Molecular fingerprinting of GPCR ligands in Cancer

描述(由申请人提供)： 癌症在这个国家仍然是一个地方性问题，对生活质量和经济都造成了损害。除了彻底的预防，通过开发新的生物学和分析技术及早发现恶性肿瘤可能提供一种拯救生命和医疗成本的最佳方法。不同的癌症拥有、响应或分泌不同的分子标记，并可能被这些分子识别，这些分子为它们的识别提供了分子签名。G蛋白偶联受体是药物治疗中最常见的靶点，调节许多重要的生理过程，包括细胞的生长和分化。内源性GPCR配体已被提出作为肿瘤生物标志物，但尚不存在用于筛选生物样本中GPCR配体的通用高通量分析方法。同时分析小样本中所有已知GPCR配体的能力可以为筛选已知生物标记物和识别或发现新的生物标记物提供一种新的高通量蛋白质组学检测技术。特别是，如果检测系统利用受体本身作为传感器，这将提供一个灵敏度和特异度都很高的有效筛选平台。这项提议的目标是为这种高通量系统提供生物臂，该系统将利用荧光成像来读出传感器的生物活性。具体目标是：(1)验证聚苯乙烯微球作为生物传感器的生长和分析平台的使用。(2)验证含有荧光阻滞剂、不同GPCR和量子微点的U2OS细胞作为条形码生物传感器的使用。(3)证明生物传感器可以是多路的，这样潜在的100多个不同的GPCR生物传感器可以用来分析100微升或更小的液滴体积。这种类型的GPCR配体生物分析平台将对基础研究和临床筛选具有实用价值。此外，目前已有用于分析生物传感器的自动成像系统，它们识别用于癌症治疗的GPCR配体的通过率可能会提高100倍以上。

DESCRIPTION (provided by applicant): Cancer remains an endemic problem in this country extracting a toll on both the quality of life and the economy. Short of outright prevention, the early detection of malignancies through the development of novel biological and analytical techniques potentially provides an optimal approach for saving life and healthcare costs. Different cancers possess, respond to, or secrete distinct molecular markers and may presumably be identified by these molecules which provide molecular signatures for their identification. G protein-coupled receptors are the most common targets of pharmacological therapy and regulate many critical physiological processes including cell growth and differentiation. Endogenous GPCR ligands have been proposed as tumor biomarkers but generalized high throughput assays to screen biological samples for GPCR ligands do not exist. The ability to analyze a small biological sample simultaneously for all known GPCR ligands could provide a novel high-throughput proteomic-based detection technique for screening known biomarkers and identifying or discovering new ones. In particular if the detection system utilizes the receptors themselves as sensors, this would provide a validated screening platform of great sensitivity and specificity. The goal of this proposal is to provide the biological arm for such a high-throughput system that will utilize fluorescence imaging as readout of sensor bioactivity. The specific aims are: (1) Validate the use of polystyrene microspheres as both a growth and analysis platform for the biosensors. (2) Validate the use of U2OS cells containing fluorescent arrestins, distinct GPCRs, and quantum microdots as barcoded biosensors. (3) Demonstrate that biosensors can be mutiplexed such that potentially upwards of 100 distinct GPCR biosensors can be used to analyze drop-sized volumes of 100 microliters or less. This type of platform for biological analysis of GPCR ligands would have utility for either basic research and clinical screening. Moreover, automated imaging systems for the analysis of the biosensors are currently available and their throughput rate for identifying GPCR ligands useful in cancer therapy could be increased upwards of 100 fold.