Varvara V. Dudenkova

Intracellular pH imaging in cancer cells in vitro and tumors in vivo using the new genetically encoded sensor SypHer2.

BACKGROUND Measuring intracellular pH (pHi) in tumors is essential for the monitoring of cancer progression and the response of cancer cells to various treatments. The purpose of the study was to develop a method for pHi mapping in living cancer cells in vitro and in tumors in vivo, using the novel genetically encoded indicator, SypHer2. METHODS A HeLa Kyoto cell line stably expressing SypHer2 in the cytoplasm was used, to perform ratiometric (dual excitation) imaging of the probe in cell culture, in 3D tumor spheroids and in tumor xenografts in living mice. RESULTS Using SypHer2, pHi was demonstrated to be 7.34±0.11 in monolayer HeLa cells in vitro under standard cultivation conditions. An increasing pHi gradient from the center to the periphery of the spheroids was displayed. We obtained fluorescence ratio maps for HeLa tumors in vivo and ex vivo. Comparison of the map with the pathomorphology and with hypoxia staining of the tumors revealed a correspondence of the zones with higher pHi to the necrotic and hypoxic areas. CONCLUSIONS Our results demonstrate that pHi imaging with the genetically encoded pHi indicator, SypHer2, can be a valuable tool for evaluating tumor progression in xenograft models. GENERAL SIGNIFICANCE We have demonstrated, for the first time, the possibility of using the genetically encoded sensor SypHer2 for ratiometric pH imaging in cancer cells in vitro and in tumors in vivo. SypHer2 shows great promise as an instrument for pHi monitoring able to provide high accuracy and spatiotemporal resolution.

Probing metabolic states of differentiating stem cells using two-photon FLIM

The ability of stem cells to differentiate into specialized cell types presents a number of opportunities for regenerative medicine, stem cell therapy and developmental biology. Because traditional assessments of stem cells are destructive, time consuming, and logistically intensive, the use of a non-invasive, label-free approach to study of cell differentiation provides a powerful tool for rapid, high-content characterization of cell and tissue cultures. Here, we elucidate the metabolic changes in MSCs during adipogenic differentiation, based on the fluorescence of the metabolic co-factors NADH, NADPH, and FAD using the methods of two-photon fluorescence microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH is required. In our study we demonstrated, for the first time, an increased contribution of protein-bound NADPH in adipocytes that is associated with lipogenesis. The optical redox ratio FAD/NAD(P)H decreased during adipogenic differentiation, and that this was likely to be explained by the intensive biosynthesis of lipids and the enhanced NADPH production associated with this. Based on the data on the fluorescence lifetime contribution of protein-bound NAD(P)H, we registered a metabolic switch from glycolysis to oxidative phosphorylation in adipocytes.

Genetically encoded far-red fluorescent sensors for caspase-3 activity.

Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new genetically encoded sensors for caspase-3 activity possessing the most red-shifted spectra to date. These consist of Förster resonance energy transfer (FRET) pairs in which a far-red fluorescent protein (mKate2 or eqFP650) is connected to the infrared fluorescent protein iRFP through a linker containing the DEVD caspase-3 cleavage site. During staurosporine-induced apoptosis of mammalian cells (HeLa and CT26), both mKate2-DEVD-iRFP and eqFP650-DEVD-iRFP sensors showed a robust response (1.6-fold increase of the donor fluorescence intensity). However, eqFP650-DEVD-iRFP displayed aggregation in some cells. For stably transfected CT26 mKate2-DEVD-iRFP cells, fluorescence lifetime imaging (FLIM) enabled us to detect caspase-3 activation due to the increase of mKate2 donor fluorescence lifetime from 1.45 to 2.05 ns. We took advantage of the strongly red-shifted spectrum of mKate2-DEVD-iRFP to perform simultaneous imaging of EGFP-Bax translocation during apoptosis. We conclude that mKate2-DEVD-iRFP is well-suited for multiparameter imaging and also potentially beneficial for in vivo imaging in animal tissues.

Multi-modal optical imaging characterization of atherosclerotic plaques

We combined cross-polarization optical coherence tomography (CP OCT) and non-linear microscopy based on second harmonic generation (SHG) and two-photon-excited fluorescence (2PEF) to assess collagen and elastin fibers and other vascular structures in the development of atherosclerosis, including identification of vulnerable plaques, which remains an important clinical problem and imaging application. CP OCTs ability to visualize tissue birefringence and cross-scattering adds new information about the microstructure and composition of the plaque. However its interpretation can be ambiguous, because backscattering contrast may have a similar appearance to the birefringence related fringes. Our results represent a step towards minimally invasive characterization and monitoring of different stages of atherosclerosis, including vulnerable plaques. CP OCT image of intimal thickening in the human coronary artery. The dark stripe in the cross-polarization channel (arrow) is a polarization fringe related to the phase retardation between two eigen polarization states. It is histologically located in the area of the lipid pool, however this stripe is a polarization artifact, rather than direct visualization of the lipid pool.

