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High-intensity irreversible electroporation targeting intracellulafigr structures enhances tumor ablation in lung cancer models

PLOS ONE

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As you see, the reviewers are recommending substantial changes, which are somewhat consistent amongst the two.  They both find some untenable assertions being made for which they present literature as support.  Further they call out some of your citations as not relevant.

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Reviewers' comments:

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-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: No

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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-->5. Review Comments to the Author

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Reviewer #1: The manuscript entitled “High-intensity irreversible electroporation targeting intracellulafigr structures enhances tumor ablation in lung cancer models” presents an innovative and timely study investigating the effects of high-intensity irreversible electroporation (IRE) on intracellular structures, particularly nuclear membranes and mitochondria, in lung cancer models. The work combines simulation, in vitro experiments, and preliminary in vivo validation, offering significant insights into potential improvements in IRE-based tumor ablation.

Strengths:

The study addresses a critical limitation of conventional IRE, i.e., incomplete intracellular damage leading to possible tumor recurrence.

The combination of computational modeling, in vitro assays, and in vivo ultrastructural analysis provides a comprehensive assessment of high-field effects.

The results are compelling, demonstrating that high electric field intensities induce significant mitochondrial dysfunction, oxidative stress, and nuclear membrane deformation, which could potentially improve tumor eradication.

The manuscript is generally well organized.

Areas for Improvement:

1. Title Typographical Error:

The title contains a typographical error:

“intracellulafigr” → should be “intracellular”

2. Clarification of Energy Equivalence:

The manuscript frequently compares low electric field (LEF) and high electric field (HEF) conditions, stating that they delivered the “same total energy.” However, it is not entirely clear how this equivalence was calculated (e.g., purely Joule heating, energy per pulse, pulse shape effects). I recommend including a table summarizing pulse conditions and explicit calculations of delivered energy.

3. Statistical Details:

Although statistical analyses are reported, several figures lack precise p-values, and it is unclear which post hoc tests were applied after ANOVA. Please clarify statistical methods and ensure consistency across all figures.

4. In Vivo Data Expansion:

The in vivo data are limited to TEM analysis. It would significantly strengthen the manuscript to include: Tumor volume measurements or necrosis quantification post-treatment, Histological analyses beyond TEM (e.g., H&E staining), Data on potential side effects or systemic responses (e.g., inflammation, weight loss)

5. Thermal Considerations:

The discussion briefly mentions temperature increases during high-field IRE. Given that high electric fields might cause localized heating in tissues, additional discussion or in vivo measurements of thermal effects would be valuable for assessing clinical safety.

6. Terminology Consistency:

The manuscript switches between terms like HEF, high-voltage IRE, and high electric field. For clarity, please adopt consistent terminology throughout.

7. Language and Minor Errors:

Besides the title typo, minor wording issues are present,

e.g.: “The viabilities of 4000 and 5000 V/cm were almost similar each other…” → should read: “The viabilities at 4000 and 5000 V/cm were similar to each other.”

In several places, “Data is represented…” should read: “Data are represented…”

Please proofread the manuscript carefully for minor grammatical errors and ensure consistent spelling of technical terms (e.g., spell out “NO” as nitric oxide on first mention).

Questions for the Authors:

1. Could you clarify precisely how “same total energy” was calculated for LEF and HEF conditions?

2. Did you measure tissue temperatures in vivo to confirm that high-field protocols remain non-thermal in tissue?

3. Were any physiological responses (e.g., muscle contractions) observed during in vivo HEF pulsing?

4. Why were in vivo analyses limited to TEM without quantifying macroscopic tumor response?

5. The data suggest suppressed transcriptional activity under HEF (e.g., low p53, ATR). Does this indicate necrosis as the dominant cell death pathway, and were any necrosis-specific markers assessed?

6. Have you considered potential systemic toxicity from high oxidative stress observed in HEF-treated cells?

7. Did you assess potential effects on normal lung tissue adjacent to treated tumors?

8. How does your proposed HEF protocol compare to existing high-frequency IRE (HFIRE) approaches in terms of efficacy and safety?

