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Development of a high-throughput agar colony formation assay to identify drug candidates against medulloblastoma
Citation
Sedeeq, M and Maklad, A and Guven, N and Azimi, I, Development of a high-throughput agar colony formation assay to identify drug candidates against medulloblastoma, Pharmaceuticals, 13, (11) Article 368. ISSN 1424-8247 (2020) [Refereed Article]
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Copyright Statement
Copyright 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license http://creativecommons.org/licenses/by/4.0/).
Abstract
Medulloblastoma (MB) is the most common malignant childhood brain cancer. High-risk
MB 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 proliferation
and 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 not
representative of tumour growth in vivo. In this study, we developed a 3D 384-well agar colony
formation assay using MB cells of molecular subgroup 3 that is associated with the highest level of
metastasis. 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 cells
in the agar matrix. The assay was optimised for seeding density, growth period, substrate incubation
time and homogeneity of the fluorescent signals within individual wells. Our data demonstrate
the feasibility to multiplex the two fluorescent substrates without detectable signal interference.
This assay was validated by assessing the concentration-dependent effect of two commonly used
chemotherapeutic 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 3D
growth of D341 MB cells, which identified modulators of T-type voltage gated and ORAI calcium
channels as selective growth modulators. Overall, this 3D assay provides a reproducible, time and
cost-effective assay for high-throughput screening to identify potential drugs against MB.
Item Details
| Item Type: | Refereed Article |
|---|---|
| Keywords: | high-throughput assay, medulloblstoma, cancer, calcium signalling |
| Research Division: | Biomedical and Clinical Sciences |
| Research Group: | Pharmacology and pharmaceutical sciences |
| Research Field: | Basic pharmacology |
| Objective Division: | Manufacturing |
| Objective Group: | Human pharmaceutical products |
| Objective Field: | Human pharmaceutical products not elsewhere classified |
| UTAS Author: | Sedeeq, M (Mr Mohammed Sedeeq) |
| UTAS Author: | Maklad, A (Mr Ahmed Maklad) |
| UTAS Author: | Guven, N (Dr Nuri Guven) |
| UTAS Author: | Azimi, I (Dr Iman Azimi) |
| ID Code: | 141750 |
| Year Published: | 2020 |
| Deposited By: | Pharmacy |
| Deposited On: | 2020-11-16 |
| Last Modified: | 2021-04-09 |
| Downloads: | 1 View Download Statistics |
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