High-Frequency Irreversible Electroporation for Treatment of Primary Liver Cancer: A Proof-of-Principle Study in Canine Hepatocellular Carcinoma.

PURPOSE: To determine the safety and feasibility of percutaneous high-frequency irreversible electroporation (HFIRE) for primary liver cancer and evaluate the HFIRE-induced local immune response. MATERIALS AND METHODS: HFIRE therapy was delivered percutaneously in 3 canine patients with resectable hepatocellular carcinoma (HCC) in the absence of intraoperative paralytic agents or cardiac synchronization. Pre- and post-HFIRE biopsy samples were processed with histopathology and immunohistochemistry for CD3, CD4, CD8, and CD79a. Blood was collected on days 0, 2, and 4 for complete blood count and chemistry. Numeric models were developed to determine the treatment-specific lethal thresholds for malignant canine liver tissue and healthy porcine liver tissue. RESULTS: HFIRE resulted in predictable ablation volumes as assessed by posttreatment CT. No detectable cardiac interference and minimal muscle contraction occurred during HFIRE. No clinically significant adverse events occurred secondary to HFIRE. Microscopically, a well-defined ablation zone surrounded by a reactive zone was evident in the majority of samples. This zone was composed primarily of maturing collagen interspersed with CD3+/CD4-/CD8- lymphocytes in a proinflammatory microenvironment. The average ablation volumes for the canine HCC patients and the healthy porcine tissue were 3.89 cm3 ± 0.74 and 1.56 cm3 ± 0.16, respectively (P = .03), and the respective average lethal thresholds were 710 V/cm ± 28.2 and 957 V/cm ± 24.4 V/cm (P = .0004). CONCLUSIONS: HFIRE can safely and effectively be delivered percutaneously, results in a predictable ablation volume, and is associated with lymphocytic tumor infiltration. This is the first step toward the use of HFIRE for treatment of unresectable liver tumors.

Experimental High-Frequency Irreversible Electroporation Using a Single-Needle Delivery Approach for Nonthermal Pancreatic Ablation In Vivo.

PURPOSE: To investigate the feasibility of single-needle high-frequency irreversible electroporation (SN-HFIRE) to create reproducible tissue ablations in an in vivo pancreatic swine model. MATERIALS AND METHODS: SN-HFIRE was performed in swine pancreas in vivo in the absence of intraoperative paralytics or cardiac synchronization using 3 different voltage waveforms (1-5-1, 2-5-2, and 5-5-5 [on-off-on times (µs)], n = 6/setting) with a total energized time of 100 µs per burst. At necropsy, ablation size/shape was determined. Immunohistochemistry was performed to quantify apoptosis using an anticleaved caspase-3 antibody. A numerical model was developed to determine lethal thresholds for each waveform in pancreas. RESULTS: Mean tissue ablation time was 5.0 ± 0.2 minutes, and no cardiac abnormalities or muscle twitch was detected. Mean ablation area significantly increased with increasing pulse width (41.0 ± 5.1 mm2 [range 32-66 mm2] vs 44 ± 2.1 mm2 [range 38-56 mm2] vs 85.0 ± 7.0 mm2 [range 63-155 mm2]; 1-5-1, 2-5-2, 5-5-5, respectively; p < 0.0002 5-5-5 vs 1-5-1 and 2-5-2). The majority of the ablation zone did not stain positive for cleaved caspase-3 (6.1 ± 2.8% [range 1.8-9.1%], 8.8 ± 1.3% [range 5.5-14.0%], and 11.0 ± 1.4% [range 7.1-14.2%] cleaved caspase-3 positive 1-5-1, 2-5-2, 5-5-5, respectively), with significantly more positive staining at the 5-5-5 pulse setting compared with 1-5-1 (p < 0.03). Numerical modeling determined a lethal threshold of 1114 ± 123 V/cm (1-5-1 waveform), 1039 ± 103 V/cm (2-5-2 waveform), and 693 ± 81 V/cm (5-5-5 waveform). CONCLUSIONS: SN-HFIRE induces rapid, predictable ablations in pancreatic tissue in vivo without the need for intraoperative paralytics or cardiac synchronization.

Cycled pulsing to mitigate thermal damage for multi-electrode irreversible electroporation therapy.

Purpose: This study evaluates the effects of various pulsing paradigms, on the irreversible electroporation (IRE) lesion, induced electric current, and temperature changes using a perfused porcine liver model. Materials and methods: A 4-monopolar electrode array delivered IRE therapy varying the pulse length and inter-pulse delay to six porcine mechanically perfused livers. Pulse paradigms included six forms of cycled pulsing schemes and the conventional pulsing scheme. Finite element models provided further insight into the effects of cycled pulsing on the temperature and thermal injury distribution. Results: 'Single pulse cycle with no interpulse delay' deposited maximum average energy (2.34 ± 0.35 kJ) and produced the largest ratio of thermally damaged tissue area and IRE ablation area from all other pulse schemes (18.22% ± 8.11, p < .0001 all pairwise comparisons). These compared favorably to the conventional algorithm (2.09 ± 0.37 kJ, 3.49% ± 2.20, p < .0001, all comparisons). Though no statistical significance was found between groups, the '5 pulse cycle, 0 s delay' pulse paradigm produced the largest average IRE ablation cross sectional area (11.81 ± 1.97 cm2), while conventional paradigm yielded an average of 8.90 ± 0.91 cm2. Finite element modeling indicated a '10 pulse cycle, 10 s delay' generated the least thermal tissue damage and '1 pulse cycle, 0 s delay' pulse cycle sequence the most (0.47 vs. 3.76 cm2), over a lengthier treatment time (16.5 vs. 6.67 minutes). Conclusions: Subdividing IRE pulses and adding delays throughout the treatment can reduce white tissue coagulation and electric current, while maintaining IRE treatment sizes.

Irreversible electroporation for the ablation of pancreatic malignancies: A patient-specific methodology.

BACKGROUND AND OBJECTIVES: Irreversible Electroporation (IRE) is a focal ablation technique highly attractive to surgical oncologists due to its non-thermal nature that allows for eradication of unresectable tumors in a minimally invasive procedure. In this study, our group sought to address the challenge of predicting the ablation volume with IRE for pancreatic procedures. METHODS: In compliance with HIPAA and hospital IRB approval, we established a pre-treatment planning methodology for IRE procedures in pancreas, which optimized treatment protocols for individual cases of locally advanced pancreatic cancer (LAPC). A new method for confirming treatment plans through intraoperative monitoring of tissue resistance was also proved feasible in three patients. RESULTS: Results from computational models showed good correlation with experimental data available in the literature. By implementing the proposed resistance measurement system 210 ± 26.1 (mean ± standard deviation) fewer pulses were delivered per electrode-pair. CONCLUSION: The proposed physics-based pre-treatment plan through finite element analysis and system for actively monitoring resistance changes can be paired to significantly reduce ablation times and risk of thermal effects during IRE procedures for LAPC.

Investigation of High Frequency Irreversible Electroporation for Canine Spontaneous Primary Lung Tumor Ablation.

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.

High-frequency irreversible electroporation improves survival and immune cell infiltration in rodents with malignant gliomas.

Background: Irreversible electroporation (IRE) has been previously investigated in preclinical trials as a treatment for intracranial malignancies. Here, we investigate next generation high-frequency irreversible electroporation (H-FIRE), as both a monotherapy and a combinatorial therapy, for the treatment of malignant gliomas. Methods: Hydrogel tissue scaffolds and numerical modeling were used to inform in-vivo H-FIRE pulsing parameters for our orthotopic tumor-bearing glioma model. Fischer rats were separated into five treatment cohorts including high-dose H-FIRE (1750V/cm), low-dose H-FIRE (600V/cm), combinatorial high-dose H-FIRE + liposomal doxorubicin, low-dose H-FIRE + liposomal doxorubicin, and standalone liposomal doxorubicin groups. Cohorts were compared against a standalone tumor-bearing sham group which received no therapeutic intervention. To further enhance the translational value of our work, we characterize the local and systemic immune responses to intracranial H-FIRE at the study timepoint. Results: The median survival for each cohort are as follows: 31 days (high-dose H-FIRE), 38 days (low-dose H-FIRE), 37.5 days (high-dose H-FIRE + liposomal doxorubicin), 27 days (low-dose H-FIRE + liposomal doxorubicin), 20 days (liposomal doxorubicin), and 26 days (sham). A statistically greater overall survival fraction was noted in the high-dose H-FIRE + liposomal doxorubicin (50%, p = 0.044), high-dose H-FIRE (28.6%, p = 0.034), and the low-dose H-FIRE (20%, p = 0.0214) compared to the sham control (0%). Compared to sham controls, brain sections of rats treated with H-FIRE demonstrated significant increases in IHC scores for CD3+ T-cells (p = 0.0014), CD79a+ B-cells (p = 0.01), IBA-1+ dendritic cells/microglia (p = 0.04), CD8+ cytotoxic T-cells (p = 0.0004), and CD86+ M1 macrophages (p = 0.01). Conclusions: H-FIRE may be used as both a monotherapy and a combinatorial therapy to improve survival in the treatment of malignant gliomas while also promoting the presence of infiltrative immune cells.