9. Was current measured during the electroporation procedures, particularly in vivo, to monitor tissue conductivity changes or electrical dose delivered? If so, could you report those results?

Overall, this is an interesting and promising manuscript addressing important challenges in cancer ablation therapy. The study would benefit from clarifying methodological details, expanding in vivo analyses, and addressing minor language issues. I look forward to the authors’ revisions.

Reviewer #2: Summary:

In this submission, Kim et al. describe a method to increase the lethality of IRE by compressing the energy of pulses into shorter pulse widths of higher intensity. It is difficult to draw conclusions from the data as the choice of parameters seems arbitrary, with many comparisons made between 4000 V of 20p at 20 us and 1000 Vof 64p of 100 us, which is not conventional IRE and the normalization was not rationalized. Since so many parameters were varied between two only two groups, the outcomes can be due to voltage, pulse number, or pulse width. Further, there are many incorrect statements with improper citations that do not support the statements, some of which talk about IRE with papers that are not electroporation at all.

Comments:

1. There is a typo in the submission title. Not sure if this will cause a problem.

2. “Spark-free” is mentioned a few times. Sparking was not necessarily a problem with IRE and H-FIRE previously, and the terminology makes it seem like you are overcoming this problem which isn’t prevalent.

3. The introduction heavily cites review articles, which do not entirely support the statements being made, particularly with statements that IRE is membrane-focused and minimally affects intracellular structures. Higher frequency pulses may not be as attenuated by the cell membrane impedance, but there is a trade off with pore formation decreasing the membrane resistance and increasing the capacitance. If the cell membrane experiences a pulse, it does not know 2 µs into the pulse if the pulse will end or if it will continue for another 98 µs. For IRE and H-FIRE during those initial 2 µs, the electric field distribution within the cell and outside should theoretically be the same. Ignoring that IRE can physically hemorrhage the cell which would affect all the contents, IRE will also damage intracellular structures, but perhaps the ratio of membrane disruption to intracellular disruption is different between different pulse widths. For the same electric field strength (i.e., 2000 V/cm), IRE will affect intracellular structures more than H-FIRE. However, in the context of this paper, the pulse widths are not short enough to be beyond the beta dispersion caused by membrane capacitance.

4. “Studies have shown that residual cancer cells, after membrane-focused IRE, exhibit increased motility, elevated expression of heat shock proteins (HSPs), and enhanced resistance to subsequent therapies[12].” Citation 12 is not an electroporation paper and does not support this claim. There is literature demonstrating the opposite, that electroporation decreases genetic expression associated with metastasis (doi: 10.1016/j.bioelechem.2025.109036.) and decreases motility of cells following treatment in brain cancers (doi: 10.1016/j.bioelechem.2025.109036. , 10.1016/j.bioelechem.2025.109005).

5. Citations 15 and 16 for high-electric-field IRE are nanosecond and H-FIRE, not IRE.

6. H-FRE has only been briefly used in human tumors, but there are in vivo studies using H-FIRE >2,300 V/cm (https://doi.org/10.34133/bmef.0169, 10.1109/TBME.2024.3468159, 10.1016/j.ebiom.2019.05.036).

7. Was the conductivity of the media measured, because my understanding is that cell culture media (i.e., RPMI, DMEM) is roughly ~1 S/m at room temperature and 1.2 S/m at 37C (10.1115/1.4053595). Also, how would half filling the cuvette reduce the sparking risk? My understanding is that sparking is due to electric field or current density intensity, which wouldn’t really change if it was ½ as tall? The electric field would be roughly the same, but the volume would be half, meaning the current should be roughly half. However, the current scale linearly with the height, meaning the current density should be the same no matter the height? If it was experimentally observed, then that is interesting and possibly due to heating?

