JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

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

Published: March 28th, 2021

DOI:

10.3791/62312

1Department of Cell Biology, School of Basic Medical Sciences, Nanjing Medical University, 2Institute for Brain Tumors & Key Laboratory of Rare Metabolic Diseases, Nanjing Medical University; Nanjing Medical University Affiliated Cancer Hospital; Key Laboratory of Human Functional Genomics of Jiangsu Province
* These authors contributed equally

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

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

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved