Astrocytes influence medulloblastoma phenotypes and CD133 surface expression

Emily Gronseth; Ankan Gupta; Chris Koceja; Suresh Kumar; Raman G. Kutty; Kevin Rarick; Ling Wang; Ramani Ramchandran

doi:10.1371/journal.pone.0235852

Peer Review History

Original SubmissionMarch 6, 2020
26 Mar 2020 Decision Letter - Gianpaolo Papaccio, Editor PONE-D-20-06629 Astrocytes influence medulloblastoma phenotypes and CD133 surface expression PLOS ONE Dear Dr. Ramchandran, 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. ============================== Albeit of interest, the paper in its present form requires to be consistently amended in several parts, following all the points highlighted by the two referees. Actually to perform a microarray does not make sense if the altered genes are not investigated and without a confirmation by RT-PCR. Moreover, the stemness must be observed by means of the specific genes including at least Nanog and OCT3/4. Discussion is largely speculative and long. Therefore, the Authors must amend each point raised in the referee's comments in their revised paper. ============================== We would appreciate receiving your revised manuscript by May 10 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. 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We look forward to receiving your revised manuscript. Kind regards, Gianpaolo Papaccio, M.D., 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 http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for including your ethics statement: AUA 320 for zebrafish animal study. No human studies in this project. 3. Please amend your current ethics statement to include the full name of the ethics committee that approved your specific study. For additional information about PLOS ONE submissions requirements for ethics oversight of animal work, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research. Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). 4. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing. 5. In your Methods section, please provide additional details regarding the cell lines used in your study. For the UW228/1 cell line, please address whether the cell line was verified, and if so, how it was verified. Please also ensure that you have cited the original paper describing this cell line. For the Daoy cell line, please include the ATCC catalog number. For more information on PLOS ONE's guidelines for research using cell lines, see https://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines. 6. 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. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [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: I Don't Know ****** 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: No 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: The manuscript is interesting and highlights the role of astrocytes in supporting medulloblastoma tumorigenesis. Although this, there are some concerns that need to be addressed. Following microarray analyses, the authors should confirm the main genes involved in adhesion by real time PCR. Moreover, they should indicate also whether there are specific pathways downregulated. The image of neurospheres must be changed. The authors must add the pictures with an original magnification grater in order to detect the differences in size following treatments. Moreover, to confirm the stemness of cells treated with ACM, the authors must evaluate stem transcriptional factors such as OCT4, Nanog and Sox2. The authors demonstrate that CD133 is associated with increased adhesion. As the microarray indicates that adhesion genes are increased, the authors should analyze the genes with the highest value for each experimental condition and evaluate how they change. When CD133 is silenced, what do it happens to these genes? The Discussion section must be revised. It is too speculative. It should be better focused on the importance of results obtained in medulloblastoma treatment. Reviewer #2: In this manuscript the Authors investigate about the effect of astrocytes conditioned media (ACM) on medulloblastoma cell in terms of adhesion, invasion and stemness. They performed basic studies in vitro showing that ACM have an effect on the adhesion, migration and spheroids formation of MB cells. Moreover they use a zebrafish model to show the effect of ACM in an in vivo model. They also knocked down CD113 to show its role in the ACM induced effects. The work is interesting in principle, however there are few concerns that need to be addressed as follows: It is not clear why the Authors performed a microarray, showing a strong alteration of adhesion gene, but did not investigate the role of any of those genes in MB to establish a mechanist effect. The Author state that ACM increased the stemness of MB cells, however this was based only on spheroid formation. For instance the spheroid analysis and images are not acceptable, more clear and higher magnification images should be provided and an algorithm to calculate sphere diameter should be used. Then, to show an increase in stemness features only spheres formation it’s not enough, the Author should at least show an increase of stem-associated-genes such as Nanog, OCT3/4, and Sox-2 or perform side population assay, or ALDH or any other assay appropriated for MB. This is valid as well for the CD133 knock down. Discussion should be more focused and less speculative. ****** 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 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. https://doi.org/10.1371/journal.pone.0235852.r001
Revision 1
15 Apr 2020 Author Response REVIEWER RESPONSE LETTER Re: PONE-D-20-06629: Astrocytes influence medulloblastoma phenotypes and CD133 surface expression Editor Comments: Albeit of interest, the paper in its present form requires to be consistently amended in several parts, following all the points highlighted by the two referees. Actually to perform a microarray does not make sense if the altered genes are not investigated and without a confirmation by RT-PCR. Response: We appreciate the concern here. The microarray was performed as a hypothesis-generating experiment. Based on this experiment, we observed that adhesion pathways (Fig. S1B) were influenced in medulloblastoma tumor cells that were conditioned in astrocyte media. Yes, we did validate the targets. In the revision, we have now included the qPCR (Fig. S1C) and western blot (Fig. S1D) results for select microarray targets related to adhesion (NFASC, L1CAM, NCAM2) that were upregulated in ACM-MB cells. Moreover, the stemness must be observed by means of the specific genes including at least Nanog and OCT3/4. Response: We performed Sox2 western blot (Fig. 3F), which is also a stem marker and found no difference between ACM-conditioned and DMEM-conditioned MB cells. Due to current COVID-19 situation, we are unable to perform other stem marker genes as requested. We have therefore made revisions in the text to de-emphasize the stemness point. Discussion is largely speculative and long. Therefore, the Authors must amend each point raised in the referee's comments in their revised paper. Response: We have made extensive changes to the discussion to make the points that are directly relevant to the data in the manuscript, and those that support or oppose existing literature on this subject. We provide below a point-by-point response to the reviewer queries. 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 http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: We have ensured that the manuscript and its file names meet PLOS ONE style requirements. 3. Please amend your current ethics statement to include the full name of the ethics committee that approved your specific study. For additional information about PLOS ONE submissions requirements for ethics oversight of animal work, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research. Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). Response: The requested changes have been made. 4. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing. Response: We plan to submit the microarray data to the GEO database and will provide the reference number upon completion. 5. In your Methods section, please provide additional details regarding the cell lines used in your study. For the UW228/1 cell line, please address whether the cell line was verified, and if so, how it was verified. Please also ensure that you have cited the original paper describing this cell line. For the Daoy cell line, please include the ATCC catalog number. For more information on PLOS ONE's guidelines for research using cell lines, see https://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines. Response: Short Tandem Repeat (STR) genetic profiling for cell authentication was performed on UW228/1 cells. Although these cells do not have a public STR profile for positive comparison, the STR analysis confirmed no match with any other cell lines within the DSMZ database in addition to confirming a single donor source. This is noted in the revised manuscript. 6. 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. Response: The requested supporting information is included. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Response: This information has been provided in the supporting information (file “S1_raw_images”). Reviewers' comments: We thank both reviewers for evaluating our manuscript and providing constructive comments. Reviewer #1: The image of neurospheres must be changed. The authors must add the pictures with an original magnification grater in order to detect the differences in size following treatments. Response: Yes, we realize this concern but due to COVID-19 related lab closures, hibernations, and other issues, it is unclear when institution will open up. Deliveries are also delayed these days, and thus the May 10 deadline is not feasible. Thus, we are unable to perform additional new experiments to generate spheres for better high power imaging. For better visualization of the spheres, we enlarged the representative images (Fig 3) as much as possible while maintaining good image quality. In terms of size determinations, we have used two independent methods of quantifications for images taken on two different scopes (Zeiss and Keyence), which has been explained in detailed later (rev. 2 query below), and showed the same result. We also excluded spheres less than 75 �m to avoid including aggregates in our quantification. Thus, we are confident with our neurosphere quantification. Moreover, to confirm the stemness of cells treated with ACM, the authors must evaluate stem transcriptional factors such as OCT4, Nanog and Sox2. Response: We performed Sox2 western blot (Fig. 3F), which is also a stem marker and found no difference between ACM-conditioned and DMEM-conditioned MB cells. Due to current COVID-19 situation, we are unable to perform other stem marker genes as requested. We have therefore made revisions in the text to de-emphasize the stemness point. The authors demonstrate that CD133 is associated with increased adhesion. As the microarray indicates that adhesion genes are increased, the authors should analyze the genes with the highest value for each experimental condition and evaluate how they change. When CD133 is silenced, what do it happens to these genes? Response: The microarray was performed as a hypothesis-generating experiment. Based on this experiment, we observed that adhesion pathways (Fig. S1B) were influenced in medulloblastoma tumor cells (Daoy) that were conditioned in astrocyte media. Yes, we did validate the targets. In the revision, we have now included the qPCR (Fig. S1C) and western blot (Fig. S1D) results for select adhesion gene targets (NFASC, L1CAM, NCAM2) upregulated in ACM-MB cells. We never claimed that the adhesion gene targets NFASC, L1CAM and NCAM2 were downstream of CD133, and thus did not see the rationale for performing this experiment. The Discussion section must be revised. It is too speculative. It should be better focused on the importance of results obtained in medulloblastoma treatment. Response: We have made extensive changes to the discussion to make the points that are directly relevant to the data in the manuscript, and those that support or oppose existing literature on this subject. Please see revised manuscript with track changes. Reviewer #2: It is not clear why the Authors performed a microarray, showing a strong alteration of adhesion gene, but did not investigate the role of any of those genes in MB to establish a mechanist effect. Response: We apologize if the microarray rationale was not clear. The microarray was performed as a hypothesis-generating experiment. Based on this experiment, we observed that adhesion pathways (Fig. S1B) were influenced in medulloblastoma (MB) tumor cells that were conditioned in astrocyte media. Yes, we did validate the adhesion gene targets. In the revision, we have now incuded the qPCR (Fig. S1C) and western blot (Fig. S1D) results for select targets (NFASC, L1CAM, NCAM2) upregulated in ACM-MB cells. We also performed knockdown of L1CAM target but did not observe changes in adhesion (data not shown). Thus, we rationalized redundancy in the up regulated adhesion molecules, and focused on a known CD133 adhesion molecule that is associated with MB progression [1]. Without the microarray data, we would not have hypothesized that the adhesion pathway was involved in astrocyte conditioned MB cell phenotype. Some of these explanations have been included in the revised MS. The Author state that ACM increased the stemness of MB cells, however this was based only on spheroid formation. For instance the spheroid analysis and images are not acceptable, more clear and higher magnification images should be provided and an algorithm to calculate sphere diameter should be used. Response: We have now de-emphasized the stemness point, because our Sox2 western blot data showed great variability between DMEM-conditioned MB cells and ACM-conditioned MB cells (Fig. 3F), which precludes any conclusion. We actually performed two independent methods of quantification for images taken under two different scopes. We initially took pictures on Zeiss scope. To measure diameter, we performed a rough calibration of pixels to micrometer (�m), and then used ImageJ for further data processing. In the second Keyence scope, we pre-programmed set points for picture, which removes bias, and used the Keyence analysis software to directly measure micrometer size of the spheres. In this second method, the diameter measurement was more straightforward, and unbiased. Further, we only calculated spheres with greater than 75 �m to avoid including aggregates. Results from both methods showed the same result. In terms of new and better images, we will need to perform the experiment again. Under normal circumstances, we would be more than happy to do it. But, unfortunately, due to COVID-19 associated lab and institution closure, we cannot perform new experiments. For the representative images in Figure 3, we have enlarged our existing pictures (as much as possible while maintaining good image quality) for better visualization of the spheres. Then, to show an increase in stemness features only spheres formation it’s not enough, the Author should at least show an increase of stem-associated-genes such as Nanog, OCT3/4, and Sox-2 or perform side population assay, or ALDH or any other assay appropriated for MB. This is valid as well for the CD133 knock down. Response: We appreciate the reviewer suggestion, and did perform Sox2 western blot (Fig. 3F), which is also a stem marker [2] and found no difference between ACM-conditioned and DMEM-conditioned MB cells. Due to current COVID-19 situation, we are unable to perform other stem marker genes as requested. We have therefore made revisions in the text to de-emphasize the stemness point. Discussion should be more focused and less speculative. Response: We have made extensive changes to the discussion to make the points that are directly relevant to the data in the manuscript, and those that support or oppose existing literature on this subject. We thank the reviewers for spending their valuable time providing us feedback on our manuscript. REFERENCES 1. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003;63(18):5821-8. PubMed PMID: 14522905. 2. Ellis P, Fagan BM, Magness ST, Hutton S, Taranova O, Hayashi S, et al. SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult. Dev Neurosci. 2004;26(2-4):148-65. PubMed PMID: 15711057. Attachments Attachment Submitted filename: Reviewer Response Letter_Gronseth et al_2.1.docx https://doi.org/10.1371/journal.pone.0235852.r002
