High-frequency Irreversible Electroporation Research Articles

Ultrasound-guided percutaneous high-frequency irreversible electroporation in porcine livers using four electrode needles: A feasibility and safety study.

Malignant liver tumors seriously endanger human health. Among different therapeutic approaches, high-frequency irreversible electroporation (H-FIRE) is a recently emerging tumor ablation technique. The objective of this study was to evaluate the feasibility and safety of ultrasound-guided percutaneous H-FIRE using four electrode needles in porcine livers. Twelve experimental pigs underwent percutaneous H-FIRE ablation using a compound steep-pulse therapeutic device. Liver tissues adjacent to the gallbladder, blood vessels, and bile ducts were selected as the ablation targets. Pigs were randomly divided into three groups: (1) immediately after ablation (N = 4), (2) 2 days after ablation (N = 4), and (3) 7 days after ablation (N = 4). Blood routine, liver and kidney function, and myocardial enzyme levels were measured before and after ablation. Ultrasound, contrast-enhanced ultrasound (CEUS), contrast-enhanced magnetic resonance imaging (MRI), and hematoxylin-eosin staining were performed to evaluate the ablation performance. Ultrasound-guided percutaneous H-FIRE ablations using four electrode needles were successfully performed in all 12 experimental pigs. The general conditions of the pigs, including postoperative activities and feeding behaviors, were normal, with no significant changes compared with the preoperative conditions. The imaging features of ultrasound, CEUS, and MRI demonstrated no significant changes in the gallbladder walls, bile ducts, or blood vessels close to the ablation areas. Laboratory tests showed that liver function indices and myocardial enzymes increased temporarily after H-FIRE ablation, but decreased to normal levels at 7 days after ablation. Histopathological examinations of porcine liver specimens showed that this technique could effectively ablate the target areas without damaging the surrounding or internal vascular systems and gallbladder. This study demonstrated the feasibility and safety of ultrasound-guided percutaneous H-FIRE ablation in porcine livers invivo, and proposed a four-needle method to optimize its clinical application.

Influence of Joule heating during single-cell electroporation simulation under IRE and H-FIRE pulses

Conventional irreversible electroporation (IRE) and high-frequency irreversible electroporation (H-FIRE) pulses have been widely employed in many biomedical and food applications. To investigate the effect of Joule heating generated by pulsed electric fields on cell electroporation, single-cell electroporation models without (EP only) and with the consideration of Joule heating (EP+T) were subjected to the equivalent energy IRE and H-FIRE pulses. The simulation results showed that with the consideration of Joule heating, the transmembrane voltage of the anodic point (TMPA) reached the electroporation threshold (∼1 V) faster, and a significant increase in the plasma membrane conductivity (σA) and pore number (kp) was found. We further evaluated the effect of the pulse burst repetition frequency (PBRF) of H-FIRE pulses on cell electroporation based on the EP+T model, and discovered kp increased with frequency when PBRF lower than 2 kHz, whereas it decreased with frequency for PBRF higher than 2 kHz. The accumulation of the ratio of the perforation area to the cell membrane area (sum of Ap/A) during the 400th pulse was also reversed when PBRF was 2 kHz. Our results indicated that Joule heating generated by repetitive pulse delivery would facilitate electroporation, and the effect of PBRF on cell electroporation was non-monotonic.

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

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. 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. 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). 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.

Abstract 4917: High-frequency irreversible electroporation ablation of glioma alters extracellular vesicles and disrupts the blood-brain barrier endothelium

