Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript is a review of work evaluating the potential of electrochemotherapy and effect on immunotherapy either alone or combined with another agent. For the most part, this is well-written and provides a fair assessment of the subject. Conclusions are good and the summary of the studies are also well done.

The only concern is that the authors state that they screened several databases using multiple search terms and only found 231 publications and of those only 9 that had the requisite criteria. This reviewer did a cursory review just of pub med and found an additional 11 publications that appear to match the stated criteria including 6 reporting on clinical results that were not included in the review. It is possible I did not understand criteria used, but if that is the case the authors nee to make that clearer.

Publications found:

Sedlar, A, et al, Radiol Oncol 2012 46:302

Falk , H, Et al, Acta Oncol, 2017 56:1126

Ursic, K, et al, J Control Release, 2021 332:623

Lampreht Tratar, U, et al Int Immunopharmacol, 2023 120:110274

Campana, LG, et al, Cancers, 2021 13:4289

Karaca, B, et al, Anticancer Drugs 2918 29:190

Heppt, MV, et al, Cancer Immunol Immunother, 2016 65:951

Mozzillo, N, et al, 2015 4:e1008842

Brizio, M, et al, 2015 Eur J Dermatol 25:271

Barca, I, Oral Maxillofac Surg Cases 2023 9:100330

Trotovsek, B, World J Gastroenterol 2021 27:8216

Author Response

REVIEWER 1

Comment 1: This manuscript is a review of work evaluating the potential of electrochemotherapy and effect on immunotherapy either alone or combined with another agent. For the most part, this is well-written and provides a fair assessment of the subject. Conclusions are good and the summary of the studies are also well done.

Answer 1:  Thank you for your positive feedback on our manuscript, especially your appreciation of the writing quality, the fair assessment of ECT and its potential in immunotherapy, as well as the strength of our conclusions and study summaries. We are glad that you found these sections well-written and well-organized.

 

Comment 2: The only concern is that the authors state that they screened several databases using multiple search terms and only found 231 publications and of those only 9 that had the requisite criteria. This reviewer did a cursory review just of pub med and found an additional 11 publications that appear to match the stated criteria including 6 reporting on clinical results that were not included in the review. It is possible I did not understand criteria used, but if that is the case the authors need to make that clearer.

Publications found:

Sedlar, A, et al, Radiol Oncol 2012 46:302

Falk , H, Et al, Acta Oncol, 2017 56:1126

Ursic, K, et al, J Control Release, 2021 332:623

Lampreht Tratar, U, et al Int Immunopharmacol, 2023 120:110274

Campana, LG, et al, Cancers, 2021 13:4289

Karaca, B, et al, Anticancer Drugs 2918 29:190

Heppt, MV, et al, Cancer Immunol Immunother, 2016 65:951

Mozzillo, N, et al, 2015 4:e1008842

Brizio, M, et al, 2015 Eur J Dermatol 25:271

Barca, I, Oral Maxillofac Surg Cases 2023 9:100330

Trotovsek, B, World J Gastroenterol 2021 27:8216

Answer 2: Thank you for your detailed feedback and for bringing additional references to our attention. We have carefully reviewed the publications you mentioned. Upon evaluation, we found that nine of these studies focus on the combination of ECT and immunotherapy, while one (Falk 2017) discusses calcium electroporation. Additionally, the Barca 2023 paper involves ECT but does not provide information on its immune effects. We realize that our inclusion and exclusion criteria may not have been sufficiently clear, and we appreciate your observation. To clarify, our review specifically focuses on the impact of ECT on immune functions, rather than the combination of ECT with immunotherapy, which we plan to cover in a separate review. Based on your suggestion, we have now added the following clarification to the Materials and Methods section of the manuscript:

"Furthermore, studies reporting the results of combined modality treatments based on the association of ECT and immunotherapy were excluded."

We sincerely thank you for highlighting these publications, as they will be valuable for our future analyses on the combined use of ECT and immunotherapy.

Reviewer 2 Report

Comments and Suggestions for Authors

This narrative review systematically explores the immunomodulatory effects of Electrochemotherapy (ECT) in cancer treatment, drawing on both preclinical and clinical studies. ECT, which combines electroporation and chemotherapy, has been shown to induce immunogenic cell death, activate dendritic cells, and release tumor antigens, potentially enhancing anti-tumor immune responses. The review synthesizes findings, positioning ECT as a promising adjunct to immunotherapy, though clinical evidence remains limited. The authors emphasize the need for further research to refine treatment protocols, investigate synergistic therapies, and elucidate the mechanisms driving systemic immune responses.

