination index analysis of the proliferation inhibition in XG387-Luc and XG328-Luc cells treated with temozolomide and UMI-77. CI <1 indicates a synergistic effect. (C,D) Synergy plots generated by Combenefit showing the interaction between temozolomide and UMI-77. Analysis of interaction resulted in HSA (high single agent) values and Bliss values, indicating synergistic efficacy as calculated from the expected. HSA and Bliss values >0 indicate synergistic effects. Each treatment was performed in triplicate with two independent experiments. Please click here to view a larger version of this figure.
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 combination with small molecular inhibitor was introduced to quantify the synergistic drug effect of two small molecular inhibitors7. The range of SI values is from -1 to 1, and the positive SI value indicates a sensitizing effect between each of the drugs. The higher the SI value achieved, the stronger the synergy was. Although the SI value alone is incapable to provide an affirmative answer about the type (synergistic, additive, or antagonistic) of drug-drug interaction, those top-ranked candidates have high probability to synergize with the drug of interest and therefore are worthy of further validation. In comparison, most of the current high-throughput drug combination screening methodologies are still laborious and involve difficult algorithms8,9.
To exemplify the feasibility of this method, a small-scale test screen was performed. As a result, it was possible to identify UMI-77, a selective MCL1 inhibitor, as the top candidate among 20 targeted agents to synergize with temozolomide in GSCs growth suppression. In fact, in a previous study, UMI-77 was also found to synergistically enhance the anti-glioma activity of temozolomide in established GBM cells10. In the current study, the synergistic interaction between UMI-77 and temozolomide was approved again in GSCs using the classical Chou-Talalay combination index, BLISS or HSA methods. Another advantage of this protocol is the usage of luciferase-tagged GSCs for measuring the viable proportion of cells. The luciferase activity of cells can be easily measured by the addition of the luciferin, the substrate of luciferase, and capture the luminescence by any instrument with the function of luminometric measurement. Because the luciferase-luciferin reaction is quick, herein it provides a cheap and quick solution in comparison with traditional MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), or CCK-8 (cell counting kit-8) assays, all of which require long incubation times. Together, the protocol presents a high-throughput screening of potential drug combination for GBM. The protocol also provides optional quick and simple solution for drug combination screen in addition to the standard synergy evaluation methods.
The authors have nothing to disclose.
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.
B-27 | Gibco | 17504-044 | 50X |
EGF | Gibco | PHG0313 | 20 ng/ml |
FGF | Gibco | PHG0263 | 20 ng/ml |
Gluta Max | Gibco | 35050061 | 100X |
Neurobasal | Gibco | 21103049 | 1X |
Penicillin-Streptomycin | HyClone | SV30010 | P: 10,000 units/ml S: 10,000 ug/ml |
Sodium Pyruvate | Gibco | 2088876 | 100 mM |
Table 1. The formulation of GSC complete culture medium. | |||
ABT-737 | MCE | Selective and BH3 mimetic Bcl-2, Bcl-xL and Bcl-w inhibitor | |
Adavosertib (MK-1775) | MCE | Wee1 inhibitor | |
Axitinib | MCE | Multi-targeted tyrosine kinase inhibitor | |
AZD5991 | MCE | Mcl-1 inhibitor | |
A 83-01 | MCE | Potent inhibitor of TGF-β type I receptor ALK5 kinase | |
CGP57380 | Selleck | Potent MNK1 inhibitor | |
Dactolisib (BEZ235) | Selleck | Dual ATP-competitive PI3K and mTOR inhibitor | |
Dasatinib | MCE | Dual Bcr-Abl and Src family tyrosine kinase inhibitor | |
Erlotinib | MCE | EGFR tyrosine kinase inhibitor | |
Gefitinib | MCE | EGFR tyrosine kinase inhibitor | |
Linifanib | MCE | Multi-target inhibitor of VEGFR and PDGFR family | |
Masitinib | MCE | Inhibitor of c-Kit | |
ML141 | Selleck | Non-competitive inhibitor of Cdc42 GTPase | |
OSI-930 | MCE | Multi-target inhibitor of Kit, KDR and CSF-1R | |
Palbociclib | MCE | Selective CDK4 and CDK6 inhibitor | |
SB 202190 | MCE | Selective p38 MAP kinase inhibitor | |
Sepantronium bromide (YM-155) | MCE | Survivin inhibitor | |
TCS 359 | Selleck | Potent FLT3 inhibitor | |
UMI-77 | MCE | Selective Mcl-1 inhibitor | |
4-Hydroxytamoxifen(Afimoxifene) | Selleck | Selective estrogen receptor (ER) modulator | |
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. |