Extended interpulse delays improve therapeutic efficacy of microsecond-duration pulsed electric fields.

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.

High-Frequency Irreversible Electroporation (H-FIRE) Induced Blood-Brain Barrier Disruption Is Mediated by Cytoskeletal Remodeling and Changes in Tight Junction Protein Regulation.

Glioblastoma is the deadliest malignant brain tumor. Its location behind the blood-brain barrier (BBB) presents a therapeutic challenge by preventing effective delivery of most chemotherapeutics. H-FIRE is a novel tumor ablation method that transiently disrupts the BBB through currently unknown mechanisms. We hypothesized that H-FIRE mediated BBB disruption (BBBD) occurs via cytoskeletal remodeling and alterations in tight junction (TJ) protein regulation. Intracranial H-FIRE was delivered to Fischer rats prior to sacrifice at 1-, 24-, 48-, 72-, and 96 h post-treatment. Cytoskeletal proteins and native and ubiquitinated TJ proteins (TJP) were evaluated using immunoprecipitation, Western blotting, and gene-expression arrays on treated and sham control brain lysates. Cytoskeletal and TJ protein expression were further evaluated with immunofluorescent microscopy. A decrease in the F/G-actin ratio, decreased TJP concentrations, and increased ubiquitination of TJP were observed 1-48 h post-H-FIRE compared to sham controls. By 72-96 h, cytoskeletal and TJP expression recovered to pretreatment levels, temporally corresponding with increased claudin-5 and zonula occludens-1 gene expression. Ingenuity pathway analysis revealed significant dysregulation of claudin genes, centered around claudin-6 in H-FIRE treated rats. In conclusion, H-FIRE is capable of permeating the BBB in a spatiotemporal manner via cytoskeletal-mediated TJP modulation. This minimally invasive technology presents with applications for localized and long-lived enhanced intracranial drug delivery.

Advancements in drug delivery methods for the treatment of brain disease.

The blood-brain barrier (BBB) presents a formidable obstacle to the effective delivery of systemically administered pharmacological agents to the brain, with ~5% of candidate drugs capable of effectively penetrating the BBB. A variety of biomaterials and therapeutic delivery devices have recently been developed that facilitate drug delivery to the brain. These technologies have addressed many of the limitations imposed by the BBB by: (1) designing or modifying the physiochemical properties of therapeutic compounds to allow for transport across the BBB; (2) bypassing the BBB by administration of drugs via alternative routes; and (3) transiently disrupting the BBB (BBBD) using biophysical therapies. Here we specifically review colloidal drug carrier delivery systems, intranasal, intrathecal, and direct interstitial drug delivery methods, focused ultrasound BBBD, and pulsed electrical field induced BBBD, as well as the key features of BBB structure and function that are the mechanistic targets of these approaches. Each of these drug delivery technologies are illustrated in the context of their potential clinical applications and limitations in companion animals with naturally occurring intracranial diseases.

A Theoretical Argument for Extended Interpulse Delays in Therapeutic High-Frequency Irreversible Electroporation Treatments.

High-frequency irreversible electroporation (H-FIRE) is a tissue ablation modality employing bursts of electrical pulses in a positive phase-interphase delay (d1)-negative phase-interpulse delay (d2) 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 d1, and extension of d2 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 (d1) and extended interpulse delays (d2) may improve therapeutic efficacy of H-FIRE as it emerges as a clinical tissue ablation modality.

Rapid Impedance Spectroscopy for Monitoring Tissue Impedance, Temperature, and Treatment Outcome During Electroporation-Based Therapies.

