Urinary 8-iso PGF2α and 2,3-dinor-8-iso PGF2α can be indexes of colitis-associated colorectal cancer in mice

Yusuke Miyazaki; Tatsuro Nakamura; Shinya Takenouchi; Akane Hayashi; Keisuke Omori; Takahisa Murata

doi:10.1371/journal.pone.0245292

Peer Review History

Original SubmissionAugust 16, 2020
3 Sep 2020 Decision Letter - Hiroyasu Nakano, Editor PONE-D-20-25696 Urinary 8-iso-PGF2α and 2,3-dinor-8-iso-PGF2α can be indexes of colitis-associated colorectal cancer in mice Lipid mediators as biomarker of colitis-associated colorectal cancer PLOS ONE Dear Dr. Murata 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. ============================== While the reviewer appreciates the importance of the study, the reviewer has several concerns that need to be addressed before publication. Specifically, the cellular source of PGF2alpha and its metabolites is not directly investigated in the study. Hence, the authors need to investigate whether colon cancer cells or cancer tissues produce PGF2alpha and its metabolites. Moreover, it would be intrigued to determine the concentrations of PGF2alpha and its metabolites in the plasma of untreated and AOM/DSS-treated mice. ============================== Please submit your revised manuscript by the end of November, 2020. 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. 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Kind regards, Hiroyasu Nakano, 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 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. As part of your revisions, please provide additional details pertaining to your animal research procedures. Please revise your Methods to address the following points: (1) sample size: how many animals per group and how did you determine the numbers (power analysis? pilot study? previously published data/findings?); (2) provide complete information about all methods undertaken to minimize pain and distress of the animals in your work. (3) Please discuss your monitoring parameters (physical and behavioral signs to assess health and well-being), in addition to your humane endpoints (criteria used to determine when to euthanize animals in cases where animals become sick/moribund). (4) Please state the rate of mortality for animals who died unexpectedly (and state cause(s) of death). (6) Please also discuss supportive care that you provided to the animals. (7) Lastly, please complete and submit the ARRIVE Guidelines checklist (Essential 10) with your revision: https://arriveguidelines.org/resources/author-checklists [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No ****** 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 ****** 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 ****** 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: Miyazaki et al established a murine model of inflammation-induced colon cancer (CAC), and searched for novel lipid biomarkers in the urine of this model mouse. They identified contents of several metabolites derived from arachidonic acid are increased in the CAC urine. Some of them (PGD and E metabolites) are also increased in the inflammation period, and not candidates for biomarkers. They finally found that 8-iso-PGF2αand 2,3-dinor-8-iso-PGF2α are increased only in the CAC period, and concluded that these two PGF2 metabolites are candidate biomarkers for colorectal cancer. They also stained the mouse cancer tissues and showed that the expression of COX-2 and NADPH oxidase is increased in the cancer cells, and this can explain the increase of the metabolites. This is an interesting study that utilizes a mouse oncogenic model to find biomarkers. The biosynthesis and metabolic inactivation of arachidonic acid-derived lipid mediators are conserved among various animal species, this strategy is reasonable. This group has a long history of measuring PG and PG metabolites, and the manuscript is simply and clearly written. The reviewer, however, has to raise several important issues to be addressed in the revised manuscript. Major points The authors claim that 8-iso-PGF2alpha and 2,3-dinor-8-iso-PGF2 are possible biomarkers based on the specific increase of these metabolites in CAC phase. They did not succeed in convincing the reviewer that the colon cancer cells can generate and metabolize PGF2 alpha. Quantification of PGF2alpha and its metabolites in the cancer tissues, hopefully, lazar-dissected tissues, is required. If they think that cancer cells produce PGF2alpha, which enters into the blood and is metabolized in the other tissues, they should measure PGF2alpha and metabolites in the plasma. Regarding the application to the human situation, they should measure these metabolites in the urine of human colon cancer patients and show some representative results. Minor points Statistic analyses are missing in Fig. 1E, F, and Fig. 2. ****** 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 [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.0245292.r001
Revision 1
21 Dec 2020 Author Response Responses to the reviewers’ comments of the manuscript “Urinary 8-iso-PGF2α and 2,3-dinor-8-iso-PGF2 can be indexes of colitis-associated colorectal cancer in mice”. Comment 1-1. 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 <Response> We revised the manuscript and supplemental files according to the PLOS ONE style templates and they correctly meet PLOS ONE’s requirement. Comment 1-2 As part of your revisions, please provide additional details pertaining to your animal research procedures. Please revise your Methods to address the following points: (1) sample size: how many animals per group and how did you determine the numbers (power analysis? pilot study? previously published data/findings?); <Response> We added the descriptions in manuscript in line 89-91 for (1); “The number of mice were 13 in vehicle group and 8 – 26 in AOM/DSS administration group depending on the phase of day 5, 26, 47 and 60.” Comment 1-3 (2) provide complete information about all methods undertaken to minimize pain and distress of the animals in your work. (3) Please discuss your monitoring parameters (physical and behavioral signs to assess health and well-being), in addition to your humane endpoints (criteria used to determine when to euthanize animals in cases where animals become sick/moribund). (4) Please state the rate of mortality for animals who died unexpectedly (and state cause(s) of death). (6) Please also discuss supportive care that you provided to the animals. <Response> We used eight weeks old C57BL/6J male mice (20-22 g). Mice were housed under