Multiple effects of electroporation on the adhesive behaviour of breast cancer cells and fibroblasts
© Pehlivanova et al; licensee BioMed Central Ltd. 2012
Received: 11 January 2012
Accepted: 22 March 2012
Published: 22 March 2012
Recently electroporation using biphasic pulses was successfully applied in clinical developments for treating tumours in humans and animals. We evaluated the effects of electrical treatment on cell adhesion behaviour of breast cancer cells and fibroblasts. By applying bipolar electrical pulses we studied short- and long-lived effects on cell adhesion and survival, actin cytoskeleton and cell adhesion contacts in adherent cancer cells and fibroblasts.
Two cancer cell lines (MDA-MB-231 and MCF-7) and one fibroblast cell line 3T3 were used. Cells were exposed to high field intensity (200 - 1000 V/cm). Cell adhesion and survival after electrical exposure were studied by crystal violet assay and MTS assay. Cytoskeleton rearrangement and cell adhesion contacts were visualized by actin staining and fluorescent microscope.
The degree of electropermeabilization of the adherent cells elevated steadily with the increasing of the field intensity. Adhesion behaviour of fibroblasts and MCF-7 was not significantly affected by electrotreatment. Interestingly, treating the loosely adhesive cancer cell line MDA-MB-231 with 200 V/cm and 500 V/cm resulted in increased cell adhesion. Cell replication of both studied cancer cell lines was disturbed after electropermeabilization. Electroporation influenced the actin cytoskeleton in cancer cells and fibroblasts in different ways. Since it disturbed temporarily the actin cytoskeleton in 3T3 cells, in cancer cells treated with lower and middle field intensity actin cytoskeleton was well presented in stress fibers, filopodia and lamellipodia. The electrotreatment for cancer cells provoked preferentially cell-cell adhesion contacts for MCF-7 and cell-ECM contacts for MDA-MB- 231.
Cell adhesion and survival as well as the type of cell adhesion (cell-ECM or cell-cell adhesion) induced by the electroporation process is cell specific. The application of suitable electric pulses can provoke changes in the cytoskeleton organization and cell adhesiveness, which could contribute to the restriction of tumour invasion and thus leads to the amplification of anti-tumour effect of electroporation-based tumour therapy.
Electroporation is a biophysical method, performed by the application of high voltage electrical pulses to cells in vitro or tissues in vivo, used to increase the cell's uptake of different molecules by permeabilization of the plasma membrane [1–4]. Most of the electropermeabilization protocols use unipolar electrical pulses [5–7], but recently the higher efficiency of biphasic pulses was confirmed [8–10] so they were successfully used in clinical developments for treating tumours in humans and animals [11–13] and for DNA transfection . The field intensity and duration of the applied electrical pulses of the electroporation (electropermeabilization) can either reversibly open nanoscale pores on the cell membrane after which the cell can survive, or irreversibly open the cell membrane, after which the cell dies . In cancer treatment, the reversible electroporation has been exploited to increase transport of chemotherapeutic drugs through the plasma membrane into the tumour cells. This process is called electrochemotherapy  and it is widely used for the treatment of accessible human tumours and tumour lesions [15–18]. Non-thermal ablation is a recently discovered new technique for treating inoperable tumours , which is based on irreversible electroporation of cells . It is believed to affect only the cell membrane and no other structure in the tissue and in this way a direct electrical filed induced cancer cell death is achieved. Moreover, not as selective as electrochemotherapy, the thermal ablation can be used as a minimally invasive surgical procedure to ablate cancer tissue without the use of potentially harmful chemotherapeutic drugs.
Apart from the effect on cell membrane (to open nanoscaled pores), the applied external electric pulses demonstrate to be able to alter the cytoskeletal reorganization which affects the cell adhesion. For instance, changes in the cytoskeletal structure have been demonstrated during processes of electrofusion  and electrotransfer . Actin cytoskeletal redistribution has been reported in directional cell electromigration induced by dc electrical field [23, 24] and in electroporation-based therapies [25, 26]. For example Kanthou et al.  studied the vascular effect of electropermeabilization as well as the changes in the cytoskeleton organization of primary endothelial cells and in the monolayer permeability. The results of Xiao et al.,  which showed that the disruption of actin skeleton of cancer cells by application of electrical pulses, prevents cells from apoptosis and necrosis were very interesting too.
Using plated/adherent cells in the experimental model we can study the cells in their intact internal structure (cytoskeleton) and the results obtained in these cells are better comparable to real in vivo situations than the results from cell suspensions . Comparing all data concerning actin cytoskeleton changes (how strong they can be and if they are reversible) in adherent cells induced by applied electrical pulses, it becomes visible that they depend mainly on the intensity of the applied field, electropulsation medium and cell type. For instance, it was shown that when culture medium was used during electroporation, the cytoskeletal structures  were best preserved. Yizraeli and Weihs  showed that fibroblasts were affected very weakly by the applied electrical field in comparison to MDA-MB- 231 cells.