Abstract High-frequency irreversible electroporation (H-FIRE) is a nonthermal tumor ablation technology that kills cancer cells via pulsed electric fields. H-FIRE has been shown to precisely and nonthermally ablate brain tumors while transiently disrupting the peritumoral blood-brain barrier (BBB). The enhanced BBB permeability post-tumor ablation can be exploited to enhance therapeutic delivery to invasive tumor margins, but the mechanisms of H-FIRE-induced BBB disruption remain incompletely characterized. We hypothesize that bystander effects of H-FIRE tumor cell ablation, specifically the release of tumor-derived extracellular vesicles (TDEVs), mediate peritumoral BBB endothelium disruption. F98 glioma and bEnd.3 cerebral endothelial cell lines modelled primary brain cancer and the BBB endothelium, respectively. F98 glioma cells were treated in vitro with sham treatment, a sub-ablative (1,500 V/cm), or an ablative (3,000 V/cm) electric field dose of H-FIRE, and TDEVs were isolated from the supernatants via filtration and ultracentrifugation. Post-H-FIRE TDEVs were characterized via nanoparticle tracking analysis and transmission electron microscopy, with ablative doses of H-FIRE resulting in decreased release of TDEVs compared to sub-ablative H-FIRE and sham treatment. Post-H-FIRE TDEVs were then applied to Transwell models of the BBB endothelium to evaluate the effect of H-FIRE-induced TDEVs on BBB endothelium permeability. TDEVs released after the ablative H-FIRE dose (3,000 V/cm) significantly increased permeability of the BBB endothelium in vitro compared to TDEVs released after sub-ablative and sham H-FIRE treatment. TDEVs were then fluorescently labelled with CFSE, and confocal microscopy was used to characterize internalization of post-H-FIRE TDEVs by cerebral endothelial cells. The TDEVs released after ablative doses of H-FIRE were significantly internalized by bEnd.3 cerebral endothelial cells, while TDEVs released after sub-ablative and sham treatment were not internalized. The proteomic payloads of the post-H-FIRE TDEVs were characterized using mass spectrometry, and clustering analysis demonstrated that the in vitro BBB-modulatory TDEVs released after 3,000 V/cm H-FIRE had distinct proteomic payloads compared to the non-BBB-modulatory TDEV populations released after sham and 1,500 V/cm H-FIRE treatment. Proteins associated with proteasomal degradation, cellular adhesion, and modulation of junctional integrity were increased in the BBB-modulatory group of TDEVs. Taken together, our results suggest that H-FIRE ablation of glioma significantly alters the proteomic cargo of TDEVs to increase internalization of TDEVs by cerebral endothelial cells and increase permeability of the BBB endothelium. Citation Format: Kelsey Murphy, Kenneth Aycock, Spencer Marsh, Alayna Hay, Christine Chang, Shay Bracha, Robert Gourdie, Rafael Davalos, John H. Rossmeisl, Nikolaos Dervisis. High-frequency irreversible electroporation ablation of glioma alters extracellular vesicles and disrupts the blood-brain barrier endothelium. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4917.

Recovery of neurosurgical high-frequency electroporation injury in the canine brain can be accelerated by 7,8-dihydroxyflavone

BackgroundAlthough traumatic brain injury (TBI) occurs in a very short time, the biological consequence of a TBI, such as Alzheimer’s disease, may last a lifetime. To date, effective interventions are not available to improve recovery from a TBI. Herein we aimed to ascertain whether recovery of neurosurgical high-frequency irreversible electroporation (HFIRE) injury in brain tissues can be accelerated by 7,8-dihydroxyflavone (7,8-DHF). MethodsThe HFIRE injury was induced in the right parietal cortex of 8 adult healthy and neurologically intact male dogs. Two weeks before HFIRE injury, each dog was administered orally with or without 7,8-DHF (30 mg/kg) once daily for consecutive 2 weeks (n = 4 for each group). The values of blood-brain barrier (BBB) disruption, brain edema, and cerebral infarction volumes were measured. The concentrations of beta-amyloid, interleukin-1β, interleukin-6 and tumor necrosis factor-α in the cerebrospinal fluid were measured biochemically. ResultsThe BBB disruption, brain edema, infarction volumes, and maximal cross-section area caused by HFIRE injury in canine brain were significantly attenuated by 7,8-DHF therapy (P < 0.0001). Additionally, 7,8-DHF significantly reduced the HFIRE-induced cerebral overproduction of beta-amyloid and proinflammatory cytokines in the cerebrospinal fluid (P < 0.0001) in dogs with HFIRE. ConclusionsRecovery of neurosurgical HFIRE injury in canine brain tissues can be accelerated by 7,8-DHT via ameliorating BBB disruption as well as cerebral overproduction of both beta-amyloid and proinflammatory cytokines.

