Electrical Membrane Breakdown (EMB): Preliminary Findings of a New Method of Non-thermal Tissue Ablation

Electrical membrane breakdown (EMB) is a novel form of non-thermal treatment that has not, to our knowledge, been previously evaluated for its potential utility as an ablation mechanism. The findings with EMB immediately after treatment were compared with other forms of ablation (cryoablation and IRE (irreversible electroporation)) in the porcine liver clinically, ultrasonographically, and by light microscopy and ultrastructural analysis. Clinically, EMB did not induce muscular contractions, in contrast with IRE. By ultrasound, all lesions were hypoechoic when compared to the untreated liver. EMB created a consistent pattern of immediate tissue destruction at the light microscopic and ultrastructural level, characterized by pulse-dose-related coagulative necrosis and nuclear pyknosis, preserved blood vessels and adjacent structures, and sharply demarcated margins. We conclude that the profile of EMB ablation is distinctive and unique, inducing necrosis by immediate electrical rupture of cell membranes non-thermally while preserving proteins and adjacent vessels with potentially superior stimulation of the immune system than other ablation modalities.


Introduction
Ablation is a surgical technique for destroying unwanted cells or organs, including abnormal growths such as cancer. Unlike surgical extirpation, ablation does not remove the treated tissue; instead, the altered cell mass persists in situ, with subsequent removal or sequestration by the body's defense and healing mechanisms. This creates an opportunity to harness the immune system to recognize the dead cells and auto-immunize the body against potential cancer neoantigens [1].
We sought to identify a novel method of ablation with a unique treatment profile unlike any other form of ablation that may be useful in combination with cancer immunotherapy: electrical tissue destruction that induces necrosis, stimulates the immune system, preserves proteins and cancer neo-antigens owing to non-thermal injury, and spares adjacent structures such as blood vessels from alteration. By preserving the protein structure and antigenicity of cancer cell membranes and intracellular contents, this novel method of non-thermal ablation should theoretically provoke the abscopal effect in distant cancer deposits (metastases) containing similar neo-antigens, thereby exploiting the immune system in killing cancer. The only other electrical ablation technique that is non-thermal (irreversible electroporation, or IRE) results in only a modest immune response owing to mechanism of cell death by apoptosis, so it would be less suitable for use in combination with immune treatment of cancer metastases [2].
In this report, we describe the immediate histopathologic and ultrastructural effects of EMB in an animal model (porcine liver) that closely mimics human tissue [3]. The findings were compared and contrasted with cryoablation, IRE, and other forms of ablation at the light microscopic and ultrastructural level to document the mechanism of cell damage. We found that EMB induced a novel pattern of cellular injury with immediate necrosis and non-thermal cell death while preserving blood vessels and nerves.

Aims of the study
• Identify the light microscopic and ultrastructural findings with electrical membrane breakdown (EMB).
• Compare the morphologic findings of EMB with cryoablation and irreversible electroporation.

Animals
Three female Yorkshire/Landrace hybrid pigs (40-60 kg) were obtained from an approved local vendor and placed in quarantine prior to experimentation. Study was conducted under the auspices of the Animal Care and Use Committee of the Celebration Florida Nicholson Center (Celebration, FL) in accordance with Good Laboratory Practice regulations as set forth by the 21 Code of Federal Regulations (CFR) Part 58 (Protocol Number 2016.08.01). Animals were placed under general anesthesia using desflourane. In addition, pancuronium (0.1 mg/kg, at a dose of 1 mg/ml) was administered through an IV prior to the procedure to reduce muscle contractions during the application of the DC electrical pulses used for creating the IRE lesions. Pancuronium (0.05 mg/ml at 1 mg/ml) was administered throughout the procedure as needed. The liver was exposed via a midline incision. The entire procedure took less than three hours per pig. Upon completion of the ablation, animals were immediately sacrificed prior to their recovery from anesthesia.

Ablation methods
Three ablation methods (EMB [electrical membrane breakdown], cryoablation, and IRE [irreversible electroporation]) were employed. EMB (electric membrane breakdown) consists of application of short, high-voltage radiofrequency pulses with instant charge reversal and a square wave form, producing high electric fields between two electrodes. Process parameters such as electric field strength, pulse width, pulse number, wave shape, and frequency were important in optimizing desired output. For our application of EMB, we used a patented device (RFEMB Holdings, LLC, Orlando, FL) [4]. Temperatures were continuously monitored using an optical temperature monitoring system from Omega Engineering, Norwalk CT. Temperatures were monitored for the center of the lesion and at each electrode. Temperature was maintained below 42 degrees centigrade at all times.
Cryoablation was performed according to the manufacturer's recommendations (Endocare, Inc., Irvine, CA). IRE (irreversible electroporation) was performed using 18 gauge stainless steel electrodes (Angiodynamics Inc, Latham, NY) with sliding insulating sheaths exposing 1 cm of each electrode, as previously described [5]. The electrodes were inserted in the liver under ultrasound monitoring with two electrodes in a roughly axial parallel configuration. Square DC pulses were applied to the liver through the electrodes using a DC pulse generator. Electrical pulses were delivered in a bipolar manner between two electrodes.

