Tamoxifen resistance alters sensitivity to 5-fluorouracil in a subset of estrogen receptor-positive breast cancer

Takayuki Watanabe; Takaaki Oba; Keiji Tanimoto; Tomohiro Shibata; Shinobu Kamijo; Ken-ichi Ito

doi:10.1371/journal.pone.0252822

Peer Review History

Original SubmissionFebruary 1, 2021
26 Feb 2021 Decision Letter - Wei Xu, Editor PONE-D-21-03437 Tamoxifen resistance alters sensitivity to 5-fluorouracil in a subset of estrogen receptor-positive breast cancer PLOS ONE Dear Dr. Ito, 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. Specifically, the reviewers suggested to examine 5-FU sensitivity in multiple breast cancer cell lines and to perform knockdown of essential enzymes. Please submit your revised manuscript by May 22nd, 2021. 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. 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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: This is a very interesting study where the authors examine the sensitivity to 5-FU after ER positive cells acquire tamoxifen resistance. Below are my comments to further complete the study and clarify the mechanisms of drug sensitivity to 5-FU: 1. One representative tamoxifen resistant clone was used in this study. However, the growth rate of parental and tamoxifen resistant sub-line is different for MCF7, T47D and BT-474. MCF7-T cells growth rate was almost doubled compared with parental, while T47D-T and BT-474-T decreased compared with parental lines. It seems that the sensitivity to 5-FU or other chemotherapy drugs correlated with the growth rate of the cells (parental and tamoxifen resistant subclone). Since there is only one cell line that has preferential sensitivity to 5-FU after acquiring tamoxifen resistance, the authors need to carefully validate observations seen in MCF7 using multiple clones (and also T47D and BT-474), to clarify whether this is due to the growth rate of the subclones or not. 2. In order to obtain the conclusion that “decrease in the target enzyme thymidine synthase, together with a drastic decrease in its catabolic enzyme, dihydropyrimidine dehydrogenase, may enhance the efficacy of 5-fluorouracil in MCF7/T cells”, the authors need to directly knockdown thymidine synthase and DPYD in parental MCF7, T47D and BT-474 cells and test for the sensitivity to 5-FU. Otherwise, this observation is merely a correlation but did not indicate the causative effect for 5-FU sensitivity. 3. If 5-aza restores the level of DPYD, did it lead to resistance to 5-FU treatment in MCF7-T cells? Reviewer #2: About one third of ER positive breast cancer patients showed tamoxifen resistance after a long-term treatment. However, few studies have investigated the response to subsequent chemotherapy in ER positive breast cancer which showed resistance to endocrine therapy. The objective of this study is to focus on whether preceding endocrine therapy could alter the sensitivity to chemotherapeutic agents in ER-positive breast cancers and find biomarkers for personalized treatment that acquired resistance endocrine therapy. The manuscript by Takayuki Watanabe et al. selected three tamoxifen resistant cell lines (T47D/T, MCF7/T and BT474/T), and then tested cells sensitivity to chemotherapeutic agents compared with wild type (wt) cells. The results showed MCF7/T cells became more sensitive to 5-fluorouracil than wt-MCF7 cells. This is because the expression of dihydropyrimidine dehydrogenase (DPYD) was decreased in MCF/T cells compared with wt-MCF7 cells. DPD is an important enzyme to degrade 5-fulorouracil to inactive metabolites. Mechanismly, they found the expression of DYPD was repressed by methylation of its promoter and post-transcriptional regulation by miRNA in MCF7/T cells. They also performed in vivo xenograft experiments to confirm capecitabine could significantly reduce tumor volume in MCF7/T compared with MCF7 cells. These new findings of this paper demonstrated that 5-fulorouracil and its derivatives may act as critical drugs in some TAM-resistant ER positive breast cancers. Overall the experimental designs are rigor with mouse models and the results support their conclusions. A few minor comments are listed below: 1. BT474 cell line is always used as a de novo tamoxifen resistant model, so it’s not appropriate for the author to select tamoxifen resistant cell line (BT474/T). 