Electrochemotherapy (ECT) is a highly effective local treatment used in clinics to treat superficial tumors, specifically various types of skin tumors when standard treatments such as surgery, chemotherapy, and radiotherapy are not sufficient or applicable.1,2 Over the past decade, ECT has also been successfully used for the treatment of deep-seated tumors, including tumors in the liver, bone, and pancreas.3,4,5,6,7,8,9
ECT essentially consists of two main steps.10,11 First, a chemotherapeutic drug is injected intratumorally or intravenously. Second, short high-intensity electric pulses that result in cell membrane electroporation are delivered to the tumor. Electroporation transiently increases the cell membrane permeability through the formation of pores/defects in the membrane and enhances the intracellular uptake of the chemotherapeutic drug. The drugs most often used in ECT are bleomycin and cisplatin, which kill cancerous cells by acting on DNA but poorly permeate the cell membrane.1,12 Electroporation potentiates the uptake, and consequently the cytotoxicity, of bleomycin by several hundred to thousand folds and of cisplatin by several ten folds compared to nonelectroporated controls.13,14,15 In addition to increased intracellular drug delivery, drug entrapment due to the blood flow modifying effect of electric pulses16, the vascular disrupting effect17,18 and immune system response19,20 were identified to critically contribute to the success of ECT.21
Electroporation can be achieved with a wide range of pulse parameters (pulse shape, polarity, duration, amplitude, number, repetition rate, etc.). In ECT, conventionally 8 square monopolar pulses of 100 μs duration at a repetition frequency of 1 Hz or 5000 Hz are applied.10,11,22 However, the use of 100 μs long pulses causes pain and muscle contractions23,24 in the patient during the treatment. Furthermore, muscle contraction might lead to the displacement of the electrodes resulting in undertreatment25 and in potential harm for the vital structures when treating deep-seated tumors.26 Thus, there is a need to use local or general anesthesia and muscle relaxants and, when performing ECT of deep-seated tumors in proximity to the heart, the pulses need to be synchronized with the heart rhythm.27,28,29,30,31 To overcome these drawbacks, recent studies suggest the use of bursts of short high-frequency bipolar pulses (1–10 μs pulse duration), which minimize pain and muscle contractions.23,32,33 Such pulses are already used for the ablation of tumors34 and cardiac tissue35,36,37 by irreversible electroporation. Furthermore,
In ECT preclinical and clinical studies have shown that immune response critically contributes to tumor eradication.19,43,44 Thus, ECT has been tested in combination with gene electrotransfer (GET) which delivers protein-encoding DNA into tumor cell/tissue to induce immune stimulation.45,46,47 Even if the combined ECT+GET treatment was applied only to some of the cutaneous metastases, this combination successfully evoked a systemic immune response and in some cases succeeded in producing a partial response or complete response of distant, non-treated nodules (i.e., abscopal effect).48 GET, which is also based on electroporation, is traditionally achieved by the application of millisecond-duration electric pulses as it is believed that different transmembrane pathways/mechanisms are involved in chemotherapeutic vs. pDNA transport.49 When ECT is used in combination with GET traditionally two different types of pulses would be necessary (conventional 8 × 100 μs pulses for ECT and millisecond duration pulses for GET).