The review offers a comprehensive analysis of ECT's immunomodulatory potential, highlighting key mechanisms like immunogenic cell death and dendritic cell activation. It provides a foundation for future research by identifying areas for optimizing ECT protocols and exploring its synergy with immunotherapy.

However, the review lacks recent studies, with no references post-2021, limiting its relevance. Furthermore, key immunomodulatory mechanisms mentioned in the abstract are not thoroughly explored in the results section.  In conclusion, the manuscript is recommended for major revisions with a possibility of acceptance based on addressing comments discussed below. 

Major Comments: 

·      Lines 70 – 87 should have the content reorganized to display the innate immune system first followed by the adaptive immune system. This would flow better for what the abscopal effect utilizes.

·      Figure 1 is rudimentary and could benefit from having more details added to the cells being seen, such as live tumor cells, treated tumor cells, etc. The figure caption should match these changes, too. Also, the anti-tumor immune response in this figure is incomplete. 

·      The dataset represented in the tables of the study do not show any papers that have not been published since 2019, but I believe there are vital studies that have been conducted since then within the realm of ECT that have occurred within the gap 2019-2024. This may be due to the “snowball technique” implemented for paper selection which may be eliminating some of these more recent studies. This can be detrimental due to gaps of progression in knowledge you may have cut out or stopped at. In addition to not showing any more recent papers, it becomes difficult to build the case that ECT has potential as an immunomodulatory therapy since the clinical papers that help build the case were published almost 10 years ago. 

·      With the snowball technique, what criteria was met or not met to have the paper seen as substantially equivalent to another for not needing to be placed into the document? 

·      I believe the manuscript should benefit from including other papers that use other approaches to ECT that are not strictly using a chemotherapy such as discussing Calcium Electroporation within the realm of ECT since it would open your paper to a broader audience and increase the papers discussed in the in vivo/clinical tables perhaps. 

·      The abstract mentions key immunomodulatory mechanisms, including immunogenic cell death, dendritic cell activation, tumor antigen release, inflammatory responses, and systemic effects like the abscopal phenomenon. However, these mechanisms are not adequately compared or thoroughly explained in the results and discussion sections based on the preclinical and clinical studies presented. 

Minor Comments: 

·      Line 47-- Please remove the word” innovative” from the sentence describing ECT. ECT has been prevalent for decades so it cannot be deemed as “innovative” or new and original.

·      Lines 45-52 -- More citations are required within the first paragraph specifically when describing the mechanism behind electroporation and how drugs are able permeate through a disrupted membrane. This background knowledge is vital to cite since this is specific knowledge elucidated within the field of electroporation.

·       In the preclinical studies section, alfa should have the symbol “α” instead. 

·      Vagueness of what is driving the immunomodulatory system should have more explanation such as in line 65 on types of immunogenic cell death i.e. necrosis or line 85-86 types of immune cells, cytokines, and chemokines are some examples. 

·      The conclusion section drives the point of “potential” behind ECT, but the suggestive language does poorly in context of the lack of recent studies to show the future moving forward for ECT. 

Comments on the Quality of English Language

Please check over for grammatical and spelling errors. There are several.

Author Response

REVIEWER 2

 

Comment 1: This narrative review systematically explores the immunomodulatory effects of Electrochemotherapy (ECT) in cancer treatment, drawing on both preclinical and clinical studies. ECT, which combines electroporation and chemotherapy, has been shown to induce immunogenic cell death, activate dendritic cells, and release tumor antigens, potentially enhancing anti-tumor immune responses. The review synthesizes findings, positioning ECT as a promising adjunct to immunotherapy, though clinical evidence remains limited. The authors emphasize the need for further research to refine treatment protocols, investigate synergistic therapies, and elucidate the mechanisms driving systemic immune responses.

Answer 1:  Thank you for your thoughtful summary of the narrative review and for recognizing the systematic exploration of the immunomodulatory effects of ECT in cancer treatment. We appreciate your acknowledgment of our efforts in synthesizing findings from both preclinical and clinical studies, and in positioning ECT as a potential adjunct to immunotherapy.

We agree that while ECT shows significant promise in enhancing anti-tumor immune responses, particularly through mechanisms such as immunogenic cell death and dendritic cell activation, the current clinical evidence remains somewhat limited. We fully support the need for further research, as noted, to optimize treatment protocols and investigate possible synergistic therapies to enhance ECT efficacy in systemic immune responses.

 

Comment 2: The review offers a comprehensive analysis of ECT immunomodulatory potential, highlighting key mechanisms like immunogenic cell death and dendritic cell activation. It provides a foundation for future research by identifying areas for optimizing ECT protocols and exploring its synergy with immunotherapy.