8. Be careful using the word significant when statistics were not done. There were not statistical analyses. E.g., “At higher voltages of 1600 V and 2000 V, significant intensification of the electric field was evident, reaching critical strengths exceeding 4000 V/cm, indicated by the red color zones”

9. “Viabilities of 20 and 40 pulses were similar due to reaching saturation effect.” This is contradictory of previous IRE and H-FIRE results that have seen a much higher saturation (10.1016/j.bioelechem.2023.108580).

10. There are many parameters being adjusted without much rationale for choices. The results needs to clearly explain why these parameters are being adjusted and general conclusion for that results section. It just seems like a lot of different things were done without a coherent story, reasoning for why, and what the results mean.

11. For the resistance changes, what is the control resistance. The figure should clearly indicate if it is an increase or decrease in resistance. Also, the cell suspensions presented are too low of cell density to see changes in resistance due to electroporation, so you are most likely measuring conductivity changes due to temperature, so “indicates that HEF may induce strong membrane polarization and destabilization, potentially increasing susceptibility to additional stress” may not be accurate. Even in super dense cell suspensions, only small changes in membrane resistance have been measured, but 1x10^5 cells /ml is orders of magnitude much too low than the >5x10^7 cell/ml to see these effects (https://doi.org/10.1007/978-3-319-26779-1_164-1, https://doi.org/10.1529/biophysj.104.048975).

12. For gene expression analysis, did the house keeper gene significantly change with treatment. If so, then you know that translation was impaired due to cell death. Using a gene that mediates cell death is not appropriate, as it is involved in the processes being induced.

13. “Typical IRE protocols primarily focus on permeabilizing the cell membrane, leaving the nuclear membrane largely intact [8]” Though the treatment done is not exactly IRE (100 µs for 100 pulses or NanoKnife 90 µs for 90 pulses), the results presented demonstrate that IRE does induce significant nuclear damage, at a lower electric field than used clinically (10.2478/raon-2025-0011).

14. This statement is incorrect and not properly cited. Citation 24 is not cancer and does not mention nucleus in the paper: “A previous study reported that up to 30% of cancer cells treated with membrane-focused IRE survive because the nuclear membrane protects genetic material and crucial cellular functions, enabling recovery and survival [24]”. They demonstrate that heterogeneous tissue conductivity causes distortions of the electric field that can drop the lethal electric field below the threshold for areas within the tissue.

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Reviewer #1: No

Reviewer #2: No

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<div>PONE-D-25-30916R1-->-->High-intensity irreversible electroporation targeting intracellular structures enhance tumor ablation in lung cancer models-->-->PLOS One

Dear Dr. Sim,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================-->-->I am returning your manuscript for revision without review because I note that points 11-14 of reviewer 2's critique have not been responded in your summary of changes to the manuscript.  One should be aware that reviewers put considerable effort into evaluation, without direct compensation.  To protect reviewer time and keeping with editor support of the reviewers, this issue is to be addressed before considering any further review of changes.  We look forward to a full revision. -->-->==============================-->-->==============================

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We look forward to receiving your revised manuscript.

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Roy P. Planalp, Ph.D.

Academic Editor

PLOS One

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High-intensity irreversible electroporation targeting intracellular structures enhance tumor ablation in lung cancer models

PONE-D-25-30916R2

Dear Dr. Sim,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->6. Review Comments to the Author

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Reviewer #1: The authors have addressed the reviewer comments satisfactorily, and the manuscript has improved significantly. I have no major remaining concerns.

I only suggest a brief clarification in the Methods section regarding the assumption of constant impedance when using the V2.t.N energy index, since tissue conductivity may change during electroporation. A short discussion of this limitation would further strengthen the methodological description.

With this minor clarification, I consider the manuscript suitable for publication.

Reviewer #2: The authors have done a great job answering the questions thoroughly and making the appropriate fixes within the manuscript.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->..-->

Reviewer #1: Yes: POOMPAVAI SADASIVAMPOOMPAVAI SADASIVAMPOOMPAVAI SADASIVAMPOOMPAVAI SADASIVAM

Reviewer #2: No

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