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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. 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.
2. Mince the GBM tissue into approximately 0.5 to 1 mm diameter piec.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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.