Correlation analysis of the proportion of monocytic myeloid-derived suppressor cells in colorectal cancer patients

Kenna Shirasuna; Masayuki Ito; Takashi Matsuda; Tsuyoshi Enomoto; Yusuke Ohara; Masayoshi Yamamoto; Satomi Nishijima; Nobuhiro Ohkohchi; Sadao Kuromitsu

doi:10.1371/journal.pone.0243643

Peer Review History

Original SubmissionMay 9, 2020
13 Aug 2020 Decision Letter - Yinyan He, Editor PONE-D-20-13755 Correlation analysis for the proportion of monocytic myeloid-derived suppressor cells in colorectal cancer patients PLOS ONE Dear Dr. Shirasuna, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Sep 27 2020 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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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: The topic is of clinical interest, since MDSCs have been shown to promote tumor growth in CRC and these cells are difficult to distinguish from normal myeloid cells. The main question is whether the authors’ assertion that iNOS and PDGF are reliable surrogate markers for m-MDSC frequency in the blood of CRC patients is valid. In the absence of any evidence of cause and effect, the results are purely correlational, and there are several potential reasons why the results may be misleading. 1. iNOS and PDGF might have nothing to do with mMDSC frequency, as there are many factors in blood that are different between healthy individuals and CRC patients. In their statistical analysis, have the authors applied the Bonferroni correction for multiple correlations to account for the sheer number of attempted correlations? 2. Can the authors demonstrate that the correlation does not hold true in CRC patients who have a low frequency of m-MDSC (comparable to healthy donors)? Apart from these major concerns, the ex vivo T cell suppression assay should be performed as a dose titration, with varying numbers of MDSCs added to the co-culture. It is difficult to interpret the data without such a titration. Also, flow plots for IFN-g secretion need to be shown ****** 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. 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Revision 1
28 Sep 2020 Author Response Dear Dr. He: Thank you for your email dated August 14, 2020 with your kind invitation to submit a revised version of our manuscript, “Correlation analysis of the proportion of monocytic myeloid-derived suppressor cells in colorectal cancer patients,” for further review. We have carefully reviewed the comments and have revised the manuscript accordingly. Our point-by-point responses to these comments start on the next page. We hope that the revised manuscript is suitable for publication in PLOS ONE and look forward to hearing from you in due course. Journal 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 Response: We followed the style template. 2. Thank you for including your competig interests statement; " The authors have declared that no competing interests exist" We note that one or more of the authors are employed by a commercial company: “Astellas Pharma, Inc.” 1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Response: We included the following statement within our amended Funding Statement: The authors declare that, other than income received from our primary employers, no financial support or compensation has been received for this research and that there are no personal conflicts of interest to declare. This research did not receive any specific grant from funding agencies in the public, commercial, or not for profit sectors. The funding organization (Astellas Pharma, Inc.) did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of author’s salaries and/or research materials. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. Response: We included the following statement within our amended Funding Statement: The funder provided support in the form of salaries for authors (KS, MI, TM, SN and SK), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. 2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Response: We included the following statement within our amended Competing Interests Statement: The authors declare no competing financial interests. This commercial affiliation does not alter our adherence to all PLOS ONE policies on sharing data and materials. Review Comments to the Author: Reviewer #1: The topic is of clinical interest, since MDSCs have been shown to promote tumor growth in CRC and these cells are difficult to distinguish from normal myeloid cells. The main question is whether the authors’ assertion that iNOS and PDGF are reliable surrogate markers for m-MDSC frequency in the blood of CRC patients is valid. In the absence of any evidence of cause and effect, the results are purely correlational, and there are several potential reasons why the results may be misleading. 1. iNOS and PDGF might have nothing to do with mMDSC frequency, as there are many factors in blood that are different between healthy individuals and CRC patients. In their statistical analysis, have the authors applied the Bonferroni correction for multiple correlations to account for the sheer number of attempted correlations? Response: We thank the reviewer for the helpful suggestion. The variables for the regression model were selected using forward and backward stepwise feature selection method from plasma protein measurements and demographic factors (i.e., sex and age). This feature selection method applies AIC (Akaike Information Criterion) statistics as criteria to select features/variables and does not use any statistical hypothesis test like Welch's t-test. That is, we did not select features/variables based on multiple statistical hypothesis testing of each measurement. Therefore, our opinion is that it is not necessary to apply a multiple comparison correction like the Bonferroni correction for this selection. We are sorry that this part was not clear in the original manuscript. We should have explained that our feature selection method applied AIC statistics. We added the description to the Results section as follows: Revised (p.14, line 243-246): Next, we used multivariate methodology. A multivariate linear regression model combining forward and backward feature selection method based on AIC (Akaike Information Criterion) for mMDSC proportion was applied, considering all variables such as age, sex, and 15 plasma proteins in CRC patients. 