Molecular fingerprinting of GPCR ligands in Cancer

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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.