PONE-D-20-06807

Circulating Blood Extracellular Vesicles as a Tool to Assess Endothelial Injury and Chemotherapy Toxicity in Adjuvant Cancer Patients

PLOS ONE

Dear Dr. Bar-Sela,

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.

Please address the very serious concerns raised by the reviewers.

Please submit your revised manuscript by Jul 24 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Jeffrey Chalmers, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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.

Reviewer #1: No

Reviewer #2: Yes

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4. 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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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Bar-Sela describes the analysis of citrated plasma from breast cancer patients for extracellular vesicles (EVs). The authors fractionate citrated plasma by centrifugation and analyze the resulting pellet using NTA, flow cytometry, and antibody array. They report differences in the fluorescence signals produced by samples stained with different antibodies, and an increase abundance of angiotensin is lysed material. The authors propose that angiostatin-bearing EVs are a useful biomarker for endothelial damage.

The authors contentions are hard to evaluate from the available data and impossible to reproduce given the information provided.

The authors have chosen to focus on a pellet of material obtained after centrifugation of previously-frozen citrated plasma for 1 hour at 20,000xg. This is a very crude fractionation and the pellet obtained will likely contain some EVs plus other sediments, but will leave significant material in the supernatant. The authors should justify their choice of fractionation method, and describe it more completely including information about starting and final volumes, concentration factor, and the centrifuge, rotor, and any braking, etc.

The authors state that EV size and concentration was evaluated by NTA, but present only a cursory description and results. The authors should present representative histograms to show the size distributions, and relevant details about the measurement, including the length of the tracking videos, the analysis algorithm used to analyze the data, and variance in their triplicate technical replicates. How did the authors evaluate the fraction of non-EV particle in their sample?

The authors use flow cytometry analyze their samples, but the data is uninterpretable from the information provided. The authors are referred to the guidelines on reporting EV flow cytometry methods and results recently published in the Journal of Extracellular Vesicles, which also includes information on essential controls and calibration.

Finally, the authors claim the angiostatin carried inside EVs is a useful potential biomarker, but the mere presence of angiostatin in a crude pellet that also contains some vesicles is unconvincing. Stronger evidence of the vesicular nature of might come from a cleaner EV prep that had been further fractionated to remove non-EV contaminants, and showing that the angiostatin is lost from the vesicle fraction after treat met with detergent, for example.

Reviewer #2: 1. Abstract: Anti tumor treatment toxicity typically connotes organ toxicities or symptom burden experience by patients. If not patient reported outcomes or actually toxicities are reported, I suggest that this not be described in this way since it is very misleading

2. Introduction: 2nd sentence in introduction is a run off sentence and needs to be edited. As stated above, I disagree with the firm association of chemotherapy toxicities with EVs. The authors have no strong data to assert this. the findings of EV in these 2 patient cohorts can be certainly described and it's very interesting. and in the discussion, conjectures can be made with chemotherapy associated complications and toxicity but the study was not poised to answer this question.

3. Methods: please move discussion of patient demographics and tumor characteristics to results section. Describe how healthy controls were obtained and under what human protocol. Was it under patient IRBs?

4. Results: please start with describing patient clinical demographics. Given long term nature of the data, are the authors interested in reporting disease outcomes in these 2 cohorts-- Since pts who had earlier recurrences may have influenced EV characteristics. If not able to do this, please acknowledge this as a limitation. comments above apply to discussion of results. We cannot draw conclusions on toxicity based on these results.

5. discussion of thrombosis factors but without reporting whether pts had thrombotic events needs to put in better context. Not enough data for the conclusions listed

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-06807R1

Circulating Blood Extracellular Vesicles as a Tool to Assess Endothelial Injury and Chemotherapy Toxicity in Adjuvant Cancer Patients

PLOS ONE

Dear Dr. Bar-Sela,

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.

Please address the issues raised.

Please submit your revised manuscript by Sep 14 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Jeffrey Chalmers, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

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.

Reviewer #1: No

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: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Bar-Sela et al describes the assessment of extracellular vesicles (EVs) as biomarkers of endothelial injury in chemotherapy. The authors analyze cell-free plasma from patients with breast cancer (BC), colon cancer (CC) and healthy controls (HC) using NTA and flow cytometry, and also fractionate plasma using differential centrifugation to prepare a 20Kxg, 60’ pellet and analyze it by protein array and Western blot. The authors report increases in the total number of particles in plasma, and an apparent increase in size, as measured by NTA in CC patient plasma compared to controls. They also report changes in the expression of several membrane surface antigens as assessed by flow cytometry immunofluorescence. They identify angiostatin present in the 20Kxg pellet, as measured by protein array, as a marker of endothelial damage from chemotherapy.

Biomarkers of drug induced toxicity are needed to help manage patient treatment, and EVs are potentially useful. The observations reported in the present manuscript are aimed at developing such biomarkers, but inadequacies in the experimental methods used and omissions in the experimental design and reporting limit their interpretation.

The authors direct their efforts at cell-free plasma, which is a noble cause but its complexity challenges methods commonly used to analyze EVs. In the case of NTA, its limitations for the analysis of plasma are well documented and are related to its lack of specificity for EVs (it measures all particles that scatter light) and the confounding effects of lipoproteins, which are present at concentrations orders of magnitude higher than EVs. For these reasons, the NTA data (which are nearly impossible to evaluate due to the oddly scaled and poorly presented histograms that should be revised) should be presented with axes labeled as “Particles” rather than “EVs”. The authors should state what metric is being reported as Particle Size (mean, median, mode, etc), and what wavelength of laser light was used for illumination.

Similarly, the essential information and controls needed to interpret the flow cytometry data are lacking. These have been recently summarized in a position paper in the Journal of Extracellular Vesicles, and include information about the instrument operation and calibration and essential controls to demonstrate that the events being detected correspond to individual EVs. The laser and filter set up of the instrument should be reported according to the MIFlowCyt guidelines (a table in Supporting Info), the fluorescence results should be reported in MESF or antibodies/particle using the appropriate commercially available standards, and specificity controls including reagent-only, serial dilutions, and detergent treatment should be presented. It is recommended that the raw flow cytometry data files be submitted to a public repository (such as flowrepository.org). Where results are presented as % positive, the authors should include the denominator (the total concentration of particles/EVs) used to calculate that percentage, as well as the fluorescence threshold (reported in MESF or antibodies/event) used to determine positivity. The inclusion of these details will greatly improve the interpretability of the data.

The observation that angiostatin is increased in both the plasma 20kxg pellet and supernatant is interesting, but the authors contention that this is EV-encapsulated is speculative given the very crude nature and lack of characterization of these plasma preparations. Additional fractionation and characterization of the samples is required to speak to how much angiostatin is present in EVs vs free in plasma, as well as the nature of those EVs that might be carrying it.

Reviewer #2: Thank you for addressing concerns and questions.

Concerns have been addressed by the writers.

I have no additional concerns

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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 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 Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Circulating Blood Extracellular Vesicles as a Tool to Assess Endothelial Injury and Chemotherapy Toxicity in Adjuvant Cancer Patients

PONE-D-20-06807R2

Dear Dr. Bar-Sela,

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.  Note, I was waiting for the other reviewer; however, I decided to go ahead with the one reviewers comments. 

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jeffrey Chalmers, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

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.

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 #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: concerns are addressed

thank you for your submission

no other comments

best of luck with your future work

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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 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 Privacy Policy.

Reviewer #2: No