Cancer stem cell-derived extracellular vesicles preferentially target MHC-II–macrophages and PD1+ T cells in the tumor microenvironment

Patricia Gonzalez-Callejo; Zihan Guo; Tahereh Ziglari; Natalie Marcia Claudio; Kayla Hoang Nguyen; Naoki Oshimori; Joaquim Seras-Franzoso; Ferdinando Pucci

doi:10.1371/journal.pone.0279400

Peer Review History

Original SubmissionJuly 28, 2022
28 Jul 2022 Author Response Attachments Attachment Submitted filename: 126756_1_rebuttal_2409020_r7x756.pdf https://doi.org/10.1371/journal.pone.0279400.r001
17 Aug 2022 Decision Letter - Daotai Nie, Editor PONE-D-22-21278Cancer stem cell-derived extracellular vesicles preferentially target MHC-II– macrophages and PD1+ T cells in the tumor microenvironmentPLOS ONE Dear Dr. Pucci, 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 address the comments from the reviewer, as well as the comment from the editor listed below:1. Figure 4Aii, immune cells are depicted in the schematic presentation, but not actually in the cartoon presented. Please double check. Please submit your revised manuscript by October 1, 2022. 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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Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 5. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical. [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 ******** 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: This work conducted by the Pucci Group suggests that EVs from different cell-of-origins within tumors, CSC defined by ALDH1A1 or SRE in this case, contribute to the heterogenicity of immune TME. The observation is interesting, the technical approaches are novel, the manuscript is well-written, and the information about experimental methods are adequate. However, addressing some points can strengthen this study and further support the conclusions. 1. The definition of CSCs by single or two markers remains controversial. Are ALDH1A+ or SRE positive cells accountable for the "cancer sternness" in HNSCC, or simply reflecting a specific and small fractions of heterogenous CSCs? That being said, indicating the orosphere forming efficiency of sorted ALDH1A1:CD63-eGFP+ mEER cells (Fig 1G) can partially answer the question. 2. The measurement of the distance between tumor cell-TMA in Fig 3 should be further clarified. How many samples were included in the experiments, and whether the assay was conduced in a double-blind manner? In addition, are ALDA1A1+ cells in the tumors derived from total tumor population (Fig 3A) closer to TAMs? A co-staining method can help to address this. This should be important since the interactions could be altered within a heterogenous intratumoral environment. 3. It should be clarified whether the attraction of TAMs or other immune cells are directly caused by EVs. More specifically, does neutralizing CD63 by antibody block the recruitment of TMAs to the peripheral or intratumoral areas? ****** 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.0279400.r002
Revision 1
5 Oct 2022 Author Response We would like to thank the Editor and the Reviewer for taking the time to read our work and to appreciate its value to the CSC and EV community. Please find below in navy font a point-by-point response to their comments. Editor: Figure 4Aii, immune cells are depicted in the schematic presentation, but not actually in the cartoon presented. Please double check. We agree that the schematic was misleading by not showing unlabeled immune cells (purple), that we now added along with labeled immune cells (red) within the CSC nice and immune cells labeled by total tumor cells EVs (green). Reviewer: The definition of CSCs by single or two markers remains controversial. Are ALDH1A+ or SRE positive cells accountable for the "cancer sternness" in HNSCC, or simply reflecting a specific and small fractions of heterogenous CSCs? That being said, indicating the orosphere forming efficiency of sorted ALDH1A1:CD63-eGFP+ mEER cells (Fig 1G) can partially answer the question. We appreciate the Reviewer’s request to add the orosphere forming efficiency, which is an important parameter for relating our work to others in the literature. We have now added the assessed orosphere forming efficiency (Fig. 1G), which is 0.66%. The measurement of the distance between tumor cell-TMA in Fig 3 should be further clarified. How many samples were included in the experiments, and whether the assay was conduced in a double-blind manner? Thank you for noticing this missing detail in the methods. We have added that the distance measurement was made blindly by 3 independent observers. This distance was quantitatively assessed in 5 random tumor sections per mice group. These details have now been included in the methods section. Representative images were displayed in Figure 3. In addition, are ALDA1A1+ cells in the tumors derived from total tumor population (Fig 3A) closer to TAMs? A co-staining method can help to address this. This should be important since the interactions could be altered within a heterogenous intratumoral environment. We appreciate this Reviewer’s knowledge of the controversial issues present in the field of cancer stemness. From the Reviewer’s comment, we understand that the concern is whether our reporters label the majority the heterogeneous CSC populations or a specific subset. We have now performed the suggested experiment and co-stained with Sox2, a major CSC reporter in squamous carcinoma (PMID: 27634934). We observed a major co-localization between our reporters and Sox2 (Fig. S3E), which indicates that we effectively tagged heterogeneous CSC subpopulations. These results, the orosphere formation efficiency (Fig. 1G) and the fact that the ALDH1A1:eGFP+ and SRE:eGFP+ CSC populations express other stemness markers (Fig. 1 and S3), together indicate that our conclusions may be broadly applicable to most CSC subsets. It should be clarified whether the attraction of TAMs or other immune cells are directly caused by EVs. More specifically, does neutralizing CD63 by antibody block the recruitment of TMAs to the peripheral or intratumoral areas? We agree with the Reviewer that understanding the mechanism of immune cell recruitment into the CSC niche is of interest. However, this goes beyond the scope of the manuscript under consideration, which is mainly focused on the characterization of the composition of the CSC niche in vivo. In addition, anti-CD63 antibodies have not been shown to deplete EVs, and likely will have the opposite effect on macrophages since opsonizing EVs with antibodies will increase their binding to macrophage Fc receptors. We hope the Reviewer will understand that answering this important question will be the focus of future work. Attachments Attachment Submitted filename: Response to Reviewers280922.docx https://doi.org/10.1371/journal.pone.0279400.r003
6 Dec 2022 Decision Letter - Daotai Nie, Editor Cancer stem cell-derived extracellular vesicles preferentially target MHC-II– macrophages and PD1+ T cells in the tumor microenvironment PONE-D-22-21278R1 Dear Dr. Pucci, 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, Daotai Nie, 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: All concerns have been adequately addressed, and the manuscript is well written. I recommend to accept and publish this study. ****** 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.0279400.r004
Formally Accepted
24 Jan 2023 Acceptance Letter - Daotai Nie, Editor PONE-D-22-21278R1 Cancer stem cell-derived extracellular vesicles preferentially target MHC-II– macrophages and PD1+ T cells in the tumor microenvironment Dear Dr. Pucci: 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. Daotai Nie Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0279400.r005

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