Dear Dr. Siddle,

Thank you very much for submitting your manuscript "The differentiation state of the Schwann cell progenitor drives phenotypic variation between two contagious cancers" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by three independent reviewers, including two (Reviewers 1 and 3) that had previously reviewed a version. You will see that the reviewers are still mixed in their appraisal of the paper. All reviewers would like to see the paper pitched in more general terms rather than so focused so as to only appeal to a very limited audience. reviewer 2 makes specific recommendations in this regard. Reviewer 3 is still quite negative about the accuracy and impact of your findings. They raise legitimate concerns about the statistics used and whether they accounted for the multiple testing. We will require you to address these criticisms in your revision, with specific mention of the types of safeguards used to guard against multiple testing artifacts. Reviewer 3 is also concerned that the level of impact of these findings may not rise to the level that we expect of papers at PLOS Pathogens, especially in light of recent findings. However, based on comments of the other two reviewers, who are also experts in this field (like Reviewer #3), editorially we do not believe this would be a significant impediment to ultimate acceptance of your paper here, if you can satisfactorily address the other comments raised. We do encourage you to cite all prior literature and draw your attention to PLOS policy of scoop protection. Therefore, in light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript may be sent to reviewers for further evaluation if needed.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the 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.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Harmit S. Malik

Guest Editor

PLOS Pathogens

Marco Vignuzzi

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In the Owen et al manuscript, the authors describe the Schwann cell origins of both lineages of Tasmanian devil facial tumour disease (DFT1 and DFT2). It had previously been shown that both lineages arose from Schwann cells, but this report goes further to show that one lineage is more differentiated than the other (DFT1 is more differentiated). Notably, this has an effect on the immune evasion strategies of these cancers, as the differentiation state affects MHC-I expression level. The authors have answered all of the reviewers' questions, removing a problematic section and adding significantly improved discussion and analysis of proteomic data.

Reviewer #2: This manuscript compares the differentiation state of Schwann cells that gave rise to two independent transmissible cancers in Tasmanian devils - DFT1 (the original lineage, described in the 1990s) and DFT2 (described in 2016). Although the gross morphology of tumors on the face of Tasmanian devils is quite similar, DFT1 and DFT2 present differently in terms of histology, and genomic differences have been identified between the two tumors. The cells of DFT1 and DFT2 also have differing levels of MHC expression, as well as responses to IFN-gamma treatment. The experiments herein, primarily a proteomics analysis, show that DFT2 emerged from a less differentiated Schwann cell than DFT1. I have not seen a previous version of the MS and note that the authors have done a good job responding to previous comments and removing less well-supported data. My main concern is that this manuscript is quite specific relating to Tasmanian devils and DFTD, and as written, may not be of general interest to the broad readership of PLoS Pathogens. As such, my general recommendation is to introduce the manuscript in a more broad way (which is done to some extent in the discussion) as to the implications for understanding the differentiation state of Schwann cell tumors. The authors mention the plasticity of Schwann cells, and perhaps a more general discussion as to how this relates to them becoming cancerous would enhance the introduction.

Reviewer #3: In the revised version of their paper, Owen et al document (1) whole-proteome differences in protein expression between representative DFT1 and DFT2 cell lines, (2) alterations in transcript abundance for selected genes in representative DFT1 and DFT2 cell lines cultured in the presence and absence of recombinant IFNg, (3) differences in the magnitude of upregulation of cell-surface B2M following IFNg treatment among several DFT2 cell lines as well as a DFT1 cell line, and (4) using immunohistochemistry, assess expression of SAHA UABC and UK in Tasmanian devil sciatic nerve and in representative DFT1 and DFT2 tumour biopsies.

In the revision, the authors have performed replicates of the qPCR experiments, and these are now considerably more robust. However, I struggle to follow a coherent story in the paper. Much of the work repeats findings already in the published literature (Patchett et al 2019, Ong et al 2021, Flies et al 2016, Siddle et al 2013, Caldwell et al 2018). The main new contribution is the proposal that DFT1 and DFT2 arose from different progenitor cell states. However, this was already hinted at in Patchett et al 2019, and, in my opinion, despite the authors’ rebuttal, I do not believe that these cancers’ progenitor cell states can be inferred from the presented work. The PCR data presented in figures 2C, 2D, 3C, 3D, S1, S2 detect subtle changes and statistical significance level does not seem to have been corrected for multiple testing. I am left unsure that differences are significant and reproducible. Furthermore, fig 3B shows that there is significant variation in cell surface B2M level among DFT2 cell lines; this makes me wonder how many of the findings from the rest of the paper are specific to the single representative cell lines selected for DFT1 and DFT2.

Overall, I don’t find the results a compelling-enough contribution to the field to warrant publication in PLOS Pathogens.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: Major issues seem to have been addressed by previous reviewers, and the authors removed the questionable data sets from the manuscript. I do not see any additional major issues of concern.

Reviewer #3: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: I could nitpick, but the manuscript is generally well-written. However, as noted above, the manuscript as written is for a specialized audience, and a more general discussion of the relevance of the data for cancer in general would broaden the scope of the paper appropriately for PLoS Pathogens.

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Dear Dr. Siddle,

We are pleased to inform you that your manuscript 'The differentiation state of the Schwann cell progenitor drives phenotypic variation between two contagious cancers' has been provisionally accepted for publication in PLOS Pathogens. Thank you for your thoughtful revisions.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Harmit S. Malik

Guest Editor

PLOS Pathogens

Marco Vignuzzi

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):