Canady Helios Cold Plasma Induces Non-Thermal (24 °C), Non-Contact Irreversible Electroporation and Selective Tumor Cell Death at Surgical Margins

Abstract

Background: The Canady Helios Cold Plasma (CHCP) system is a non-thermal, non-contact cold atmospheric plasma technology that generates transient electric fields and reactive species capable of disrupting cancer cell membranes. This study investigated the voltage-dependent membrane irreversible electroporation (IRE) dynamics induced by CHCP across biologically distinct breast cancer subtypes.

Methods: Four breast cancer cell lines, triple-negative (MDA-MB-231 and Hs578T), ER⁺/PR⁺/HER2^- (MCF-7), and ER⁺/PR⁺/HER2⁺ (BT-474), were exposed to CHCP for 5 min at 25 V (~1675 V/cm PTEF) or 30 V (~2010 V/cm), either directly or with Plasma Activated Media (PAM). Membrane permeability was assessed by propidium iodide (PI) uptake over 120 min. Morphological changes were evaluated microscopically. Functional electroporation was examined via BCL2A1-targeting siRNA delivery and clonogenic survival. Ex vivo analyses of Phase I clinical trial tumor specimens (NCT04267575) were performed to characterize CHCP-induced tissue responses.

Results: CHCP produced voltage- and time-dependent membrane permeabilization in all breast cancer cell lines, with 30 V generating robust and sustained PI uptake compared to transient effects at 25 V. Treated cells exhibited morphological features consistent with membrane disruption. CHCP enabled intracellular siRNA delivery and significantly reduced clonogenic potential, confirming functional pore formation. Ex vivo CHCP treatment selectively damaged tumor cells while sparing adjacent non-cancerous tissue.

Conclusions: This study demonstrates CHCP as a non-thermal (24 °C), non-contact plasma-based IRE platform which induces controlled membrane permeabilization and selective cancer cell death. CHCP offers a translational strategy to eradicate residual tumor cells at the surgical margins, and prevent local recurrence, positioning it as a versatile adjunct in precision surgical oncology.

Keywords: breast cancer; cancer treatment; cold atmospheric plasma; irreversible electroporation (IRE); non-contact; non-thermal (24 °C); plasma treated electromagnetic field (PTEF); surgical margin treatment.

Figure 1

Illustration showing the CHCP system.

Figure 2

Schematic representation of the Plasma Treated Electromagnetic Field (PTEF) generated by the Canady Helios Cold Plasma (CHCP) system.

Figure 3

Analysis of electroporation effects of CHCP treatment on triple-negative breast cancer MDA-MB-231 cells using PI intake intensity measurements. Cells were treated with CHCP at 30 V, CHCP at 25 V, or helium for 5 min, followed by analysis at multiple incubation time points (0, 30, and 60 min) to assess the plasma-induced membrane permeability. The percentage of PI intensity indicates the degree of cell membrane electroporation, with the data representing the mean ± SEM from n = 3 biological replicates, each containing triplicate technical measurements. Statistical significance was analyzed between CHCP-treated and helium-treated samples using Student’s t-test, where at 30 V, at zero, 0.5, and 1 h, it was p = 0.016, p = 0.020, and p = 0.062, respectively. At 25 V, at zero, 0.5, and 1 h, it was p = 0.001, p =0.008, and p = 0.618, respectively. Data are represented by * p < 0.05 and ** p < 0.01. Although the bar graph for the 30 V group at the 60-min time point appears comparable to or greater than the 25 V group at earlier incubation periods, one of the three replicate measurements for the 30 V 60 min group was similar to the helium control, resulting in higher variance within this dataset. Consequently, statistical significance was not achieved for this time point, and the SEM error bar appears relatively longer in the graph.

Figure 4

Analysis of electroporation effects of CHCP treatment on ER⁺ PR⁺ HER2⁻ breast cancer MCF-7 cells using PI intake intensity measurements. Cells were treated with CHCP at 30 V (~2010 V/cm PTEF) and 25 V for 5 min, followed by analysis at multiple incubation time points (0, 30, and 60 min) to assess the plasma-induced membrane permeability. The percentage of PI intensity indicates the degree of cell membrane electroporation, with data representing the mean ± SEM from n = 3 biological replicates, each containing triplicate technical measurements. Statistical significance was analyzed between CHCP-treated and helium-treated samples using Student’s t-test, where at 30 V, at zero, 0.5, and 1 h, it was p = 0.0015, p = 0.0089, and p = 0.013, respectively. At 25 V, at zero, 0.5, and 1 h, it was p = 0.014, p = 0.203, and p = 0.168, respectively. Data are represented by * p < 0.05 and ** p < 0.01.

