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Abstract

Medulloblastoma (MB) is the most common malignant childhood brain cancer. High-riskMB tumours have a high incidence of metastasis and result in poor patient survival. Drug screens,commonly used to identify potential novel therapeutic agents against MB, focus on 2D cell proliferationand viability assays given that these assays are easily adaptable to high-throughput regimes. However,2D models fail to address invasive characteristics that are crucial to MB metastasis and are thus notrepresentative of tumour growth in vivo. In this study, we developed a 3D 384-well agar colonyformation assay using MB cells of molecular subgroup 3 that is associated with the highest level ofmetastasis. Two fluorescence substrates, resazurin and glycyl-phenylalanyl-aminofluorocoumarin(GF-AFC) that measure cell viability via distinct mechanisms were used to assess the growth of MB cellsin the agar matrix. The assay was optimised for seeding density, growth period, substrate incubationtime and homogeneity of the fluorescent signals within individual wells. Our data demonstratethe feasibility to multiplex the two fluorescent substrates without detectable signal interference.This assay was validated by assessing the concentration-dependent effect of two commonly usedchemotherapeutic agents clinically used for MB treatment, vincristine and lomustine. Subsequently,a panel of plasma membrane calcium channel modulators was screened for their effect on the 3Dgrowth of D341 MB cells, which identified modulators of T-type voltage gated and ORAI calciumchannels as selective growth modulators. Overall, this 3D assay provides a reproducible, time andcost-effective assay for high-throughput screening to identify potential drugs against MB.

Item Type:	Article
Authors/Creators:	Sedeeq, M and Maklad, A and Guven, N and Azimi, I
Keywords:	high-throughput assay, medulloblstoma, cancer, calcium signalling
Journal or Publication Title:	Pharmaceuticals
Publisher:	MDPI AG
ISSN:	1424-8247
DOI / ID Number:	10.3390/ph13110368
Related URLs:	Google Scholar: Guven, N UTAS Profile: Guven, N Google Scholar: Azimi, I
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