Answer 2: Thank you for your encouraging remarks regarding the comprehensive analysis of ECT immunomodulatory potential presented in the manuscript. We are pleased that you found the exploration of key mechanisms, such as immunogenic cell death and dendritic cell activation, to be insightful. Our intent was indeed to provide a strong foundation for future research by identifying critical areas where ECT protocols can be optimized and where potential synergies with immunotherapy can be further explored. We appreciate your recognition of these efforts and are grateful for your thoughtful evaluation.

 

Comment 3: However, the review lacks recent studies, with no references post-2021, limiting its relevance. Furthermore, key immunomodulatory mechanisms mentioned in the abstract are not thoroughly explored in the results section.  In conclusion, the manuscript is recommended for major revisions with a possibility of acceptance based on addressing comments discussed below.

Answer 3: Thank you for your constructive feedback, and we sincerely apologize for the oversight regarding the inclusion of more recent studies. We acknowledge that our initial search was incomplete. After conducting a repeated and more thorough search, we have identified five additional preclinical studies. These new studies are as follows:

• Sersa G, Miklavcˇicˇ D, Cemazar M, Belehradek Jr J, Jarm T, Mir LM. Electrochemotherapy with CDDP on LPB sarcoma: comparison of the anti-tumor effectiveness in immunocompotent and immunodeficient mice. Bioelectrochemistry and Bioenergetics. 1997;43:279-83..
• Sedlar A, Dolinsek T, Markelc B, Prosen L, Kranjc S, Bosnjak M, Blagus T, Cemazar M, Sersa G. Potentiation of electrochemotherapy by intramuscular IL-12 gene electrotransfer in murine sarcoma and carcinoma with different immunogenicity. Radiol Oncol. 2012;46:302-11.
• Polajzer T, Jarm T, Miklavcic D. Analysis of damage-associated molecular pattern molecules due to electroporation of cells in vitro. Radiol Oncol. 2020;54:317-328.
• Ursic K, Kos S, Kamensek U, Cemazar M, Miceska S, Markelc B, Bucek S, Staresinic B, Kloboves Prevodnik V, Heller R, Sersa G. Potentiation of electrochemotherapy effectiveness by immunostimulation with IL-12 gene electrotransfer in mice is dependent on tumor immune status. J Control Release. 2021;332:623-635.
• Kesar U, Markelc B, Jesenko T, Ursic Valentinuzzi K, Cemazar M, Strojan P, Sersa G. Effects of Electrochemotherapy on Immunologically Important Modifications in Tumor Cells. Vaccines (Basel). 2023;11:925.

These studies have been incorporated into our review, and the reference list has been updated accordingly. Additionally, we have revised and simplified the two tables on preclinical studies into a single, more streamlined Table 1 to provide a clearer and more comprehensive overview of the findings.

New Table 1:

Authors, Year	Aims	Methods	Results	Conclusions
Sersa et al., 1996 [18]	Determine the effects of ECT with bleomycin on immune responsiveness.	ECT applied to SA-1 tumors in mice, evaluated phagocytic activity, blast transformation of spleen cells, and oxidative burst.	Increased oxidative burst in monocytes and higher T-lymphocyte activity 7-14 days post-treatment.	ECT affects immune system function, enhancing natural resistance and immune responsiveness.
Sersa et al., 1997 [19]	Increase antitumor effectiveness of ECT by adding TNF-α.	Combination of ECT with bleomycin and TNF-α in SA-1 tumors in mice, administered intratumorally or peritumorally.	Enhanced antitumor effectiveness when combined with TNF- α; independent of interaction with bleomycin or electric pulses.	TNF- α  has an immunomodulatory effect, improving the antitumor activity of ECT and adding a systemic component.
Sersa et al., 1997 [24]	Compare ECT with cisplatin in immunocompetent and immunodeficient mice.	ECT applied to LPB sarcoma in immunocompetent and immunodeficient mice, assessed tumor growth delay and cure rates.	Tumor growth delay was twice as long in immunocompetent mice, and a higher tumor cure rate was achieved compared to immunodeficient mice.	The immune system is critical for the success of ECT with cisplatin, indicating the importance of immune responsiveness.
Roux et al., 2008 [20]	Investigate whether ECT can induce systemic immunity when combined with CpG.	ECT combined with CpG oligodeoxynucleotide injections in murine models, analyzed tumor responses, cellular recruitment, and TLR9 expression.	Induced systemic T-dependent antitumor response and triggered tumor-specific T-cell effectors, leading to both local and distant antitumor effects.	ECT combined with CpG enhances both local and systemic antitumor effects, suggesting a potential vaccination strategy.
Sedlar et al., 2012 [25]	Test the combination of ECT with cisplatin and IL-12 gene electrotransfer.	ECT with cisplatin combined with IL-12 gene electrotransfer in sarcoma and carcinoma models, evaluated in vitro and in vivo for immune and tumor responses.	Combined therapy increased tumor cell kill, growth delay, and cure rates, especially in more immunogenic sarcomas, with an IL-12 dose-dependent response.	IL-12 gene electrotransfer enhances the effectiveness of ECT, particularly in more immunogenic tumors, emphasizing the importance of immune competence.
Calvet et al., 2014 [21]	Evaluate ECT’s ability to induce immunogenic cell death (ICD) in cancer cells.	In vitro analysis of CT26 murine colon cancer cells treated with ECT and electric pulses, assayed for immunostimulatory markers (CRT, ATP, HMGB1).	ECT induced ICD markers such as CRT exposure and ATP release. Combination with bleomycin necessary for HMGB1 release and effective antitumor immune response.	ECT not only causes direct cytotoxicity but also induces a systemic anticancer immune response via ICD activation.
Tremble et al., 2018 [22]	Examine the effect of ICOS activation with ECT.	Murine models of primary and metastatic tumors treated with ECT and ICOS activation.	Combined therapy led to effective tumor clearance, especially in primary tumors, and survival after tumor rechallenge, with immunological memory.	The combination of ECT with ICOS activation enhances immunological responses, promoting local and distant tumor clearance.
Ursic et al., 2018 [23]	Compare effectiveness of oxaliplatin and cisplatin in ECT for melanoma.	ECT applied to murine B16F10 melanoma using oxaliplatin and cisplatin, analyzing immunogenic cell death and immune cell infiltration.	Both oxaliplatin and cisplatin induced comparable immunogenic cell death and lymphocyte infiltration, with oxaliplatin requiring a higher dose for equal effectiveness.	Oxaliplatin is as effective as cisplatin in ECT, both inducing similar immune responses.
Tremble et al., 2019 [16]	Explore the efficacy of ECT with cisplatin in murine lung and colorectal cancer models.	ECT with cisplatin applied to lung and colorectal cancer models, assessed tumor growth, metastatic potential, and immune cell recruitment.	Cisplatin-based ECT reduced tumor growth and metastatic potential, mobilized immune cells, and showed abscopal effects on untreated tumors.	Cisplatin is effective in ECT for reducing tumor burden and inducing an immune response, with potential for abscopal effects.
Polajzer et al., 2020 [26]	Analyze DAMP release after electroporation of cells.	Electroporation applied to hamster ovary cells, measured DAMP (ATP, calreticulin, nucleic acids) release and correlated with cell survival.	DAMP release correlated with cell death; increased release observed with higher pulse amplitudes and time intervals.	DAMP release serves as a marker for predicting cell death and immune activation following electroporation.
Ursic et al., 2021 [27]	Investigate IL-12's potentiation of ECT effectiveness based on tumor immune status.	ECT with cisplatin, oxaliplatin, or bleomycin combined with IL-12 gene electrotransfer in different murine tumor models.	IL-12 enhanced ECT’s effectiveness in poorly immunogenic tumors but had less impact on more immunogenic tumors, with the greatest effect observed with cisplatin.	The effectiveness of combination therapy depends on tumor immune status, suggesting tailored approaches for better therapeutic outcomes.
Kesar et al., 2023 [28]	Evaluate immunogenic modifications in tumor cells induced by ECT.	ECT applied to murine tumor cell lines (B16-F10, 4T1, CT26), measured DAMP release and expression of immune markers (MHC I, II, PD-L1, CD40).	ECT induced ICD-related DAMPs and immune marker changes in a cell line- and drug-specific manner, demonstrating varying effects across tumor types and drugs.	ECT induces immunogenic modifications in tumor cells, highlighting its potential to enhance antitumor immunity, which varies by tumor type and chemotherapeutic agent.

 

Furthermore, we added the following paragraph to the results section:

“Sersa et al. (1997) conducted a study comparing the anti-tumor effectiveness of electrochemotherapy (ECT) with cisplatin in immunocompetent and immunodeficient mice with LPB sarcoma. The tumor growth delay in immunocompetent mice was about twice as long as in immunodeficient mice, and tumor cures were only achieved in immunocompetent mice. These results emphasize the crucial role of the immune system in the success of ECT with cisplatin.”

“Sedlar et al. (2012) explored the effects of combining ECT with intramuscular interleukin-12 (IL-12) gene electrotransfer in murine sarcoma and carcinoma models. ECT was more effective in the more immunogenic sarcoma model, with 17% tumor cures. IL-12 gene electrotransfer further increased the tumor response, particularly in the sarcoma model, emphasizing the importance of tumor immunogenicity in treatment effectiveness.”