2. Fig. 1 looks crowded and confused, drawing a curve to show the response to different concentrations in each WT and resistant cell line (just like figure 2) will be more nice and clearly. 3. For the result of Fig. 4, the author should show a little more background information about these enzymes, thymidine synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase, explain the correlation between the expression of these enzymes and the cells sensitive to 5-fluorouracil. What’s more, the author should also test the metabolites of 5-fulorouracil in wt-MCF7 and MCF7/T cells. 4. In Fig 8, the staining of ER and DPD by IHC is unclear, the author should repeat and show a clear picture. ****** 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. https://doi.org/10.1371/journal.pone.0252822.r001
Revision 1
10 May 2021 Author Response Point-by-point responses to the reviewers’ comments We thank the reviewers for their helpful and insightful comments. We revised our manuscript in accordance with these comments, and we believe our manuscript has greatly improved, thanks to the reviewers’ feedback. We hope that the newly submitted manuscript is now suitable for publication in PLOS ONE. Reviewer #1: This is a very interesting study where the authors examine the sensitivity to 5-FU after ER positive cells acquire tamoxifen resistance. Below are my comments to further complete the study and clarify the mechanisms of drug sensitivity to 5-FU: 1. One representative tamoxifen resistant clone was used in this study. However, the growth rate of parental and tamoxifen resistant sub-line is different for MCF7, T47D and BT-474. MCF7-T cells growth rate was almost doubled compared with parental, while T47D-T and BT-474-T decreased compared with parental lines. It seems that the sensitivity to 5-FU or other chemotherapy drugs correlated with the growth rate of the cells (parental and tamoxifen resistant subclone). Since there is only one cell line that has preferential sensitivity to 5-FU after acquiring tamoxifen resistance, the authors need to carefully validate observations seen in MCF7 using multiple clones (and also T47D and BT-474), to clarify whether this is due to the growth rate of the subclones or not. Response: We appreciate the reviewer’s insightful comments. To evaluate tamoxifen (TAM) resistance more objectively, we have re-examined TAM sensitivity for wild-type and TAM-resistant sublines with a WST assay and demonstrated the results in Figure 1A in the revised manuscript. In addition, we have also presented TAM IC50s for wild-type and TAM-resistant sublines in Table 1. From these results, we consider the difference of TAM sensitivity between wild types and our established resistant sublines to not be solely due to differences in the growth rate. We have described the results mentioned above in the first paragraph on page 12 of the revised manuscript. In the cell proliferation assay presented in Figure 1A of the originally submitted version, the initial cell number was different for each cell line (ex. wt-MCF7, 14 × 104; MCF7/T, 45 × 104). Therefore, a large difference was observed in the number cells grown after 120 h. However, when calculating the growth rate, there was no remarkable difference between wild-type and TAM-resistant sublines. The growth curve presented as Figure 1A in the originally submitted version is attached as Supplementary figure 1, with the vertical axis corrected to the growth rate for reference. Regarding MCF7, we had established several TAM-resistant sublines. We have included the data obtained using one subline, MCF7/T2, as Supplementary figure 2. As shown in Supplementary figure 2A, MCF7-T2 showed an increased sensitivity to 5-fluorouracil, equivalent to MCF7/T, while decreased expression of DPYD mRNA was observed in MCF7/T2, as demonstrated in Supplementary figure 2B. We consider these results to show that a decrease in DPYD mRNA expression may be one of the changes induced in the development of TAM resistance in MCF7. We have described the results mentioned above in the second paragraph on page 14 and in the second paragraph on page 18 of the revised manuscript. 