A Single-Cell Electroporation Model for Quantitatively Estimating the Pore Area Ratio by High-Frequency Irreversible Electroporation

The electroporation technique utilizes pulsed electric fields to induce porous defects in the cell membrane, and the technique can be used for delivering drugs into cells and killing cancer cells. To develop an electric pulse protocol in the clinic with this technique, the key issue is to understand the evolution of pores in the cell membrane during the process of electroporation. This paper presents a study to address this issue. Specifically, a mathematical model of single-cell electroporation (SCE) was developed, which includes pore area ratio (PAR) as an indicator of the electroporation dynamics and area weight for considering the 3D nature of cells. The model was employed to simulate the electroporation of a single cell with different high-frequency irreversible electroporation (H-FIRE) protocols. The simulation result has found that the change of PAR with respect to the time duration of electroporation follows a sigmoid pattern to increase under specific protocols, which is called the cumulative effect of PAR. Subsequently, the relationship between the protocol of H-FIRE, described by a set of pulse parameters such as pulse width, pulse delay, electric field strength, and pulse burst duration, and the cumulative effect of PAR was established, which thereby allows designing the protocol to kill cells effectively. The study concluded that the proposed SCE model, along with the cumulative effect of PAR, is useful in designing H-FIRE protocols for the ablation of cancer tumors in the clinic.

Differential effect of high-frequency electroporation on myocardium vs. non-myocardial tissues.

Pulsed-field ablation (PFA) is an emerging non-thermal ablation method based on the biophysical phenomenon of electroporation. Data on PFA cardiac selectivity nature and tissue-specific thresholds are lacking. We aim to compare the in vivo differential effect of high-frequency irreversible electroporation (HF-IRE) protocols on various tissues. Twenty-three Sprague-Dawle rodents were allocated into three different protocols of 300, 600, and 900 V, respectively, while delivering twenty 100 µs bursts of a 150 kHz biphasic square wave to five tissues; cardiac muscle, skeletal muscle, liver, carotid artery and sciatic nerve. Lesions were evaluated quantitatively by histologic analysis and by morphometric evaluation. There were eight, seven and eight animals in the 300, 600, and 900 V protocols, respectively. High-frequency electroporation protocols showed a graded effect on myocardial tissue with larger lesions in the 900 V protocol compared with the other two protocols as demonstrated by width (P = 0.02), length (P = 0.01) and fibrosis ratio (P = 0.001). This effect was not observed for other tissues with attenuated degree of damage. No damage to the carotid artery was observed in all protocols. Partial damage to the sciatic nerve was observed in only two samples (25%) in the 600 V group and in one sample (14.3%) in the 900 V group. Electroporation effect is tissue-specific such that myocardium is more prone to electroporation damage compared with neural and vascular tissues. Our results suggest no neural or vascular damage with using a low-amplitude HF-IRE protocol. Further investigation is warranted to better identify other tissue-specific thresholds.

Extended Focal Ablation of Localized Prostate Cancer With High-Frequency Irreversible Electroporation

