Understanding the role of calcium-mediated cell death in high-frequency irreversible electroporation.

High-frequency irreversible electroporation (H-FIRE) is an emerging electroporation-based therapy used to ablate cancerous tissue. Treatment consists of delivering short, bipolar pulses (1-10µs) in a series of 80-100 bursts (1 burst/s, 100µs on-time). Reducing pulse duration leads to reduced treatment volumes compared to traditional IRE, therefore larger voltages must be applied to generate ablations comparable in size. We show that adjuvant calcium enhances ablation area in vitro for H-FIRE treatments of several pulse durations (1, 2, 5, 10µs). Furthermore, H-FIRE treatment using 10µs pulses delivered with 1mM CaCl2 results in cell death thresholds (771±129V/cm) comparable to IRE thresholds without calcium (698±103V/cm). Quantifying the reversible electroporation threshold revealed that CaCl2 enhances the permeabilization of cells compared to a NaCl control. Gene expression analysis determined that CaCl2 upregulates expression of eIFB5 and 60S ribosomal subunit genes while downregulating NOX1/4, leading to increased signaling in pathways that may cause necroptosis. The opposite was found for control treatment without CaCl2 suggesting cells experience an increase in pro survival signaling. Our study is the first to identify key genes and signaling pathways responsible for differences in cell response to H-FIRE treatment with and without calcium.

Sonoluminescence characterization of inertial cavitation inside a BSA phantom treated by pulsed HIFU.

The aim of this study was to investigate the inertial cavitation inside a phantom treated by pulsed HIFU (pHIFU). Basic bovine serum albumin (BSA) phantoms without any inherent ultrasound contrast agents (UCAs) or phase-shift nano-emulsions (PSNEs) were used. During the treatment, sonoluminescence (SL) recordings were performed to characterize the spatial distribution of inertial cavitation adjacent to the focal region. High-speed photographs and thermal coagulations, comparing with the SL results, were also recorded and presented. A series of pulse parameters (pulse duration (PD) was between 1 and 23 cycles and pulse repetition frequency (PRF) was between 0.5kHz and 100kHz) were performed to make a systematic investigation under certain acoustic power (APW). Continuous HIFU (cHIFU) investigation was also performed to serve as control group. It was found that, when APW was 19.5W, pHIFU with short PD was much easier to form SL adjacent to the focal region inside the phantom, while it was difficult for cHIFU to generate cavitation bubbles. With appropriate PD and PRF, the residual bubbles of the previous pulses could be stimulated by the incident pulses to oscillate in a higher level and even violently collapse, resulting to enhanced physical thermogenesis. The experimental results showed that the most violent inertial cavitation occurs when PD was set to 6 cycles (5µs) and PRF to 10kHz, while the highest level of thermal coagulation was observed when PD was set to 10 cycles. The cavitational and thermal characteristics were in good correspondence, exhibiting significant potentiality regarding to inject-free cavitation bubble enhanced thermal ablation under lower APW, compared to the conventional thermotherapy.