Cell culture
The study was conducted on three human-derived cell lines from patients with pancreatic ductal adenocarcinoma: resistant to daunorubicin (EPP85-181RDB), resistant to mitoxantrone (EPP85-181RNOV) and parental (EPP85-181P). For the study, we chose the chemotherapy resistant cell lines with high expression of MDR proteins. The primary culture cell lines were kindly shared by Dr. H. Lage (Charité University Hospital, Institute of Pathology, Berlin, Germany). The cultures were maintained at 37°C under high humidity in the automated CO2 incubator (Binder). Cells were cultivated in the modified Leibovit’z (L-15) medium (Gibco, Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum, 1% antibiotics (100 IU/ml penicillin and 0.1 mg/ml streptomycin), 1.5% sodium bicarbonate (7.5%, Gibco), 0.1% glucose (45%, Sigma), 2.5 mM ultraglutamine (Lonza, Basel, Switzerland), 0.2% insulin (10 mg/mL, Sigma) and 30 TIU/L aprotinin (BioShop, Canada). Cells were collected from flasks using Trypsin (ThermoFisher Scientific).
Spheroid 3D cell culture
The cells were detached from flasks and resuspended in Phosphate EP buffer (pH = 7.4, 1 mM MgCl2 and 250 mM sucrose). The experiments were performed on 2 × 106 cells / ml concentration. Next, the cells were resuspended in L-15 medium. Afterwards, the cells were carefully counted in Kova’s chambers and further, 5000 cells were seeded in each well of Grainer Facilitate 96-well 3D cell culture plates. The cells were captured under the standard cell culture conditions. After 40 h of incubation, we examined the formation of the spheroids. Each day from then, we captured photos of the spheroids on light microscope. Further analysis included the calculation of the microtumor size and area.
PEF treatment
50 μl of cells in the concentration of 2 × 106/ml, suspended in 10 mM phosphate buffer (pH = 7.4, 1 mM MgCl2 and 250 mM sucrose, were placed in 1 mm cuvettes with aluminum electrodes (BTX, Syngen Biotech, Poland). The square wave electroporator (100 ns–1 ms) developed in the Institute of High Magnetic Fields (VGTU, Vilnius, Lithuania) was used to deliver electric pulses. The cells were exposed to 8 kV/cm x 200 ns x 100 pulses delivered at 10 kHz frequency (0.085 J, 1.6 kJ/L). Pulse shapes and amplitudes were controlled with an MDO3052 oscilloscope (Tektronix, Beaverton, OR). Subsequently, the cells were suspended in a culture medium and placed in 96 or 6 well plates on cover glasses.
Drug preparation
Paclitaxel (Abcam, 10 mg, ab120143) solutions were prepared from the DMSO stock solutions of 5 µM. The required concentrations were achieved with a dilution of stock in EP buffer to 50 nM concentration. Drug was added to the cells 24 h after nsPEF treatment to avoid transporting the drug through the cell membrane. This attempt was made to analyze the vulnerability of the cells to paclitaxel and not its transport through the membrane. 24 h was considered sufficient time for the closure of reversible pores and fixation of nsPEF-induced changes in the cell membrane.
Holotomographic microscopy
Live holotomography was performed using a 3D Cell Explorer microscope (Nanolive SA, Ecublens, Switzerland). Pancreatic cancer cells were imaged using 35 mm Ibidi glass-bottom μ-Dish dishes (Ibidi GmbH, Germany). During imaging, temperature was set to 37 °C and controlled using an Ibidi Heating System (Ibidi GmbH, Germany), while the sufficient amount of CO2 was maintained by using a CO2-independent culture medium (Sigma-Aldrich). Initially, we placed the electroporation needles surrounding the visualized cell groups. Afterwards, we started to recorded a video and after a short while treated cells with PEFs. The snapshots were taken each 1 minute for at least 13 minutes. Further, the data from the microscope was gathered and analyzed using STEVE Software.
Concerning the Yo-Pro-1 dye uptake studies, the cells were permeabilized according to the description of permeabilization of cell suspension. However, before PEFs delivery, the green-fluorescent YO-PRO-1 stain (Y3603, Thermo Fisher Scientific, Waltham, MA) in the concentration of 1 μl/ml was added to EP buffer. YO-PRO-1 cellular uptake reflects the degree of plasma membrane’s permeabilization. The fluorescence of YO-PRO-1 was excited with 488 nm wavelength and measured on Nanolive 3D Cell Explorer microscope.