Focal therapy of prostate cancer must balance the oncologic outcome and functional outcome. High-frequency irreversible electroporation (H-FIRE) can destroy cancer cells while selectively preserving surrounding nerves and blood vessels, but no clinical trials have been conducted, to our knowledge. To evaluate the efficacy and safety of H-FIRE in the treatment of localized prostate cancer (PCa). This was a single-group, objective performance criteria, nonrandomized controlled trial. Recruitment began on May 2, 2018, and ended March 27, 2019. The follow-up duration was 6 months. This was a multicenter trial conducted at 4 tertiary teaching hospitals in China. Patients with low or intermediate risk of biochemical recurrence of localized and locally advanced PCa were eligible. Key inclusion criteria were serum prostate-specific antigen (PSA) level less than 20 ng/mL, clinical stage of T2c or less, and Gleason score of 7 or less. Data were analyzed from January 20 to February 20, 2021. H-FIRE ablation of all lesions identified with biopsy. The primary end point was 6-month clinically significant PCa (csPCa), which was defined as any biopsy core with Gleason score of greater than or equal to 7, or Gleason score of 6 plus maximum cancer core length of greater than 3 mm or an increase from the original cancer burden. Secondary outcomes were calculated in patients who actually received H-FIRE treatment. A total of 117 patients (median [IQR] age, 67 [62-73] years) were recruited from 4 centers, and 109 patients (27 [24.8%] low risk and 82 [75.2%] intermediate risk) actually received H-FIRE. Median (IQR) PSA level was 9.0 (6.0-12.7) ng/mL. Among the 100 patients who underwent biopsy at 6 months, the 6-month csPCa rate was 6.0% (95% CI, 2.2%-12.6%; P < .001; 1 in the treatment zone and 5 outside the treatment zone). Superiority criteria vs the historical control of 20% was achieved. PCa was detected in 14 patients, with a Gleason score of 7 in 2 patients and 6 in the remaining 12 patients. At 6 months, median (IQR) PSA level was 1.08 (0.4-3.2) ng/mL, median (IQR) International Prostate Symptom Score was 4.5 (2.0-9.5), and median (IQR) International Index of Erectile Function 5 score was 2.0 (0.5-12.5). Superiority vs the 20% historical control was also met in the subgroup analysis that only included the 57 patients with Gleason score of 7 at baseline (3.5% 6-month csPCa; 95% CI, 0.4%-12.1%). The rate of 6-month csPCa with H-FIRE ablation was lower than the historical control using other energy platforms. ClinicalTrials.gov Identifier: NCT03838432.

Polymer Nanoparticles Enhance Irreversible Electroporation In Vitro

Expanding the volume of an irreversible electroporation treatment typically necessitates an increase in pulse voltage, number, duration, or repetition. This study investigates the addition of polyethylenimine nanoparticles (PEI-NP) to pulsed electric field treatments, determining their combined effect on ablation size and voltages. U118 cells in an in vitro 3D cell culture model were treated with one of three pulse parameters (with and without PEI-NPs) which are representative of irreversible electroporation (IRE), high frequency irreversible electroporation (H-FIRE), or nanosecond pulsed electric fields (nsPEF). The size of the ablations were compared and mapped onto an electric field model to describe the electric field required to induce cell death. Analysis was conducted to determine the role of PEI-NPs in altering media conductivity, the potential for PEI-NP degradation following pulsed electric field treatment, and PEI-NP uptake. Results show there was a statistically significant increase in ablation diameter for IRE and H-FIRE pulses with PEI-NPs. There was no increase in ablation size for nsPEF with PEI-NPs. This all occurs with no change in cell media conductivity, no observable degradation of PEI-NPs, and moderate particle uptake. These results demonstrate the synergy of a combined cationic polymer nanoparticle and pulsed electric field treatment for the ablation of cancer cells. These results set the foundation for polymer nanoparticles engineered specifically for irreversible electroporation.

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.

Lethal Electric Field Thresholds for Cerebral Cells With Irreversible Electroporation and H-FIRE Protocols: An In Vitro Three-Dimensional Cell Model Study

The lethal electric field (LEF) thresholds for three typical cerebral cells, including a malignant glioblastoma (GBM) cell line and two cell lines from the healthy blood-brain barrier (BBB), treated by irreversible electroporation (IRE) or high-frequency irreversible electroporation (H-FIRE) protocols were investigated in an in vitro three-dimensional (3D) cell model. A conventional IRE protocol (90 pulses, 1 Hz, and 100-μs pulse duration) and three novel H-FIRE protocols (1-3-1, 0.5-1-0.5, and 1-1-1) were used to treat the cerebral cells in both 3D single-cell and two-cell models. The electrical conductivity of the 3D cell model under different electric field strengths were characterized with the method of electrochemical impedance spectroscopy (EIS). Based on EIS, a numerical electrothermal model of electroporation was built for the determination of the LEF threshold with different protocols and temperature monitoring. Cell viability was assessed by fluorescence staining 6 h after the treatment. The results showed no thermal lethal effect on cells when these protocols were used. The LEF threshold for GBM cells was significantly lower than that of the healthy BBB cells. These results suggest the possibility of selective ablation of human cerebral GBM by IRE and H-FIRE treatments with no injury or reversible injury to healthy cells, and the potential use of IRE or H-FIRE for transient disruption of the BBB to allow chemotherapy to reach the tumor.