2. In order to obtain the conclusion that “decrease in the target enzyme thymidine synthase, together with a drastic decrease in its catabolic enzyme, dihydropyrimidine dehydrogenase, may enhance the efficacy of 5-fluorouracil in MCF7/T cells”, the authors need to directly knockdown thymidine synthase and DPYD in parental MCF7, T47D and BT-474 cells and test for the sensitivity to 5-FU. Otherwise, this observation is merely a correlation but did not indicate the causative effect for 5-FU sensitivity. Response: We appreciate the reviewer’s insightful comments. Per the reviewer’s suggestion, we tested whether knockdown of thymidine synthase (TS) or dihydropyrimidine dehydrogenase (DPYD) would alter the sensitivity to 5-fluorouracil in wt-MCF7 cells. Although the reviewer mentioned that we should test the knockdown of TS and DPYD in wt-T47D and at-BT474 cells as well, we performed the additional experiments in wt-MCF7 cells alone because the baseline expression level of DPYD mRNA was not high enough in wt-T47D and at-BT474 cells to inhibit its expression by siRNA. As demonstrated in Supplementary figure 3, inhibition of TS and DPYD expression by siRNA was confirmed at the mRNA level for wt-MCF7 cells (A, B). siRNA targeting of DPYD sensitized the wt-MCF7 cells to 5-fluorouracil, while siRNA targeting of TS did not alter the sensitivity to 5-fluorouracil (C). We have described the results mentioned above in the third paragraph on page 18 of the revised manuscript. 3. If 5-aza restores the level of DPYD, did it lead to resistance to 5-FU treatment in MCF7-T cells? Response: We appreciate the reviewer’s insightful comments. Per the reviewer’s suggestion, we tested whether treatment with 5-azacytidine alter the sensitivity to 5-fluorouracil of TAM-resistant MCF7 (MCF7/T) cells. We have included the results of the WST assay as Figure 6B. As demonstrated in Figure 6A, when the MCF7/T cells were treated with 5 μM of 5-azacytidine, a decrease of 5-fluorouracil sensitivity was observed in parallel with an increased DPYD mRNA expression. These data indicate the possibility that hypermethylation-mediated modulation of DPYD mRNA expression may partly be responsible for altering the sensitivity of MCF/T cells to 5-fluorouracil. We have described the results mentioned above in the second paragraph on page 21. Reviewer #2: About one third of ER positive breast cancer patients showed tamoxifen resistance after a long-term treatment. However, few studies have investigated the response to subsequent chemotherapy in ER positive breast cancer which showed resistance to endocrine therapy. The objective of this study is to focus on whether preceding endocrine therapy could alter the sensitivity to chemotherapeutic agents in ER-positive breast cancers and find biomarkers for personalized treatment that acquired resistance endocrine therapy. The manuscript by Takayuki Watanabe et al. selected three tamoxifen resistant cell lines (T47D/T, MCF7/T and BT474/T), and then tested cells sensitivity to chemotherapeutic agents compared with wild type (wt) cells. The results showed MCF7/T cells became more sensitive to 5-fluorouracil than wt-MCF7 cells. This is because the expression of dihydropyrimidine dehydrogenase (DPYD) was decreased in MCF/T cells compared with wt-MCF7 cells. DPD is an important enzyme to degrade 5-fulorouracil to inactive metabolites. Mechanismly, they found the expression of DYPD was repressed by methylation of its promoter and post-transcriptional regulation by miRNA in MCF7/T cells. They also performed in vivo xenograft experiments to confirm capecitabine could significantly reduce tumor volume in MCF7/T compared with MCF7 cells. These new findings of this paper demonstrated that 5-fulorouracil and its derivatives may act as critical drugs in some TAM-resistant ER positive breast cancers. Overall the experimental designs are rigor with mouse models and the results support their conclusions. A few minor comments are listed below: 1. BT474 cell line is always used as a de novo tamoxifen resistant model, so it’s not appropriate for the author to select tamoxifen resistant cell line (BT474/T). Response: We appreciate the reviewer’s comments. As the reviewer stated, the IC50 for TAM of BT474 cells was higher than those of wt-MCF7 and wt-T47D cells (Table 1); however, we successfully established TAM-resistant sublines for BT474 cells, wherein the IC50 was 2.0 times higher than that of the wild type. Hence, we would like to keep the results obtained for BT474 cells in the revised manuscript. 