5 May 2020 Decision Letter - Gianpaolo Papaccio, Editor PONE-D-20-06629R1 Astrocytes influence medulloblastoma phenotypes and CD133 surface expression PLOS ONE Dear Dr. Ramchandran, 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. ============================== The Authors only partly positively responded to the comments previously raised by the two referees. In their answer the Authors wrote that they were unable to perform extra experiments due to the SARS-COV-2 pandemia. This Editor understand that the problem is real but asks the Authors to obtain much more time for research completion. Therefore The Authors are allowed to ask for more time to send their revised manuscript. ============================== We would appreciate receiving your revised manuscript by Jun 19 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, Gianpaolo Papaccio, M.D., Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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: (No Response) ******** 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: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #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: No 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: The Authors do not fully address my previous concerns. They state that, due to current COVID-19 situation, they are unable to perform experiments requested by this reviewer. I recommend asking to editor more time to carry out the experiments in order to improve the manuscript. Reviewer #2: the author have addressed part of the comments raised by this reviewer, accoridng to author's responce they cannot perform required experiment because of the covid-19 spread and lab closure. Due to this situation I believ it will be more approriate a time extention so the revisin can be perform at the best. ****** 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. https://doi.org/10.1371/journal.pone.0235852.r003
Revision 2
10 Jun 2020 Author Response REVIEWER RESPONSE LETTER Re: PONE-D-20-06629: Astrocytes influence medulloblastoma phenotypes and CD133 surface expression Editor Comments: The Authors only partly positively responded to the comments previously raised by the two referees. In their answer the Authors wrote that they were unable to perform extra experiments due to the SARS-COV-2 pandemia. This Editor understand that the problem is real but asks the Authors to obtain much more time for research completion. Therefore, the authors are allowed to ask for more time to send their revised manuscript. Reviewer #1: The Authors do not fully address my previous concerns. They state that, due to current COVID-19 situation, they are unable to perform experiments requested by this reviewer. I recommend asking to editor more time to carry out the experiments in order to improve the manuscript. Reviewer #2: the author have addressed part of the comments raised by this reviewer, according to author's responce they cannot perform required experiment because of the covid-19 spread and lab closure. Due to this situation I believe it will be more appropriate a time extension so the revising can be performed at the best. Response: Thank you for the additional time that the editor provided to perform additional experiments and make more revisions to the manuscript. As per the reviewer and editor requests, we have now performed additional experiments and included new data in the following panels Figs. 3C, 3D, and 3F. New images for neurospheres were requested. We performed a new experiment to generate newer images under higher power to honor this request. All of our previous images were low power. This new higher magnified image data is now included in figure 3C with scale bars. The neurosphere sizes are clearly larger in the astrocyte-conditioned media (ACM) compared to DMEM conditions. Further, the ACM-induced neurospheres are similar in size to DMEM:F12 positive control conditions. The new images were quantified, and the quantification is also provided in the figure 3D. Western blots for additional stem cell proteins (Nanog & Oct4) were requested in addition to previously provided Nestin (Fig. 3E) and Sox2 (Fig. 3G) data. We performed westerns for Oct-4 and Nanog, and have provided new data for Oct4 (Fig. 3F) along with quantification. The Oct-4A blot shows that under both ACM and DMEM:F12 positive control conditions, Oct-4A protein (**p<0.001) expression was higher compared to DMEM conditions. We checked Nanog protein as well, but the antibody did not work well, and thus no conclusion can be further derived from this blot. Collectively, of the three stemness proteins that were detected in MB cells, we noticed 2 (Nestin and Oct-4A) out of 3 expression to be higher in ACM conditions compared to DMEM conditions. Stemness write-up de-emphasis. We carefully searched for the entire manuscript for the stemness or stem cell word, and only found that we utilized these words in reference to describing CD133 as a stem cell marker or describing Nestin, Sox2 or Oct-4A proteins in the results section. The Discussion section was extensively revised in the previous submission, and all references to stemness was removed then. Attachments Attachment Submitted filename: Reviewer Response Letter2_Gronseth et al.docx* https://doi.org/10.1371/journal.pone.0235852.r004
24 Jun 2020 Decision Letter - Gianpaolo Papaccio, Editor Astrocytes influence medulloblastoma phenotypes and CD133 surface expression PONE-D-20-06629R2 Dear Dr. Ramchandran, 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, Gianpaolo Papaccio, M.D., 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 #1: All comments have been addressed 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #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: (No Response) Reviewer #2: The Authors have adressed al the concerns raised by this reviewer. Now the manuscript is greatly improved. ****** 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 https://doi.org/10.1371/journal.pone.0235852.r005
Formally Accepted
25 Jun 2020 Acceptance Letter - Gianpaolo Papaccio, Editor PONE-D-20-06629R2 Astrocytes influence medulloblastoma phenotypes and CD133 surface expression Dear Dr. Ramchandran: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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 Prof. Gianpaolo Papaccio Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0235852.r006

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