Experimental and Numerical Investigation of Parameters Affecting High-Frequency Irreversible Electroporation for Prostate Cancer Ablation.

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.

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 s (1028 V/cm), 5-2-5 s (721 V/cm), 10-1-10 s (547 V/cm), 2-5-2 s (1043 V/cm), and 5-5-5 s (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.

Compact High-Voltage AC Generator with Pulse Transformer for High-Frequency Irreversible Electroporation (H-FIRE)

This paper is focused on a design of a high-voltage (HV) generator, which is proposed for a high-frequency irreversible electroporation (H-FIRE). The generator produces bursts of bipolar symmetrical pulses. Most HV sources used for cell electroporation are based on a controlled discharge of a capacitor into a resistive load. This solution is very simple, but it is associated with a certain risk of an uncontrolled discharge of the capacitor. We present a different type of the generator, where a DC-AC inverter with pulse transformer is used and where the mentioned risk is eliminated. Our generator is able to deliver bursts with variable length from 50 to 150 μs and a gap between bursts can be set from 0.5 to 1.5 s. Pulse frequency can be varied from 65 to 470 kHz and the output voltage is controlled in two ranges from 0 to 1.3 kV or from 0 to 2.5 kV. Results are presented with resistive load and with tissue impedance load.

High-frequency irreversible electroporation brain tumor ablation: exploring the dynamics of cell death and recovery

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.

Comparison of analysis methods for determination of dynamic tissue conductivity during microseconds-long pulsed electric fields

Objective: High-frequency irreversible electroporation (H-FIRE) is an emerging therapy which uses bursts of high-voltage, bipolar pulses to ablate tissue. This paper aims to quantify electric-field-dependent material properties of select tissues for computational models. The results show that tissues treated with 5 and 10 μs H-FIRE can be approximated as purely resistive, simplifying analysis and numerical modeling. Methods: Parallel-plate electrodes were used to apply H-FIRE waveforms (5-5-5 and 10-1-10) to samples of porcine pancreas, liver, prostate, brain, and patient-derived prostate tumor. Resistance of each sample during pulsing was calculated from captured current waveforms using two methods for comparison: one assuming a purely resistive response from tissue and the other assuming dispersion. Results: Conductivity versus electric field (EF) behavior is reported for all five tissue types. There was little dependence of conductivity on EF magnitude for most tissues except for prostate and prostate tumor tissue. Additionally, the study found that differences between the two resistance analysis methods were less than 10% except for prostate (<30%) and liver and pancreas at lower EF magnitudes. Conclusion: Prostate and prostate tumor tissues are expected to undergo more EF redistribution during H-FIRE as compared with the other tissues. Also, in most cases under study, the results suggest that frequency effects are minimal using these particular waveforms. Significance: Currently, high-frequency effects on tissue properties that occur during microseconds-long bipolar pulses are not yet clear and quantified. This work reports these properties while also assessing whether approximation of the tissue as resistive is appropriate.

A Multicenter Single-Arm Objective Performance Criteria Trial to Determine the Efficacy and Safety of High-Frequency Irreversible Electroporation as Primary Treatment for Localized Prostate Cancer: A Study Protocol.