In adherent cells, the basic actin-rich cell-extracellular matrix (ECM) ensembles are stress fibers, lamellipodia and filopodia, which play an important role in cell attachment and migration [29–31]. Actin can also be arranged into peculiar dot-like structures called podosomes which perform a role in cell migration and motility and in ECM degradation [32, 33]. In cancer cells the presence of adhesion contacts is not a prerequisite for growth and survival . Among the main features of the cancer cells are the breakdown of adherent connections (cell-cell contacts) and also the cytoskeleton organization (cell-ECM contacts) . The change in the adhesive behaviour of cancer cells determines their modified morphology and migration behaviour and predetermines their invasive properties during all stages of tumourogenesis [36, 37]. Thus, changing the cell's adhesion ability by electroporation it could be a very important prerequisite to inhibit cancer cells motility, invasion and metastasis.
The surrounding stroma of many tumours, for instance breast tumours, consists mainly of fibroblasts , which play prominent role in the development and progression of the tumour [39–41]. Therefore stromal fibroblasts have to be considered as a possible target in electropulse tumour treatment and may affect the outcome of any treatment applied to a definite region .
Regardless of some articles, concerning the changes in the cell cytoskeleton provoked by applied electrical pulses [21, 22, 25], little is known about the influence of these changes during the process of electroporation on actin cytoskeleton of adherent cancer cells.
Therefore, the aim of the present paper is to study the effect of the applied biphasic electrical pulses (200 - 1000 V/cm) on the adhesive behaviour of two breast cancer cell lines and a non-transformed fibroblast cell line in order to elucidate the effect of electrical field on tumour progression. MDA-MB-231 was chosen as a cell model for invasive and metastatic breast cancer cells and MCF-7 cell line was used as an example of a fast growing non-invasive breast cancer cell line. The non-tumorogenic cell line 3T3 (mouse fibroblasts) was chosen to present the somatic fibroblasts surrounding the tumour.
Materials and methods
Chemicals and proteins
Propidium iodide (PI) (Sigma-Aldrich Company Ltd, St. Louis, Missouri, USA) was prepared in phosphate buffered saline (PBS), pH 7.4 in a concentration of 0.1 mM.
Crystal violet was from Sigma, (St. Louis, Missouri, USA) and was used in concentration of 0.1% in PBS, pH 7.4. BODIPY 558/568-conjugated phalloidin (B3475, Invitrogen GmbH, Karlsruhe, Germany) was used in a concentration of 0.132 μM. Human plasma Fibronectin (FN, Roche Applied Science, Penzberg, Germany) in a concentration of 20 μg/ml was used for coating of surfaces for the investigation with cells.
MDA-MB-231 (ATCC, Manassas, VA, USA) were grown in RPMI 1640 medium (PAA, The Cell Culture Company, Cat. № E15-039, Germany) with 10% fetal calf serum (FCS) and supplements (insulin, L-glutamine, sodium pyruvate, antibiotic, NEAA (non essential amino acids) at 37°C, 5% CO2 and humidified atmosphere.
MCF-7 (ATCC, Manassas, VA, USA) cell line was cultivated in modified Eagle's medium (DMEM), (PAA: The Cell Culture Company, Cat. № E15-009, Germany) containing 10% FCS and supplements L-glutamine, sodium pyruvate, antibiotic, NEAA at 37°C, 5% CO2 and humidified atmosphere. 3T3 cell line (mouse fibroblasts) (ATCC, Manassas, VA, USA) was cultivated in Eagle's Minimum Essential Medium (MEM), (PAA, The Cell Culture Company, Cat. № E15-825, Germany) containing 10% FCS and supplements (L-glutamine, sodium pyruvate, antibiotic, NEAA at 37°C and 5% CO2.
When the cells are 80 - 90% confluent they were harvested with 0.05% trypsin/0.6 mM ethylenediaminetetraacetic acid (EDTA) (Sigma, Deisenhofen, Germany) at 37°C. Trypsin was neutralized with FCS. The cells were centrifugated (at 1 × 103 RPM for 5 min) and resuspended in the appropriate cultivation medium. They were seeded directly on the bottom of 24 well plates (Greiner Bio-One GmbH, Solingen, Germany) or on cover slides (18/18 mm, Superior-Marienfeld, Germany), which were coated with FN. Before protein coating the cover slides were rinsed once with 70% ethanol and twice in sterile distilled H2O and placed in 6 well plates (Greiner Bio-One GmbH, Solingen, Germany). The cells were incubated for 24 hours at 37°C and 5% CO2 to reach stable adhesion prior to the electrical treatment. Electroporation was carried out in basal cell medium (without phenol red and supplements). Immediately after electroporation the basal cell medium was replaced by supplemented medium with 10% FCS for the additional incubations.