“Polajzer et al. (2020) studied the release of damage-associated molecular patterns (DAMPs) following electroporation in vitro. Using hamster ovary cells, the study found that higher pulse amplitudes led to increased DAMP release, which correlated with cell death. These findings suggest that DAMPs could serve as markers to predict cell death and immune activation after electroporation.”

“Ursic et al. (2021) investigated the combination of ECT with IL-12 gene electrotransfer in murine tumor models with varying immunogenicity. In poorly immunogenic melanoma, IL-12 gene electrotransfer potentiated the effects of ECT, leading to a complete response in 38% of cases and inducing an abscopal effect. However, more immunogenic tumors, like 4T1 mammary carcinoma and CT26 colorectal carcinoma, responded better to ECT alone, indicating that the effectiveness of combination therapy depends on tumor immune status.”

“Kesar et al. (2023) examined the immunogenic effects of ECT in murine tumor cell lines, analyzing the release of DAMPs and the expression of immune markers. The study found that ECT induced immunogenic cell death (ICD) and altered the expression of immune markers, though the effects were tumor-type and chemotherapeutic agent-specific.”

Finally, we have also simplified the table on clinical studies for the sake of consistency and homogeneity. This new version provides a clearer and more structured presentation of the data, aligning it with the format of the revised preclinical studies table.

New Table 2

Authors/Year	Aims	Methods	Results	Conclusions
Gerlini et al., 2013 [29]	Investigate whether ECT can induce anti-tumor immunity by examining dendritic cells (DCs) recruitment in melanoma metastases.	Biopsies from 9 melanoma patients were taken before and after ECT, followed by immunofluorescence analysis to study DC-related markers in the inflammatory infiltrate.	ECT caused a significant reduction in Langerhans cells (LCs) at day 7, followed by their replacement at day 14. Plasmacytoid and dermal DCs increased significantly after ECT, with some showing activation markers.	ECT promotes migration of LCs to lymph nodes and recruits plasmacytoid and dermal DCs to the lesion site, suggesting potential for in situ DC vaccination strategies.
Di Gennaro et al., 2016 [30]	Examine the effect of ECT on T cell subsets in melanoma metastases, focusing on regulatory T cells and CD8+ T cells.	Fluorescent immunohistochemistry was performed on biopsies from 10 melanoma patients before and after ECT to analyze T cell subsets and markers of immunogenic cell death.	ECT led to a significant decrease in regulatory T cells (Tregs) and an increase in CD8+ T cells at day 14, along with the presence of calreticulin, indicating immunogenic cell death in metastatic cells.	ECT induces local immune responses, characterized by reduced Tregs and increased CD8+ T cell recruitment, suggesting potential for combining ECT with immunotherapy for long-lasting anti-tumor immunity.

 

Moreover, we have ensured that the key immunomodulatory mechanisms mentioned in the abstract are now more thoroughly discussed and explored in the Results section. For specific details on how these mechanisms have been addressed, please refer to our responses to the individual comments below.

 

Comment 4: Lines 70 – 87 should have the content reorganized to display the innate immune system first followed by the adaptive immune system. This would flow better for what the abscopal effect utilizes.

Answer 4: Thank you for your suggestion. We agree that this adjustment would better reflect the natural progression of immune activation, especially in the context of the abscopal effect.

We have revised the section accordingly to ensure that the innate immune system is discussed before the adaptive immune response, improving the logical flow of the narrative. We believe this enhances the clarity and comprehension of how these systems interact in the context of ECT-induced immune responses.

The section corresponding to lines 70–87 in the document discusses the mechanisms of ECT in activating the immune system, including both the innate and adaptive immune responses. Here is the relevant content, reorganized to follow the suggested flow:

Revised Section:

“ECT has the potential to exert immunomodulatory activity through several mechanisms. The innate immune response is first triggered when dying tumor cells release damage-associated molecular patterns, such as intracellular adenosine triphosphate (ATP) and High Mobility Group Box 1 (HMGB1) protein. These molecules attract immune cells, including macrophages and neutrophils, to the site of tumor destruction, promoting an inflammatory response.

ECT also induces the activation of dendritic cells (DCs), key antigen-presenting cells that play a crucial role in bridging innate and adaptive immunity. Activated DCs process tumor antigens and present them to T cells, initiating an adaptive immune response against the tumor. As tumor cells are destroyed, antigens are released and can be taken up by antigen-presenting cells like DCs, further supporting the development of a specific anti-tumor immune response.

