Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy

Summary

Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting endogenous fluorescence. Viscosity is measured using a fluorescent molecular rotor.

Abstract

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and physiological state of living cells. Plasma membranes of tumor cells are known to have significant alterations in their composition, structure, and functional characteristics. Along with dysregulated metabolism of glucose and lipids, these specific membrane properties help tumor cells to adapt to the hostile microenvironment and develop resistance to drug therapies. Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting fluorescence of endogenous metabolic cofactors, such as reduced nicotinamide adenine dinucleotide NAD(P)H and oxidized flavins. Viscosity is measured using a fluorescent molecular rotor, a synthetic viscosity-sensitive dye, with a strong fluorescence lifetime dependence on the viscosity of the immediate environment. In combination, these techniques enable us to better understand the links between membrane state and metabolic profile of cancer cells and to visualize the changes induced by chemotherapy.

Introduction

Malignant transformation of cells is accompanied by multiple alterations in their morphological and physiological state. Rapid and uncontrolled growth of cancer cells requires fundamental re-organization of biochemical pathways responsible for energy production and biosynthesis. The characteristic hallmarks of cancer metabolism are enhanced rate of glycolysis, even under the normal oxygen concentrations (the Warburg effect), the use of amino acids, fatty acids, and lactate as alternative fuels, high ROS production in the presence of high antioxidant levels, and increased biosynthesis of fatty acids^1,2. It is now appreciated that cancer cell metabolism is highly flexible, which allows them to adapt to the unfavorable and heterogeneous environment and provides an additional survival advantage³.

Altered metabolism supports the specific organization and composition of membranes of tumor cells. The lipid profile of the plasma membrane in cancer cells quantitatively differs from the non-cancerous cells. The main changes in the lipidome are the increased level of phospholipids including phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, the decreased level of sphingomyelin, increased amount of cholesterol, and a lower degree of unsaturation of fatty acids, to name a few^4,5,6. Therefore, physical properties of the membrane, such as membrane viscosity, the inverse of fluidity, inevitably change. Since viscosity determines the permeability of biological membranes and controls the activity of membrane-associated proteins (enzymes, transporters, receptors), its homeostatic regulation is vital for cell functioning. At the same time, the modification of viscosity through the adjustment of membrane lipid profile is important for cell migration/invasion and survival upon conditional changes.

Fluorescence lifetime imaging microscopy (FLIM) has emerged as a powerful approach for the non-invasive assessment of multiple parameters in living cells, using endogenous fluorescence or exogenous probes⁷. FLIM is commonly realized on a multiphoton laser scanning microscope, which provides (sub)cellular resolution. Being equipped with the time-correlated single-photon counting (TCSPC) module, it enables time-resolved measurements of fluorescence with high accuracy⁸.

Probing of cellular metabolism by FLIM is based on the fluorescence measurement of endogenous cofactors of dehydrogenases, the reduced nicotinamide adenine dinucleotide (phosphate) NAD(P)H and oxidized flavins – flavin adenine dinucleotide FAD and flavin mononucleotide FMN, that act as electron carriers in a number of biochemical reactions^7,9,10. The detected fluorescence of NAD(P)H is from NADH and its phosphorylated form, NADPH, as they are spectrally almost identical. Typically, fluorescence decays of NAD(P)H and flavins fit to a bi-exponential function. In the case of NAD(P)H, the first component (~0.3-0.5 ns, ~70%-80%) is attributed to its free state, associated with glycolysis, and the second component (~1.2-2.5 ns, ~20%-30%) to its protein-bound state, associated with mitochondrial respiration. In the case of flavins, the short component (~0.3-0.4 ns, ~75%-85%) can be assigned to the quenched state of FAD and the long component (~2.5-2.8 ns, ~15%-25%) to unquenched FAD, FMN, and riboflavin. Alterations in the relative levels of glycolysis, glutaminolysis, oxidative phosphorylation, and fatty acid synthesis result in the changes in the short- and long lifetime fractions of the cofactors. Additionally, the fluorescence intensity ratio of these fluorophores (the redox ratio) reflects the cellular redox status and is also used as a metabolic indicator. Although the redox ratio presents a simpler metric, compared with fluorescence lifetime, in terms of data acquisition, FLIM is advantageous to estimate NAD(P)H and FAD, because fluorescence lifetime is an intrinsic characteristic of the fluorophore and almost not influenced by such factors as excitation power, photobleaching, focusing, light scattering and absorption, especially in tissues, unlike the emission intensity.

