PONE-D-21-18814

Microglial-stimulation of Glioblastoma Invasion Involves the EGFR ligand Amphiregulin

PLOS ONE

Dear Dr. Coniglio,

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 Sep 09 2021 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. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Arun Rishi, Ph.D.

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

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

2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information on the animal research and ensure you have included details on (1) methods of sacrifice, and (2) efforts to alleviate suffering.

3. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information on the (1) Recombinant human CSF-1 and (2) CSF-1R receptor inhibitor, [4-Cyano-1H-pyrrole-2-carboxylic acid [4-(4-methyl-piperazin-1-yl)-2-(4-methyl-piperidin-1-yl)-phenyl]-amide].

4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section."

5. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

6. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

7. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical.

8. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels

9 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.

[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: Yes

**********

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: Overall opinion and general observations of the manuscript PONE-D-21-18814

Summary

This is a comprehensive study regarding the mechanisms glioblastoma (GBM) cells use to invade normal brain. It is known that microglia stimulate GBM cell invasion, and that this process is dependent on CSF-1R signaling. However, to have discovered more in-depth mechanism and pro-invasive factors upregulated in microglia, particularly amphiregulin (AREG), is interesting since targeting AREG/EGFR in the tumor could inhibit tumor invasion. Moreover, these extensive results are promising in terms of targeted therapies for GBM.

However, there are some concerns with regard to specific statements.

1. Term "multiforme" is no longer in use. According to the WHO 2016 classification of diffuse gliomas this term is omitted. Moreover, in Abstract you used this term with glioma, which is inappropriate.

2. You have cited plenty of references, however, only less than 20 is issued since 2015. For example, in Introduction you cited 8 references that showed that microglia are enhancers of glioma cell invasion, and none of them is issued since 2015.

3. The last paragraph in Introduction should be the aim of your research, not to conclude what you have found. Otherwise, it looks like Conclusion. This paragraph needs to be changed.

4. I wondered about the concentration of CSF-1R inhibitor (JnJ). Why did you use 10 nM?

5. Can you elaborate the place of inoculation of GBM cells? Why did you use those coordinates for induction of glioblastoma? Does it have impact on your model of glioblastoma?

6. Please, indicate magnification you used in Fig.3B and Fig.4. Also, if possible, add scale bar in both microphotographs. Explain what arrows indicate.

7. In Fig.4 the number of invaded cells in group treated with AREG blocking Ab is higher than in U87 alone, while in microphotographs it looks comparable between these two groups. Please, use consistent pictures.

Reviewer #2: This is an interesting article that builds on groups' prior published work from 2012, showing that CSF1-R inhibition in macrophage/microglia results in decreased glioma invasion. In the current manuscript, the authors show the mechanism by which CSF-1R signaling in microglia results in upregulation of AREG, leading to glioma cell invasion. They further show that interfering with AREG either by using RNAi or blocking antibodies attenuate macrophages/microglia's ability to promote glioma invasion. Overall, it is a well-controlled and concise in vitro study. There are a few issues that need to be fixed before considering it for publishing.

Figure 1B is missing the error bar for the microglia alone group, or if it is normalized per microglia alone group, in which case it should be fold change on Y-axis. This goes for Figure 2 as well.

Figure 2 in the text states four independent experiments – are those from four independent tumors? Need to be clarified.

In the text, it states, "Interestingly, we found that recombinant CSF-1 alone was insufficient to stimulate AREG mRNA in microglia to levels seen with GLCM (Fig 2B). Unfortunately, Figure 2B does not support the statement, and multiple comparison test should be used to see whether induction of AREG by CSF-1 stimulation and GLCM is in Figure 2B.

Images for invasion in all the figures are not very impressive, and some high-resolution inserts would be helpful.

Glioma Multiforme term is no longer used needs to be just glioblastoma.

**********

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? 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.

Attachments

Attachment

Submitted filename: Reviewer comments - PLOS ONE.docx

Microglial Stimulation of Glioma Invasion Involves the EGFR ligand Amphiregulin

PONE-D-21-18814R1

Dear Dr. Coniglio,

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 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,

Arun Rishi, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: