High throughput bioluminescence screening for glioma therapeutics

描述（由申请人提供）： 肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducing ligand，TRAIL）被认为是一种潜在的抗肿瘤药物，然而，相当多的肿瘤细胞，尤其是多形性胶质母细胞瘤（glioblastoma multiforme，GBM）对TRAIL诱导的凋亡具有抵抗性。该提案的目的是评估天然分泌的Gaussia荧光素酶（Gluc）作为高通量药物筛选的报告基因，以鉴定使胶质瘤细胞对TRAIL敏感的小分子。不同的神经胶质瘤细胞将通过基因转移工程化以表达Gluc，其表达以及因此细胞活力可以通过使用96-威尔斯格式板光度计对条件培养基的等分试样进行生物发光测量来随时间监测。最初，将针对96孔板格式优化Gluc测定的不同组分，包括孔间变化以及底物随时间的稳定性和最佳底物剂量。接下来，将测试不同的神经胶质瘤细胞系，包括从患者肿瘤切片解离的原代GBM细胞对TRAIL的敏感性或抗性。最后，将使用小分子文库筛选原代GBM细胞中使其对TRAIL敏感的药物，其中一些是FDA批准的已知穿过血脑屏障的药物。使原代GBM细胞对TRAIL敏感的候选药物将在体外剂量和时间依赖性实验中进一步表征，并可能在我们的实验性胶质瘤-Gluc模型中进行体内表征。4秒的Gluc测定时间使其成为HTS的宝贵工具，并且可以轻松应用于384孔板格式，以在相对较短的时间内筛选数千种化合物。

DESCRIPTION (provided by applicant): Tumor Necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anti-cancer agent, however, considerable numbers of cancer cells, especially glioblastoma multiforme (GBM), are resistant to apoptosis induction by TRAIL. The aim of this proposal is to evaluate the naturally secreted Gaussia Luciferase (Gluc) as a reporter for high throughput drug screening in order to identify small molecules that sensitize glioma cells to TRAIL. Different glioma cells will be engineered by gene transfer to express Gluc which expression, and therefore cell viability, can be monitored over time by subjecting an aliquot of the conditioned medium to bioluminescence measurements using a 96-wells format plate luminometer. Initially, different components of the Gluc assay including well-to-well variation, as well as substrate stability over time and optimal substrate dose will be optimized for a 96-well plate format. Next, different glioma cell lines, including primary GBM cells dissociated from patient tumor sections, will be tested for their sensitivity or resistance to TRAIL. Finally, primary GBM cells will be screened for drugs that will sensitize them to TRAIL using small molecule libraries some of which are FDA-approved drugs known to cross the blood-brain barrier. The drug candidates that sensitize primary GBM cells to TRAIL will be further characterized in dose- and time-dependent experiments in vitro and potentially in our experimental glioma-Gluc models, in vivo. The Gluc assay time of 4 seconds makes it a valuable tool for HTS and can be easily applied into 384-well plate format to screen thousands of compounds over a relatively short period of time.