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.
Nonlinear optical microscopy combined with fluorescence lifetime imaging is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a noninvasive investigation of the biological tissue with subcellular resolution. Cancer exhibits altered cellular metabolism, which affects the autofluorescence of metabolic cofactors NAD(P)H and FAD. In this study features of tumor metabolism in different systems of organization (from cell culture to patient lesion) was showed. The observed differences in the relative contributions of free NAD(P)H and FAD testify to an increased a glycolytic metabolism in cancer cells compare to fibroblasts. In 3D spheroids, the cells of the proliferating zone had greater a1 and lower tm values than the cells of the quiescent zone, which likely is a consequence of their higher glycolytic rate. During the growth of colorectal cancer in the experimental mouse model, the contribution of the free component of NAD(P)H was increased. Dysplastic nevus and melanoma is characterized by raised contribution of free NADH compare to healthy skin. Therefore, melanoma cells had very short value of τ1.
The study of metabolic and oxygen states of cells in a tumor in vivo is crucial for understanding of the mechanisms responsible for the tumor development and provides background for the relevant tumor’s treatment. Here, we show that a specially designed implantable fiber-optical probe provides a promising tool for optical interrogation of metabolic and oxygen states of a tumor in vivo. In our experiments, the excitation light from a ps diode laser source is delivered to the sample through an exchangeable tip via a multimode fiber, and the emission light is transferred to the detector by another multimode fiber. Fluorescence lifetime of nicotinamid adenine dinucleotide (NAD(P)H) and phosphorescence lifetime of an oxygen sensor based on iridium (III) complex of enzothienylpyridine (BTPDM1) are explored both in model experiment in solutions, and in living mice. The luminescence spectroscopy data is substantiated with immunohistochemistry experiments. To the best of our knowledge, the measurements of both metabolic status and oxygenation of tumor in vivo by fluorescence/phosphorescence lifetime spectroscopy with a fiber-optic probe are done for the first time.
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.
The aim of the study was to estimate energy metabolism in human cervical cancer cells HeLa Kyoto after chemotherapy in vitro and in vivo using two-photon fluorescence lifetime microscopy (FLIM). Cellular metabolism was examined by monitoring of the fluorescence intensities and lifetimes of metabolic cofactors NAD(P)H and FAD. Cancer metabolism was analyzed in dynamics after treatment with cisplatin. Two-photon fluorescence and second harmonic generation microscopies as well as standard histopathology with hematoxylin and eosin were used to characterize cancer tissue structure. We showed an increase of the optical redox ratio FAD/NAD(P)H in cancer cells in vitro and decrease of the relative contribution of free NAD(P)H (ɑ1) in vitro and in vivo, which presumably indicate a shift to more oxidative metabolism after chemotherapy. These data demonstrate the possibility to detect response of cancer cells to chemotherapy using optical metabolic imaging.
Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities
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.
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.
Despite of the success of photodynamic therapy (PDT) in cancer treatment, the problems of low selective accumulation of a photosensitizer in a tumor and skin phototoxicity have not resolved yet. The idea of encoding of a photosensitizer in genome of cancer cells is attractive, particularly because it can provide highly selective light induced cell killing. This work is aimed at the development of new approach to PDT of cancer, namely to using genetically encoded photosensitizers. A phototoxicity of red fluorescent GFP-like protein KillerRed and FMN-binding protein miniSOG was investigated on HeLa tumor xenografts in nude mice. The tumors were generated by subcutaneous injection of HeLa cells stably expressing the phototoxic proteins. The tumors were irradiated with 594 nm or 473 nm laser at 150 mW/cm2 for 20 or 30 min, repeatedly. Fluorescence intensity of the tumors was measured in vivo before and after each treatment procedure. Detailed pathomorphological analysis was performed 24 h after the therapy. On the epi-fluorescence images in vivo photobleaching of both proteins was observed indicating photodynamic reaction. Substantial pathomorphological abnormalities were found in the treated KillerRed-expressing tumor tissue, such as vacuolization of cytoplasm, cellular and nuclear membrane destruction, activation of apoptosis. In contrast, miniSOG-expressing tumors displayed no reaction to PDT, presumably due to the lack of FMN cofactor needed for fluorescence recovery of the flavoprotein. The results are of interest for photodynamic therapy as a proof of possibility to induce photodamages in cancer cells in vivo using genetically encoded photosensitizers.