Additionally, ECT can lead to the local release of cytokines and chemokines, contributing to immune cell recruitment and the generation of a broader immune response. This local immune response may occasionally manifest in systemic immune activity, known as the abscopal effect, where distant untreated tumor sites are affected by immune activation.”

 

Comment 5: Figure 1 is rudimentary and could benefit from having more details added to the cells being seen, such as live tumor cells, treated tumor cells, etc. The figure caption should match these changes, too. Also, the anti-tumor immune response in this figure is incomplete.

Answer 5: Thank you for your insightful feedback on Figure 1. We agree that adding more details to represent the different cell types, such as live tumor cells, treated tumor cells, and a more complete depiction of the anti-tumor immune response, would enhance the clarity and educational value of the figure. We have updated Figure 1 accordingly, ensuring that the various cells involved in the immune response are clearly identified. Additionally, the figure now includes a more comprehensive depiction of the anti-tumor immune response. The caption has also been revised to match these changes.

 

1. Electric pulse  is applied to the tumor mass and the chemotherapy drug can be delivered within the tumor cell, amplifying the cytotoxic effect
2. Dying tumor cells release signals that alert the immune system including damage-associated molecular patterns (DAMP) release
3. DAMPs attract immune cells to the site of tumor destruction and promote an inflammatory response
4. At the same time ECT can lead to the recruitment and activation of dendritic cells (DCs)
5. Activated DCs process tumor antigens and present them to T cells
6. Anti-tumor immune response and tumor elimination

 

Comment 6: The dataset represented in the tables of the study do not show any papers that have not been published since 2019, but I believe there are vital studies that have been conducted since then within the realm of ECT that have occurred within the gap 2019-2024. This may be due to the “snowball technique” implemented for paper selection which may be eliminating some of these more recent studies. This can be detrimental due to gaps of progression in knowledge you may have cut out or stopped at. In addition to not showing any more recent papers, it becomes difficult to build the case that ECT has potential as an immunomodulatory therapy since the clinical papers that help build the case were published almost 10 years ago.

Answer 6: Thank you once again for your observation regarding the inclusion of more recent studies. We agree that recent literature is essential for demonstrating the progress in the field of ECT and its potential as an immunomodulatory therapy. As mentioned in our previous response, we have now conducted an updated and more comprehensive literature search that has included studies published between 2019 and 2024. These additional studies have been integrated into the manuscript, and the tables have been updated to reflect this more recent research. This ensures that the progression of knowledge in ECT and its potential immunomodulatory effects is more accurately represented. We hope these updates address your concerns and strengthen the overall relevance of our review. Moreover, please consider also response 2 to the reviewer 1.

 

Comment 7: With the snowball technique, what criteria was met or not met to have the paper seen as substantially equivalent to another for not needing to be placed into the document?

Answer 7: Thank you for your insightful comment regarding the snowball technique and its application in our study. In this updated bibliographic search, we employed both backward and forward snowballing methods. In our previous search, we used the backward snowballing technique, where we analyzed the reference lists of the works initially selected from the database search. This allowed us to identify earlier published papers on the same topic. We examined their titles and abstracts, and, when relevant, we reviewed them in full. In our current search, we also applied the forward snowballing technique. Specifically, using PubMed "cited by" function, we examined all the papers that had cited the initially selected publications. This extended our search to more recent works.

To clarify this approach, we have added the following sentence to the Methods section: "In particular, we used both the 'backward' and 'forward' snowballing methods."

Additionally, we have included the following reference to support the use of the forward snowballing technique:

"Felizardo KR, Mendes E, Kalinowski M, Souza ÉF, Vijaykumar NL. Using forward snowballing to update systematic reviews in software engineering. In Proceedings of the 10th ACM/IEEE International Symposium on Empirical Software Engineering and Measurement 2016 Sep 8 (pp. 1-6)."

Thank you once again for your helpful suggestion, which has allowed us to improve the robustness and comprehensiveness of our bibliographic search.

 

Comment 8: I believe the manuscript should benefit from including other papers that use other approaches to ECT that are not strictly using a chemotherapy such as discussing Calcium Electroporation within the realm of ECT since it would open your paper to a broader audience and increase the papers discussed in the in vivo/clinical tables perhaps.

Answer 8: Thank you for your valuable suggestion. We agree that calcium electroporation is an interesting and innovative topic. However, for reasons of homogeneity in this manuscript, we have chosen to focus specifically on the combination of electroporation with chemotherapeutic drugs. As we mentioned in our response to comment 2 of reviewer 1, this review serves as a preparatory step for another ongoing review, which will explore broader combinations of electroporation, including its use with chemotherapeutic drugs, other agents, and immunotherapy. In that upcoming review, we certainly intend to include calcium electroporation as an important aspect of the discussion.  We appreciate your understanding and thank you again for your insightful feedback.

