The clinicopathologic significance of Tks5 expression of peritoneal mesothelial cells in gastric cancer patients

Atsushi Sugimoto; Tomohisa Okuno; Gen Tsujio; Tomohiro Sera; Yurie Yamamoto; Koji Maruo; Shuhei Kushiyama; Sadaaki Nishimura; Kenji Kuroda; Shingo Togano; Yuichiro Miki; Mami Yoshii; Tatsuro Tamura; Takahiro Toyokawa; Hiroaki Tanaka; Kazuya Muguruma; Masaichi Ohira; Masakazu Yashiro

doi:10.1371/journal.pone.0253702

Peer Review History

Original SubmissionJanuary 5, 2021
18 Mar 2021 Decision Letter - Surinder K. Batra, Editor PONE-D-21-00313 The Clinicopathologic Significance of Tks5 Expression of Peritoneal Mesothelial Cells in Gastric Cancer Patients PLOS ONE Dear Dr. Yashiro, 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 below by the reviewer's during the review process. Please submit your revised manuscript by Apr 30 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. 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At this time, please address the following queries: Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” If any authors received a salary from any of your funders, please state which authors and which funders. If you did not receive any funding for this study, please state: “The authors received no specific funing for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [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: Partly 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: Peritoneal dissemination is a frequent metastatic pattern of gastric cancer. Peritoneal mesothelial cells activated by gastric cancer cells play an important role in peritoneal carcinomatosis by providing the invasive front. The current work relies on a previously published report on an adaptor protein, Tks5. Tks5 activity has been shown to facilitate peritoneal mesothelial cell migratory capacity and invasiveness. In the current work, the authors have investigated the clinicopathological correlation of Tks5 in PMC in a gastric cancer patient cohort. 1) General comments: The overall flow of the paper is satisfactory. However, the authors are encouraged to address the concerns and revise their manuscript before resubmission. 2) Detailed comments: Abstract: Abstract is compact and easy to understand Introduction: Satisfactory and easy to understand Materials and methods: satisfactory Results: Authors are encouraged to address the following concerns 1. Provide a better image/improved staining quality for Tks5 2. Functional assay to provide insight on how Tks5 is activated in PMC- whether GC cells induce the expression in PMC? What type of stimulus can activate Tks5 expression in PMC? 3. What is the expression status of Tks5 in healthy peritoneum? 4. Functional data to identify Tks5 dependent gene regulation. How the overall gene landscape is altered in absence of Tks5. Does Tks5 regulate EMT gene signatures or other markers that can affect immune cells in the TME? 5. Does Tks5 play any role in chemoresistance or angiogenesis? Discussion: Based on current data, it is easy to understand Conclusion: Based on the current data provided, it is easy to understand References: good Images/tables (if any): Not satisfied with the staining quality. Authors are encouraged to provide a better image/improved staining quality in the revised version. Language quality: Easy to understand. Good flow throughout the text. There are a few typographical/grammar errors. Authors are encouraged to correct them in the revised version of the manuscript. Statistic results (if applicable): Satisfactory Reviewer #2: The study is well planned and interesting, however this reviewer has a major concern related to non-specific staining visible in IHC images. The IHC images shown in Figure 1 shows staining of both the mesothelial cells and the underlying stroma. The intensity of the stromal staining is also decreasing as the score for mesothelial cells goes down. Hence, the authors should include some additional control to show the specificity of the staining. Staining with a different antibody might validate the earlier analysis. ****** 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: PloS One review II (1).doc https://doi.org/10.1371/journal.pone.0253702.r001
Revision 1
5 May 2021 Author Response May 5, 2021 Professor Surinder K. Batra Academic Editor PLOS ONE Dear Professor Surinder K. Batra; Re; Manuscript: PONE-D-21-00313 Title: The Clinicopathologic Significance of Tks5 Expression of Peritoneal Mesothelial Cells in Gastric Cancer Patients Authors: Atsushi Sugimoto, et al. We greatly appreciate your invitation for us to revise our article “The Clinicopathologic Significance of Tks5 Expression of Peritoneal Mesothelial Cells in Gastric Cancer Patients”. We would like to thank you for a number of comments and suggestions for improvement in our manuscript. We have carefully considered the referees’ comments and have made point-by-point responses as described below. Also, we highlight all changes in the revised manuscript. This manuscript is not being considered in whole or in part by any other journal. All authors are aware of the content of this manuscript. We hope you will seriously consider this report for publication in PLOS ONE. Sincerely, Masakazu Yashiro, MD Molecular Oncology and Therapeutics, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan TEL; (+81) 6-6645-3838 FAX; (+81) 6-6646-6450 We have responded to the Reviewer #1 comments, as follows. Reviewer #1 Thank you very much for the careful review of the Reviewer #1. We corrected several points according to the descriptions by the Reviewer #1, as described below. We indicated the changes point by point and highlighted them in the revised paper. 