2. Can the authors demonstrate that the correlation does not hold true in CRC patients who have a low frequency of m-MDSC (comparable to healthy donors)? Response: We thank the reviewer for the suggestion. We did not mean that there is no correlation in CRC patients with a low proportion of mMDSCs. Fig5A shows the relationship between predicted values (horizontal axis) and measured values (vertical axis) in the proportion of mMDSC in CRC patients. This figure reveals a trend: the lower the level of measured value, the lower is the predicted value. However, it would be difficult to present data only for CRC patients with a low frequency of mMDSCs in a statistically meaningful way due to the small number of samples. Reviewer #1: Apart from these major concerns, the ex vivo T cell suppression assay should be performed as a dose titration, with varying numbers of MDSCs added to the co-culture. It is difficult to interpret the data without such a titration. Response: We thank the reviewer for the helpful suggestion. We agree with you and have incorporated this suggestion throughout our paper. We have conducted in vitro suppression assays with titrated numbers of mMDSCs from both CRC patients (n = 3) and healthy donors (n = 3). When autologous mMDSCs were added in a mMDSC:CD14− cell ratio of 0.25:1, 0.5:1 and 1:1, IFN-γ production of CD14− cells was inhibited by mMDSCs at a ratio of 1:1 and 0.5:1, and the loss of IFN-γ suppressive activity was observed as mMDSCs were titrated down in CRC patients. We also found that mMDSCs showed the similar suppressive activity of IFN-γ production in healthy donors compared with those in CRC patients. The data are shown in Fig. 4A of the revised manuscript. Revised Fig4A Revised (p.7, line 134-135) mMDSC, CD14− cell, and T cell isolation for in vitro mMDSC functional assay Revised (p.8, line 142-143) CD14− cells were isolated from PBMCs using CD14 selection Micro beads in parallel with CD14+ cell isolation as above. Revised (p.8, line 149-p.9, line 157) Autologous mMDSC subsets were added at different ratios to CD14− cells (5 × 104 cells/well) in 96-well flat bottom plates (Iwaki, Tokyo, Japan) in RPMI 1640 media containing 10% fetal calf serum. Cells were incubated with anti-CD2/anti-CD3/anti-CD28 antibody conjugated beads (Miltenyi Biotech) at a 1:1 CD14− to bead ratio. Plates were incubated at 37°C in a humidified 5% CO2 incubator for 5 d. After culture, supernatants were collected and IFN-γ concentration was measured using a human IFN-γ AlphaLISA Detection Kit (Perkin Elmer, Waltham, MA, USA). For further study, autologous mMDSC subsets (5 × 104 cells/well) were added in a mMDSC:T cell ratio of 1:1 in 96-well flat bottom plates (Iwaki, Tokyo, Japan) in RPMI 1640 media containing 10% fetal calf serum. Revised (p.11, line 201-p.12, line 215) Comparison of in vitro suppressive function of mMDSCs from CRC patients with those from healthy donors To confirm that peripheral blood mMDSCs from CRC patients and healthy donors suppressed T-cell activation, we isolated mMDSCs, autologous CD14− cells, and pan-T cells from PBMCs and co-cultured under stimulation with anti-CD2/anti-CD3/anti-CD28 antibody conjugated beads for 5 d. Then we assessed the suppressive function of mMDSCs in vitro. For mMDSC titration assay (CRC patients: n = 3, healthy donors: n = 3), when autologous mMDSCs were added in a mMDSC:CD14− cell ratio of 0.25:1, 0.5:1 and 1:1, IFN-γ production of CD14− cells was inhibited by mMDSCs at a ratio of 1:1 and 0.5:1, and the loss of IFN-γ suppressive activity was observed as mMDSCs were titrated down in both CRC patients and healthy donors (Fig. 4A). For further assay (CRC patients: n = 9, healthy donors: n = 5), when autologous mMDSCs were co-cultured with pan-T cells at a ratio of 1:1 (mMDSCs:pan-T cells), IFN-γ production of pan-T-cells was decreased in 4 out of 5 healthy donors and 8 out of 9 CRC patients, confirming mMDSCs’ suppressive function irrespective of disease state (Fig. 4B and Table 2). Figure 4. In vitro suppressive activity of mMDSCs Revised (p.16, line 279-285) We first isolated CD14+ cells by magnetic sorting and then HLA-DR−/low cells by flow cytometry and used these isolated cells as mMDSCs for in vitro co-culture assay with autologous CD14− cells or T cells isolated separately using magnetic sorting. As a result, mMDSC-mediated suppressive activity of IFN-γ production of CD14− cells increased with increasing numbers of mMDSCs in CRC patients. INF-γ production by T cells was reduced by mMDSCs in 8 out of 9 CRC patients at a 1:1 ratio. Interestingly, we found that mMDSCs showed similar suppressive activity of IFN-γ production in healthy donors. Revised (p.20, line 349-352) Figure 4. In vitro suppressive activity of mMDSCs (A) CD14− cells or (B) T cells from healthy donors and CRC patients were stimulated with anti-CD2/anti-CD3/anti-CD28 antibody conjugated beads in the absence or presence of autologous mMDSCs. Culture supernatant was collected at 5 d to measure IFN-γ concentration. Reviewer #1: Also, flow plots for IFN-g secretion need to be shown Response: We thank the reviewer for the helpful suggestion. We understand that monitoring IFN-γ secretion by flow cytometry is one of the methods in suppression assay. However, in our study, we only measured IFN-γ production by ELISA (AlphaLISA Detection Kit, Perkin Elmer, Waltham, MA, USA). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0243643.r002
25 Nov 2020 Decision Letter - Lucienne Chatenoud, Editor Correlation analysis of the proportion of monocytic myeloid-derived suppressor cells in colorectal cancer patients PONE-D-20-13755R1 Dear Dr. Shirasuna, We’re pleased to inform you that the revised version of 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. Yours Sincerely, Lucienne Chatenoud 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.0243643.r003
Formally Accepted
27 Nov 2020 Acceptance Letter - Lucienne Chatenoud, Editor PONE-D-20-13755R1 Correlation analysis of the proportion of monocytic myeloid-derived suppressor cells in colorectal cancer patients Dear Dr. Shirasuna: 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 Lucienne Chatenoud Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0243643.r004

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