OBJECTIVE: Electroporation-based therapies (EBTs) employ high voltage pulsed electric fields (PEFs) to permeabilize tumor tissue; this results in changes in electrical properties detectable using electrical impedance spectroscopy (EIS). Currently, commercial potentiostats for EIS are limited by impedance spectrum acquisition time (  âˆ¼ 10 s); this timeframe is much larger than pulse periods used with EBTs (  âˆ¼ 1 s). In this study, we utilize rapid EIS techniques to develop a methodology for characterizing electroporation (EP) and thermal effects associated with high-frequency irreversible EP (H-FIRE) in real-time by monitoring inter-burst impedance changes. METHODS: A charge-balanced, bipolar rectangular chirp signal is proposed for rapid EIS. Validation of rapid EIS measurements against a commercial potentiostat was conducted in potato tissue using flat-plate electrodes and thereafter for the measurement of impedance changes throughout IRE treatment. Flat-plate electrodes were then utilized to uniformly heat potato tissue; throughout high-voltage H-FIRE treatment, low-voltage inter-burst impedance measurements were used to continually monitor impedance change and to identify a frequency at which thermal effects are delineated from EP effects. RESULTS: Inter-burst impedance measurements (1.8 kHz - 4.93 MHz) were accomplished at 216 discrete frequencies. Impedance measurements at frequencies above  âˆ¼ 1 MHz served to delineate thermal and EP effects in measured impedance. CONCLUSION: We demonstrate rapid-capture ( 1 s) EIS which enables monitoring of inter-burst impedance in real-time. For the first time, we show impedance analysis at high frequencies can delineate thermal effects from EP effects in measured impedance. SIGNIFICANCE: The proposed waveform demonstrates the potential to perform inter-burst EIS using PEFs compatible with existing pulse generator topologies.

Multi-Tissue Analysis on the Impact of Electroporation on Electrical and Thermal Properties.

OBJECTIVE: Tissue electroporation is achieved by applying a series of electric pulses to destabilize cell membranes within the target tissue. The treatment volume is dictated by the electric field distribution, which depends on the pulse parameters and tissue type and can be readily predicted using numerical methods. These models require the relevant tissue properties to be known beforehand. This study aims to quantify electrical and thermal properties for three different tissue types relevant to current clinical electroporation. METHODS: Pancreatic, brain, and liver tissue were harvested from pigs, then treated with IRE pulses in a parallel-plate configuration. Resulting current and temperature readings were used to calculate the conductivity and its temperature dependence for each tissue type. Finally, a computational model was constructed to examine the impact of differences between tissue types. RESULTS: Baseline conductivity values (mean 0.11, 0.14, and 0.12 S/m) and temperature coefficients of conductivity (mean 2.0, 2.3, and 1.2 % per degree Celsius) were calculated for pancreas, brain, and liver, respectively. The accompanying computational models suggest field distribution and thermal damage volumes are dependent on tissue type. CONCLUSION: The three tissue types show similar electrical and thermal responses to IRE, though brain tissue exhibits the greatest differences. The results also show that tissue type plays a role in the expected ablation and thermal damage volumes. SIGNIFICANCE: The conductivity and its changes due to heating are expected to have a marked impact on the ablation volume. Incorporating these tissue properties aids in the prediction and optimization of electroporation-based therapies.

An Investigation for Large Volume, Focal Blood-Brain Barrier Disruption with High-Frequency Pulsed Electric Fields.

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.

Properties of tissue within prostate tumors and treatment planning implications for ablation therapies.

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.

Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation.

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.

Patient Derived Xenografts Expand Human Primary Pancreatic Tumor Tissue Availability for ex vivo Irreversible Electroporation Testing.

New methods of tumor ablation have shown exciting efficacy in pre-clinical models but often demonstrate limited success in the clinic. Due to a lack of quality or quantity in primary malignant tissue specimens, therapeutic development and optimization studies are typically conducted on healthy tissue or cell-line derived rodent tumors that don't allow for high resolution modeling of mechanical, chemical, and biological properties. These surrogates do not accurately recapitulate many critical components of the tumor microenvironment that can impact in situ treatment success. Here, we propose utilizing patient-derived xenograft (PDX) models to propagate clinically relevant tumor specimens for the optimization and development of novel tumor ablation modalities. Specimens from three individual pancreatic ductal adenocarcinoma (PDAC) patients were utilized to generate PDX models. This process generated 15-18 tumors that were allowed to expand to 1.5 cm in diameter over the course of 50-70 days. The PDX tumors were morphologically and pathologically identical to primary tumor tissue. Likewise, the PDX tumors were also found to be physiologically superior to other in vitro and ex vivo models based on immortalized cell lines. We utilized the PDX tumors to refine and optimize irreversible electroporation (IRE) treatment parameters. IRE, a novel, non-thermal tumor ablation modality, is being evaluated in a diverse range of cancer clinical trials including pancreatic cancer. The PDX tumors were compared against either Pan02 mouse derived tumors or resected tissue from human PDAC patients. The PDX tumors demonstrated similar changes in electrical conductivity and Joule heating following IRE treatment. Computational modeling revealed a high similarity in the predicted ablation size of the PDX tumors that closely correlate with the data generated with the primary human pancreatic tumor tissue. Gene expression analysis revealed that IRE treatment resulted in an increase in biological pathway signaling associated with interferon gamma signaling, necrosis and mitochondria dysfunction, suggesting potential co-therapy targets. Together, these findings highlight the utility of the PDX system in tumor ablation modeling for IRE and increasing clinical application efficacy. It is also feasible that the use of PDX models will significantly benefit other ablation modality testing beyond IRE.

