Search

Irreversible electroporation (IRE) is a minimally thermal tissue ablation modality used to treat solid tumors adjacent to critical structures. Widespread clinical adoption of IRE has been limited due to complicated anesthetic management requirements and technical demands associated with placing multiple needle electrodes in anatomically challenging environments. High-frequency irreversible electroporation (H-FIRE) delivered using a novel single-insertion bipolar probe system could potentially overcome these limitations, but ablation volumes have remained small using this approach. While H-FIRE is minimally thermal in mode of action, high voltages or multiple pulse trains can lead to unwanted Joule heating. In this work, we improve the H-FIRE waveform design to increase the safe operating voltage using a single-insertion bipolar probe before electrical arcing occurs. By uniformly increasing interphase ( d 1 ) and interpulse ( d 2 ) delays, we achieved higher maximum operating voltages for all pulse lengths. Additionally, increasing pulse length led to higher operating voltages up to a certain delay length (  ∼ 25 μs), after which shorter pulses enabled higher voltages. We then delivered novel H-FIRE waveforms via an actively cooled single-insertion bipolar probe in swine liver in vivo to determine the upper limits to ablation volume possible using a single-needle H-FIRE device. Ablations up to 4.62 ± 0.12cm x 1.83 ± 0.05cm were generated in 5 minutes without a requirement for cardiac synchronization during treatment. Ablations were minimally thermal, easily visualized with ultrasound, and stimulated an immune response 24 hours post H-FIRE delivery. These data suggest H-FIRE can rapidly produce clinically relevant, minimally thermal ablations with a more user-friendly electrode design.

In this study, the feasibility of treating canine primary lung tumors with high-frequency irreversible electroporation (H-FIRE) was investigated as a novel lung cancer treatment option. H-FIRE is a minimally invasive tissue ablation modality that delivers bipolar pulsed electric fields to targeted cells, generating nanopores in cell membranes and rendering targeted cells nonviable. In the current study, canine patients ( n = 5) with primary lung tumors underwent H-FIRE treatment with an applied voltage of 2250 V using a 2-5-2 µs H-FIRE waveform to achieve partial tumor ablation prior to the surgical resection of the primary tumor. Surgically resected tumor samples were evaluated histologically for tumor ablation, and with immunohistochemical (IHC) staining to identify cell death (activated caspase-3) and macrophages (IBA-1, CD206, and iNOS). Changes in immunity and inflammatory gene signatures were also evaluated in tumor samples. H-FIRE ablation was evident by the microscopic observation of discrete foci of acute hemorrhage and necrosis, and in a subset of tumors ( n = 2), we observed a greater intensity of cleaved caspase-3 staining in tumor cells within treated tumor regions compared to adjacent untreated tumor tissue. At the study evaluation timepoint of 2 h post H-FIRE, we observed differential gene expression changes in the genes IDO1 , IL6 , TNF , CD209 , and FOXP3 in treated tumor regions relative to paired untreated tumor regions. Additionally, we preliminarily evaluated the technical feasibility of delivering H-FIRE percutaneously under CT guidance to canine lung tumor patients ( n = 2). Overall, H-FIRE treatment was well tolerated with no adverse clinical events, and our results suggest H-FIRE potentially altered the tumor immune microenvironment.

Pancreatic cancer is a significant cause of cancer-related mortality and often presents with limited treatment options. Pancreatic tumors are also notorious for their immunosuppressive microenvironment. Irreversible electroporation (IRE) is a non-thermal tumor ablation modality that employs high-voltage microsecond pulses to transiently permeabilize cell membranes, ultimately inducing cell death. However, the understanding of IRE's impact beyond the initiation of focal cell death in tumor tissue remains limited. In this study, we demonstrate that IRE triggers a unique mix of cell death pathways and orchestrates a shift in the local tumor microenvironment driven, in part, by reducing the myeloid-derived suppressor cell (MDSC) and regulatory T cell populations and increasing cytotoxic T lymphocytes and neutrophils. We further show that IRE drives induce cell cycle arrest at the G0/G1 phase in vitro and promote inflammatory cell death pathways consistent with pyroptosis and programmed necrosis in vivo . IRE-treated mice exhibited a substantial extension in progression-free survival. However, within a span of 14 days, the tumor immune cell populations reverted to their pre-treatment composition, which resulted in an attenuation of the systemic immune response targeting contralateral tumors and ultimately resulting in tumor regrowth. Mechanistically, we show that IRE augments IFN- γ signaling, resulting in the up-regulation of the PD-L1 checkpoint in pancreatic cancer cells. Together, these findings shed light on potential mechanisms of tumor regrowth following IRE treatment and offer insights into co-therapeutic targets to improve treatment strategies., Competing Interests: IA, and RD are inventors on pending and issued patents related to the work. The authors declare no additional conflicts of interest. IA and RD have had projects supported, in part, by industry funding from business entities utilizing or developing IRE-based technologies for commercial applications. Those relevant to the current study include AngioDynamics, Galvanize Therapeutics, and ManaMed Tech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Imran, Brock, Beitel-White, Powar, Orr, Aycock, Alinezhadbalalami, Salameh, Eversole, Tintera, Markov Madanick, Hendricks-Wenger, Coutermarsh-Ott, Davalos and Allen.)

