CC BY
20B-I-8
Multiparametric FLIM for cancer study using endogenous fluorescence and genetically encoded sensors
M. Shirmanova1, A. Gavrina1, A. Polozova1, L. Shimolina1, I. Druzhkova1, N. Ignatova1, V.
Dudenkova1, M. Lukina1, V. Shcheslavskiy12, K. Lukyanov3, V. Belousov4, E. Zagaynova15
1-Privolzhsky Research Medical University, Nizhny Novgorod, Russia 2-Becker & Hickl GmbH, Berlin, Germany 3-Skolkovo Institute of Science and Technology, Moscow, Russia 4-Pirogov Russian National Research Medical University, Moscow, Russia 5-Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia Shirmanovam@gmail.com
Nowadays, fluorescent proteins, the majority of which belong to the GFP family, represent an indispensable instrument for cancer research. A significant progress has been made in the development of genetically encoded fluorescence lifetime-based sensors that enable long-term, quantitative assessments of various parameters inside living cells using fluorescence lifetime imaging (FLIM) techniques. In addition, one of the most useful applications of FLIM is probing of cellular metabolism using endogenous fluorescence from the reduced nicotinamide adenine dinucleotide (phosphate) NAD(P)H and oxidized flavin adenine dinucleotide FAD. NAD(P)H and FAD act as electron carriers in a number of biochemical reactions and their fluorescence lifetimes are largely determined by protein binding. Unlike fluorescence intensity, fluorescence lifetime allows to overcome difficulties associated with an unknown and/or unstable fluorophore concentration, photobleaching, instrument configurations, absorption and scattering events [1].
In our work, we have developed methodologies for multiparametric imaging of cancer cells and tumors using simultaneous detection of endogenous fluorescence in the blue spectral range and far-red genetically encoded sensors.
To monitor non-invasively the cell cycle, genetically encoded indicator FUCCI-Red was used [2]. FUCCI-Red utilizes two red fluorescent proteins with different fluorescence lifetimes, mCherry and mKate2. These proteins carry cell cycle-dependent degradation motifs to resolve G1 and S/G2/M phases. The cell cycle was visualized in monolayer cell culture, tumor spheroids and animal tumors in vivo by FLIM-microscopy. To visualize apoptosis, the FRET (Fortser Resonance Energy Transfer-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, was applied [3]. Caspase-3 activity was detected by measuring fluorescence lifetime of the donor protein mKate2. Due to the loss of energy transfer to the acceptor iRFP upon caspase-3 activation, fluorescence lifetime of the donor mKate2 increases. The execution of apoptosis was investigated in cultured cancer cells treated with different agents. Fluorescence lifetime-based mapping of cytosolic pH in cancer cells in vitro and tumor xenografts in vivo was performed using new SypHer-based genetically encoded sensor. Fluorescence lifetime of the sensor increases with increase of pH. pH measurements were done at the cellular level with FLIM-microscopy and at the level of a whole tumor with macroscopic FLIM.
The possibility of simultaneous imaging of the indicated parameters (cell cycle, apoptosis, pH) using the genetically encoded sensors and cellular metabolic state using fluorescence of metabolic cofactors was demonstrated in vitro and in vivo. In general, such multiparameter imaging improves our understanding of biological behavior of cancer cells.
The studies were supported by the Russian Science Foundation (project № 20-65-46018).
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2. Shirmanova, M.V., Gorbachev, D.A., Sarkisyan, K.S. et al. FUCCI-Red: a single-color cell cycle indicator for fluorescence lifetime imaging. Cell. Mol. Life Sci. (2021).
3. T.F. Sergeeva, M.V. Shirmanova, O.A. Zlobovskaya, et al., Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis. BBA - Molecular Cell Research, 1864(3): 604-611 (2017).