Changing the conventional 8 × 100 μs pulses to an alternative type of pulse such as high-frequency bipolar pulses or millisecond pulses could thus be advantageous in ECT. However, it is not well understood whether the use of alternative types of pulses would compromise the efficiency of the ECT treatment. Recently,
In this study, we expanded upon Vižintin
We used Chinese hamster ovary cell line (CHO-K1; cat. no. 85051005, European Collection of Authenticated Cell Cultures, United Kingdom). Cells were grown in 25 cm2 culture flasks (no. 90026, TPP, Switzerland) for 2–4 days in an incubator at 37°C, in a humidified atmosphere with 5% CO2. CHO cells were cultured in Ham-F12 growth medium (cat.no. N6658, Sigma Aldrich, Germany) supplemented with 10% fetal bovine serum (cat. No. F9665, Sigma Aldrich, Germany), L-glutamine (cat. No. G7513, Sigma Aldrich, Germany), antibiotics penicillin/streptomycin (cat.no. P0781, Sigma Aldrich, Germany), and gentamycin (cat.no. G1397, Sigma Aldrich, Germany). The cell suspension was prepared by detaching the cells in the exponential growth phase with 10× trypsin-EDTA (cat. no. T4174, Sigma Aldrich, Germany), diluted 1:9 in Hank’s basal salt solution (cat. no. H4641, Sigma Aldrich, Germany). After no more than 2 minutes, trypsin was inactivated by adding Ham-F12, and cells were transferred to a 50 ml centrifuge tube. Then, the cells were centrifuged (5 min, 180 g, 21°C) and re-suspended in Dulbecco’s Modified Eagle Medium (DMEM, cat. no. D5671, Sigma-Aldrich, Missouri, United States) supplemented with 10% FBS (cat. no. F9665, Sigma-Aldrich), 2.0 mM L-glutamine, 1 U/ml penicillin-streptomycin and 50 μg/ml gentamycin. The CHO cells were re-suspended at concentrations of 4 × 106 cells/ml (permeability and survival experiments for determination of the optimal electric field) and 4.2 × 106 cells/ml (for the clonogenic assay experiments and intracellular platinum concentration experiments).
Three different types of pulses were used to perform experiments. For brevity, we refer to the three types of pulses used as 50 × 50 HF pulses, 8 × 100 μs pulses, and 8 × 5 ms pulses, and they are described in detail as follows. (i) The first type consisted of high-frequency bipolar pulses, specifically 50 bursts with a repetition frequency of 1 Hz. Each burst contained 50 short bipolar pulses having a pulse duration of 2 μs for the positive as well as for the negative pulse. The interpulse delay between consecutive bipolar pulses was 2 μs (Figure 1A). These high-frequency bipolar pulses were delivered by the pulse generator L-POR V0.1 (mPOR, Slovenia) at various voltages ranging from 80 V to 320 V with a step of 40 V. (ii) The second type of pulses consists of eight 100 μs monopolar pulses delivered at a repetition frequency of 1 Hz. These pulses were delivered by a prototype pulse generator based on H-bridge digital amplifier with 1 kV MOSFETs developed in our lab and described previously.39 The voltage of these pulses varied from 80 V to 320 V with a step of 40 V, Figure 1B. (iii) The third type consists of eight 5 ms long monopolar pulses, delivered at a repetition frequency of 1 Hz. These pulses were delivered by BTX Gemini X2 pulse generator (Harvard Apparatus, USA). Note that the pulse on time (the time when the voltage was different than zero) was 20 ms for 50 × 50 HF pulses, 800 μs for 8 × 100 μs, and 40 ms for 8 × 5 ms pulses. The voltage of these pulses varied from 80 V to 160 V with a step of 20 V (Figure 1B). The electric pulses were applied to cells in suspension placed in 2 mm aluminium cuvette. To ensure the quality of the delivered pulses the voltage and the current were monitored in all experiments with an oscilloscope Wavesurfer 422, 200 MHz, a differential voltage probe ADP305, and a current probe CP030 (from LeCroy, USA), according to the recommendations.53
To select the optimal electric field strength, i.e., where the highest cell membrane permeability and highest survival are achieved, we determined the so-called permeability and survival curves for each of the tested types of pulses. The selected optimal electric field strength was later used in the experiments with cisplatin.