The efficiency of electroporation treatments depends on the application of a critical electric field over the targeted tissue volume. Both the electric field and temperature distribution strongly depend on the tissue-specific electrical properties, which both differ between patients in healthy and malignant tissues and change in an electric field-dependent manner from the electroporation process itself. Therefore, tissue property estimations are paramount for treatment planning with electroporation therapies. Ex vivo methods to find electrical tissue properties often misrepresent the targeted tissue, especially when translating results to tumors. A voltage ramp is an in situ method that applies a series of increasing electric potentials across treatment electrodes and measures the resulting current. Here, we develop a robust deep neural network, trained on finite element model simulations, to directly predict tissue properties from a measured voltage ramp. There was minimal test error (R 2 >0.94;p<0.0001) in three important electric tissue properties. Further, our model was validated to correctly predict the complete dynamic conductivity curve in a previously characterized ex vivo liver model (R 2 >0.93;p<0.0001) within 100 s from probe insertion, showing great utility for a clinical application. Lastly, we characterize the first reported electrical tissue properties of lung tumors from five canine patients (R 2 >0.99;p<0.0001). We believe this platform can be incorporated prior to treatment to quickly ascertain patient-specific tissue properties required for electroporation treatment planning models or real-time treatment prediction algorithms. Further, this method can be used over traditional ex vivo methods for in situ tissue characterization with clinically relevant geometries., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors have patents related to the paper. R.V.D. has ownership interest in startup companies within the field of bioelectrics. In addition, R.V.D. also receives royalty income from technologies he has invented and serves as a consultant., (Copyright © 2023. Published by Elsevier B.V.)

This study introduces a new method of targeting acidosis (low pH) within the tumor microenvironment (TME) through the use of cathodic electrochemical reactions (CER). Low pH is oncogenic by supporting immunosuppression. Electrochemical reactions create local pH effects when a current passes through an electrolytic substrate such as biological tissue. Electrolysis has been used with electroporation (destabilization of the lipid bilayer via an applied electric potential) to increase cell death areas. However, the regulated increase of pH through only the cathode electrode has been ignored as a possible method to alleviate TME acidosis, which could provide substantial immunotherapeutic benefits. Here, we show through ex vivo modeling that CERs can intentionally elevate pH to an anti-tumor level and that increased alkalinity promotes activation of naïve macrophages. This study shows the potential of CERs to improve acidity within the TME and that it has the potential to be paired with existing electric field-based cancer therapies or as a stand-alone therapy., (© 2023. The Author(s) under exclusive licence to Biomedical Engineering Society.)

The nonthermal mechanism for irreversible electroporation has been paramount for treating tumors and cardiac tissue in anatomically sensitive areas, where there is concern about damage to nearby bowels, ducts, blood vessels, or nerves. However, Joule heating still occurs as a secondary effect of applying current through a resistive tissue and must be minimized to maintain the benefits of electroporation at high voltages. Numerous thermal mitigation protocols have been proposed to minimize temperature rise, but intraoperative temperature monitoring is still needed. We show that an accurate and robust temperature prediction AI model can be developed using estimated tissue properties (bulk and dynamic conductivity), known geometric properties (probe spacing), and easily measurable treatment parameters (applied voltage, current, and pulse number). We develop the 2-layer neural network on realistic 2D finite element model simulations with conditions encompassing most electroporation applications. Calculating feature contributions, we found that temperature prediction is mostly dependent on current and pulse number and show that the model remains accurate when incorrect tissue properties are intentionally used as input parameters. Lastly, we show that the model can predict temperature rise within ex vivo perfused porcine livers, with error <0.5 °C. This model, using easily acquired parameters, is shown to predict temperature rise in over 1000 unique test conditions with <1 °C error and no observable outliers. We believe the use of simple, readily available input parameters would allow this model to be incorporated in many already available electroporation systems for real-time temperature estimations., Competing Interests: Declaration of competing interest The authors have patents related to the paper. R.V.D. has ownership interest for startup companies within the field of bioelectrics. In addition, R.V.D. also receives royalty income from technologies he has invented and serves as a consultant., (Copyright © 2023. Published by Elsevier Ltd.)

