CC BY
13B-O-12
BIOMEDICAL PHOTONICS
Imaging of plasma membrane microviscosity in cancer cells during chemotherapy using molecular rotor and FLIM
L. Shimolina1, A. Hlynova1, I. Druzhkova1, N. Ignatova1, M. Kuimova3, E. Zagaynovau, M. Shirmanova1
1-Privolzhsky Research Medical University, 603005 Minin and Pozharsky Sq., 10/1, Nizhny Novgorod, Russia 2- Nizhny Novgorod State University, 603950 Gagarin Av., 23, Nizhny Novgorod, Russia 3- Imperial College London, Faculty of Natural Sciences, Department of Chemistry, SW7 2AZ,
London, United Kingdom shimolina.l.e@gmai.com
The microscopic viscosity plays an essential role in cellular biophysics by controlling the rates of diffusion and bimolecular reactions within the cell interior [1]. Understanding the role of the plasma membrane in the response of cancer cells to chemotherapy is important, since the cell membrane is actively involved in the transport of drugs and the regulation of the unfolding of biological processes [2-3]. Recent studies suggest that tumor response to chemotherapy is determined by not only interaction of the drug with the primary target (e.g. nuclear) but can include multiple physiological and physicochemical changes [4]. The study of the effects of chemotherapeutic drugs on the viscosity of living cells is important for better understanding the mechanisms of the drug action and evaluating the effectiveness of therapy.
The present work is aimed to study of plasma membrane's microviscosity in cancer cells using the fluorescent molecular rotor BODIPY 2 and fluorescence lifetime imaging FLIM microscopy during chemotherapy with platinum drugs.
The study was performed on cultured cancer cells CT26 (mouse colorectal cancer), HCT116 (human colorectal cancer) and oxaliplatin-resistant cell line - HCT116-OXAR. The molecular rotor fluorescence lifetime was recorded using an LSM 880 confocal microscope (Carl Zeiss, Germany) equipped with a TCSPC-based FLIM module (Becker&Hickl Inc., Germany). Microviscosity was measured in individual cell plasma membranes using a BODIPY2 fluorescent molecular rotor (eg 850 nm, em 500-550 nm). The cells were treated with cisplatin (Teva, Israel) at a dose of 2.6 ^M (IC50) for CT26, and oxaliplatin (Teva, Israel) at a dose of 2.0 ^M (IC50) for HCT116.
We showed a significant increase in membrane viscosity in viable cells CT26 in 24 h after cisplatin treatment from 322 ± 21 cP up to 400 ± 27 cP [2]. Treatment of HCT116 cells with oxaliplatin resulted in an increase in membrane microviscosity from 437 ± 77 cP to 593 ± 139 cP after 24 h of incubation with chemotherapy drug [3]. To validate the obtained effects on membrane viscosity, an analysis was made of the viscosity changes in chemoresistant HCT116-OXAR cells under the action of oxaliplatin. Incubation with oxaliplatin did not affect the membrane microviscosity, the values were ~450 cP. We suggest that the registered increase in membrane microviscosity at late time point of incubation with platinum-containing drugs (24 h) is part of the response of the tumor cell to treatment, and is not associated with direct interaction of the drug with the membrane.
The presented study is important for a deep understanding of the mechanisms platinum drug action and to assess the effectiveness of chemotherapy. This work is supported by the Russian Science Foundation under grant No: 20-14-00111.
[1] L. Shimolina et.al, Imaging tumor microscopic viscosity in vivo using molecular rotors, Scientific Reports, 7, 41097, (2017).
[2] L. Shimolina et.al, Mapping cisplatin-induced viscosity alterations in cancer cells using molecular rotor and fluorescence lifetime imaging microscopy, Journal of biomedical optics, 25(12), 126004, (2020).
[3] L. Shimolina et.al, The Role of Plasma Membrane Viscosity in the Response and Resistance of Cancer Cells to Oxaliplatin, Cancers, 13(24), 6165, (2021).
[4] Rebillard A. et al. Cisplatin-induced apoptosis involves membrane fluidification via inhibition of NHE1 in human colon cancer cells, Cancer Res. , 67, 7865-7874, (2007).
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