DynamicMonitoringofCellularRemodelingInducedbytheTransformingGrowth1

The plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states such as cancer and fibrosis. In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-β1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial–mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate.

Key Words: real-time cell analysis - cell plasticity - epithelial–mesenchymal transition - transforming growth factor-β1 - F-actin - cytoskeleton remodeling

Introduction

The phenomenon of plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states. Epithelial–mesenchymal transition (EMT) is a crucial process in embryogenesis, but it also occurs during progression of tumors derived from epithelial cells (for review, see (1)). The transforming growth factor-β1 (TGF-β1) is an important growth factor inducing remodeling of epithelial cells. TGF-β1 induces a complex change of the gene expression profile, which leads to the induction of cell cycle arrest, increased cell migration, and spreading (2–4). In general, determination of the quality and quantity of remodeling of epithelial cells is a complex issue. It usually includes quantification of expression of epithelial and mesenchymal markers (E-cadherin, N-cadherin, and vimentin), visualization of cytoskeletal rebuilding (F-actin), migration, and invasive assay (wound healing and migration through Matrigel matrix; (5)). Conventionally, most of the approaches mentioned are based on a time-consuming end-point analysis of the state of whole cell populations combined with advanced techniques of analysis of individual cells with the use of flow cytometry or digital microscopic techniques and image analysis. However, neither the episodic nor the spatial resolution of these techniques is capable of registering very small and fast changes in cellular morphology. Currently, label-free and noninvasive methods based on electronic cell sensor arrays were suggested for the monitoring of cell physiology, particularly adhesion, spreading, and transient changes in cell morphology (6–9). To widely accept this methodological approach and to correctly and precisely interpret data for these measurements is crucial to obtain precise correlation with cell morphology and overall phenotype using a relevant reference method. However, well-described models applying this methodological approach with different cell lines and various cell plasticity modulating conditions are missing. Here, we showed that the impedance-based real-time cell analyzer (RTCA) allows dynamic monitoring and quantification of cell remodeling during TGF-β1-induced EMT in non-transformed prostate epithelial cells. This novel application of such approach demonstrated a great medium-throughput potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate.

Materials and methods

Cells

BPH-1 cells were obtained from the German Collection of Microorganisms and Cell Cultures and cultivated in RPMI 1640, supplemented with 20% bovine fetal serum (both PAA), 5?μg/ml transferrin, 5 ng/ml sodium selenite, and 5?μg/ml insulin (Invitrogen). The cell lines were cultivated in Nunc (Thermo Fisher Scientific) cultivation dishes, flasks, and plates in a humidified incubator at 37°C in an atmosphere of 5% CO₂.

Real-time cell impedance analysis

Acea E-plates? 96 were used for noninvasive real-time measurement with the use of an xCELLigence RTCA SP system including RTCA Software version 1.1 (both Roche). First, a standard background measurement was performed using 100 μl of complete cultivation media. BPH-1 cells were trypsinized, quantified, and seeded in additional 100 μl of cultivation media in a final concentration of 30,000 cells per cm². The cells were monitored continually every 1 min in the first 45 min after the seeding and then every 1 h for a period of 96 h. Recombinant TGF-β1 (Millipore) treatment with various concentrations in triplicate was performed 24 h after the seeding of the cells. Formation of contractile microfilaments was blocked by cytochalasin B (CB), Helminthosporium dematioideum (Calbiochem) dissolved in methanol (MeOH). The cells were pretreated with TGF-β1 (10 ng/ml) for 68 h and treated with CB (10 μg/ml) for another 3 h. The cells were monitored continually every 15 s after the CB addition. In this case, data are presented as a normalized cell index (CI; normalized at the time of 68 h). Cultivation of the cells and their treatment were performed under standard conditions (37°C/5% CO₂).

Cell counts

The numbers of trypsinized BPH-1 cells in the culture were determined using a Coulter Counter? ZM (Beckman-Coulter).

ATP assay

Intracellular ATP was detected in BPH-1 cells by the commercial ATP cellular kit (Biothema, Sweden). The cells were incubated according to the experimental procedure, the supernatant was removed, and the cells were lysed by the Somatic cell ATP releasing reagent (Sigma-Aldrich). Then, 50 μl of lysate was mixed with 20 μl of ATP reagent containing D-luciferin, luciferase, and stabilizers. Intracellular ATP contents were determined using a microplate luminometer LM-01T (Immunotech).

Fluorescent and light microscopy

F-actin was visualized after the staining of paraformaldehyde (2%) fixed and permeabilized BPH-1 cells with phalloidin-fluorescein isothiocyanate (Sigma-Aldrich) using a fluorescent microscope (Olympus IX-70, Fluoview II CCD camera). Nuclear counterstaining was performed by using 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Fluka). Cell morphology was documented by phase contrast on the same microscope.

Western blot

BPH-1 cells were treated by various concentrations of TGF-β1 for different time intervals and harvested in radioimmunoprecipitation assay buffer (50 mM Tris–HCl pH?7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, protease inhibitor cocktail, and phosphatase inhibitor cocktail set II (Merck)). Protein concentration was determined using detergent-compatible protein assay (Bio-Rad). The cell lysates were sonicated (5 s, Sonifier? B-12, Branson Ultrasonics Corp), spun, and mixed with 3× sodium dodecyl sulfate (SDS) loading buffer (240 mM Tris–HCl pH?6.8, 6% SDS, 0.02% bromphenol blue, 30% glycerol, 3% β-mercaptoethanol). Equivalent quantities of protein (20?μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore) using established procedures. The membranes were blocked in Tris-buffer saline (20 mM Tris–HCl pH?7.2, 140 mM NaCl) containing 0.1% Tween 20 and 5% non-fat milk. The levels of phosphorylated (Ser465/467) and total Smad2, and expression of vimentin, a characteristic mesenchymal marker, were analyzed with specific primary antibodies (Cell Signaling and Sigma-Aldrich). Anti-β-actin (A5441) was from Sigma-Aldrich; horseradish peroxidase-conjugated anti-mouse IgG (#NA931) and anti-rabbit IgG (#NA934) were from GE Healthcare. Detection of antibody reactivity was performed using Immobilon Western HRP Substrate (Millipore). Densitometric measurements were performed using ImageJ software (NIH) and normalized to the expression of β-actin.