Molecular fingerprinting of GPCR ligands in Cancer

描述（由申请人提供）： 癌症仍然是这个国家的一个地方性问题，对生活质量和经济都造成了损失。在缺乏彻底预防的情况下，通过开发新的生物和分析技术早期发现恶性肿瘤可能为挽救生命和节省医疗费用提供最佳方法。不同的癌症具有、响应于或分泌不同的分子标记物，并且可能通过这些分子来鉴定，这些分子为它们的鉴定提供分子特征。G蛋白偶联受体是药物治疗的最常见靶点，并调节许多关键的生理过程，包括细胞生长和分化。已经提出内源性GPCR配体作为肿瘤生物标志物，但是用于筛选生物样品的GPCR配体的通用高通量测定尚不存在。同时分析所有已知GPCR配体的小生物样品的能力可以提供用于筛选已知生物标志物和鉴定或发现新生物标志物的新型高通量基于蛋白质组学的检测技术。特别地，如果检测系统利用受体本身作为传感器，这将提供具有高灵敏度和特异性的经验证的筛选平台。该提案的目标是为这样的高通量系统提供生物手臂，该系统将利用荧光成像作为传感器生物活性的读出。具体目标是：（1）探索聚苯乙烯微球作为生物传感器生长和分析平台的应用。(2)使用含有荧光抑制蛋白、不同GPCR和量子微粒的U2OS细胞作为条形码生物传感器。(3)证明生物传感器可以被复合，使得可能超过100个不同的GPCR生物传感器可以用于分析100微升或更少的液滴大小的体积。这种类型的GPCR配体生物分析平台将用于基础研究和临床筛选。此外，用于分析生物传感器的自动化成像系统目前是可用的，并且它们用于鉴定在癌症治疗中有用的GPCR配体的吞吐率可以增加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.