IntroductionThe classical pathway for the therapy of low- to intermediate-risk localized prostate cancer is radical prostatectomy or radiation therapy, which has shown a high incidence of complications, including erectile dysfunction, urinary incontinence, and bowel injury. An alternative pathway is to perform an ablation by some energy to the localized lesion, known as focal therapy. High-frequency irreversible electroporation (H-FIRE) is nonthermal energy that can be used in cancer ablation to deliver pulsed high-voltage but low-energy electric current to the cell membrane and to invoke cell death. An H-FIRE pathway has been reported to be tissue-selective, which leads to fewer side effects.Methods and AnalysisThis is a multicenter and single-arm objective performance criteria (OPC) study, in which all men with localized prostate cancer are allocated to H-FIRE ablation. This trial will assess the efficacy and safety of the H-FIRE ablation for prostate cancer. Efficacy will be assessed by prostate biopsy 6 months after treatment while safety will be assessed by adverse event reports and questionnaires. The main inclusion criteria are moderate to low-risk prostate cancer in NCCN risk classification and had no previous therapy for prostate cancer. A sample size of 110 participants is required. The primary objective is to determine whether the detection rate of clinically significant cancer by prostate biopsy is less than 20% after the H-FIRE ablation.Ethics and DisseminationThis study has obtained ethical approval by the ethics committee of all participating centers. The results of the study will be submitted for dissemination and publication in peer-reviewed journals.ConclusionsThis multicenter single-arm objective performance criteria trial will evaluate the efficacy and safety of the use of high-frequency irreversible electroporation in treating prostate cancer.Strengths and Limitations of This StudyA comprehensive evaluation of imaging and histopathology is used to determine the effect of treatment. Questionnaires were used to assess the treatment side effects. Multicenter and pragmatic designs capacitate higher generalizability. A limitation of this trial is that the prostate biopsy as an endpoint may not be as accurate as of the specimen from prostate prostatectomy. Another limitation is the 6-month follow-up time, making this trial challenging to come to firm conclusions regarding the efficacy and safety of IRE in the long term.Clinical Trial Registration ClinicalTrials.gov, NCT03838432

Machine Learning for H-FIRE Protocols: Tuning Parameters for High-Frequency Irreversible Electroporation by Machine Learning

Microwave (MW) technologies are increasingly being employed in biomedicine and health care. Applications such as MW ablation (MWA), MW sensors for the realtime monitoring of physiological parameters, and lab-on-a-chip devices are just some of the examples in a wide variety of possibilities. Recently, high-frequency irreversible electroporation (H-FIRE) has been attracting more attention due to its important applications in oncology. It uses pulsed electrical fields (PEFs) to induce a controlled but irreversible process of permeabilization of cell membranes, thus triggering a substantial alteration in the physiological equilibria of cells, ultimately leading to their death. In this article we proposed an ML-based approach that exploits an ANN to estimate ablation occurrence, ablation area, and E-field lethal threshold (i.e., the three parameters highly impacting each H-FIRE therapeutic application). The ANN was modeled and trained by examining a large set of scientific publications describing H-FIRE experiments and their outcomes. Starting from these data, the ANN was capable of providing a satisfactory estimation accuracy on the aforementioned parameters. The achieved results, although very promising, could be further improved by widening the initial data set (i.e., by gathering details about a larger number of H-FIRE experiments) as well as by evaluating more complex ANN models. The presented tool has powerful potential to achieve fast, personalized protocols in H-FIRE therapy, streamlining the long and complex treatment planning process based on standard electromagnetic and multiphysics simulation methods for a new era of H-FIRE applications.