To determine the permeability curve, the cell suspension was mixed with YO-PRO-1 iodide (cat. no Y3603, Thermo Fisher Scientific, Massachusetts, USA) to a final concentration of 1 μM. 150 μl of cells-YO-PRO-1 mixture was transferred in a 2 mm aluminum cuvette and then pulses were applied. 20 μl of the treated sample was transferred to a 1.5 ml centrifuge tube. Three minutes after pulse delivery the treated sample was diluted in 150 μl of DMEM and vortexed. The uptake of YO-PRO-1 was measured on the flow cytometer (Attune NxT; Life Technologies, Carlsbad, CA, USA). Cells were excited with a blue laser at 488 nm, and the emitted fluorescence was detected through a 530/30 nm band-pass filter. For each measurement, we acquired 10,000 events. Single cells were separated from all events by gating. Obtained data were analyzed using the Attune NxT software. The percentage of permeabilized cells was determined from the histogram of YO-PRO-1 fluorescence (see Supplementary Info S1).
To determine the survival curve, 150 μl of cell suspension was transferred to a 2 mm aluminum cuvette and then pulses were applied. 20 μl of the treated sample was transferred to a 1.5 ml centrifuge tube, and 25 minutes after pulse delivery, the samples were diluted in 380 μl Ham-F12. The cell suspension was gently mixed and 100 μl were transferred per well of a 96-well plate in triplicates. After 24 h of incubation in a humidified atmosphere at 37°C and 5% CO2, the MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS), Promega, USA54) was performed. The MTS assay was used to quantify the number of viable cells by evaluating their metabolic activity by measuring the formazan absorbance at 490 nm on a microplate reader (Tecan Infinite 200 pro; Tecan, Grödig, Austria). Cell survival was determined by first subtracting the background (signal from blank wells containing medium without cells) from all measurements and then normalizing the absorbance of the treated samples to the absorbance of the control sample.
We determined cisplatin cytotoxicity in combination with electroporation pulses using the clonogenic assay which is based on the ability of a single cell to divide and grow into colonies.55 On the day of the experiment, saline solution was used to dilute cisplatin (Cisplatin Kabi, 1 mg/ml, Fresenius Kabi, Germany or Cisplatin Accord, 1 mg/ml, Accord, UK) and prepare working solutions. The final concentrations of cisplatin during electroporation were 0 μM, 10 μM, 30 μM, and 50 μM. First, 150 μl of cell suspension with added specific cisplatin concentration was transferred to a 2 mm aluminium cuvette and then the suspension was exposed to the selected type of pulses of the optimal electric field as described in the subsection
Cells were prepared and treated with electric pulses in the presence of different concentrations of cisplatin, as described in previous section 25 minutes after pulse delivery, each sample was diluted in Ham-F12, centrifuged, and washed twice. The cell pellet was separated from the supernatant and the intracellular concentration of platinum was analyzed using inductively coupled plasma mass spectrometry (ICP-MS). To aid sample digestion, 0.1 ml of H2O2 and 0.1 ml of HNO3 (both from Merck, Darmstadt, Germany), were added to the cell pellets. The tubes were then sealed with caps and Teflon tape and left overnight at 80°C. Following digestion, 1.8 ml of Milli-Q water (Direct-Q 5 Ultrapure water system; Merck Millipore, Massachusetts, USA) was added. The platinum content in the samples was then measured using ICP-MS (7900 ICP-MS; Agilent Technologies, California, USA) with 193Ir (Merck, Darmstadt, Germany) used as an internal standard during the measurement.
To determine the amount of Pt per cell, the number of cells in the pellet was divided with the measured Pt in the cell pellet of each sample. To assess the number of cisplatin molecules per cell, it was assumed that 1 mol of Pt is equivalent to 1 mol of cisplatin. Control samples (not electroporated cells that were not incubated with cisplatin) were used for blank subtraction for all cisplatin-treated samples. To reduce cross-contamination of the instrument during the measurement, a mixture containing 1% HNO3 and 1% HCl (Merck, Darmstadt, Germany) was used as a rinse between the sample runs.