Temporal Characterization of Blood-Brain Barrier Disruption with High-Frequency Electroporation.

Treatment of intracranial disorders suffers from the inability to accumulate therapeutic drug concentrations due to protection from the blood-brain barrier (BBB). Electroporation-based therapies have demonstrated the capability of permeating the BBB, but knowledge of the longevity of BBB disruption (BBBD) is limited. In this study, we quantify the temporal, high-frequency electroporation (HFE)-mediated BBBD in an in vivo healthy rat brain model. 40 male Fisher rats underwent HFE treatment; two blunt tipped monopolar electrodes were advanced into the brain and 200 bursts of HFE were delivered at a voltage-to-distance ratio of 600 V/cm. BBBD was verified with contrast enhanced T1W MRI (gadopentetate dimeglumine) and pathologically (Evans blue dye) at time points of 1, 24, 48, 72, and 96 h after HFE. Contrast enhanced T1W scans demonstrated BBBD for 1 to 72 h after HFE but intact BBB at 96 h. Histologically, tissue damage was restricted to electrode insertion tracks. BBBD was induced with minimal muscle contractions and minimal cell death attributed to HFE. Numerical modeling indicated that brief BBBD was induced with low magnitude electric fields, and BBBD duration increased with field strength. These data suggest the spatiotemporal characteristics of HFE-mediated BBBD may be modulated with the locally applied electric field.

High-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity.

BACKGROUND: Despite promising treatments for breast cancer, mortality rates remain high and treatments for metastatic disease are limited. High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation technique that utilizes high-frequency bipolar electric pulses to destabilize cancer cell membranes and induce cell death. However, there is currently a paucity of data pertaining to immune system activation following H-FIRE and other electroporation based tumor ablation techniques. METHODS: Here, we utilized the mouse 4T1 mammary tumor model to evaluate H-FIRE treatment parameters on cancer progression and immune system activation in vitro and in vivo. FINDINGS: H-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment. We further show that local treatment with H-FIRE significantly reduces 4T1 metastases. H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo. Our data indicate that the level of tumor ablation correlates with increased activation of cellular immunity. Likewise, we show that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression. INTERPRETATION: Cell death and tumor ablation following H-FIRE treatment activates the local innate immune system, which shifts the tumor microenvironment from an anti-inflammatory state to a pro-inflammatory state. The non-thermal damage to the cancer cells and increased innate immune system stimulation improves antigen presentation, resulting in the engagement of the adaptive immune system and improved systemic anti-tumor immunity.

Characterization of Nonlinearity and Dispersion in Tissue Impedance During High-Frequency Electroporation.

OBJECTIVE: The use of high-voltage, high-frequency bipolar pulses (HFBPs) is an emerging electroporation-based therapy for the treatment of solid tumors. In this study, we quantify the extent of nonlinearity and dispersion during the HFBP treatment. METHODS: We utilize flat-plate electrodes to capture the impedance of the porcine liver tissue during the delivery of a burst of HFBPs of widths 1 and 2  $\mu$s at different pulse amplitudes. Next, we fit the impedance data to a frequency-dependent parallel RC network to determine the conductivity and permittivity of the tissue as a function of frequency, for different applied electric fields. Finally, we present a simple model to approximate the field distribution in the tissue using the conductivity function at a frequency that could minimize the errors due to approximation with a nondispersive model. RESULTS: The conductivity/permittivity of the tissue was plotted as a function of frequency for different electric fields. It was found that the extent of dispersion reduces with higher applied electric field magnitudes. CONCLUSION: This is the first study to quantify dispersion and nonlinearity in the tissue during the HFBP treatment. The data have been used to predict the field distribution in a numerical model of the liver tissue utilizing two needle electrodes. SIGNIFICANCE: The data and technique developed in this study to monitor the electrical properties of tissue during treatment can be used to generate treatment-planning models for future high-frequency electroporation therapies as well as provide insights regarding treatment effect.