Functional evaluation of bioactive compounds using cell-based assays is key in discovering new and improved drugs to address our societies growing medical needs. In a large number of academic and private R&D facilities around the world, high-content microscopy screening is used to complement and often outperform the conventional luminescence and fluorescence plate-reader assays. The combination of fluorescent-probe technology, modern optical microscopes and innovative functional assays allows monitoring highly dynamic events in living cells with exquisite temporal and spatial resolution.
Drug candidates and treatment regimens are commonly tested on living cells maintained at atmospheric oxygenation levels (i.e. at 21% O2) while in reality, cells in our bodies never experience such high oxygen levels. Rather, most cells experience 2-5% of O2 and cancer cells in solid tumors are generally hypoxic, i.e. they function at < 1% of O2.
Jalink lab along with various other research groups, has focused on implementing FRET (Förster Resonance Energy Transfer) based biosensors as sensitive tools in live cell microscopy. FRET is a powerful, time-proven technique to study dynamic protein-protein interactions and also a great readout for biosensors, which can be designed to study various steps of signal transduction cascades. FLIM (Fluorescence Lifetime Imaging) is a robust and inherently quantitative method for FRET detection: interaction between donor and acceptor shortens the excited-state lifetime and is linearly related to FRET efficiency. Thus, FLIM is ideally suited to quantitatively study baseline and stimulated FRET values in individual cells and among different cell populations, yielding data that are directly comparable between different laboratories around the world. As mentioned above, cells can experience hypoxia for different reasons, importantly abnormal cell growth in tumors. Consequently, genetically engineered cancer cells expressing FRET biosensors are valuable tools to study cell signaling alterations due to oxygen variations.
Given the time and effort invested worldwide in the improvement of drugs that target cell signaling (anti-cancer, schizophrenia, diabetes, etc.), there is an urgent need to come up with laboratory models that better recapitulate the in vivo setting while maintaining the accessibility and scalability necessary to investigate the effects of large panels of (candidate) drugs on diverse cellular functions. The overall aim of this project was to address this timely need by establishing an innovative microscopy platform with fully adjustable atmospheric conditions (O2, N2, CO2) and test it by initiating studies into cellular signals and sensitivity to biologically active compounds at hypoxia in relevant physiological models. The project had the following research objectives:
1. Finalizing the working prototype design and testing the hypoxia setup for FRET and FLIM.
2. Testing how well various existing fluorescent protein-based FRET biosensors perform at low levels of O2.
3. Studying G protein coupled receptor activation in living cells under hypoxia.
At the end of the project, we conclude that FLIM-FRET biosensors are promising tools to study hypoxia effects on cell behavior. mTurquoise2 is a reliable fluorescent protein under low oxygen conditions and can be trusted in hypoxia experiments. A method to externally control drug concentrations in the hypoxia chamber without breaking hypoxic conditions was developed and successfully implemented for studies of receptor mediated signaling.
