Periodic Reporting for period 1 - MICROX (Microscopy of living cancer cells at physiological oxygen levels: the MICROX platform)
Periodo di rendicontazione: 2019-09-15 al 2022-09-14
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.
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.
As the first step of the project, we designed and performed a focused set of control experiments aiming to prove that the FLIM-FRET readouts correspond to changes in intracellular cAMP and to demonstrate that the sensor’s integrity and functionality remain intact under hypoxia. We carried out the characterization studies in three different cell lines (human cervical carcinoma HeLa, human melanoma MelJuSo, and mouse melanoma B16F10 cells) stably expressing the Epac based FRET biosensor for cAMP. From this work we concluded the following:
• the donor fluorophore mTurquoise2 of the biosensor is able to mature adequately under hypoxia and the FRET sensor readout in FLIM is reliable under various O2 levels;
• the cAMP sensitive FRET sensor (Jalink lab construct H201 and others) is functional after hours under hypoxia;
• this holds true for live cell imaging in at least three mammalian cell lines.
We next set out to evaluate whether there are differences in GPCR-mediated cAMP pathway activation under hypoxic conditions compared to normoxic conditions. Meljuso and HeLa cells stably expressing the cAMP FRET biosensor (Epac-SH189) were imaged whilst increasing concentrations of stimulatory receptor ligands were added onto the cells through the fluid inlet and outlet system attached to the hypoxia chamber. We focused on the cAMP pathway activation by either the β adrenergic receptor agonist isoproterenol or the prostanoid receptor agonist prostaglandin E1.
Contrary to indications obtained in pilots using ratiometric methods, when measured using inherently quantitative FLIM/FRET methods hypoxia has no effect on receptor mediated cAMP production stimulated by isoproterenol and PGE1 in Hela or MelJuSo cells.
Next, we tested whether the activity of phosphodiesterases (PDEs) is influenced by oxygen availability. This assay has previously been successfully used to study the activity of individual PDEs in HeLa cells in our and is described in detail in: https://www.nature.com/articles/s41598-021-00098-9(si apre in una nuova finestra)
Following addition of isoproterenol and propranolol, identical cAMP breakdown times between normoxia and hypoxia were observed. Therefore, this experiment shows cAMP breakdown time by PDEs in HeLa cells is not influenced by hypoxia.
To stress the versatility and the benefits of fluorescence lifetime imaging we wrote a book chapter in Springer’s Methods in Molecular Biology on the imaging approach developed in the lab accompanied with a detailed protocol for a demo-experiment:
https://link.springer.com/protocol/10.1007/978-1-0716-2245-2_7(si apre in una nuova finestra)
All of the Research objectives were met during the project. In total two scientific publications have been published during the project and one manuscript is in preparation.
• the donor fluorophore mTurquoise2 of the biosensor is able to mature adequately under hypoxia and the FRET sensor readout in FLIM is reliable under various O2 levels;
• the cAMP sensitive FRET sensor (Jalink lab construct H201 and others) is functional after hours under hypoxia;
• this holds true for live cell imaging in at least three mammalian cell lines.
We next set out to evaluate whether there are differences in GPCR-mediated cAMP pathway activation under hypoxic conditions compared to normoxic conditions. Meljuso and HeLa cells stably expressing the cAMP FRET biosensor (Epac-SH189) were imaged whilst increasing concentrations of stimulatory receptor ligands were added onto the cells through the fluid inlet and outlet system attached to the hypoxia chamber. We focused on the cAMP pathway activation by either the β adrenergic receptor agonist isoproterenol or the prostanoid receptor agonist prostaglandin E1.
Contrary to indications obtained in pilots using ratiometric methods, when measured using inherently quantitative FLIM/FRET methods hypoxia has no effect on receptor mediated cAMP production stimulated by isoproterenol and PGE1 in Hela or MelJuSo cells.
Next, we tested whether the activity of phosphodiesterases (PDEs) is influenced by oxygen availability. This assay has previously been successfully used to study the activity of individual PDEs in HeLa cells in our and is described in detail in: https://www.nature.com/articles/s41598-021-00098-9(si apre in una nuova finestra)
Following addition of isoproterenol and propranolol, identical cAMP breakdown times between normoxia and hypoxia were observed. Therefore, this experiment shows cAMP breakdown time by PDEs in HeLa cells is not influenced by hypoxia.
To stress the versatility and the benefits of fluorescence lifetime imaging we wrote a book chapter in Springer’s Methods in Molecular Biology on the imaging approach developed in the lab accompanied with a detailed protocol for a demo-experiment:
https://link.springer.com/protocol/10.1007/978-1-0716-2245-2_7(si apre in una nuova finestra)
All of the Research objectives were met during the project. In total two scientific publications have been published during the project and one manuscript is in preparation.
During this project at the Netherlands Cancer Institute, a state-of-the-art prototype of a live cell imaging dedicated hypoxia chamber was designed, built and evaluated. The figure below shows a cross-section of this cylindrical hypoxia chamber in which gas conditions can be changed within minutes. The setup reached absolute hypoxia with reliable oxygen levels below 0.5%, clearly outperforming the currently available commercial solutions, e.g. by our commercial partner can reach stable oxygen levels down to 1.5%.
Importantly, our developments have already attracted interest and formed scientific collaboration, as one further manuscript will likely be prepared together with members of the Sanquin research foundation (Amsterdam) who used our hypoxia chamber for experiments involving activation of blood cells and formation of neutrophil extracellular traps (NETs) in hypoxic conditions.
Jalink lab is welcoming aspiring collaborators to contact us, discuss and plan experiments using this setup for imaging under hypoxia.
Importantly, our developments have already attracted interest and formed scientific collaboration, as one further manuscript will likely be prepared together with members of the Sanquin research foundation (Amsterdam) who used our hypoxia chamber for experiments involving activation of blood cells and formation of neutrophil extracellular traps (NETs) in hypoxic conditions.
Jalink lab is welcoming aspiring collaborators to contact us, discuss and plan experiments using this setup for imaging under hypoxia.