Determination of electro-permeabilization
The electro-permeabilization of plasma membrane was measured by cellular uptake of PI. 3T3 cells at cell density of 1.5 × 105 cells ml-1 were cultivated on cover glasses in 6 well plates. After 24 hours the adhered cells were electroporated in a basal cell medium containing 0.1 mM PI (Sigma, Deisenhofen, Germany) under the above electrical parameters. After the exposure to electrical pulses, the cells were incubated for 15 min at 37°C. This incubation time was shown to be the optimal, since it allows resealing of the plasma membrane and it does not affect cell viability due to the evaporation of the medium and lack of nutrients . To visualize the PI uptake, the cover glasses were washed with PBS, pH 7.4 and then were fixed with 3% paraformaldehyde (PFA) for 15 minutes at room temperature. After three washes with PBS and distilled H2O, the slides were mounted on objective glasses using Mowiol and were visualized using fluorescent inverted microscope (Leica DMI3000 B, Leica Microsystems GmbH, Germany) with objective HI PLAN 40×/0.50, filter set I3 S and light source from Hg 100 W lamp. The images were taken by camera Moticam 2500, 5.0 M Pixels USB 2.0. For each used voltage 5 images from different fields of the sample were produced. The middle section of the cover glass, which is situated in the centre of the electrical field, was examined.
Cell adhesion (crystal violet assay)
Cell adhesion assay was adapted to that described by Hernandez et al. . 200 μl cell suspension with density of 1.5 × 105 cells ml-1 and 10% FCS were seeded in each of the 24 well plates (Greiner Bio-One GmbH, Solingen, Germany). After 24-hour incubation at 37°C and 5% CO2 the adhered cells were electroporated in a basal cell medium. No electrical pulses were applied to the cells in the control. After the electrical treatment, the cells were incubated for additional 2 and 24 hours. The well plates were washed three times with PBS, pH 7.4 to remove the non-adhered cells and then the adhered cells were fixed with 500 μl 3% solution of PFA for 20 minutes at room temperature. The cells were washed twice with distilled H2O, stained with 0.1% solution of crystal violet for 20 minutes at room temperature, washed again with distilled H2O and dried for 24 hours at 37°C. 100 μl 0.1 M HCl was added in each well. The relative number of the adhered cells was defined colorimetrically by the intensity of the solution of crystal violet at 630 nm using a microplate reader (Multiskan Spectrum, Thermo Electron Corp., Finland). Three independent experiments with three repeats were performed for each cell line.
The survival of cells after electrotreatment was determined as described by Cemazar et al. . We used CellTiter 96 AQueous One Solution Cell Proliferation MTS assay (Promega, Madison, WI, USA) to determine cell survival. Cells were electrotreated as described for cell adhesion assay. After the electrical treatment, the cells were incubated for 2 and 24 hours. Then, 50 μl of MTS reagent was added directly to the adherent cells. They were incubated for 2 hours at 37°C and recorded the absorbance at 490 nm with 96-well plate reader Tecan Infinite F200 PRO (Tecan Austria GmbH, Salzburg). The survival of the cells treated with different electrical field intensities was presented as a relative number of adhered cells (O.D. at 490 nm). Three independent experiments were performed for each cell line.
For both cell adhesion and survival experiments a fraction of cells was calculated, where the cells were under the influence of the electrical field in the sample. We assumed that the cells were randomly adhered on the bottom of the well. The electrodes used for electroporation (the distance between the electrodes was 10 mm and the length of the electrodes used for these experiments was 9 mm) cover a frame between them with area (Se) of 90 mm2. The total surface area (St) of the bottom of the 24 well plates was equal to 132.66 mm2. Thus, approximately 70% of all cells adhered on the well were under the applied electrical field.
3T3, MDA-MB-231 and MCF-7 with cell density of 1.5 × 105 cells ml-1 were cultivated on cover glasses (18/18 mm) placed in 6 well plates. After 24-hour incubation the cells were electroporated in a basal cell medium and were cultivated additionally for a period of 2, 24 and 48 hours in full cell medium. After the incubation period, non-adhered cells were removed by triple rinsing with PBS, pH 7.4. The adhered cells were fixed with 1 ml 3% solution of PFA for 15 minutes at room temperature. The fixed cells were permeabilized using 1 ml 0.5% solution of Triton X-100 for 5 minutes and then incubated with 1 ml 1% solution of bovine serum albumin (BSA) for 15 minutes. The samples were washed three times with PBS, pH 7.4 and then incubated for 30 minutes at room temperature with BODIPY 558/568 phalloidin. Again, the samples were washed three times with PBS and once with distilled water, and then were installed on objective glasses by Mowiol. Preparations were analyzed using inverted fluorescent microscope (Leica DMI3000 B, Leica Microsystems GmbH, Germany) with object HCX PL FLUOTAR 63×/1.25 oil.
