Biophysical properties of living cells largely determine their vital activity and functionality. In cancer, the physical state of the plasma membrane of cells is important for the invasion and metastasis. Cellular-scale viscoelasticity affects cell morphology, motility, interaction with the extracellular matrix, and resistance to mechanical stress. However, the links between membrane fluidity and cellular mechanics are poorly understood. Here, we present the in vitro study of microviscosity and viscoelastic properties of colorectal cancer cells. Measuring microviscosity of membranes at the micrometer scale was performed using fluorescence lifetime imaging microscopy FLIM with a viscosity sensitive probe. Atomic force microscopy AFM was used to evaluate the mechanical properties of cells. Additionally, the lipid profile of cells plasma membranes was analyzed using time-of-flight secondary ion mass spectrometry. A good positive correlation was found between cell stiffness (the Young’s modulus) and the plasma membrane microviscosity of cancer cells. Of the five cell lines, HT29 cells, which has an epithelial phenotype, had the most fluid membranes and the lowest stiffness values; the highest microviscosity and stiffness values were recorded for the SW480 cell line, which is characterized by a mesenchymal phenotype. The obtained results indicate that cell biomechanics is determined by the two sets of parameters that are interconnected in tumor cells and are involved in their migratory behavior.
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.
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.
Collageneus tissues manifest strongly pronounced viscoelastic behavior. Namely, viscosity leads to time-dependence of
the deformation processes. This concerns both compression (loading) and unloading of the tissue. Both processes can be
characterized by a relaxation time - the time during which the strain changes e-times. We demonstrate the applicability
of the OCT-based 2D relaxography to characterize local relaxation time of collageneous tissues. The developed
technique can be used for further investigation of the viscoelastic properties of healthy and pathological collageneous
tissues.
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.
OCT-based local strain relaxation/creep evaluation is an emerging tool for tissue viscoelasticity characterization. We present a tool for 2D visualization of local strain relaxation and creep time/rate inside the tissue.
The aim of the present work was to study energy metabolism in human cervical carcinoma (HeLa) cells in vitro and in vivo using two-photon FLIM. Cellular metabolism was examined by monitoring of the fluorescence lifetimes of free and protein-bound forms of NAD(P)H and FAD and their relative contributions. Two-photon fluorescence and second harmonic generation microscopy as well as standard histopathology with hematoxylin and eosin were used to characterize tissue structure. Cellular metabolism was analyzed in cancer cells co-cultured with human fibroblasts and in tumor xenografts transplanted to nude mice. In the HeLa-huFB co-culture we observed a metabolic shift from OXPHOS toward glycolysis in cancer cells, and from glycolysis to OXPHOS in fibroblasts, starting from Day 2 of co-culturing. In the tumor tissue we detected metabolic heterogeneity with more glycolytic metabolism of cancer cells in the stroma-rich zones. The results of the study are of a great importance for understanding metabolic behavior of tumors and for development of anticancer drugs targeted to metabolic pathways.
The development of tumor therapies based on the activation of antitumor immunity requires tumor models that are highly immunogenic. The immunologic response to fluorescent proteins, green fluorescent protein (GFP), or enhanced GFP (EGFP) was demonstrated in different cancer models. However, for live animal imaging, red and far-red fluorescent proteins are preferable, but their immunogenicity has not been studied. We assessed the immunogenicity of the red fluorescent protein, KillerRed (KR), in CT26 murine colon carcinoma. We showed a slower growth and a lower tumor incidence of KR-expressing tumors in comparison with nonexpressing ones. We found that KR-expressing lung metastases and rechallenged tumors were not formed in mice that had been surgically cured of KR-expressing primary tumors. The effect of low-dose cyclophosphamide (CY) treatment was also tested, as this is known to activate antitumor immune responses. The low-dose CY therapy of CT26-KR tumors resulted in inhibition of tumor growth and improved mouse survival. In summary, we have established a highly immunogenic tumor model that could be valuable for investigations of the mechanisms of antitumor immunity and the development of new therapeutic approaches.
This work is aimed at the development of new approach to register intracellular pH with genetically encoded ratiometric sensor. Intracellular pH of cancer cells was studied in vitro and in vivo. Changes of intracellular pH under conditions of co-culturing with fibroblast were investigated.
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