Multiple effects of electroporation on the adhesive behaviour of breast cancer cells and fibroblasts

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.

Chemipulse III electroporation apparatus was produced at the Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria. The following electrical parameters were used for the experiments - 8 biphasic pulses; 50 + 50 μs with 20 μs interval between both phases and pause between bipolar pulses of 880 μs, the voltage was between 200 - 1000 V. At that field conditions Daskalov and Bankov have proved that the effectiveness of the electroporation is high [42]. The apparatus was supplied with a battery and during the manipulations it was not connected to the mains supply. The induction of 200 - 1000 V voltage was done in two parallel stainless steel electrodes (Figure 1) with a 10 mm distance between them and a length of 22 mm (when cells are cultivated on cover glasses) or 9 mm (when cells are cultivated directly on the bottom of 24 well plates). A uniform electric field was generated by approximation ignoring edge effects. A scheme of the experimental dish and shape of the electrical pulses is shown in Figure 1.

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.

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 [43]. 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 assay was adapted to that described by Hernandez et al. [44]. 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. [45]. We used CellTiter 96 AQ_ueous 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 (S_e) of 90 mm². The total surface area (S_t) 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.

Penetration of PI into the cells was used to define the success of permeabilization of the cell membrane by the process of electroporation. The results showed membrane permeabilization at all three intensities of the electrical field (Figure 2, B-D). For PI staining pictures were made at the same parameters of the fluorescent microscope for the exposure of the sample as time of exposure, brightness, contrast, etc. It was visible that cells treated with 200 V/cm and 500 V/cm were less electroporated than those at 1000 V/cm (Figure 2). The results showed that the content of PI in the cells increase at a higher electrical filed intensity. Each picture in Figure 2 is a representative of five different pictures taken at different electrical intensity.

Cell adhesion is an important process in cancer insemination. We have tested short- and long-lived effects on cell adhesion caused by electropermeabilization.

The colorimetric method of crystal violet staining was used for quantitative estimation of cell adhesion and by MTS assay the cell survival after electropermeabilization was determined. All cells were cultivated 24 h prior electroporation on FN coated cover glasses. In Figure 3 adhesion and survival of electroporated 3T3 cells after 2 (Figure 3A) and 24 (Figure 3B) hours are shown. Cell adhesion of 3T3 cells was not affected significantly by the electrical treatment (Figure 3) over the entire examined time. Decrease in cell adhesion was detected only at 1000 V/cm 24 hours after electroporation in comparison to those at 200 V/cm (asterisks in Figure 3B). The replication of cells was not affected by electropermeabilization since after 24 hours the relative number (O.D. at 630 nm) of non-treated cells (control) and electrotreated cells increased between 30% - 40%. Minor decrease of cell survival of all treated cells 2 hours after electroporation was detected. There was no difference in the cell survival between treated cells and the control 24 hours later. The invasive tumour cell line MDA-MB-231 showed different cell behaviour when treated with high voltage electrical pulses (Figure 4A and 4B). It is well known that MDA-MB-231 cells exhibit week adhesive behaviour in general [46]. Two hours after the electrical treatment, cell adhesion at the higher field intensities (1000 V/cm) decreased significantly compared to the control and cells treated with 200 V/cm and 500 V/cm. Cell survival did not decrease significantly when increasing the intensity of the electrical pulses (Figure 4A). Surprisingly, even after 24-hour-electrotreatment cell adhesion at 200 V/cm and 500 V/cm showed some increase (comparing to the control) and only the cell adhesion at 1000 V/cm was diminished in comparison to those at 500 V/cm (Figure 4B). Cell survival after 24 hours seemed to be not affected by electroporation. The non-invasive tumour cell line MCF-7 showed general tendency to decreasing of cell adhesion 2 hours after electroporation (Figure 5A). The adhesion was getting lower with the intensity increase of the electrical field and reached the minimum at 1000 V/cm (Figure 5A). The trend of the cell survival resembles the one of the 3T3 cells: the cell survival slowly decreases. After 24 hours the cell adhesion and survival were not affected by electrotreatment (Figure 5B). Our results are in agreement with Yizraeli and Weihs [28], who showed that fibroblasts is not affected significantly by electrical treatment in comparison to cancerous cells (MDA-MB-231). In general, for two cancer cell lines the electropermeabilization seems to slower down the cell replication since 24 hours later there was no significant increase in the relative cell number. In contrast to the work of Cemazar et al., [45] which showed a significant decrease in MCF-7 cell survival between 200 V/cm and 1000 V/cm, our results did not indicate a decrease in cell adhesion and survival between the above values of the electrical field intensity. Most probably the preadsorption step with FN is the factor which plays a protective role for cell adhesion and survival against the electrotreatment.

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 [47]. 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.