Introduction:: Irreversible electroporation (IRE) is a minimally invasive, non-thermal tissue ablation modality in which high voltage, short duration (100μs) electrical pulses are employed to permeabilize the lipid bilayer and disrupt cellular homeostasis [1]. In a clinical setting, IRE has been shown to induce muscle contractions, requiring a neuroparalytic during treatment as well as cardiac synchronization. A second generation therapy termed high frequency irreversible electroporation (HFIRE) was developed to mitigate these pitfalls. HFIRE uses a bipolar waveform with short bursts of 0.5-10μs length pulses in which the polarity is switched after each subsequent pulse [2]. It has been demonstrated that both IRE and HFIRE may trigger an immune response induced by both accidental cell death (necrosis) or regulated cell death (apoptosis, pyroptosis, and necroptosis)[3]. Different mechanisms of cell death may lead to varying types and/or levels of immunogenicity. Damage associated molecular patterns (DAMPs) specific to a type of cell death can indicate the extent of irreversible damage done to the tissue area of interest. By correlating the level of DAMPs release for each type of cell death for each pulse width, we can determine what cell death mechanism is most apparent in certain areas of treatment relative to the electrodes. Different types of cell death may be favorable for patient specific needs if a certain level of immune response is desired. Understanding how pulse width modulates the cell death response could increase the efficacy of personalized treatment planning with electroporation therapy.

Materials and Methods:: Human malignant glioblastoma cells (U251) were cultured in a well-plate to create a 2D monolayer. The cells were treated with varying pulse widths through a two-needle electrode configuration. 24-hours post-treatment a live/dead fluorescent dye was used to stain the cells and measure the resultant lesion area. Electric field thresholds (EFT) were determined based on the area of cell death and were used to normalize the applied field among the waveforms. Normalization allows for an equal level of cell death to occur across the varying pulse widths. Four electric field strengths (0.5x, 1x, 1.5x, and 2x) were tested with each pulse width to quantify any effect due to the electric field magnitude. All samples were pulsed in a uniform electric field between parallel plate electrodes in a low conductivity calcium-based buffer [4]. Three assays were performed: ATP, Caspase 3/7, and Caspase 1. An ATP assay was performed to assess an overall immunogenic response. The supernatant was collected from each sample for a 0.5 and 24 hour test point to be measured for ATP concentration. A colorimetric Caspase 3/7 assay was used to quantify apoptotic cell death and a fluorometric Caspase 1 assay was used for pyroptotic cell death. The collected samples were adjusted to have the same protein levels and the assay protocols were carried out according to the manufacturer. Each assay was run for all waveforms and electric field intensity combinations.

Results, Conclusions, and Discussions:: We found that the majority of ATP was released almost immediately after treatment at our 0.5 hour test point while minimal excess ATP is released up to 24 hours post-treatment. Cellular ATP depletion after IRE and H-FIRE has been shown to occur as the ATP leaks through the permeabilized membrane [5]. We found that the ATP released was positively correlated to both the applied electric field magnitude and pulse width. We normalized the baseline applied electric field to induce equal amounts of cell death between each pulse width. The increase in DAMP at comparable viabilities indicates that we can modulate ablations with certain cell death mechanisms.

We found that Caspase 3/7 activation was significantly higher than the control for the two shortest pulse widths (1μs and 5μs) at electric fields well above lethal threshold (2*EFT). Minimal caspase 3/7 activation was observed with the higher pulse widths which coincides with previous studies [6]. Caspase 1 activation displayed opposite trends from that of caspase 3/7. Caspase 1 activation was significantly higher at sublethal electric fields.

Our results indicate higher levels of apoptotic cell death occur at lower pulse widths, increasing in electric field magnitude, and higher levels of pyroptotic cell death occur at higher pulse widths and lower electric field strengths. This suggests that during treatment there are multiple layers of cell death; apoptosis occurs close to the electrodes where higher electric fields are present while pyroptosis occurs further from the electrodes where there are lower electric fields. We can optimize the pulse parameters based on our results to achieve ablations with favorable modes of cell death. This can significantly improve electroporation based therapies for patient-specific treatment planning where inflammation is desired.


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1. Edd, J. F., Horowitz, L., Davalos, R. V., Mir, L. M. & Rubinsky, B. In vivo results of a new focal tissue ablation technique: irreversible electroporation. IEEE Transactions on Biomedical Engineering 53, 1409–1415 (2006).

2. Arena, C. B. et al. High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction. BioMedical Engineering OnLine 10, 102 (2011).
3. Polajzer, T., Jarm, T. & Miklavcic, D. Analysis of damage-associated molecular pattern molecules due to electroporation of cells in vitro. Radiology and Oncology 54, 317–328 (2020).



4. Wasson, E. M. et al. Understanding the role of calcium-mediated cell death in high-frequency irreversible electroporation. Bioelectrochemistry 131, 107369 (2020).



5. Acute ATP loss during irreversible electroporation mediates caspase independent cell death - ScienceDirect.


6. Mercadal, B. et al. Dynamics of Cell Death After Conventional IRE and H-FIRE Treatments. Ann Biomed Eng 48, 1451–1462 (2020).