Development of a miniaturized analysis platform for quantitative and time-resolved high throughput-analysis of AKT-mTOR signaling kinetics in cancer cells
Protein kinase networks such as the mammalian target of rapamycin (mTOR) network control virtually all metabolic processes, and are recognized as central determinants and drug targets for neurodegenerative, metabolic and tumor disorders. Systems biology uses dynamic, data driven computational models to simulate and analyze signaling network kinetics and to unravel their complex dysregulation in disease processes such as tumor growth. Ultimately, systems approaches aim at individualized and targeted drug interventions. Yet, the currently available methods which are pursued to obtain parameterization data for computational models, are strongly limited concerning their accuracy: the detection of kinase phosphorylations by immunoblotting and ELISA offers comparably low sensitivity and dynamic range, with high variance, low throughput and the requirement for high volumes of samples and reagents. This restricts the number of time points and replicates to be analyzed and thus limits data accuracy. In addition, measurement of clinical samples is often unfeasible, as they are available only in minute amounts. Using the mTOR network as an example, the present project aims to develop a high throughput method to quantitatively measure phosphorylation kinetics by plug-based immunoassays at nanoliter scale. The here developed high throughput microfluidic platform will open new perspectives to systems biology in that computer models can be parameterized and validated with a higher density of measurements across a kinetic curve with more biological and technical replicates. This will increase the speed, accuracy and statistical power of model parameterization and analyses.
This project is performed in collaboration with
IMTEK - Department of Microsystems Engineering, University of Freiburg, Germany (Prof. Dr. Rühe, Dr. Brandstetter, Lukas Metzler)