Generation of Tumor-activated T cells Using Electroporation

Expansion of cytotoxic T lymphocytes (CTLs) is a crucial step in almost all cancer immunotherapeutic methods. Current techniques for expansion of tumor-reactive CTLs present major limitations. This study introduces a novel method to effectively produce and expand tumor-activated CTLs using high-voltage pulsed electric fields. We hypothesize that utilizing high-voltage pulsed electric fields may be an ideal method to activate and expand CTLs due to their non-thermal celldeath mechanism.Tumor cells were subjected to high-frequency irreversible electroporation (HFIRE) with various electric field magnitudes (1250, 2500 V/cm) and pulse widths (1, 5, and 10 µs), or irreversible electroporation (IRE) at 1250 V/cm. The treated tumor cells were subsequently cocultured with CD4+ and CD8+ T cells along with antigen-presenting cells.We show that tumor-activated CTLs can be produced and expanded when exposed to treated tumor cells. Our results suggest that CTLs are more effectively expanded when pulsed with HFIRE conditions that induce significant cell death (longer pulse widths and higher voltages). Activated CD8+ T cells demonstrate cytotoxicity to untreated tumor cells suggesting effector function of the activated CTLs. The activated CTLs produced with our technique could be used for clinical applications with the goal of targeting and eliminating the tumor.

Comparison between high-frequency irreversible electroporation and irreversible electroporation ablation of small swine liver: follow-up of DCE-MRI and pathological observations.

Background:High-frequency irreversible electroporation (H-FIRE) is a novel, next-generation nanoknife technology with the advantage of relieving irreversible electroporation (IRE)-induced muscle contractions. However, the difference between IRE and H-FIRE with distinct ablation parameters was not clearly defined. This study aimed to compare the efficacy of the two treatments in vivo.Methods:Ten Bama miniature swine were divided into two group: five in the 1-day group and five in the 7-day group. The efficacy of IRE and H-FIRE ablation was compared by volume transfer constant (Krans), rate constant (Kep) and extravascular extracellular volume fraction (Ve) value of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), size of the ablation zone, and histologic analysis. Each animal underwent the IRE and H-FIRE. Temperatures of the electrodes were measured during ablation. DCE-MRI images were obtained 1, 4, and 7 days after ablation in the 7-day group. All animals in the two groups were euthanized 1 day or 7 days after ablation, and subsequently, IRE and H-FIRE treated liver tissues were collected for histological examination. Student's t test or Mann-Whitney U test was applied for comparing any two groups. One-way analysis of variance (ANOVA) test and Welch's ANOVA test followed by Holm-Sidak's multiple comparisons test, one-way ANOVA with repeated measures followed by Bonferroni test, or Kruskal-Wallis H test followed by Dunn's multiple comparison test was used for multiple group comparisons and post hoc analyses. Pearson correlation coefficient test was conducted to analyze the relationship between two variables.Results:Higher Ve was seen in IRE zone than in H-FIRE zone (0.14 ± 0.02 vs. 0.08 ± 0.05, t = 2.408, P = 0.043) on day 4, but no significant difference was seen in Ktrans or Kep between IRE and H-FIRE zones at all time points (all P > 0.05). For IRE zone, the greatest Ktrans was seen on day 7, which was significantly higher than that on day 1 (P = 0.033). The ablation zone size of H-FIRE was significantly larger than IRE 1 day (4.74 ± 0.88 cm2vs. 3.20 ± 0.77 cm2, t = 3.241, P = 0.009) and 4 days (2.22 ± 0.83 cm2vs. 1.30 ± 0.50 cm2, t = 2.343, P = 0.041) after treatment. Apoptotic index (0.05 ± 0.02 vs. 0.73 ± 0.06 vs. 0.68 ± 0.07, F = 241.300, P < 0.001) and heat shock protein 70 (HSP70) (0.03 ± 0.01 vs. 0.46 ± 0.09 vs. and 0.42 ± 0.07, F = 64.490, P < 0.001) were significantly different between the untreated, IRE and H-FIRE zones, but no significant difference was seen in apoptotic index or HSP70 between IRE and H-FIRE zone (both P > 0.05). Electrode temperature variations were not significantly different between the two zones (18.00 ± 3.77°C vs. 16.20 ± 7.45°C, t = 0.682, P = 0.504). The Ktrans value (r = 0.940, P = 0.017) and the Kep value (r = 0.895, P = 0.040) of the H-FIRE zone were positively correlated with the number of hepatocytes in the ablation zone.Conclusions:H-FIRE showed a comparable ablation effect to IRE. DCE-MRI has the potential to monitor the changes of H-FIRE ablation zone.