Revisiting the NPcis mouse model: A new tool to model plexiform neurofibroma

Camille Plante; Teddy Mohamad; Dhanushka Hewa Bostanthirige; Michel Renaud; Harsimran Sidhu; Michel ElChoueiry; Jean-Paul Sabo Vatasescu; Mikael Poirier; Sameh Geha; Jean-Philippe Brosseau

doi:10.1371/journal.pone.0301040

Peer Review History

Original SubmissionJune 30, 2023
12 Dec 2023 Decision Letter - Giulio Piluso, Editor PONE-D-23-19723Revisiting the NPcis mouse model: a new tool to model plexiform neurofibromaPLOS ONE Dear Dr. Brosseau, 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 submit your revised manuscript by Jan 26 2024 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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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] 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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 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 ****** 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: Because the existing mouse models of neurofibroma and MPNST do not recapitulate the key clinical and molecular features of the corresponding human tumors, the authors aimed to establish a model the recapitulated the human disease clinically and molecularly. Therefore they revisited the NP cis model. The paper is interesting well written and clear. The Authors also discussed the current state of NF mouse models, highlighting model relationship to human tumors. Reviewer #2: In this manuscript Plante et al re-characterized the CisNf1+/-;P53+/- (NPcis) mouse. Consistent with the published data, the NPcis mouse developed malignant peripheral nerve sheath tumor (GEM-PNST). The authors also observed lesions reminiscent of a neurofibroma in a peripheral nerve, a cutaneous neurofibroma, and para-spinal neurofibroma on rare occasions in NPcis mice by a systematic histological analysis. They also observed that 10% of the mice developed GEM-PNSTs spontaneously. More interestingly, about half of the sciatic nerves from NPcis mice developed plexiform neurofibromas (PNFs) within 1-6 months when intentionally injured. Thus,this might provided a procedure to turn the widely used NPcis sarcoma model into a model recapitulating PNF. The authors have provided extensive of histology/immunohistochemistry data. Overall, this is a very well organized manuscript. Most of the data are convincing. However, some of the following concerns diminished my enthusiasm for publication at current version: 1. Nf-/- Schwann cells are the pathological cells for PNFs. In the NPcis nerve injury model, is there loss of heterozygosity (LOH)? Is there any difference between the mice with PNF and without PNF in the level of LOH? What about the environment change, especially the macrophages . 2. Is there any histological difference on PNFs developed by longitudinal or cross section procedure? 3. Some of the NPcis mice also develop brain tumors. This can cause mouse death too. It is important to be included in the characteristics. ****** 6. 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? 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Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0301040.r001
Revision 1
1 Mar 2024 Author Response OBJECT : Resubmission of Plante et al. PONE-D-23-19723 Dear Editors, Thank you for the constructive comments and opportunity to address the editorial and reviewer`s comments on our manuscript entitled: ``Revisiting the NPcis mouse model: a new tool to model plexiform neurofibroma``. Please find below the point-by-point response. EDITORIAL COMMENTS 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf We now use the PLoS ONE template 2. Note from Emily Chenette, Editor in Chief of PLOS ONE, and Iain Hrynaszkiewicz, Director of Open Research Solutions at PLOS: Did you know that depositing data in a repository is associated with up to a 25% citation advantage (https://doi.org/10.1371/journal.pone.0230416)? If you’ve not already done so, consider depositing your raw data in a repository to ensure your work is read, appreciated and cited by the largest possible audience. You’ll also earn an Accessible Data icon on your published paper if you deposit your data in any participating repository (https://plos.org/open-science/open-data/#accessible-data). Thank you for the suggestion. 3. Thank you for stating the following financial disclosure: "Fonds de recherche du Québec - Santé. Grant # 281660" Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. The funder had no role, therefore thank you for amending the funder role in the online submission form as ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" 4. Please expand the acronym “FRQS” (as indicated in your financial disclosure) so that it states the name of your funders in full. This information should be included in your cover letter; we will change the online submission form on your behalf. FRQS stands for ``Fonds de Recherche du Québec – Santé``. Thank you for amending it in the online submission form. 5. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. We moved any ethic statement to the Method section. 6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. We now follow the PLoS ONE template. The list of Supporting Information is now found at the end of the manuscript. 7. