PONE-D-19-28381

Effect of post-implant exercise on tumour growth rate, perfusion and hypoxia in mice

PLOS ONE

Dear Dr. Dachs,

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.

While both reviewers found this an interesting study, there were a number of issues raised which should be addressed if the authors intend to submit a revised manuscript. 

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Salvatore V Pizzo

Academic Editor

PLOS ONE

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2. Please include a caption for figure 5.

3. Thank you for stating the following in the Financial Disclosure section:

This research was funded in part by the Mackenzie Charitable Foundation, NZ. We would also like to acknowledge Dr. Andreas Moeller (QIMR Berghofer, Australia) for the kind gift of the EO771 cells and Andrew Dachs (Decision Consulting Ltd, NZ) for developing the magnetic counter for the mouse wheels. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We note that you received funding from a commercial source: Decision Consulting Ltd, NZ

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

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: Yes

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: This is a nice study evaluating the effect of exercise (running wheel) on tumor growth and vascular characteristics using B16F10 and E0771 models. Strengths of the work include the use of orthotopic models, appropriate sample size and statistical analysis, and the use of experimental design that minimizes animal stress. Also, this work is important because it reports a non-significant impact of exercise on tumor growth/vascular biology, which is important in the current climate of exercise oncology research where negative results are often ignored. Overall, I would like to see the study published, but do have some concerns which need to be addressed.

1. Abstract Line 20 “This has only been shown in breast and prostate cancer models…” seems a little inaccurate. Several studies have shown increases in tumor vessel perfusion and/or hypoxia. Perhaps soften the wording to "has been shown most convincingly in breast and prostate cancer models..."

2. Mice received an exercise wheel on the day of tumor transplant. It’s not clear to me what clinical situation this is meant to mimic. Typically, patients are either already exercisers who then develop cancer (modeled by pre-implantation training) or will begin exercise after a cancer diagnosis (modeled by exercise beginning after a detectable/measurable tumor is present). Exercise beginning on the day of tumor cell inoculation is not an obvious model for me. Please explain in your introduction or discussion, or if there is no explanation, please list as a study limitation in the discussion.

3. Were mice that were euthanized early included in the analysis of vasculature and hypoxia?

4. Others have shown in B16F10 tumors that exercise did not change vessel density, but did change the average length of the vessels or the number of open lumens. Did the authors evaluate these parameters in either tumor model?

5. There appears to be significant variation in the distance run between individual mice. Is it possible to analyze tumor growth or vascular characteristics in a way that takes this variation into consideration? For example, perhaps tumors behave differently in low exercising vs high exercising mice. Is there any correlation between amount of daily running and survival time, hypoxia, or vessel number?

6. I am concerned about the conclusions that were drawn regarding vascular structure, function, etc. based on images that are very difficult to see and interpret. In figure 3a, the images are extremely blurry. It would be nice to see both a low magnification and high magnification image for each condition. Also, how do the authors know that the Hoecsht 3342 is demonstrating perfused tumor vessels and not leakage outside of vessels? These images would be strengthened greatly by the addition of CD31 or VE-cadherin immunoflourescent staining so that the location of Hoecsht 3342 and of hypoxyprobe-1 relative to endothelium can truly be assessed. (3 color staining showing Hoecsht, hypoxia, and endothelium together)

7. Similarly, it’s not clear to me how the hypoxic area was determined? What software was used to determine the % hypoxic area? Is this a ratio of pimonidazole positive: total nuclei or pimonidazole: total tissue area? It seems that comparing to the total nuclei would be most appropriate because some of the tissue area may be necrotic, but if this is not possible due to software limitations, please explain the process for determining the % hypoxic area.

8. The figure legend for Figure 5 is labeled as Figure 1.

Reviewer #2: This paper describes the results of a study of post-implantation exercise in syngeneic mouse tumour graft using B16-F10 melanoma cells and EO771 breast cancer cells. No differences were observed including in measures of tumour growth, and hypoxic fraction and vascular density at sacrifice.

The work appears to have been conducted carefully, and should be published – not least in the interests of avoiding publication bias. The discussion is well balanced.

I have a few relatively minor points for clarification.

In figure 1, the y-axis is labelled as cumulative survival. This is potentially a little confusing as all animals were subjected to pre-specified euthanasia end-points (I think). In this case, it would be better to simply refer to % reaching end-point. Could the authors also clarify whether all end-points were reached due to tumour size, or where some were due to weight loss or other criteria?

In figure 2, the mean distance run does not appear to be reduced with tumour growth. Does this suggest a relative low systemic effect of the malignancy? Could the authors comment?

In figure 3, the hypoxic area is extremely variable. Could the authors describe this further? In any one tumour, were the 10 random fields similar? Or was the variability observed within tumours?

In figures 3 and 4, as indicated in the legends, there are slightly different numbers. Could the authors indicate why?

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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 to be viewed.]

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PONE-D-19-28381R1

Effect of post-implant exercise on tumour growth rate, perfusion and hypoxia in mice

PLOS ONE

Dear Dr. Dachs,

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.

As you will see, both reviewers were in agreement that this manuscript is ready to be accepted for publication.  However, Reviewer No. 2 would like to see some clarifications made in the Figure Legends. If the authors agree to this issue, then we would certainly welcome a revised manuscript ready for publication. 

We would appreciate receiving your revised manuscript by Mar 13 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Salvatore V Pizzo

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: 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?

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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

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: 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

**********

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: Congratulations on this work. I'm very excited to see it out in publication! Nice use of good models and good statistics!

Reviewer #2: (No Response)

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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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

Effect of post-implant exercise on tumour growth rate, perfusion and hypoxia in mice

PONE-D-19-28381R2

Dear Dr. Dachs,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Salvatore V Pizzo

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: