. Significance: Despite the importance of the cell membrane in regulation of drug activity, the influence of drug treatments on its physical properties is still poorly understood. The combination of fluorescence lifetime imaging microscopy (FLIM) with specific viscosity-sensitive fluorescent molecular rotors allows the quantification of membrane viscosity with high spatiotemporal resolution, down to the individual cell organelles. Aim: The aim of our work was to analyze microviscosity of the plasma membrane of living cancer cells during chemotherapy with cisplatin using FLIM and correlate the observed changes with lipid composition and cell’s response to treatment. Approach: FLIM together with viscosity-sensitive boron dipyrromethene-based fluorescent molecular rotor was used to map the fluidity of the cell’s membrane. Chemical analysis of membrane lipid composition was performed with time-of-flight secondary ion mass spectrometry (ToF-SIMS). Results: We detected a significant steady increase in membrane viscosity in viable cancer cells, both in cell monolayers and tumor spheroids, upon prolonged treatment with cisplatin, as well as in cisplatin-adapted cell line. ToF-SIMS revealed correlative changes in lipid profile of cisplatin-treated cells. Conclusions: These results suggest an involvement of membrane viscosity in the cell adaptation to the drug and in the acquisition of drug resistance.

Exploring metabolism in human tumors at the cellular level remains a challenge. The reduced form of metabolic cofactor NAD(P)H is one of the major intrinsic fluorescent components in tissues and a valuable indicator of cellular metabolic activity. Fluorescence lifetime imaging (FLIM) enables resolution of both the free and protein-bound fractions of this cofactor, and thus, high sensitivity detection of relative changes in the NAD(P)H-dependent metabolic pathways in real time. However, the clinical use of this technique is still very limited. The applications of metabolic FLIM could be usefully expanded by probing cellular metabolism in tissues ex vivo. For this, however, the development of appropriate tissue preservation protocols is required in order to maintain the optical metabolic characteristics in the ex vivo sample in a state similar to those of the tumor in vivo. Using mouse tumor models of different histological types—colorectal cancer, lung carcinoma and melanoma—we tested eight different methods of tissue handling by comparing NAD(P)H fluorescence decay parameters ex vivo and in vivo as measured with two-photon excited FLIM microscopy. It was found that the samples placed in 10% BSA on ice immediately after excision maintained the same fluorescence lifetimes and free/bound ratios as measured in vivo for at least 3 hours. This protocol was subsequently used for metabolic assessments in fresh postoperative samples from colorectal cancer patients. A high degree of inter- and intra-tumor heterogeneity with a trend to a more oxidative metabolism was detected in T3 colorectal tumors in comparison with normal tumor-distant colon samples. These results suggest that the methodology developed on the basis of FLIM of NAD(P)H in tissues ex vivo show promise for interrogating the metabolic state of patients’ tumors.

Abnormal levels of viscosity in tissues and cells are known to be associated with disease and malfunction. While methods to measure bulk macroscopic viscosity of bio-tissues are well developed, imaging viscosity at the microscopic scale remains a challenge, especially in vivo. Molecular rotors are small synthetic viscosity-sensitive fluorophores in which fluorescence parameters are strongly correlated to the microviscosity of their immediate environment. Hence, molecular rotors represent a promising instrument for mapping of viscosity in living cells and tissues at the microscopic level. Quantitative measurements of viscosity can be achieved by recording time-resolved fluorescence decays of molecular rotor using fluorescence lifetime imaging microscopy (FLIM), which is also suitable for dynamic viscosity mapping, both in cellulo and in vivo. Among tools of experimental oncology, 3D tumour cultures, or spheroids, are considered a more adequate in vitro model compared to a cellular monolayer, and represent a less labour-intensive and more unified approach compared to animal tumour models. This chapter describes a methodology for microviscosity imaging in tumour spheroids using BODIPY-based molecular rotors and two photon-excited FLIM.

Maintenance of the biophysical properties of membranes is essential for cell survival upon external perturbations. However, the links between a fluid membrane state and the drug resistance of cancer cells remain elusive. Here, we investigated the role of membrane viscosity and lipid composition in the responses of cancer cells to oxaliplatin and the development of chemoresistance. Plasma membrane viscosity was monitored in live colorectal cancer cells and tumor xenografts using two-photon excited fluorescence lifetime imaging microscopy (FLIM) using the fluorescent molecular rotor BODIPY 2. The lipid profile was analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS). It was found that the plasma membrane viscosity increased upon oxaliplatin treatment, both in vitro and in vivo, and that this correlated with lower phosphatidylcholine and higher cholesterol content. The emergence of resistance to oxaliplatin was accompanied by homeostatic adaptation of the membrane lipidome, and the recovery of lower viscosity. These results suggest that maintaining a constant plasma membrane viscosity via remodeling of the lipid profile is crucial for drug resistance in cancer.

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The investigations reported here were designed to determine whether the bulk plasma membrane is involved in mechanisms of acquired resistance of colorectal cancer cells to 5-fluorouracil (5-FU). Fluorescence lifetime imaging microscopy (FLIM) of live cultured cells stained with viscosity-sensitive probe BODIPY 2 was exploited to non-invasively assess viscosity in the course of treatment and adaptation to the drug. In parallel, lipid composition of membranes was examined with the time-of-flight secondary ion mass spectrometry (ToF-SIMS). Our results showed that a single treatment with 5-FU induced only temporal changes of viscosity in 5-FU sensitive cells immediately after adding the drug. Acquisition of chemoresistance was accompanied by persistent increase of viscosity, which was preserved upon treatment without any changes. Lipidomic analysis revealed that the resistant cells had a lower level of monounsaturated fatty acids and increased sphingomyelin or decreased phosphatidylcholine in their membranes, which partly explain increase of the viscosity. Thus, we propose that a high membrane viscosity mediates the acquisition of resistance to 5-FU.