2. Fig. 1 looks crowded and confused, drawing a curve to show the response to different concentrations in each WT and resistant cell line (just like figure 2) will be more nice and clearly. Response: We agree with the reviewer’s point. We have re-examined TAM sensitivity for wild-type and TAM-resistant sublines with the WST assay and have demonstrated the results as Figure 1A in the revised manuscript. The growth curve presented as Figure 1A in the originally submitted version is attached as Supplementary figure 1, with the vertical axis corrected to the growth rate for reference. We have described the results in the first paragraph on page 12 of the revised manuscript. 3. For the result of Fig. 4, the author should show a little more background information about these enzymes, thymidine synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase, explain the correlation between the expression of these enzymes and the cells sensitive to 5-fluorouracil. What’s more, the author should also test the metabolites of 5-fulorouracil in wt-MCF7 and MCF7/T cells. Response: We appreciate the reviewer’s insightful comments. 5-fluorouracil is converted to fluorodeoxyuridine (FdUrd) by thymidine phosphorylase, and is then phosphorylated by thymidine kinase to fluorodeoxyuridine monophosphate (FdUMP). Inhibition of thymidylate synthase by FdUMP is one of the principal mechanisms of 5-fluorouracil’s action. However, 5-fluorouracil is enzymatically inactivated by dihydropyrimidine dehydrogenase to form dihydrofluorouracil (DHFU) (DPD). Subsequently, DHFU is metabolized to α-fluoro-ureidopropionic acid (FUPA), then 2-fluoro-β-alanine (FBAL), releasing ammonia and carbon dioxide. Based on this background, we analyzed the mRNA expression of thymidylate synthase, thymidine phosphorylase, and DPYD in wild-type and TAM-resistant sublines. In accordance with the helpful suggestion by the reviewer, we have provided concise background information about the metabolism of 5-fluorouracil, as mentioned in the second paragraph on page 17 of the Results. We have edited the description of interpretation in the second paragraph on page 27 in the Discussion as well. Moreover, we analyzed the metabolites of 5-fluorouracil in wt-MCF7 and MCF7/T cells, following the reviewer’s suggestion. We quantitated the two 5-fluorouracil metabolites (FdUrd and FBAL) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We observed that the amount of 5-fluorouracil’s active metabolite, FdUrd, was higher in MCF7-T cells compared to wt-MCF7 cells, while that of FBAL was lower. These data demonstrate high sensitivity to 5-fluorouracil observed in MCF7/T cells. We have included these data as Supplementary figure 4 in the revised manuscript. We have described the results mentioned above in the third paragraph on page 18 of the revised manuscript. 4. In Fig 8, the staining of ER and DPD by IHC is unclear, the author should repeat and show a clear picture. Response: We appreciate the reviewer’s helpful comment. We have replaced the pictures in Figure 8A with higher resolution images. Attachments Attachment Submitted filename: Response to the reviewers comments.docx https://doi.org/10.1371/journal.pone.0252822.r002
24 May 2021 Decision Letter - Wei Xu, Editor Tamoxifen resistance alters sensitivity to 5-fluorouracil in a subset of estrogen receptor-positive breast cancer PONE-D-21-03437R1 Dear Dr. Ito, 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, Wei Xu Academic Editor PLOS ONE 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: 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: (No Response) Reviewer #2: All comments had been addressed, the manuscript had been revised according to the comments. The manuscript could be accepted. ****** 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.0252822.r003
Formally Accepted
31 May 2021 Acceptance Letter - Wei Xu, Editor PONE-D-21-03437R1 Tamoxifen resistance alters sensitivity to 5-fluorouracil in a subset of estrogen receptor-positive breast cancer Dear Dr. Ito: 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 Dr. Wei Xu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0252822.r004

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