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.
Highlights
Medulloblastoma (MB), the most common paediatric malignant brain tumour, represents 20% of all childhood brain cancers [1]
Z’-factor is a statistical method used to judge the quality of a given bioassay, and its suitability for high-throughput screening (HTS)
Resazurin can be readily reduced to resorufin in the presence of interfering chemical species without the presence of any cellular activity, which is of particular relevance for compounds with thiol and carboxylic acid moieties [22]
Summary
Medulloblastoma (MB), the most common paediatric malignant brain tumour, represents 20% of all childhood brain cancers [1]. MB is highly aggressive and often associated with high mortality rates. The tumour usually arises in the posterior fossa region of the brain as densely packed cells with high invasive ability allowing them to spread to other areas of the central nervous system [1]. Current treatment strategies for MB involve surgical resection, radiotherapy and chemotherapy as the most effective form of therapy available. These treatment modalities improve survival rate to 60%, it is associated with severe complications. The cytotoxic effects of these therapies have a devastating effect on the developing brain and often leave MB survivors with severe treatment-related morbidities including neurological, intellectual and physical disabilities [2,3]. Since previous efforts to reduce the morbidity and mortality associated with MB have been restricted by the harsh nature of conventional treatments, there is a clear need for a better treatment options [3,4,5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