One of the convenient ways to map viscosity in living cells and tissues at the microscopic level is based on the use of fluorescent molecular rotors, small synthetic viscosity-sensitive dyes, in which fluorescence parameters strongly depend on the local viscosity^11,12. In a viscous medium, the fluorescence lifetime of the rotor increases due to the slowing down of the intramolecular twisting or rotation. Among molecular rotors, the derivatives of boron dipyrromethene (BODIPY) are well suited for sensing viscosity in biological systems as they have a good dynamic range of fluorescence lifetimes in physiological range of viscosities, temperature independence, monoexponential fluorescence decays that allow straightforward data interpretation, sufficient water-solubility and low cytotoxicity^13,14. Quantitative assessments of microviscosity using BODIPY-based rotors and FLIM has been previously demonstrated on cancer cell in vitro, multicellular tumor spheroids and mouse tumor in vivo^15,16.

Here, we present a detailed description of sequential probing methodologies for studying cellular metabolism and plasma membrane viscosity in cancer cells in vitro by FLIM. To avoid contamination of the relatively weak endogenous fluorescence with the fluorescence of the BODIPY-based rotor, imaging of the same layer of cells is performed sequentially with the fluorescence of NAD(P)H and FAD imaged first. Fluorescence lifetimes of the cofactors are measured in the cytoplasm, and the fluorescence lifetime of the rotor is measured in the plasma membranes of cells by the manual selection of corresponding zones as regions of interest. The protocol was applied to correlate metabolic state and viscosity for different cancer cell lines and to assess the changes after chemotherapy.

The protocol for FLIM sample preparation does not differ from that for confocal fluorescence microscopy. Once data has been acquired, the main task is to extract the fluorescence lifetime from the raw data. The performance of the protocol is demonstrated using HCT116 (human colorectal carcinoma), CT26 (murine colon carcinoma), HeLa (human cervical carcinoma), and huFB (human skin fibroblasts) cells.

Protocol

1. Description of the minimal setup to perform FLIM To perform this experiment, ensure the required setup is available: an inverted confocal microscope, a pulsed laser, typically a ps or fs, with the synchronization signal, a fast photon counting detector (time response 150 ps) and photon counting electronics, available output and input ports for the detector and the laser, respectively, on the microscope, the scan clock pulses from the microscope scan controller, the scan head of the microscope with the lase…

Representative Results

Using the protocol described here, we have visualized the metabolic cofactors and microscopic membrane viscosity in live cultured cells using FLIM. The measurements have been done in different cancer cell lines – human colorectal carcinoma HCT116, murine colon carcinoma CT26, human cervical cancer HeLa Kyoto, and human skin fibroblasts huFB. Fluorescence intensity-based redox ratio FAD/NAD(P)H and fluorescence lifetimes of NAD(P…

Discussion

This protocol illustrates the possibilities of FLIM for multiparametric, functional, and biophysical analysis of cancer cells. The combination of the optical metabolic imaging based on endogenous fluorescence and the measurements of plasma membrane viscosity using exogenous labeling with fluorescent molecular rotor enables us to characterize the interconnections between these two parameters in live cancer cells in a cell culture and follow the changes in response to chemotherapy.

Two-photon ex…

Materials

Item/Device
Cell culture incubator	Sanyo		37°C, 5% CO2, humidified atmosphere
Centrifuge 5702 R	Eppendorf	5703000010
imageJ 1.53c	Wayne Rasband (NIH)
FLIM module Simple Tau 152 TCSPC (in LSM 880)	Becker & Hickl GmbH
Laminar flow hood	ThermoFisher Scientific
Leica microscope DFC290	Leica Microsystems
LSM 880 confocal microscope	Carl Zeiss
Ti:Sapphire femtosecond laser Mai Tai	Spectra Physics
Microscope incubator XLmulti S DARK LS	PeCon GmbH	273-800 050
Mechanical pipettor	Sartorius mLINE		volume 0.5-10 μL; 20-200 μL; 100-1000 μL
Oil-immersion objective C-Apochromat 40×/1.2 NA W Korr  (in LSM 880)	Carl Zeiss	421767-9971-790
Power-Tau 152 module with the detector HPM-100-40	Becker&Hickl GmbH
SPCImage software	Becker & Hickl GmbH		SPC 9.8; SPCImage 8.3
ZEN software	Carl Zeiss		ZEN 2.1 SP3 (black), Version 14.0.0.201
Reagent/Material
5-fluorouracil	Medac GmbH	3728044
DMEM	Gibco, Life Technologies	31885023
DMSO	PanEco	F135
FBS	Hyclone	A3160801
FluoroBright DMEM	Gibco, Life Technologies	A1896701
Hank’s solution without Ca2+/Mg2+	Gibco, Life Technologies	14175
l-Glutamine	PanEco	F032
Mammalian cells			HCT116, CT26, HeLa Kyoto, huFB
Molecular rotor BODIPY 2			Synthesized and Supplied by Marina Kuimova Group, Imperial College London
Penicillin/streptomycin	PanEco	A065
Tissue culture dish with cover glass-bottom FluoroDishes	World Precision Instruments, Inc
Trypsin- EDTA 0.25%	PanEco	P034
Versen buffer	PanEco	R080p