standard laboratory conditions (22±1°C, 12-hour light/dark cycles, food and water ad libitum). Animal care and handling procedures were in accordance with International Association for the Study of Pain (IASP) guidelines for the use of animals in pain research and the protocols for animal care and use were approved by the Institutional Animal Care and Use Committee at the University of Tokyo (P11-576 and P08-258). All effort was made to limit the number of animals used. We set humane endpoint to prevent or alleviate pain and/or distress when 20% body weight loss, disability in food intake and water drinking are observed. However, all the mice used in the current study did not exceed the criteria and did not die unexpectedly. We revised the manuscript as follows: Line 108 for (2); “Mice were properly euthanized by cervical dislocation.” Line 93 for (3); “These parameters were also used in previously published paper [19] and appropriate to assess animals’ health and well-being.” Line 173 for (4); “Almost all of the animals have not died unexpectedly.” Line xx for (6); Comment 1-4 (7) Lastly, please complete and submit the ARRIVE Guidelines checklist (Essential 10) with your revision: https://arriveguidelines.org/resources/author-checklists <Response> We filled in the ARRIVE Guidelines checklist. And we added the following descriptions in manuscript; “Seven- to nine-weeks old female wild type C57BL/6 mice were used.” In line 82; “No randomization was used to allocate mice to any groups and confounders were not controlled.” In line 88; “No criteria for including and excluding were set and no animals and data points were excluded.” in line 161. Reviewer #1: Comment 2-1 Miyazaki et al established a murine model of inflammation-induced colon cancer (CAC), and searched for novel lipid biomarkers in the urine of this model mouse. They identified contents of several metabolites derived from arachidonic acid are increased in the CAC urine. Some of them (PGD and E metabolites) are also increased in the inflammation period, and not candidates for biomarkers. They finally found that 8-iso-PGF2αand 2,3-dinor-8-iso-PGF2α are increased only in the CAC period, and concluded that these two PGF2 metabolites are candidate biomarkers for colorectal cancer. They also stained the mouse cancer tissues and showed that the expression of COX-2 and NADPH oxidase is increased in the cancer cells, and this can explain the increase of the metabolites. This is an interesting study that utilizes a mouse oncogenic model to find biomarkers. The biosynthesis and metabolic inactivation of arachidonic acid-derived lipid mediators are conserved among various animal species, this strategy is reasonable. This group has a long history of measuring PG and PG metabolites, and the manuscript is simply and clearly written. The reviewer, however, has to raise several important issues to be addressed in the revised manuscript. Major points The authors claim that 8-iso-PGF2alpha and 2,3-dinor-8-iso-PGF2 are possible biomarkers based on the specific increase of these metabolites in CAC phase. They did not succeed in convincing the reviewer that the colon cancer cells can generate and metabolize PGF2 alpha. Quantification of PGF2alpha and its metabolites in the cancer tissues, hopefully, lazar-dissected tissues, is required. If they think that cancer cells produce PGF2alpha, which enters into the blood and is metabolized in the other tissues, they should measure PGF2alpha and metabolites in the plasma. <Response> Thank you. 8-iso-PGF2� is an oxidized metabolite of arachidonic acid not PGF2��and 2,3-dinor-8-iso-PGF2� is a metabolite of 8-iso-PGF2�. As following your suggestion, we measured the concentrations of these two metabolites in plasma and polyps of AOM+DSS-treated mice (day 60). As shown in below Figure, the levels of 2, 3-dinor-8-iso PGF2α (left figure) in plasma and that of 8-iso-PGF2α (right figure) in colon tissues were unexpectedly comparable between control and CAC mice. The concentrations of 8-iso-PGF2α in all plasma samples and that of 2, 3-dinor-8-iso PGF2α in all colon samples were below the lower limit of quantification (LLOQ). From these results, we could not find the increase levels of F2-IsoPs in plasma and CAC region in this study. This may be because the local production of 8-iso-PGF2� by ROS-generating cells was too small to quantify in mice. Indeed, the levels of 8-iso-PGF2α in some colon tissues and polyp were below the LLOQ (n=1/4: control, n=3/8: inflamed colon, n=4/7: polyp). It needs more sensitive methods for measuring local F2-IsoPs levels. In other words, urine is more appropriate for detecting CAC biomarkers than plasma. We added these results as Figure 4E-F and added the descriptions in the material and method (line 137-146), results (line 255-264), and discussion section (line 298-303). Comment 2-2 Regarding the application to the human situation, they should measure these metabolites in the urine of human colon cancer patients and show some representative results. <Response> We attempted to obtain urine sample from patients in cooperating with Prof. Mamoru Watanabe, predecessor of Advanced Clinical Center for IBD in Tokyo Medical and Dental University Hospital in Japan. However, we could not obtain the data for this manuscript because it will take time to approve clinical studies and sampling. Please understand the situation. Comment 2-3 Statistic analyses are missing in Fig. 1E, F, and Fig. 2. <Response> We performed statistical analysis and added in Fig. 1E, F, and Fig.2. Attachments Attachment Submitted filename: 20201216 Response to reviewers.docx https://doi.org/10.1371/journal.pone.0245292.r002
26 Dec 2020 Decision Letter - Hiroyasu Nakano, Editor Urinary 8-iso PGF2α and 2,3-dinor-8-iso PGF2α can be indexes of colitis-associated colorectal cancer in mice PONE-D-20-25696R1 Dear Dr. Murata 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, Hiroyasu Nakano, 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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) ****** 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 https://doi.org/10.1371/journal.pone.0245292.r003
Formally Accepted
18 Jan 2021 Acceptance Letter - Hiroyasu Nakano, Editor PONE-D-20-25696R1 Urinary 8-iso PGF_2α and 2,3-dinor-8-iso PGF_2α can be indexes of colitis-associated colorectal cancer in mice Dear Dr. Murata: 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 Professor Hiroyasu Nakano Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0245292.r004

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