Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-β1

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.


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)(3)(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)(7)(8)(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.

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), 5µg/ml transferrin, 5 ng/ml sodium selenite, and 5µg/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% CO 2 .

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 cm 2 . 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% CO 2 ).

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).

Results and discussion
Data acquisition demonstrated a linear increasing of the CI values in control cells during the time interval observed. However, this linear trend was significantly changed by TGF-β1 in less than 12 h after the treatment in a concentration-dependent manner (Fig. 1a). Concentrations of 1 and 10 ng/ml induced a significant steep increase in CI values, which reached a plateau in 48 h. The impedance-based determination is by its nature dependent on the number of adherent cells. Thus, we compared CI determination with the analysis of cell numbers to clarify the contribution of changes in cell numbers and the morphological alternation of cells to detected CI values. In parallel with the E-plates® 96, the cells were seeded at the same density on 40 mm dishes and 4-well plates, and treated with TGF-β1 in the same experimental design. At various time intervals after the treatment, the numbers of trypsinized cells were determined using a Coulter Counter simultaneously with the determination of metabolically active viable cells based on determination of intracellular ATP in cell lysate. Our data, shown in Fig. 1b, c, demonstrate that TGF-β1 induced antiproliferative effects in BPH-1 cells in a time-and concentration-dependent manner. These trends are in negative correlation with CI values acquired with the use of RTCA. Taken together, these data showed that the TGF-β1 induced antiproliferative effects in BPH-1 cells are paralleled by an increase of cell impedance. It is a well-known fact that when stimulated with TGF-β1, epithelial cells undergo EMT and exhibit a significant formation of actin stress fibers that emanate from focal adhesions (10 ). In this case, our results demonstrate the usual uncertainty of image analysis which is limited by confluence of the cells. The control cells reached a relatively high density after 72 h of cultivation.
Based on the F-action visualization and cell morphology analysis, there is no significant difference between control cells and cells treated with 0.1 ng/ml of TGF-β1. However, it is evident that TGF-β1 at 1 and 10 ng/ml concentrations induced formation of F-actin stress fibers and increased cell spreading (Fig. 2). These results positively correlate with the activation of Smad-dependent signaling and EMT by TGF-β1 (Fig. 3). Our results showed transient phosphorylation of Smad2 induced by both 1 and 10 ng/ml of TGF-β1, which is followed by massive induction of vimentin expression. TGF-β1 at a concentration of 0.1 ng/ml did not induce vimentin expression and only slightly induced phosphorylation of Smad2. Furthermore, we wanted to examine the influence of formation of stress fibers on TGF-β1-induced increase of CI values. CB, a potent inhibitor of the formation of contractile microfilaments, induced inhibition of F-actin formation in both control and TGF-β1-treated BPH-1 cells (Fig. 4a). This inhibition was paralleled by a rapid decrease of normalized CI values in both groups (Fig. 4b). However, the drop of CI values induced by CB was much stronger in the case of TGF-β1-pretreated BPH-1 cells, with high abundance of F-actin stress fibers in comparison with CB-treated control cells. These results demonstrate that TGF-β1-induced increase of CI values is at least partially dependent on stress fiber formation. We can summarize that TGF-β1-induced EMT in BPH-1 cells is associated with inhibition of proliferation, rebuilding of cytoskeleton, formation of stress fibers, and increase of spreading. These cellular changes are associated with a significant increase in CI values analyzed by the RTCA system. Until now, most of the applications of RTCA focused on analysis of adhesion or spreading were designed to study immediate interaction of cells with substrate in relatively short time intervals (6,11 ). Here, our data suggest a novel application and a great potential of real-time impedance measurement for dynamic monitoring of cellular remodeling and plasticity, induced after adhesion of the cells to the substrate and formation of stress fibers in longer time intervals. Furthermore, these measurements also show a potential misinterpretation of the data in experimental setups using impedance-based methods for the detection of cell proliferation without simultaneous monitoring of cellular morphological changes.