Statistical analysis was performed using Prism 9.4.1 (GraphPad Software, USA). For permeability and survival experiments we performed oneway ANOVA if the normality test passed, or the ANOVA on ranks if the normality test failed with the Shapiro-Wilk test. For cisplatin cytotoxicity and cisplatin uptake experiments, we performed two-way ANOVA (independent variables: cisplatin concentration and pulse type). The normality test passed with the D’Agostino-Pearson test. Statistically significant difference was analyzed with respect to the control group (no cisplatin, no pulses) for all experiments. In the figures, the asterisk (*) indicates p < 0.05.
To model the intracellular uptake of cisplatin molecules, we used the phenomenological model developed by Sweeney
The source current
The term
The increase of the transmembrane voltage results in the formation of small pores/defects (first porous state
We implemented the model using a custom script in Matlab 2019b (MathWorks, Natick, MA, USA) and verified that the model in its original form reproduces the published results.56 The model was developed using the same cell line as in this study (CHO-K1) but based on quantitative measurements of propidium iodide uptake. Therefore, we modified the parameters related to the transported molecule, in our case cisplatin. Specifically, we changed the value of the solute radius
The term
Model parameters
Electroporation threshold voltage | 258 mV | 56 | |
Membrane thickness | 5 nm | 56 | |
Cell radius | 7.5 μm | 56 | |
Membrane time constant | 1 µs | 56 | |
Membrane permittivity | 12 × 8.85 × 10−12 F/m | 56 | |
Solute radius | 0.58 nm | 58 | |
Defect radius | 0.8 nm | 56 | |
Solute radius/Defect radius | 0.7250 | 56 | |
Solute diffusivity | 1.670 × 10−9 m2/s | 58,59 | |
Parameter in N formation rate | 2 × 10−6 | 56 | |
N relaxation rate | 4 × 10−8 | 56 | |
Relative permeabilzed conductance | 1 × 106 | 56 | |
Parameter in M formation rate | 1 × 10−3 | 56 | |
M relaxation rate | 4 × 10−9 | 56 | |
Permeability coefficient | 8.45 × 10−4 | 56 | |
Electroporation medium conductivity | 1.4 S/m | * |
Measured conductivity of DMEM using a conductometer (Mettler Toledo, S230)
The output of the model is the time course of the intracellular concentration of cisplatin
First, we performed experiments to determine the optimal electric field strength for each of the three tested types of pulses, to be later used in the experiments with cisplatin. As the optimal electric field strength, we consider the one in which the highest permeability and highest cell survival are achieved.
Figure 3 shows the experimentally determined permeability (dashed) and survival (solid) curves as a function of the applied electric field for A) 50 × 50 HF pulses, B) 8 × 100 μs pulses, and C) 8×5 ms pulses. Permeability curves show how the percentage of permeabilized cells increases with increasing electric field strength, whereas the survival curves show how the percentage of viable cells decreases with increasing electric field strength. The chosen optimal electric fields (i.e., highest permeability and highest survival) are 1.4 kV/cm for 50 × 50 HF pulses, 1.2 kV/cm for 8 × 100 μs pulses, and 0.6 kV/cm for 8 × 5 ms pulses.
We next used the clonogenic assay to determine the cytotoxicity of cisplatin when exposing cells to the three types of pulses at their optimal electric field strength. Figure 4A shows how cell survival decreases as the extracellular concentration of cisplatin increases. In the absence of applied pulses, the tested cisplatin concentrations (0 μM, 10 μM, 30 μM, and 50 μM) do not affect cell viability (black curve). However, cytotoxicity is strongly potentiated with all three types of pulses, decreasing the cell survival to ~0.8% for 50 × 50 HF pulses, ~2.7% for 8 × 100 μs pulses, and ~4% for 8 × 5 ms pulses at the highest cisplatin concentration (50 μM) − note the logarithmic scale. Results for all three types of pulses are similar and are not statistically significantly different. Qualitatively similar results were obtained when measuring cell viability with the metabolic MTS assay (see Supplementary Info S2). However, as well known, the MTS assay reported better cell viability than the clonogenic assay for the same experimental conditions.52,60
We also measured the mass of intracellular Pt for each tested condition using ICP-MS and determined the average number of intracellular cisplatin molecules per cell, assuming that 1 mol of Pt is equivalent to 1 mol of cisplatin (Figure 4B). When no electric pulses are applied (black line), the number of cisplatin molecules increases slightly with increasing cisplatin concentration due to passive (i.e., diffusion), and active (i.e., membrane transporters61,62,63, endocytosis, pinocytosis, macrocytosis64,65) transport of cisplatin. However, when electric pulses are applied, the number of cisplatin molecules increases considerably. The greatest increase is observed for 8 × 5 ms pulses (up to 6.7 × 107 at 50 μM). Roughly 2 times lower increase is observed for both 50 × 50 HF pulses and 8 × 100 μs pulses. There is a statistically significant difference between 8 × 5 ms pulses and the other two types of tested pulses when extracellular cisplatin concentration is 50 μM.