 

Comment 9: The abstract mentions key immunomodulatory mechanisms, including immunogenic cell death, dendritic cell activation, tumor antigen release, inflammatory responses, and systemic effects like the abscopal phenomenon. However, these mechanisms are not adequately compared or thoroughly explained in the results and discussion sections based on the preclinical and clinical studies presented.

Answer 9: Thank you for your detailed observation regarding the abstract and its discussion of key immunomodulatory mechanisms. We agree that these mechanisms should be more thoroughly compared and explained in the results and discussion sections, based on the preclinical and clinical studies we presented.

To address this, we have revised the Results and Discussion sections to provide a more detailed comparison and explanation of these mechanisms. Specifically, we have expanded the discussion on how the preclinical and clinical studies demonstrate these key immunomodulatory effects, and we now better clarify the role of each mechanism in the context of the immune response elicited by ECT. The revised sections highlight the similarities and differences observed between the preclinical and clinical evidence, offering a more comprehensive analysis.

The updated Table 1 now reflects a clearer summary of the most relevant preclinical studies, which demonstrate how ECT can trigger immunogenic cell death (ICD), recruit dendritic cells (DCs), and initiate both local and systemic immune responses. Similarly, Table 2 has been simplified for consistency and homogeneity with the preclinical studies, providing a structured presentation of clinical findings related to the immunomodulatory potential of ECT.

Revised Discussion Section:

“ECT has demonstrated immunomodulatory effects across multiple preclinical and clinical studies, highlighting its capacity to activate both local and systemic immune responses. Preclinical studies consistently show that ECT can trigger immunogenic cell death (ICD) through the release of damage-associated molecular patterns (DAMPs), such as ATP and HMGB1, which play a key role in immune activation. For example, Calvet et al. (2014) demonstrated that ECT induced ICD markers and promoted an effective antitumor immune response, particularly when combined with bleomycin. Similarly, Polajzer et al. (2020) showed that DAMP release increased with higher pulse amplitudes and was closely linked to cell death, further supporting ECT role in initiating immune activation. Studies like those of Sersa et al. (1996) and (1997) emphasized the importance of the immune system in enhancing the antitumor effectiveness of ECT, with stronger responses seen in immunocompetent mice compared to immunodeficient ones. The addition of immunomodulatory agents, such as TNF-α and IL-12, was shown to further augment these effects by increasing tumor cell kill rates and systemic immune responses (Sersa et al., 1997; Sedlar et al., 2012).

In addition to enhancing local immune responses, several studies have demonstrated ECT potential to generate systemic effects. Roux et al. (2008) showed that combining ECT with CpG oligodeoxynucleotides led to both local tumor regression and rejection of distant untreated tumors, suggesting that ECT can induce systemic T-cell–mediated responses. Similar results were observed in Tremble et al. (2019), where ECT combined with cisplatin mobilized immune cells and showed abscopal effects, reducing growth in untreated tumors. These findings were further supported by studies that combined ECT with IL-12 gene electrotransfer, demonstrating that IL-12 could potentiate the systemic immune effects of ECT, particularly in less immunogenic tumor models (Ursic et al., 2021).

Clinical studies have echoed these immunomodulatory effects, as seen in Gerlini et al. (2013), where ECT induced dendritic cell recruitment and activation in melanoma metastases, highlighting its potential for in situ DC vaccination strategies. Moreover, Di Gennaro et al. (2016) demonstrated that ECT in melanoma patients reduced regulatory T cells (Tregs) and increased CD8+ T cells, indicating that ECT promotes a favorable immune environment for tumor clearance and could be effectively combined with immunotherapies to achieve durable responses. Together, these studies underscore the importance of the immune system in determining the success of ECT and point to the potential of combining ECT with immunomodulatory agents to further enhance both local and systemic antitumor effects.”

 

Comment 10:

Line 47-- Please remove the word” innovative” from the sentence describing ECT. ECT has been prevalent for decades so it cannot be deemed as “innovative” or new and original.

Answer 10:

Thank you for pointing this out. We agree that the term "innovative" is not appropriate given the established history of ECT. We have removed the word "innovative" from the sentence in line 47 to more accurately reflect the long-standing use of ECT.

 

Comment 11:

Lines 45-52 -- More citations are required within the first paragraph specifically when describing the mechanism behind electroporation and how drugs are able permeate through a disrupted membrane. This background knowledge is vital to cite since this is specific knowledge elucidated within the field of electroporation.