Tissue electroporation is the basis of several therapies. Electroporation is performed by briefly exposing tissues to high electric fields. It is generally accepted that electroporation is effective where an electric field magnitude threshold is overreached. However, it is difficult to preoperatively estimate the field distribution because it is highly dependent on anatomy and treatment parameters., Objective: We developed PIRET, a platform to predict the treatment volume in electroporation-based therapies., Methods: The platform seamlessly integrates tools to build patient-specific models where the electric field is simulated to predict the treatment volume. Patient anatomy is segmented from medical images and 3D reconstruction aids in placing the electrodes and setting up treatment parameters., Results: Four canine patients that had been treated with high-frequency irreversible electroporation were retrospectively planned with PIRET and with a workflow commonly used in previous studies, which uses different general-purpose segmentation (3D Slicer) and modeling software (3Matic and COMSOL Multiphysics). PIRET outperformed the other workflow by 65 minutes (× 1.7 faster), thanks to the improved user experience during treatment setup and model building. Both approaches computed similarly accurate electric field distributions, with average Dice scores higher than 0.93., Conclusion: A platform which integrates all the required tools for electroporation treatment planning is presented. Treatment plan can be performed rapidly with minimal user interaction in a stand-alone platform., Significance: This platform is, to the best of our knowledge, the most complete software for treatment planning of irreversible electroporation. It can potentially be used for other electroporation applications.

To evaluate the feasibility of real-time temperature monitoring during an electroporation-based therapy procedure, a data-driven state-space model was developed. Agar phantoms mimicking low conductivity (LC) and high conductivity (HC) tissues were tested under the influences of high (HV) and low (LV) applied voltages. Real-time changes in impedance, measured by Fourier Analysis SpecTroscopy (FAST) along with the known tissue conductivity and applied voltages, were used to train the model. A theoretical finite element model was used for external validation of the model, producing model fits of 95.8, 88.4, 90.7, and 93.7% at 4 mm and 93.2, 58.9, 90.0, and 90.1% at 10 mm for the HV-HC, LV-LC, HV-LC, and LV-HC groups, respectively. The proposed model suggests that real-time temperature monitoring may be achieved with good accuracy through the use of real-time impedance monitoring.

Irreversible electroporation (IRE), or pulsed field ablation, employs microsecond-duration pulsed electric fields to generate targeted cellular damage without injury to the underlying tissue architecture. Biphasic, burst-type waveforms (termed high-frequency IRE, or H-FIRE) have garnered attention for their ability to elicit clinically relevant ablation volumes while reducing several undesirable side effects (muscle contractions/electrochemical effects) seen with monophasic pulses. Pulse width is generally the main (or only) parameter considered during burst construction, with little attention given to the delays within the burst. In this work, we tested the hypothesis that H-FIRE waveforms could be further optimized by manipulating only the interpulse delay between biphasic pulses within each burst. Using benchtop, ex vivo, and in vivo models, we demonstrate that extended interpulse delays (i.e., ~100 μs) reduce the severity of induced muscle contractions, alleviate mechanical tissue destruction, and minimize the chances of electrical arcing. Clinical Relevance- This proof-of-concept study shows that H-FIRE waveforms with extended interpulse delays provide several therapeutic benefits over conventional waveforms.

While the primary goal of focal therapy for prostate cancer (PCa) is conserving patient quality of life by reducing oncological burden, available modalities use thermal energy or whole-gland radiation which can damage critical neurovascular structures within the prostate and increase risk of genitourinary dysfunction. High-frequency irreversible electroporation (H-FIRE) is a promising alternative ablation modality that utilizes bursts of pulsed electric fields (PEFs) to destroy aberrant cells via targeted membrane damage. Due to its nonthermal mechanism, H-FIRE offers several advantages over state-of-the-art treatments, but waveforms have not been optimized for treatment of PCa. In this study, we characterize lethal electric field thresholds (EFTs) for H-FIRE waveforms with three different pulse widths as well as three interpulse delays in vitro and compare them to conventional irreversible electroporation (IRE). Experiments were performed in non-neoplastic and malignant prostate cells to determine the effect of waveforms on both targeted (malignant) and adjacent (non-neoplastic) tissue. A numerical modeling approach was developed to estimate the clinical effects of each waveform including extent of nonthermal ablation, undesired thermal damage, and nerve excitation. Our findings indicate that H-FIRE waveforms with pulse durations of 5 and 10 μs provide large ablations comparable to IRE with tolerable levels of thermal damage and minimized muscle contractions. Lower duration (2 μs) H-FIRE waveforms exhibit the least amount of muscle contractions but require increased voltages which may be accompanied by unwanted thermal damage., (Copyright © 2022 by ASME.)