To determine the number of intracellular cisplatin molecules needed to achieve a cytotoxic effect, we combined the data from Figures 4A and 4B and plotted cell survival as a function of the number of cisplatin molecules in Figure 4C. Consistent with previous observations13,52, electroporation potentiates the cytotoxicity of cisplatin, as much lower survival is obtained with any of the three types of pulses compared with control for the same number of cisplatin molecules. The curves for 50 × 50 HF pulses and 8 × 100 μs pulses are almost overlapping, demonstrating that practically the same number of cisplatin molecules results in the same cytotoxic effect. Interestingly, in spite of two times higher Pt continent for 8 × 5 ms pulses the cytotoxicity is lower than when using 50 × 50 HF pulses or 8 × 100 μs pulses.
A previous study by Vižintin
Experimental data in previous section suggests that any type of pulses can be used for ECT, if it results in the same average number of internalized cisplatin molecules. Therefore, it would be useful to have a mathematical model for predicting the uptake of cisplatin molecules as a function of the pulse parameters. Figure 5 compares the measured uptake of cisplatin with prediction from a phenomenological model developed by Sweeney
In this study, we investigated how different types of pulses affect ECT
Different types of pulses can be considered equivalent for electroporation when the electric field strength is adjusted for each type of pulses separately.66 We thus first determined, for each selected type of pulses, how the electric field strength affects the percentage of cells that become permeable to YO-PRO-1 and the percentage of cells that survive the exposure to electric pulses in the absence of cisplatin. YO-PRO-1 is a nucleic acid stain that allows rapid screening of permeabilized cells using flow cytometry. The size of YO-PRO-1 (630 Da) is somewhat larger, but nevertheless comparable to cisplatin (300 Da). Therefore, cells that become permeable to YO-PRO-1 are expected to also become permeable to cisplatin. Cell survival was measured 24 hours after electroporation with the metabolic MTS assay. An
We then performed
By combining the results on cisplatin cytotoxicity and cisplatin uptake, we were able to determine the number of internalized cisplatin molecules needed to achieve a cytotoxic effect. For all tested types of pulses, this number was in the range of 2−7 ×107 cisplatin molecules per cell. Same range was obtained by Vižintin
ECT has been demonstrated as a locally effective treatment of tumors of various histotypes.72 The consistent clinical success of ECT has been achieved through the meticulous development of pulse protocols, electrodes, and the publication of Standard Operating Procedures for cutaneous and subcutaneous tumors.11 It has been later demonstrated that also deep-seated tumors can be successfully treated by ECT provided the tumor is covered by sufficiently high electric fields either as intraoperative5 or percutaneous procedure.31,73,74,75 Accordingly, Standard Operating Procedures have been updated.10 With good success and acceptance by patients, larger tumors and patients with more extensive diseases were treated. Pain and muscle contraction-related high voltage pulse delivery became the most often reported side effects and alternative pulse waveforms that would maintain ECT efficacy but reduce pain and muscle contraction.24 In this respect, high-frequency bipolar short pulses 33,34 and also nanosecond pulses 76,77 were suggested. Furthermore, recent studies investigate how high-frequency bipolar pulses and nanosecond pulses affect ECT. Our previous
A study by Vižintin
Considerable efforts are also focused on making ECT a systemic treatment by combining it with immunotherapy.20,48,80,81 Electrochemotherapy can induce immunogenic cell death through the release of damage-associated molecular patterns (DAMP) which serve as a signal to stimulate the immune system.82,83,84 Massive liberation of tumor antigens together with DAMPs can activate the antigen-presenting dendritic cells.85,86 Multiple studies in canine87,88,89, in mice47 and human patients90 have thus been testing ECT in combination with gene electrotransfer (GET) of plasmid DNA encoding for interleukin-12 (IL-12), which stimulates the immune system.45,46,47 Traditionally in GET millisecond duration pulses are used to deliver DNA into the cells.91,92,93 Thus, when ECT is used in combination with GET two different types of pulses 8 × 100 μs pulses and millisecond pulses are used, respectively. However, it might be beneficial to use the same type of pulses when ECT is combined with GET as this would allow the use of simpler pulse generators.
