A multiplexed high-throughput platform to report pharmacologic alteration of cancer stem cell membrane potential and cell cycle state - IMAT R61 Abstract In addition to its well-recognized roles in neuronal computation and cardiac contractions, the voltage across the plasma membrane has been implicated as a fundamental regulator of differentiation and proliferation. The concurrent regulation of proliferation and stem cell states by membrane voltage contributes to normal tissue morphogenesis, development, and diseases such as cancer. Membrane hyperpolarization tends to promote increased differentiation and reduced proliferation while depolarization is associated with immature and proliferative stem-like cell states. Of clinical relevance, cancer cells and cancer stem-like cells exhibit depolarized membrane voltage values compared with the normal differentiated cells from which they arise. Despite the strong association between voltage and cancer cell phenotypes, voltage modulation has not been effectively exploited as a drug target against cancer, largely reflecting the technological challenges with membrane voltage measurements in physiologically relevant contexts. Current techniques to measure membrane voltage, including electrode arrays and voltage sensitive dyes, have limitations in the number of experimental observations possible, compatibility with complementary phenotypic readouts, the duration of sampling, and the inability to perform longitudinal cell analyses. These technologic challenges motivate our hypothesis that integration of genetically encoded voltage and cell cycle sensors will provide a robust and novel high-throughput platform to identify modulators of membrane voltage and cell proliferation. We propose to develop, optimize, and validate a cell-based technology platform integrating JEDI, a novel voltage indicator, and FUCCI cell-cycle sensors to enable real time high-throughput analysis of concurrent changes in voltage and cell proliferation in living cells. To build the platform, we propose to demonstrate high-throughput imaging of voltage dynamics (Aim 1) and cell cycle states (Aim 2) in glioblastoma stem cells. We next propose to deploy these technologies for high-throughput quantification of the impact of ion channel-modulating drugs on the membrane voltage and cell-cycle changes (Aim 3). This novel platform is expected, for the first time, to provide a tool for real-time live cell readouts of concurrent changes in membrane voltage and cell proliferation. Besides its implications for the treatment of cancer, we anticipate this platform to have far reaching basic science and clinical translational applications in neuroscience, stem cell and iPSC biology, cell-based therapies, and cardiovascular diseases.