Improved therapeutics for malignant brain tumors are urgently needed. High-frequency irreversible electroporation (H-FIRE) is a minimally invasive, nonthermal tissue ablation technique, which utilizes high-frequency, bipolar electric pulses to precisely kill tumor cells. The mechanisms of H-FIRE-induced tumor cell death and potential for cellular recovery are incompletely characterized. We hypothesized that tumor cells treated with specific H-FIRE electric field doses can survive and retain proliferative capacity. F98 glioma and LL/2 Lewis lung carcinoma cell suspensions were treated with H-FIRE to model primary and metastatic brain cancer, respectively. Cell membrane permeability, apoptosis, metabolic viability, and proliferative capacity were temporally measured using exclusion dyes, condensed chromatin staining, WST-8 fluorescence, and clonogenic assays, respectively. Both tumor cell lines exhibited dose-dependent permeabilization, with 1,500 V/cm permitting and 3,000 V/cm inhibiting membrane recovery 24 h post-treatment. Cells treated with 1,500 V/cm demonstrated significant and progressive recovery of apoptosis and metabolic activity, in contrast to cells treated with higher H-FIRE doses. Cancer cells treated with recovery-permitting doses of H-FIRE maintained while those treated with recovery-inhibiting doses lost proliferative capacity. Taken together, our data suggest that H-FIRE induces reversible and irreversible cellular damage in a dose-dependent manner, and the presence of dose-dependent recovery mechanisms permits tumor cell proliferation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Davalos, Aycock, Dervisis, and Rossmeisl have pending and issued patents related to electroporation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Irreversible electroporation (IRE), also referred to as nonthermal pulsed field ablation (PFA), is an attractive focal ablation modality for solid tumors and cardiac tissue due to its ability to destroy aberrant cells with limited disruption of the underlying tissue architecture. Despite its nonthermal cell death mechanism, application of electrical energy results in Joule heating that, if ignored, can cause undesired thermal injury. Engineered thermal mitigation (TM) technologies including phase change materials (PCMs) and active cooling (AC) have been reported and tested as a potential means to limit thermal damage. However, several variables affect TM performance including the pulsing paradigm, electrode geometry, PCM composition, and chosen active cooling parameters, meaning direct comparisons between approaches are lacking. In this study, we developed a computational model of conventional bipolar and monopolar probes with solid, PCM-filled, or actively cooled cores to simulate clinical IRE treatments in pancreatic tissue. This approach reveals that probes with integrated PCM cores can be tuned to drastically limit thermal damage compared to existing solid probes. Furthermore, actively cooled probes provide additional control over thermal effects within the probe vicinity and can altogether abrogate thermal damage. In practice, such differences in performance must be weighed against the increased time, expense, and effort required for modified probes compared to existing solid probes., (Copyright © 2022 by ASME.)

The treatment of CNS disorders suffers from the inability to deliver large therapeutic agents to the brain parenchyma due to protection from the blood-brain barrier (BBB). Herein, we investigated high-frequency pulsed electric field (HF-PEF) therapy of various pulse widths and interphase delays for BBB disruption while selectively minimizing cell ablation. Eighteen male Fisher rats underwent craniectomy procedures and two blunt-tipped electrodes were advanced into the brain for pulsing. BBB disruption was verified with contrast T1W MRI and pathologically with Evans blue dye. High-frequency irreversible electroporation cell death of healthy rodent astrocytes was investigated in vitro using a collagen hydrogel tissue mimic. Numerical analysis was conducted to determine the electric fields in which BBB disruption and cell ablation occur. Differences between the BBB disruption and ablation thresholds for each waveform are as follows: 2-2-2 μs (1028 V/cm), 5-2-5 μs (721 V/cm), 10-1-10 μs (547 V/cm), 2-5-2 μs (1043 V/cm), and 5-5-5 μs (751 V/cm). These data suggest that HF-PEFs can be fine-tuned to modulate the extent of cell death while maximizing peri-ablative BBB disruption. Furthermore, numerical modeling elucidated the diffuse field gradients of a single-needle grounding pad configuration to favor large-volume BBB disruption, while the monopolar probe configuration is more amenable to ablation and reversible electroporation effects.