In order to capitalize on a significant body of clinical evidence we tested equivalence of such pulses by
For the success of ECT, all the tumor needs to be covered by an electric field of sufficient amplitude to permeabilize the cells/tissue, and a sufficient amount of chemotherapeutic drug is needed in the tumor.
In the Standard Operating Procedures all information related to the types of electrodes i.e., of fixed geometry, pulse parameters, and pulse generators10,11,22 which guarantee a complete coverage of the tumor are provided. However, to treat deep-seated tumors long needles with variable configuration electrodes are used.95 Thus, there is a need to determine the optimal position of the electrodes and the optimal pulse parameters for complete coverage of the tumor tissue. It is not trivial to determine how the electric field is distributed in biological tissues due to tissue-specific properties, the use of different types of electrode geometries, and pulse parameters. Treatment planning using numerical models helps clinicians to determine the optimal parameters to treat a specific tumor. Currently, in treatment planning a fixed threshold electric field is used to deem tissue permeabilized or not.3,96 However, just a high-enough electric field does not guarantee cell death as simultaneously a high-enough extracellular cisplatin concentration is needed to obtain enough internalized cisplatin molecules for cell death. Our model presents a missing link in the complete model for treatment planning, connecting the external electric field, the number of internalized cisplatin molecules, and cell death. The first building block for the multiscale model of tissue electroporation was published in Dermol-Černe
A mathematical model that can also predict the uptake of molecules such as chemotherapeutics drugs (e.g., cisplatin and bleomycin) is needed to be determined for treatment planning for different pulse types is useful. Now we only need the final piece, and this is a model of cisplatin transport across the cell membrane as a function of electric pulses. Our previous study demonstrated that existing mechanistic models of electroporation have limited reliability for predicting the transmembrane transport of small molecules across a wide range of pulse parameters.97 We observed that the contribution of electrophoretic transport during pulse delivery is often overestimated. Therefore, we decided to test a phenomenological model developed by Sweeney
The drawback of our study is the use of only one cell line i.e., the Chinese hamster ovary cells (CHO-K1) non-cancerous cells to perform
It has been shown that the immune system plays an important role in the efficiency of ECT. Electroporation can potentiate the cytotoxicity and uptake of cisplatin but can also stimulate the immune response by releasing damage-associated molecular patterns (DAMP). In our
We have focused on equivalent drug delivery to cells
Our study focused on the effect of different types of electric pulses in ECT, particularly in terms of cisplatin uptake and cisplatin cytotoxicity, using CHO cells
In addition, we experimentally determine the number of cisplatin molecules needed to achieve a cytotoxic effect which is in the range of 2−7 × 107 cisplatin molecules per cell in agreement with previous study.52 We also used a mathematical model describing electroporation and transmembrane molecular transport, as a tool to predict the number of cisplatin molecules into individual cells when different types of pulses need to be tested. The future goal is to improve treatment planning by including a model that predicts the uptake of molecules such as cisplatin or bleomycin.