Figure 5

Analysis of electroporation effects of CHCP treatment on triple-negative breast cancer Hs578T cells using PI intake intensity measurements. Cells were treated with CHCP at 30 V and 25 V for 5 min, followed by analysis at multiple incubation time points (0, 30, and 60 min) to assess the plasma-induced membrane permeability. The percentage of PI intensity indicates the degree of cell membrane electroporation, with data representing the mean ± SEM from n = 3 biological replicates, each containing triplicate technical measurements. Statistical significance was analyzed between CHCP-treated and helium-treated samples using Student’s t-test, where at 30 V, at zero, 0.5, and 1 h, it was p = 0.0030, p = 0.0105, and p = 0.00014, respectively. At 25 V, at zero, 0.5, and 1 h, it was p = 0.0087, p = 0.03, and p = 0.783, respectively. Data are represented by * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 6

Analysis of electroporation effects of CHCP treatment on ER⁺ PR⁺ HER2⁺ breast cancer BT-474 cells using PI intake intensity measurements. Cells were treated with CHCP at 30 V and 25 V for 5 min, followed by analysis at multiple incubation time points (0, 30, and 60 min) to assess the plasma-induced membrane permeability. The percentage of PI intensity indicates the degree of cell membrane electroporation, with data representing the mean ± SEM from n = 3 biological replicates, each containing triplicate technical measurements. Statistical significance was analyzed between CHCP-treated and helium-treated samples using Student’s t-test, where at 30 V, at zero, 0.5, and 1 h, it was p = 0.0005, p = 0.0010, and p = 0.0001, respectively. At 25 V, at zero, 0.5, and 1 h, it was p = 0.0008, p = 0.0135, and p = 0.273, respectively. Data are represented by * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 7

Analysis of temperature during CHCP treatment on breast cancer cells. Breast cancer cell lines MDA-MB-231, MCF-7, and Hs578T were treated with CHCP at 15 V, 20 V, 25 V, and 30 V for 5 min and temperature measurements of media were recorded before, during, and after the treatment. The figure shows the bar graphs showing the temperatures. The error bars represent means ± SEM. (n = 3). No Treatment (NT) and helium control (He) are at zero volts.

Figure 8

Analysis of electroporation effects of CHCP treatment on breast cancer cells by silencing of BCL2A1 mRNA. Transfection of siBCL2A1 by CHCP treatment. Breast cancer cell lines MDA-MB-231, MCF-7, and Hs578T were treated with CHCP at 15 V, 20 V, 25 V, and 30 V for 5 min. Bar graphs show the expression of BCL2A1 mRNA with esiRNA for BCL2A1 silencing with CAP treatment. The data represent the mean ± SEM from n = 3 biological replicates, each containing triplicate technical measurements. (MDA-MD-231 at 30 V, p = 0.0055; MCF-7 at 25 V, p = 0.012, and 30 V, p = 5.76253 × 10⁻⁰⁶; Hs578T at 25 V, p = 0.0005, and 30 V, p = 0.02; Student’s t-test for siBCL2A1 versus control siRNA. Data are represented by * p < 0.05, ** p < 0.01 and *** p < 0.001.)

Figure 9

Analysis of electroporation effects of CHCP treatment on breast cancer cells on colony formation. Breast cancer cell lines MDA-MB-231, MCF-7, and Hs578T were treated with CHCP at 15 V, 20 V, 25 V, and 30 V for 5 min and incubated for 30, 60, and 120 min, followed by colony formation assays. The figure shows bar graphs showing the number of colonies with CAP treatment. The data represent the mean ± SEM from n = 3 biological replicates, each containing triplicate technical measurements. Significance was set at p < 0.05, using one-way ANOVA followed by post-hoc Tukey’s tests for treated samples versus the helium control. All p-values < 0 (*), indicating they were highly significant.

Figure 10

Light micrographs of H&E-stained Zone 0 tissue samples from patient (A) R0009 (metastatic pleomorphic sarcoma of the left distal femur) and (B) R0004 (metastatic recurrent non-small cell lung adenocarcinoma of the left hip/proximal femur) with or without CHCP treatment, imaged at 63× objective magnification. Black arrows indicate viable untreated tumor cells, red arrows denote CHCP-treated tumor cells exhibiting apoptosis with cellular cytosolic content leakage, and green arrows represent normal cells. Scale bars = 0.2 mm.

Figure 11

Schematic representation of the Canady Helios Cold Plasma (CHCP) treatment workflow. The illustration depicts (i) surgical resection of the tumor using standard oncologic procedures, (ii) collection of specimens from the tumor core, surgical margin (Zone 0), peritumoral tissue (Zone 1), and adjacent normal tissue, and (iii) intraoperative application of the CHCP spray directly to the surgical margin. (iv) A representative intraoperative image shows CHCP treatment of a pancreatic surgical margin following a Whipple procedure. (v) The schematic further highlights the CHCP-generated PTEF, where E denotes the electric field, B the magnetic field, and C the three-dimensional vector reference of the magnetic field. Reactive oxygen and nitrogen species (ROS/RNS), including hydroxyl radicals, are shown entering the cancer cell membrane through transient “pores” formed by CHCP-induced electroporation, ultimately disrupting cellular integrity and promoting selective tumor cell death.