Ultrasound
Ultrasound imaging was performed with Noblus Hitachi Ultrasound machine (Hitachi Healthcare Americas, Twinsburg, OH) using a multifrequency endfire intracavitary transducer. Ultrasonographic studies were obtained directly from the liver immediately after each ablation and before each animal was sacrificed to determine if the effects of EMB can be monitored by medical imaging.

Pathology and ultra-structural studies
Upon completion of each ablation method, superficial 2.0 X 2.0 X 1.0 cm slices of liver tissue were obtained for pathologic analysis to encompass the maximum area of gross abnormality; elapsed time from treatment to biopsy was less than 30 min. In all instances, tissue was trimmed and then immediately immersed in 10% neutral phosphate buffered formalin for light microscopic analysis; in six instances, tissue was also minced into 1 mm blocks and immersed immediately in a mixture of 4% formaldehyde and 1% glutaraldehyde for ultrastructural analysis. A slice of non-ablated liver was separately submitted as a presumptive normal control.
For light microscopy, tissue specimens were routinely processed through graded alcohols and xylene, embedded in paraffin, sliced at 3 microns, and stained with hematoxylin and eosin.
For ultrastructural studies, specimens were routinely processed, stained with uranyl acetate, and examined in a JEOL (Tokyo, Japan) transmission electron microscopy at magnifications ranging from 5000X to 60,000X.

Clinical findings
For IRE, we were unable to make the neuromuscular blockade complete with standard dosages of medication during the application of the DC pulses. Conversely, EMB showed no evidence of muscular contractions at the same voltages even in the absence of neuromuscular blockade.

Ultrasound findings
All lesions were hypoechoic when compared to the untreated liver, irrespective of the modality employed.

Light microscopic findings
Normal porcine liver: The normal porcine liver was remarkably similar in histologic architecture and cellular detail to the human liver, as previously described [3], consisting of lobules of confluent hexagonal plates of hepatocytes with innermost central veins. At the vertices of the lobules, the portal triads included the bile ducts, hepatic arteries and hepatic portal veins. Inflammation was absent ( a Submitted for light microscopy only; ultrastructural findings derived from the literature. b The cell and nuclear membranes appeared intact by routine light microscopy and transmission electron microscopy; it should be noted that electroporation is best visualized by scanning electron microscope, so such findings would not be evident in our studies.  EMB (1500 pulses) 90 μs pulse width, 250 kHZ, 2000 V: The lobular architecture was disorganized and chaotic, with marked vascular congestion, although vascular structures appeared intact. There was no inflammation. Hepatocyte nuclei were shrunken and hyperchromatic, cellular organelles such as mitochrondria were distorted, and cell membranes were indistinct. At the margins, there was a sharp demarcation between altered and un-altered tissue (less than 50 microns).
EMB (4000 pulses) 90 μs pulse width, 250 kHZ, 2000 V: There was massive cell death (coagulative necrosis) with loss of the lobular architecture accompanied by marked vascular congestion. Vessels and bile ducts in the portal triads were preserved, as were the central veins, although venous thrombosis was focally present. No inflammation was observed. Hepatocyte nuclei were pyknotic, mitochondria and cellular organelles were destroyed, and cell membranes were obliterated. No cytoplasmic vacuoles, monophasic glycogen granules, or electrondense lysosomes were observed. At the margins, there was a sharp demarcation between altered and un-altered tissue (Figure 1). Cryoablation: The liver showed mummification (coagulative necrosis), with complete cell death, marked vascular congestion and occasional scattered intermediate size and large vacuoles within hepatocytes; There was no thrombosis, but vessels were mummified, including pyknotic nuclei as well as loss of staining of nuclei, creating pale "ghost"nuclei. At the margins, there was a thin line of demarcation between altered and un-altered tissue (Figure 2A and B). IRE (99 pulses) DC pulses, 90 μs pulse width 2000V: There were minimal changes, including vascular congestion and minimal distortion of scattered mitochondrial cristae. No vacuolization was observed. All adjacent structures, including blood vessels, were intact ( Figure 3).

Ultra structural findings
EMB (100 pulses): Architecture and cell and nuclear membranes were preserved, with vascular congestion and the appearance of occasional scattered microvesicles at the periphery of intact cells ( Figure 4A). Mitochondria were mildly distorted. EMB (1500 pulses): Architecture was distorted, accompanied by prominent vascular congestion. The most striking finding was alteration of mitochondria, with marked enlargement and destruction of cristae in about half of cells. Cytoplasmic blebs were occasionally observed at the edge of some hepatocytes.  EMB (4000 pulses): There was marked distortion of all cells and internal organelles, including disorganization, complete loss of cell junctions, mummification of cytoplasmic contents with loss of resolution and early vacuolization of mitochondria, and loss of microvilli around bile ductules ( Figure 4B). Cell membranes were fuzzy and indistinct.
Cryoablation: There was coagulative necrosis, characterized by architectural distortion and loss of cell integrity, including mummification of cytoplasmic contents and marked alteration of mitochondrial contours and cristae ( Figure 5). There was also prominent vascular congestion.

