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 n....

Protocol

1. Description of the minimal setup to perform FLIM

1. 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.......

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

Name	Company	Catalog Number	Comments
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