Development of a medium-throughput colony formation screen for identification of novel radiosensitising drugs for glioblastoma


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Natividad Gomez-Roman1,Mark R Jackson1,Sandeep Chahal1,Anthony J Chalmers1
1University of Glasgow

Abstract

Background

Glioblastoma (GBM) is a lethal malignancy of the brain.  Standard of care treatment comprising surgery and radiochemotherapy confers median survival of just 15 months.  The clinical failure of many molecular targeted agents identified using conventional two-dimensional in vitro culture has rendered such models unsuitable for drug discovery.  We previously described a low-throughput three-dimensional (3D) scaffold GBM model that recapitulates key clinical features of GBM including, responses to molecular therapies.  Here, we report the development of a 3D in vitro system with greater clinical-relevance that is compatible with combination drug-screening applications.

Method

Use of 3D-Alvetex™ scaffolds in 96-well format allowed for the medium-throughput determination of clonogenicity, a widely-used gold-standard survival endpoint.  Images were acquired using a custom-mounted Nikon camera; automated image segmentation, colony counting and data analysis were performed in ImageJ, OpenCFU, and R software, respectively.  Single agent drug activity was determined (0Gy). When combined with radiation (3Gy), a significant reduction in IC50 indicated a drug-radiation therapeutic interaction.

Results

Automated colony detection was reproducible and correlated with manual counting.  Moreover, the radiation response of patient-derived GBM cells was found to be comparable to that previously reported in 3D system.  To further validate this novel approach, the single agent and radiosensitising potential of a panel of DNA damage response (DDR) inhibitors was characterised.

DDR target

Drug

IC50 0Gy (µM)

IC50 3Gy (µM)

P-value

PARP

Talazoparib

0.031

0.003

<0.001

Chk1

AZD7762

0.158

0.052

<0.001

Chk1

SCH900776

1.049

0.282

0.0016

ATR

AZD6738

0.728

0.237

0.0023

ATR

VX970

0.722

0.204

0.0057

DNA-PKcs

NU7441

3.083

0.193

<0.001

Conclusion

Gold-standard clonogenic survival was measured in a medium-throughput 3D culture system.  The therapeutic potential of a panel of DDR inhibitors was confirmed in primary GBM cells.  This novel methodology has the potential to accelerate the identification of bona fide therapeutic and radiosensiting agents in cancers of unmet need.