Discussion
Our results showed that EMB created a consistent pattern of immediate tissue destruction at the light microscopic and ultrastructural level, characterized by pulse dose-related coagulative necrosis and nuclear pyknosis. Unlike cryoablation and most other forms of tissue ablation, EMB preserved blood vessels, bile ducts, and nerves,   indicating preferential destruction of hepatocytes. To our knowledge, these findings are the first to demonstrate the potential utility of EMB as a new form of non-thermal tissue ablation that may have clinical value.
We confirmed that IRE, the only other electrical non-thermal form of tissue ablation, did not induce necrosis or pyknotic (morphologically dead or dying) nuclei immediately after treatment, similar to previous descriptions [6,7]. These findings reflected differences in mechanism of cell death between EMB and IRE (immediate necrosis vs. apoptosis, respectively) ( Table 2). However, both treatments preserved surrounding tissues, thereby avoiding the heat sink effect [8] and both were sharply demarcated, with no apparent transition zone with alteration at the margins.
EMB did not induce muscular contractions, even in the absence of neuromuscular blockade, in contrast with IRE. This is an important distinction, as IRE requires the administration of general anesthesia and paralytic agents in order to eliminate muscle contractions [9].
There are numerous methods of ablation that vary by mechanism of tissue destruction (chemical, thermal, electrical), rapidity of cell death (apoptosis: slow [1-3 days]; necrosis: immediate), effect on native proteins (intact or denatured); differential sparing of adjacent structures such as blood vessels and nerves (intact or ablated); and likely impact on the immune system (non-stimulatory or stimulatory) ( Table 3). The four requirements for activation of the acquired immune system are antigen presentation, antigen recognition by T-cells, interaction of costimulatory molecules, and the presence of danger signals [10] and the method of ablation likely influences these factors. For example, protein denaturation by ablation may interfere with antigen presentation or recognition. Thermal ablation methods such as hyperthermia and cryoablation under clinically used protocols irreversibly alter the three dimensional protein structures and destroy the structure of antigenic determinants [11], whereas non-thermal methods of ablation such as IRE and EMB theoretically preserve native proteins and cancer neoantigens. Ablation may also result in rapid cell death (necrosis) or slow programmed cell death (apoptosis), but only necrosis is thought to stimulate a substantial immune response. Mehta et al. noted that apoptotic cell death eliminated danger signals (e.g., production of heat shock proteins) and blocked antigen presentation because phagocytosis shielded intracellular contents; [12] also; dendritic cells that take ingest apoptotic cells do not mature, may have suppressed cytokine production, and may trigger clonal deletion and anergy. Conversely, necrosis caused cell rupture with subsequent spilling of intracellular contents into the extracellular environment and likely migration and activation of dendritic cells with subsequent stimulation of cytotoxic T cells.
Our study has multiple limitations. In this preliminary report, we restricted study to a single species and single tissue type, and employed only a modest number of animals and specimens. Despite the wellrecognized histologic similarity between the porcine and human liver, our findings in the pig may not apply in humans. We limited our study to the changes immediately following treatment, but it would be useful to have multiple time points after treatment to determine the full extent of damage and thereby confirm the mechanisms of cell death. We employed only a single skilled operator (Dr. Onik), and these results may differ according to the expertise of the surgeon. Finally, only a limited number of EMB variables were studied (e.g., duration of treatment, wave shapes created, combinations of treatments, etc.) so these results should be considered preliminary, albeit novel. It should also be noted that we have not directly studied the effect of our treatments on cancer neo-antigens and protein structure, so any hypotheses regarding potential utility of ablation in harnessing the immune response should be considered speculative [13][14][15][16][17][18][19][20].

EMB
Acute a Coagulation necrosis with nuclear pyknosis; blood vessels and nerves preserved Delayed b Unknown; assume resorption of necrotic tissue or conversion to fibrosis. IRE: Irreversible electroporation; N-TIRE: Non-thermal IRE; EMB: Electrical membrane breakdown. a Acute refers to immediate post-operative period (up to 1 hour after treatment); b Delayed refers to late changes (hours to days or more after treatment), allowing for effects of regeneration.

Conclusion
EMB is a novel form of non-thermal tissue ablation that results in immediate coagulative necrosis while preserving adjacent structures. It is distinguished from IRE by a unique mechanism of non-thermal cell destruction and characteristic electrical properties that avoid muscular contractions during treatment.

Declaration of Conflicting Interests
DGB, GO, and JM are affiliated and have a personal financial interest, including patents, in RFEMB Holdings, LLC, a company that was established to commercialize the technology of electrical membrane breakdown (EMB). Each of these authors has signed the following: "I had full access to all of the data in this study and I take complete responsibility for the integrity of the data and the accuracy of the data analysis." ZW and MBF declare that they have no conflicting interests.

Funding Source
Funding was provided by RFEMB Holdings, LLC, Orlando, FL.