Results and Discussion
Degree of cell electroporation
Cell adhesion and survival
Cell adhesion is an important process in cancer insemination. We have tested short- and long-lived effects on cell adhesion caused by electropermeabilization.
It is well known that the actin cytoskeleton plays a fundamental role in cell adhesion, migration and growth and in cancerogenesis these processes are significantly unregulated . The investigation of the influence of high electrical pulses on the organization of actin cytoskeleton was conducted with two breast tumour cell lines and one non-transformed cell line - 3T3 fibroblasts. The alteration in actin cytoskeleton was followed up to 48 hours after the electrotreatment in order to monitor how stable the changes are.
a) 3T3 cells
The disturbance of the cytoskeleton of the adherent cells leads to changes in cell attachment, diminished cell motility and survival. The dominant type of stromal cells surrounding each tumour is from fibroblast origin . Many investigations conducted in vitro and/or in vivo show that fibroblasts can support the growth of tumour cells [39–41]. We could suggest that the destabilization of actin cytoskeleton of the fibroblasts under the influence of high electrical pulses could lead to an additional positive effect of the applied electrochemotherapy leading to restriction of tumour expansion.
c) MCF-7 cells
In general, for cancer cells electroporation did not cause a significant disturbance in actin cytoskeleton structures. Well visible actin stress fiberes as well as lamellipodia and filopodia could be seen in electroporated cancer cells predominantly on the lower and middle field intensity. That fact could be positive to provoke cell death by application of electrical field as Xiao et al.,  suggested. In his work he showed that the destruction of the cell cytoskeleton of tumor cells prevents them from necrosis and apoptosis when electrical field is applied.
It is important to note that according to the obtained results for the actin organization after electroporation for MDA-MB-231 cells became predominant after a time cell-substrate adhesion contacts, since electrotreated MCF-7 cells expressed preferentially cell-cell contacts. Thus, it could be assumed that the type of cell adhesion (cell-substrate or cell-cell adhesion) induced by the electroporation process is cell specific. As other investigators  have found out that fact could be a result of the involvement of different signal pathways and signal molecules in the process of cell adhesion. Using different signalling inhibitors Wang and colleagues  described the ability of invasive and metastatic breast cancer cells either to be reverted to a near-normal phenotype or to cause cell death. Based on the received results we could suggest that the application of high voltage electrical pulses to the transformed MDA-MB-231 cells could lead to their regression to a less or non-transformed cell phenotype since the consolidation of cell-substrate contacts leads to a reduction of cell motility and invasiveness .
Also, in MCF-7 cells, an alteration in cell adhesiveness and cell phenotype is observed but the changes are related to the decreased cell-substrate contacts and amplified the ability of the electroporated cells to form stable cell aggregates and cell-cell contacts. It can be suggested that this tendency could lead to a formation of a cell phenotype with decreased cell motility and invasiveness.
Cell adhesion and survival of fibroblasts and MCF-7 are not affected significantly by the applied electroporation. While, the electrotreatment of invasive breast cancer cell line MDA-MB-231 induces an increase in cell adhesion at lower field intensities and decreased cell adhesion at 1000 V/cm. The cell replication by both cancer cell lines is disturbed by electropermeabilization.
Actin cytoskeleton is differently influenced by electroporation in fibroblasts and cancer cells. In 3T3 cells actin cytoskeleton is temporary disturbed, since in cancer cells treated with lower and middle field intensities actin cytoskeleton is well presented in stress fibers, lamelopodia and fillopodia.
The type of cell adhesion (cell-substrate or cell-cell adhesion) induced by the electroporation process is cell specific. While by MDA-MB-231 dominates the cellsubstrate adhesion, in MCF-7 cells cell-cell adhesion is more often found.
The present work deals with the changes in adhesive behaviour of fibroblasts and transformed cell lines during the process of electroporation. It raises many questions concerning which signalling pathways are involved in cell adhesion in breast cancer cells and how they can be influenced and/or blocked in order to reach a phenotypic reversion of cancerous cells. The obtained results can be helpful for understanding and multiplying the effect of electrochemotherapy by choosing the suitable electrical parameters.
This work is supported by a grant of the Bulgarian Scientific Fund No DO 02/178 and project No BG051PO001-3.3.04/42 funded by the European Social Fund. We would like to thank Mrs. Radoslavova for editing and proof-reading of the paper in English.
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