Photobleaching and phototoxicity of KillerRed in tumor spheroids induced by continuous wave and pulsed laser illumination

The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light-dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm(2) and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm(2) and light doses of 140 J/cm(2) , 170 J/cm(2) and 200 J/cm(2). Irradiation of KillerRed-expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP-like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed. Representative fluorescence image of two KillerRed-expressing spheroids before and immediately after CW irradiation.

Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis

A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis.

The metabolic interaction of cancer cells and fibroblasts – coupling between NAD(P)H and FAD, intracellular pH and hydrogen peroxide

ABSTRACT Alteration in the cellular energy metabolism is a principal feature of tumors. An important role in modifying cancer cell metabolism belongs to the cancer-associated fibroblasts. However, the regulation of their interaction has been poorly studied to date. In this study we monitored the metabolic status of both cell types by using the optical redox ratio and the fluorescence lifetimes of the metabolic co-factors NAD(P)H and FAD, in addition to the intracellular pH and the hydrogen peroxide levels in the cancer cells, using genetically encoded sensors. In the co-culture of human cervical carcinoma cells HeLa and human fibroblasts we observed a metabolic shift from oxidative phosphorylation toward glycolysis in cancer cells, and from glycolysis toward OXPHOS in fibroblasts, starting from Day 2 of co-culturing. The metabolic switch was accompanied by hydrogen peroxide production and slight acidification of the cytosol in the cancer cells in comparison with that of the corresponding monoculture. Therefore, our HeLa-huFb system demonstrated metabolic behavior similar to Warburg type tumors. To our knowledge, this is the first time that these 3 parameters have been investigated together in a model of tumor-stroma co-evolution. We propose that determination of the start-point of the metabolic alterations and understanding of the mechanisms of their realization can open a new ways for cancer treatment.

Comparative Analysis of Proliferation and Viability of Multipotent Mesenchymal Stromal Cells in 3D Scaffolds with Different Architectonics.

3D biodegradable materials (scaffolds) containing bioactive hydroxyapatite molecules fabricated by foaming in supercritical carbon dioxide and by selective laser sintering were used for culturing of mesenchymal stromal cells from the human adipose tissue. Experiments showed that stromal cells from the human adipose tissue adhered and proliferated on all studied types of structures. Addition of hyproxyapatite to the scaffold stimulated proliferation of stromal adipose tissue cells.

Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation

BackgroundMetabolic plasticity and the versatility of different lineages of stem cells as they satisfy their energy demands are not completely understood. In this study we investigated the metabolic changes in mesenchymal stem cells (MSCs) undergoing differentiation in two directions, osteogenic and chondrogenic, using two-photon fluorescence microscopy combined with FLIM.MethodsDifferentiation was induced by incubating the human bone marrow MSCs in osteogenic or chondrogenic mediums. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. The cells were imaged before the induction of differentiation (day 0) and on days 7, 14, and 21 of osteogenic and chondrogenic differentiation.ResultsBased on the data for the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound NAD(P)H, we registered a metabolic shift toward a more glycolytic status in the process of MSC differentiation. The difference was that, in osteogenic differentiation, an increase in oxidative phosphorylation preceded the shift to the glycolytic status in the process of such MSC differentiation. The fluorescence lifetime characteristics of FAD indicated the stimulation of an unknown metabolic pathway, where protein-bound FAD participates.ConclusionsIn this study, probing of the metabolic status of MSCs during osteogenic and chondrogenic differentiation was implemented for the first time with the use of optical metabolic imaging of the two cofactors - NAD(P)H and FAD. Our data suggest that biosynthetic processes, associated, presumably, with the synthesis of collagen, drive energy metabolism in differentiating cells, and promote a metabolic shift from a more oxidative to a more glycolytic state.

Chemotherapy with cisplatin: insights into intracellular pH and metabolic landscape of cancer cells in vitro and in vivo

Although cisplatin plays a central role in cancer chemotherapy, the mechanisms of cell response to this drug have been unexplored. The present study demonstrates the relationships between the intracellular pH (pHi), cell bioenergetics and the response of cervical cancer to cisplatin. pHi was measured using genetically encoded sensor SypHer2 and metabolic state was accessed by fluorescence intensities and lifetimes of endogenous cofactors NAD(P)H and FAD. Our data support the notion that cisplatin induces acidification of the cytoplasm early after the treatment. We revealed in vitro that a capacity of cells to recover and maintain alkaline pHi after the initial acidification is the crucial factor in mediating the cellular decision to survive and proliferate at a vastly reduced rate or to undergo cell death. Additionally, we showed for the first time that pHi acidification occurs after prolonged therapy in vitro and in vivo, and this, likely, favors metabolic reorganization of cells. A metabolic shift from glycolysis towards oxidative metabolism accompanied the cisplatin-induced inhibition of cancer cell growth in vitro and in vivo. Overall, these findings contribute to an understanding of the mechanisms underlying the responsiveness of an individual cell and tumor to therapy and are valuable for developing new therapeutic strategies.