Molecular dynamics simulation of membrane response
The molecular dynamics simulations were performed with GROMACS 2018.3 software [54] on the calculational cluster in the Department of Theoretical Chemistry and Physics at the Lorraine University. The models for simulations were built with CHARMM-GUI web software and visually inspected with VMD software [55], [56]. The simulated systems were composed of a membrane in the ionic water solution. The membrane was composed of ~256 lipids per membrane layer. All the systems were built considering mixture of lipids-cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine – POPC. Two systems representing 10% and 30% cholesterol were modeled, each with POPC. Before the simulation, the system was solvated in physiological conditions of NaCl water (TIP3) solution. Six (5 mM) calcium ions were present in each system. Both water compartments were separated by the introduction of the vacuum above and below the system. The whole simulation was performed in the periodic boundary conditions. The simulation proceeded with the CHARMM36 force field [55]. The systems were minimized, equilibrated (100 ns, NPT conditions: Nose-Hoover thermostat and Berendsen barostat) and simulated for 10 ns in the NVT ensemble to obtain the value of the surface tension in the air-water interphase. Afterwards, the system was simulated for 100 ns under various ionic gradient conditions. The gradient was introduced by moving the ions throughout the membrane. The electroporation simulation was carried under NPγT (constant surface tension, number of molecules, pressure and temperature) conditions. Moreover, we reduced the surface tension of the membrane in one of the studied systems. After the simulation, the system was evaluated if the pore was formed during the time of the simulation and the visual representations of the membrane were presented on the layouts.
Confocal microscopy
To visualize the actin filaments, pan-cadherin, N-cadherin and nuclei of the cells, an Olympus Fluoview FV1000 confocal laser scanning microscope (Olympus) was used. confocal microscope was used. Control and nsPEF-treated cells were seeded on microscopic glass plates. Following a 24 h of incubation, the cells were fixed with a 4% formaline for 10 minutes and washed 3 times with PBS. Afterwards, the cells were incubated with 5% FBS in 4% Triton X-100 diluted in PBS to block the unspecific interactions in further steps. Actin filaments were stained with Invitrogen™ Alexa Fluor™ 488 Phalloidin (2 μg/ml, Life Sciences – Thermo Fisher Scientific) according to the manufacturer protocol. Fluoroshield™ with DAPI (4,6-diamidino-2-phenylindole, Sigma-Aldrich) was applied to visualize the nuclei and to mount the cells. Primary antibodies were used: mouse monoclonal anti-CD91, PE, eBioscience™ diluted (1:200) in 4% Triton X-100 in PBS, mouse monoclonal anti-CD243, PerCP-eFluor™, eBioscience™ (ThermoFisher Scientific) diluted in 4% Triton X-100 in PBS (1:200), rabbit polyclonal anti-pan-Cadherin (Invitrogen) diluted (1:300) in 4% Triton X-100 in PBS, and rabbit monoclonal anti-N-cadherin (Invitrogen) diluted (1:300). All antibodies were diluted with a 4% Triton X-100 in PBS. As a secondary antibody, anti-rabbit antibody was used conjugated with AlexaFluor488TM diluted (1:250) in 4% Triton X-100 in PBS was used. For the imaging, an Olympus Fluoview FV1000 confocal laser scanning microscope (Olympus) was used. The images were recorded by employing the Plan-Apochromat ×60 objective. Any contrast and brightness adjustments were performed in FV10-ASW_Viewer or in ImageJ. The images were analyzed with the use of ImageJ to evaluate the expression pattern of the proteins in the cross-section of the cells.
Electric field distribution
The spatial electric field distribution has been simulated in OpenEP software. A two-dimensional model of the cuvette electrodes (aluminum, 1 mm gap) a has been designed and the conductivity of the surrounding medium (phosphate buffered saline (PBS)) of 1.5 S/m has been selected. The 8 kV/cm electric field distribution was simulated and presented in the visual way.
Viability assays
For Presto Blue viability studies we have added the reagent in 1:10 (reagent to culture medium) ratio to each well and fluorescence was measured, after 30 minutes of incubation at 37 °C, using a GloMax Discover microplate reader (Promega; Exc. 520 nm/Em. 580–640 nm). The results were expressed as the percentage of viable cells relative to untreated control cells.
CellcyteX studies
CellcyteX live-cell imaging system acquires phase contrast images, which are analyzed with detection software. Detection software was used to determine cell viability by measuring cell confluency and comparing the results with cell count and nuclei studies. The system was used to assess the kinetics of cells’ growth after treatment with nsPEF and Paclitaxel. In the study we aimed to establish the optimal time for the viability tests and examine if the antiproliferative effect of nsPEF is permanent. Control and PEF-treated cells were seeded on 96-well plates. After 24 hours at 37°C paclitaxel was added to wells and cells were further incubated at 37 °C in the incubator set to work with CellcyteX operating Cellcyte Studio software. The experiment was performed in replicates and each well consisted of four, square zones, in which the cells were counted. Further, the images were analyzed with Aivia Software. The results were expressed as the percentage of viable cells relative to untreated control cells.
Statistical analysis
The experiments were performed in at least 3 replicates. Data were expressed as mean ± SD and analyzed by one-way ANOVA (in GraphPad Prism 8), with p < 0.05 being considered statistically significant.