Sedeeq Mohammed, Maklad Ahmed, Gueven Nuri, Azimi Iman
School of Pharmacy and Pharmacology, College of Health and Medicine, University of Tasmania, Hobart TAS 7005, Tasmania, Australia.
Pharmaceuticals (Basel). 2020 Nov 5;13(11):368. doi: 10.3390/ph13110368.
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.
髓母细胞瘤（MB）是儿童最常见的恶性脑癌。高危MB肿瘤转移发生率高，患者生存率低。药物筛选通常用于确定针对MB的潜在新型治疗药物，鉴于这些检测方法易于适应高通量检测，所以主要集中在二维细胞增殖和活力检测上。然而，二维模型无法解决对MB转移至关重要的侵袭特性，因此不能代表体内肿瘤生长情况。在本研究中，我们使用与最高转移水平相关的分子亚组3的MB细胞开发了一种三维384孔琼脂集落形成检测方法。使用两种通过不同机制测量细胞活力的荧光底物，即刃天青和甘氨酰 - 苯丙氨酰 - 氨基荧光香豆素（GF - AFC）来评估MB细胞在琼脂基质中的生长。该检测方法针对接种密度、生长期、底物孵育时间以及各个孔内荧光信号的均匀性进行了优化。我们的数据证明了复用这两种荧光底物而无明显信号干扰的可行性。通过评估临床上用于MB治疗的两种常用化疗药物长春新碱和洛莫司汀的浓度依赖性效应，验证了该检测方法。随后，筛选了一组质膜钙通道调节剂对D341 MB细胞三维生长的影响，确定了T型电压门控钙通道和ORAI钙通道的调节剂为选择性生长调节剂。总体而言，这种三维检测方法为高通量筛选提供了一种可重复、省时且经济高效的检测方法，以识别针对MB的潜在药物。
