Quantitative Spatiotemporal Analysis of Intracellular Kinase Activity in Metastatic Breast Cancer Cells Using a Microfluidic-Based Lateral Diffusion Assay.

Mass transport by diffusion helps shape extracellular gradients of soluble signaling molecules in tumor microenvironments and other physiological settings. Microfluidic technologies are conducive to generating predictable chemical gradients. Yet, they often require specialized fluid-handling expertise and external pumping systems. We designed and implemented a simple microfluidic-based lateral diffusion assay (LDA) that enables reproducible and predictable biomolecular gradients without pumps and is devoid of any confounding pressure-driven flow. Using breast cancer cells that coexpress a kinase translocation reporter (KTR) for Akt, we demonstrate quantitative, real-time analysis of intracellular kinase signaling in response to diffusion-limited extracellular gradients of epidermal growth factor (EGF) in the LDA. We observed temporally and spatially staggered Akt activation and deactivation in KTR cells, with these signaling dynamics correlating to the rate of EGF delivery across zonal boundaries or EGF flux. We identified a threshold EGF concentration required for Akt activation in the median cell population and showed that this threshold concentration increases with cell density. Using mathematical modeling that incorporated empirically derived parameters, we accurately predicted individual cell Akt activation patterns among different EGF source concentrations and cell densities. Finally, we showed that both activation and deactivation patterns depend on the rate of the EGF concentration change, revealing the pivotal role of EGF flux in controlling signaling dynamics. Together, these findings establish the LDA as a powerful and accessible platform for dissecting the dynamics of extracellular gradients in controlling intracellular signaling.