Irreversible electroporation (IRE) is a promising alternative therapy for the local treatment of prostate tumors. The procedure involves the direct insertion of needle electrodes into the target zone, and subsequent delivery of short but high-voltage pulses. Successful outcomes rely on adequate exposure of the tumor to a threshold electrical field. To aid in predicting this exposure, computational models have been developed, yet often do not incorporate the appropriate tissue-specific properties. This work aims to quantify electrical conductivity behavior during IRE for three types of tissue present in the target area of a prostate cancer ablation: the tumor tissue itself, the surrounding healthy tissue, and potential areas of necrosis within the tumor. Animal tissues were used as a stand-in for primary samples. The patient-derived prostate tumor tissue showed very similar responses to healthy porcine prostate tissue. An examination of necrotic tissue inside the tumors revealed a large difference, however, and a computational model showed that a necrotic core with differing electrical properties can cause unexpected inhomogeneities within the treatment region.

High-frequency irreversible electroporation (H-FIRE) is a tissue ablation modality employing bursts of electrical pulses in a positive phase-interphase delay (d 1 )-negative phase-interpulse delay (d 2 ) pattern. Despite accumulating evidence suggesting the significance of these delays, their effects on therapeutic outcomes from clinically-relevant H-FIRE waveforms have not been studied extensively., Objective: We sought to determine whether modifications to the delays within H-FIRE bursts could yield a more desirable clinical outcome in terms of ablation volume versus extent of tissue excitation., Methods: We used a modified spatially extended nonlinear node (SENN) nerve fiber model to evaluate excitation thresholds for H-FIRE bursts with varying delays. We then calculated non-thermal tissue ablation, thermal damage, and excitation in a clinically relevant numerical model., Results: Excitation thresholds were maximized by shortening d 1 , and extension of d 2 up to 1,000 μs increased excitation thresholds by at least 60% versus symmetric bursts. In the ablation model, long interpulse delays lowered the effective frequency of burst waveforms, modulating field redistribution and reducing heat production. Finally, we demonstrate mathematically that variable delays allow for increased voltages and larger ablations with similar extents of excitation as symmetric waveforms., Conclusion: Interphase and interpulse delays play a significant role in outcomes resulting from H-FIRE treatment., Significance: Waveforms with short interphase delays (d 1 ) and extended interpulse delays (d 2 ) may improve therapeutic efficacy of H-FIRE as it emerges as a clinical tissue ablation modality.

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients' anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.

High-frequency irreversible electroporation (H-FIRE) has emerged as an alternative to conventional irreversible electroporation (IRE) to overcome the issues associated with neuromuscular electrical stimulation that appear in IRE treatments. In H-FIRE, the monopolar pulses typically used in IRE are replaced with bursts of short bipolar pulses. Currently, very little is known regarding how the use of a different waveform affects the cell death dynamics and mechanisms. In this study, human pancreatic adenocarcinoma cells were treated with a typical IRE protocol and various H-FIRE schemes with the same energized time. Cell viability, membrane integrity and Caspase 3/7 activity were assessed at different times after the treatment. In both treatments, we identified two different death dynamics (immediate and delayed) and we quantified the electric field ranges that lead to each of them. While in the typical IRE protocol, the electric field range leading to a delayed cell death is very narrow, this range is wider in H-FIRE and can be increased by reducing the pulse length. Membrane integrity in cells suffering a delayed cell death shows a similar time evolution in all treatments, however, Caspase 3/7 expression was only observed in cells treated with H-FIRE.

Irreversible electroporation (IRE) has established a clinical niche as an alternative to thermal ablation for the eradication of unresectable tumors, particularly those near critical vascular structures. IRE has been used in over 50 independent clinical trials and has shown clinical success when used as a standalone treatment and as a single component within combinatorial treatment paradigms. Recently, many studies evaluating IRE in larger patient cohorts and alongside other novel therapies have been reported. Here, we present the basic principles of reversible electroporation and IRE followed by a review of preclinical and clinical data with a focus on tumors in three organ systems in which IRE has shown great promise: the prostate, pancreas, and liver. Finally, we discuss alternative and future developments, which will likely further advance the use of IRE in the clinic., Competing Interests: R.V.D. is an inventor on pending and issued patents related to irreversible electroporation. The authors declare no other conflicts of interest., (© Kenneth N. Aycock and Rafael V. Davalos 2019; Published by Mary Ann Liebert, Inc.)