SignificanceAutofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications.AimWe present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells.ApproachWe use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids.ResultsThe use of a multispectral detector in combination with an excitation at a single wavelength of 750 nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins.ConclusionsThe approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750 nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids.

The modifications in biomechanical properties of cells and tissue are important in cancer progression, including its different aspects, e.g. invasion, migration, adhesion, signaling, interactions with microenvironment, and immune response. However, our understanding of the changes in physical characteristics of cells, and especially the molecular basis for these changes, caused by malignant transformation remains rather limited. While the differences in stiffness and viscoelasticity of tumor and normal cells have been well documented at the cellular scale, subcellular and molecular alterations have been poorly characterised. In our work, we investigated the stiffness and viscoelastic parameters, mainly determined by the actin cortex, and the microviscosity of the plasma membrane, mainly determined by its lipid profile, in normal and cancer cells. The mechanical properties of the cells were assessed using atomic force microscopy (AFM). The microviscosity of the membrane was visualized by fluorescence lifetime imaging microscopy (FLIM) with the viscosity-sensitive probe BODIPY2. Chemical analysis of cell membranes was performed by secondary ion time-of-flight mass spectrometry (ToF-SIMS). The MCF-10 A (normal epithelial cells) and MCF-7 (human breast cancer) cell lines were used in the study. It was shown that cancer cells were more deformable due to a less organized and more isotropic filamentous structure of the actin cytoskeleton. At the same time they had more viscous plasma membranes, compared to normal cells, in both in vitro and tissue conditions. In the membrane lipid profile, increased signals of sphingomyelin and saturated fatty acids and decreased signals of polyunsaturated fatty acids were detected in cancer cells, which explain the higher microviscosity of their membranes. The obtained data indicate a complex reorganization of cell biomechanics at the cellular, subcellular and molecular levels during malignant transformation, which is important for better understanding of fundamental mechanisms of tumor development.