Recent studies suggest that cancer cell response to cisplatin can not be fully described in terms of only interaction of the drug with DNA, but can include effects associated with other cellular targets. The study of effects of chemotherapeutic drugs on the viscosity of plasma membrane is important for better understanding the mechanisms of the drug action and evaluating the effectiveness of therapy. The aim of this work was to analyze microviscosity of plasma membrane of cancer cells during chemotherapy with cisplatin. For imaging viscosity at the microscopic level fluorescent molecular rotor BODIPY2 and fluorescence lifetime imaging microscopy (FLIM) were used. We detected a significant increase in membrane viscosity in viable human cervical cancer cells HeLa, both in cell monolayer and tumor spheroids after cisplatin treatment. Measuring viscosity in cisplatin-resistant cell line showed that viscosity increases when cells acquire chemoresistance. These results suggest that microviscosity of membrane plays a role in the cytotoxicity of cisplatin and its mapping may provide a powerful tool for investigation of tumor responses to chemotherapy and mechanisms of drug resistance.
Colorectal cancer is one of the most commonly diagnosed and poorly responding to chemotherapy types of cancer, which emphasizes the importance of personalized approach to treatment selection. Short-term primary cell cultures established from patients’ tumors represent a valuable model for testing drug response. In this study, we developed protocols for generation of the short-term primary cell cultures from colorectal cancer tissue and assessment their chemosensitivity using MTT test. Additionally, we showed the possibility of metabolic analysis of patient-derived cancer cells using fluorescence lifetime imaging (FLIM) of autofluorescent cofactor NAD(P)H. Since FLIM of NAD(P)H demonstrates the potential to detect early responses to cancer treatment, we assume that this method, alone or in combination with MTT assay, can be used for choosing the optimal chemotherapy for patients.
Cancer-associated fibroblasts (CAFs) are one of the key determinants in the malignant progression of cancer. The subject of this research was metabolic reorganization of CAFs and their participation in collagen cross-linking process. The metabolic differences between normal fibroblasts and CAFs were elucidated using two-photon fluorescence lifetime imaging microscopy (FLIM). Collagen structure in 3D model was assessed using second harmonic generation (SHG) microscopy. We show increased metabolic activity of fibroblasts derived from patient’s colon tumor with a shift to more oxidative metabolism compare to dermal fibroblasts. The results of the study of collagen suggest that CAFs may contribute to the tumor progression through the facilitation of collagen alignment. In general, our findings support the idea of the strong association between cancer cells and fibroblasts and extensive involvement of CAFs in modulation of tumor microenvironment.
Fluorescence lifetime imaging microscopy (FLIM) is a promising non-invasive highly sensitive technique for probing multiple physiological and physicochemical parameters in living cells and tissues. The present study is focused on the investigation of bioenergetics and microscopic viscosity of cultured cancer cells and animal tumors using FLIM during natural growth and chemotherapy. Fluorescence lifetime measurements of the metabolic cofactor NAD(P)H revealed a decrease of the relative amplitude of free NAD(P)H after cisplatin treatment, indicating a change towards a more oxidative metabolic state. Microviscosity mapping performed with the use of fluorescent molecular rotor BODIPY-2 showed a pronounced increase in the plasma membrane viscosity in cancer cells exposed to cisplatin. Although biochemical mechanisms underlying the metabolic and viscosity alterations during chemotherapy have yet to be clarified, our data suggest that the cisplatin-induced changes in cellular metabolism and membrane viscosity play a role in the cytotoxicity of the drug. The results of the study contribute to an understanding of mechanisms of cisplatin action and will be useful for development new approach for assessing response to a therapy.