Answer 11:

Thank you for your insightful feedback. We agree that the background knowledge describing the mechanism behind electroporation and how drugs permeate through the disrupted membrane requires additional citations to provide proper context and credibility. We have now expanded the introduction section and added the relevant references to support these statements and to ensure that this section is appropriately cited, reflecting the established understanding within the field of electroporation.

New paragraph in the introduction section:

“Electroporation, a biophysical technique that employs short, high-voltage electrical pulses, temporarily disrupts the lipid bilayer of cell membranes, creating transient pores. These pores increase the permeability of the cell membrane, allowing otherwise impermeable molecules, such as chemotherapeutic agents, to enter the cell more efficiently. In the context of cancer treatment, this process is known as ECT, which enhances the intracellular concentration of cytotoxic drugs in tumor cells. The most commonly used drugs in ECT, such as bleomycin or cisplatin, are particularly suited for this technique as they have poor membrane permeability on their own but exhibit potent cytotoxicity once inside the cell.

The process begins with the administration of the chemotherapeutic agent, either intravenously or intratumorally, followed by the application of electrical pulses directly to the tumor site. These pulses are carefully controlled in terms of duration, amplitude, and frequency to ensure optimal permeabilization of the tumor cells without causing excessive damage to surrounding healthy tissue. Once the electric field is applied, the temporarily permeabilized cells allow the chemotherapeutic drugs to enter at significantly higher concentrations than would occur without electroporation.

ECT efficacy stems from its dual mechanism of action. First, the electroporation process increases the intracellular uptake of the chemotherapeutic agents, enhancing their cytotoxic potential. Second, the drugs themselves exert their antineoplastic effects, such as causing DNA strand breaks (in the case of bleomycin) or forming DNA adducts that inhibit DNA repair (as seen with cisplatin). Together, these mechanisms lead to enhanced tumor cell death.

Moreover, ECT offers a targeted treatment approach, minimizing systemic toxicity by confining the cytotoxic effects to the tumor region. This localized approach makes ECT particularly effective in treating cutaneous and subcutaneous tumors.”

New references:

Dev SB, Hofmann GA. Electrochemotherapy--a novel method of cancer treatment. Cancer Treat Rev. 1994;20:105-15.

Escoffre JM, Rols MP. Electrochemotherapy: progress and prospects. Curr Pharm Des. 2012;18:3406-15.

Gehl J. Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand. 2003;177:437-47.

 

Comment 12: In the preclinical studies section, alfa should have the symbol “α” instead

Answer 12: Thank you for pointing out this detail. We have replaced "alfa" with the correct symbol "α" in the newly updated Table 1 and in the preclinical studies section, ensuring consistency throughout the manuscript.

 

Comment 13: Vagueness of what is driving the immunomodulatory system should have more explanation such as in line 65 on types of immunogenic cell death i.e. necrosis or line 85-86 types of immune cells, cytokines, and chemokines are some examples.

Answer 13: Thank you for your valuable feedback. Please refer to answer 4.  

 

Comment 14: The conclusion section drives the point of “potential” behind ECT, but the suggestive language does poorly in context of the lack of recent studies to show the future moving forward for ECT.

Answer 14: Thank you for your insightful comment regarding the conclusion. We agree that the use of suggestive language, particularly around the "potential" of ECT, could be strengthened to better reflect the current state of research and future directions. To address this, we have revised the conclusion to emphasize the need for further research and clinical trials, while also highlighting the advancements that have been made in recent studies. This update ensures a more balanced view of the future of ECT, acknowledging both its promising aspects and the need for more evidence-based studies.

Revised conclusion section:

“In conclusion, ECT demonstrates significant immunomodulatory effects that could complement current cancer therapies. While preclinical and early clinical studies have highlighted ECT potential to induce immunogenic cell death, activate immune cells, and trigger systemic immune responses, including the abscopal effect, recent research remains limited in terms of large-scale clinical validation. The promising results observed in smaller studies underscore the need for well-designed, large-scale clinical trials to better understand the mechanisms underlying ECT effects, optimize treatment protocols, and explore combinations with immunotherapy and other systemic treatments.

Future research should focus on standardizing ECT protocols and evaluating its long-term efficacy across diverse tumor types and patient populations. Despite the gaps in the current body of research, the integration of ECT into multimodal cancer treatment regimens represents a valuable avenue for enhancing therapeutic outcomes.”

 

Comment 15: Please check over for grammatical and spelling errors. There are several.

Answer 15: Thank you for pointing out the grammatical and spelling issues in the manuscript. We appreciate your careful review and have thoroughly revised the text to address these errors. Based on your suggestion, we have made several corrections to improve clarity and readability.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I have no further comments.