1. Provide a better image/improved staining quality for Tks5. We improved staining and replaced the picture with the better images of Tks5 staining (Figure 1). 2. Functional assay to provide insight on how Tks5 is activated in PMC- whether GC cells induce the expression in PMC? What type of stimulus can activate Tks5 expression in PMC? It has been reported that the Tks5 is activated by transforming growth factor-β (TGFβ) via Src phosphorylation [20]. We previously reported that gastric cancer cells increased the Tks5 expression of peritoneal mesothelial cells (PMCs), which stimulated the invasion activity of PMCs and resulted in the stimulation of invasiveness of gastric cancer cells [13]. Since most of gastric cancer cells frequently produce TGFβ [21], TGFβ from gastric cancer cells might activate Tks5 the PMCs via TGFβ-Src-Tks5 signaling. We added these comments in the discussion. (on page 8 line 15- 21). 3. What is the expression status of Tks5 in healthy peritoneum? Tks5 expression was examined using 17 cases of normal peritoneum. The weak expression of Tks5 was found in the cytoplasm of the monolayer PMCs of the normal peritoneum (Supplement Figure 1). Tks5 expression of PMCs was statistically compared between gastric cancer cases and normal cases. Of the total of 17 normal cases, 6 cases (35.3%) were Tks5-positive and 11 cases (64.7 %) were Tks5-negative. Tks5-positive expression of PMCs in GC cases were significantly (p = 0.022) more frequent than that in normal cases (Supplement Table 1). We added these comments in the materials and methods, the results and the discussion. (on page 6 line 10-11; on page 4 line 28-29; on page 6 line 8-15). 4. Functional data to identify Tks5 dependent gene regulation. How the overall gene landscape is altered in absence of Tks5. Does Tks5 regulate EMT gene signatures or other markers that can affect immune cells in the TME? Sundararajan V et al. reported that Tks5 represented a mediator of invasive behavior of cancer cells through the co-expression with ZEB1 which is one of epithelial to mesenchymal transition (EMT) inducers [26]. We added these comments in the discussion. (on page 9 line 4-6). 5. Does Tks5 play any role in chemoresistance or angiogenesis? Bayarmagnai B et al. reported that Tks5 localization is elevated in G1 phase of cell cycle, and anti-proliferative drugs, many of which are arresting cancer cells in G1 phase, may result in higher invasion and metastasis. [28]. We added comments in the discussion. (on page 9 line 7-9). Blouw B et al. reported that Tks5 and podosome formation play as mediators of tumor angiogenesis [25]. We added some comments in the discussion. (on page 9 line 9-10). We have responded to the Reviewer #2 comments, as follows. Reviewer #2 Thank you very much for the careful review of the Reviewer #2. We correct several points according to the descriptions by the Reviewer #2, as follows. 1. The study is well planned and interesting, however this reviewer has a major concern related to non-specific staining visible in IHC images. The IHC images shown in Figure 1 shows staining of both the mesothelial cells and the underlying stroma. The intensity of the stromal staining is also decreasing as the score for mesothelial cells goes down. Hence, the authors should include some additional control to show the specificity of the staining. Staining with a different antibody might validate the earlier analysis. We added additional control to show the specificity of the staining including normal peritoneum and an anti-Calretinin antibody, a specific marker for PMCs. Tks5 expression was examined using 17 cases of normal peritoneum. The weak expression of Tks5 was found in the cytoplasm of the monolayer PMCs of the normal peritoneum (Supplement Figure 1A). The Calretinin expression was found at PMCs with Tks5 expression the surface of peritoneum of gastric cancer cases (Supplement Figure 1B). We replaced the picture with the clearer photo for Tks5 staining in Figure 1. We added some comments in the result. (on page 6 line 8-12). https://doi.org/10.1371/journal.pone.0253702.r002
11 Jun 2021 Decision Letter - Surinder K. Batra, Editor The Clinicopathologic Significance of Tks5 Expression of Peritoneal Mesothelial Cells in Gastric Cancer Patients PONE-D-21-00313R1 Dear Dr. Yashiro, 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, Surinder K. Batra 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: 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: After revision the quality of the images and data has been substantially improved. Addition of new sections in the text has also improved the overall quality. ****** 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.0253702.r003
Formally Accepted
5 Jul 2021 Acceptance Letter - Surinder K. Batra, Editor PONE-D-21-00313R1 The Clinicopathologic Significance of Tks5 Expression of Peritoneal Mesothelial Cells in Gastric Cancer Patients Dear Dr. Yashiro: 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. Surinder K. Batra Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0253702.r004

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