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. We fully revised the reference list. REVIEWERS` COMMENTS Reviewer #1: Because the existing mouse models of neurofibroma and MPNST do not recapitulate the key clinical and molecular features of the corresponding human tumors, the authors aimed to establish a model that recapitulated the human disease clinically and molecularly. Therefore, they revisited the NP cis model. The paper is interesting, well written and clear. The Authors also discussed the current state of NF mouse models, highlighting model relationship to human tumors. We thank the reviewer for the enthusiasm about our manuscript. Reviewer #2: In this manuscript Plante et al re-characterized the CisNf1+/-;P53+/- (NPcis) mouse. Consistent with the published data, the NPcis mouse developed malignant peripheral nerve sheath tumor (GEM-PNST). The authors also observed lesions reminiscent of a neurofibroma in a peripheral nerve, a cutaneous neurofibroma, and para-spinal neurofibroma on rare occasions in NPcis mice by a systematic histological analysis. They also observed that 10% of the mice developed GEM-PNSTs spontaneously. More interestingly, about half of the sciatic nerves from NPcis mice developed plexiform neurofibromas (PNFs) within 1-6 months when intentionally injured. Thus, this might provide a procedure to turn the widely used NPcis sarcoma model into a model recapitulating PNF. The authors have provided extensive of histology/immunohistochemistry data. Overall, this is a very well-organized manuscript. Most of the data are convincing. However, some of the following concerns diminished my enthusiasm for publication at current version: Again, we thank the reviewer for the enthusiasm about our manuscript. 1. Nf-/- Schwann cells are the pathological cells for PNFs. In the NPcis nerve injury model, is there loss of heterozygosity (LOH)? Is there any difference between the mice with PNF and without PNF in the level of LOH? What about the environment change, especially the macrophages. All excellent points. Ideally, we would have performed DNA sequencing. Unfortunately, the only material output from this study is FFPE slides. -To determine whether or not there is LOH, we did the following two experiments which are featured in a brand-new Fig. 7 along with S6 and S7 Fig: a) We performed immunostaining using NF1 and p53 antibodies on normal sciatic nerves (no injury) from wild-type mice (n=8), injury-induced sciatic nerves from NPcis mice that develop pNF (n=12), injury-induced sciatic nerves from NPcis mice that did not develop (n=9) pNF and spontaneous sarcoma from NPcis mice (n=23) “Fig 7a”. Then, the staining intensity was scored and output as a bar graph representing the percentage of tissues assigned to each staining intensity level. The results indicate that NF1 and p53 expression are largely preserved in non-malignant tissues whereas it is lowly expressed in malignant tissues as expected “Fig 7b”. b) We also performed qPCR using Nf1 and p53 specific primers on normal sciatic nerves (no injury) from wild-type mice (n=2), injury-induced sciatic nerves from NPcis mice that develop pNF (n=2), injury-induced sciatic nerves from NPcis mice that did not develop (n=2) pNF and spontaneous sarcoma from NPcis mice (n=2). Again, the results indicate that Nf1 and p53 expression are largely preserved in non-malignant tissues. More precisely, Nf1 expression is not significantly different between NPcis nerves and pNF but it is for p53. As expected, both Nf1 and p53 expression is significantly decreased in sarcoma compared to normal nerve from NPcis mice “Fig 7c”. Thus, it suggests that loss of heterozygosity of Nf1 and p53 did not took place in injury-induced pNF although alternative strategies to lower p53 expression may exist. -To determine any changes in macrophages, we performed IHC using Iba1 antibodies on injury-induced sciatic nerves from NPcis mice that develop pNF (n=7), injury-induced sciatic nerves from NPcis mice that did not develop (n=8) pNF. As expected we found positive staining in the injury-induced pNF and minimal staining in the injury-induced sciatic nerves that did not develop pNF. This new result is now presented as S5 Fig. 2. Is there any histological difference on PNFs developed by longitudinal or cross section procedure? We revisited the H&E from injury-induced neurofibroma from the ``needle`` and the ``cut`` to evaluate any difference in the histologies. This comparison is now shown in S4 Fig. 3. Some of the NPcis mice also develop brain tumors. This can cause mouse death too. It is important to be included in the characteristics. It is true that NPcis develop a plethora of manifestations, including brain tumors. However, we strictly focused on the benign manifestations of the skin (cNF) and peripheral nervous system (pNF) in this study. Attachments Attachment Submitted filename: Plante Rebuttal letter v2.pdf https://doi.org/10.1371/journal.pone.0301040.r002
11 Mar 2024 Decision Letter - Giulio Piluso, Editor Revisiting the NPcis mouse model: a new tool to model plexiform neurofibroma PONE-D-23-19723R1 Dear Dr. Brosseau, 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. 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at http://www.editorialmanager.com/pone/ and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Giulio Piluso, M.Sc. 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: I Don't Know ****** 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: The authors have addressed all of my concerns and the current version is now acceptable for publication. ****** 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 ******** https://doi.org/10.1371/journal.pone.0301040.r003
Formally Accepted
22 May 2024 Acceptance Letter - Giulio Piluso, Editor PONE-D-23-19723R1 PLOS ONE Dear Dr. Brosseau, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Giulio Piluso Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0301040.r004

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