Unveiling immune cell response disparities in human primary cancer-associated fibroblasts between two- and three-dimensional cultures

Jian-Ping Yang; Nikhil Nitin Kulkarni; Masashi Yamaji; Tsubasa Shiraishi; Thang Pham; Han Do; Nicole Aiello; Michael Shaw; Toshihiro Nakamura; Akiko Abiru; Narender R. Gavva; Shane R. Horman

doi:10.1371/journal.pone.0314227

Peer Review History

Original SubmissionAugust 22, 2024
18 Sep 2024 Decision Letter - Zhiming Li, Editor PONE-D-24-36264Unveiling Immune Cell Response Disparities in Human Primary Cancer-Associated Fibroblasts between Two- and Three-Dimensional CulturesPLOS ONE Dear Dr. Yang, Thank you for submitting your manuscript to PLOS ONE, which has been evaluated by two independent reviewers. Both of the referees are interested in the presented study, but also raised some concerns that should be addressed, including revising the introduction and discussion sections, and addtional experiments. 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 Nov 02 2024 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. 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If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. [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 Reviewer #2: Yes ******** 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: Yang et al has tried to demonstrate the disparities in human primary cancer associated fibroblast by using 2D and 3D culture. The study design is good but there are some lacks in proper introduction or rational for the study. Authors are suggested to revise the manuscript after incorporated following comments. 1. Studies conducted with 2D and 3D culture of fibroblast cells have been performed using various cancer models, it has been known for a ling time. Authors should highlight the strength of their study over previously published studies. Authors should mention these studies in the introduction section, At line number 82 which can give a broad idea for the readers. a. PMID: 24124550 b. PMID: 34102862 2. The table 1 and 2 can be added into the supplementary data. 3. The lines 258-262 of the result section can be added in the method/introductory section. 4. Authors have focused the co-culture experiment using pancreatic CAF cells, is there any reason to choose these cells? If yes, then authors are suggested to mention the rational in the introduction section with proper citations. 5. The figure 4H shows the high expression of TNFRSF21 in both 2D and 3D culture which is a pro-inflammatory molecule and enhances the NF-kBeta signaling. What does authors comment on this particular result? 6. Previously conducted studies have also addressed the role of M1 and M2 macrophages and their activation, authors are suggested to cite the following publications at line 415. a. PMID: 36769180 b. PMID: 38461312 7. Authors should briefly highlight the pros and cons of fibroblast 2D and 3D culture from previous conducted cancer model studies in discussion section. Reviewer #2: This study examines how 2D and 3D culturing conditions affect cancer-associated fibroblasts (CAFs) in a tumor microenvironment (TME). In 2D cultures, CAFs displayed a myofibroblast (myCAF) subtype, while in 3D tumor spheroids, they shifted to a more inflammatory (iCAF) state. Using RNA sequencing and proteomics, researchers found that 3D CAFs enhanced tumor growth and shielded cancer cells from natural killer (NK) cell attacks. Additionally, 3D CAF-secreted proteins promoted monocyte migration and differentiation into immunosuppressive macrophages. The findings suggest that 3D TME models offer a more clinically relevant system for drug discovery in cancer treatment. To make this manuscript more informative, please consider to perform following experiments. For example, characteristic changes in CAFs in 3D culture need to be assessed in in vivo setting, using PDAC model mouse. Cytokine production, immune cell infiltration and their characteristics must be evaluated. In addition, authors are encouraged to confirm these findings also in human PDAC samples. For 3D culture, authors should use adequate scaffold such as collagen and other ECM to recapitulate cancer stroma. These settings will further reinforce authors’ claim. Above mentioned experiments seem indispensable to make this manuscript suitable for publication. Please consider to perform these experiments accordingly. ****** 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: Yes: NAMRATA ANAND Reviewer #2: Yes: Shin Hamada ******** [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. 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Revision 1
27 Oct 2024 Author Response We are grateful for your and the reviewers’ comments, and the positive evaluation of our work. The followings are our response to the reviewers’ comments: Reviewer #1: Yang et al has tried to demonstrate the disparities in human primary cancer associated fibroblast by using 2D and 3D culture. The study design is good but there are some lacks in proper introduction or rational for the study. Authors are suggested to revise the manuscript after incorporated following comments. 1. Studies conducted with 2D and 3D culture of fibroblast cells have been performed using various cancer models, it has been known for a ling time. Authors should highlight the strength of their study over previously published studies. Authors should mention these studies in the introduction section, At line number 82 which can give a broad idea for the readers. a. PMID: 24124550 b. PMID: 34102862 We thank the reviewer for the additional references and have cited the two papers at line number 82. 2. The table 1 and 2 can be added into the supplementary data. We agree with the reviewer and table 1 and 2 have been moved to the supplementary data. 3. The lines 258-262 of the result section can be added in the method/introductory section. We thank the reviewer for this suggestion but respectfully disagree and feel that these statements are best left in the results section as the first two sentences are a transition into why we used the ensuing methodology. 4. Authors have focused the co-culture experiment using pancreatic CAF cells, is there any reason to choose these cells? If yes, then authors are suggested to mention the rational in the introduction section with proper citations. We thank the reviewer for pointing out this important point of the research so we have added the following statement:“The microenvironment of pancreatic ductal adenocarcinoma (PDAC) is distinguished by a dense, fibrotic stroma, with cancer-associated fibroblasts (CAFs) serving as pivotal contributors” into line 259 as the rational for this study and the corresponding reference PMID: 36358721 is cited accordingly. 5. The figure 4H shows the high expression of TNFRSF21 in both 2D and 3D culture which is a pro-inflammatory molecule and enhances the NF-kBeta signaling. What does authors comment on this particular result? The authors thank the reviewer for pointing out this interesting observation. We added “On the other hand, the expression of a pro-inflammatory molecule TNFRSF21 are considerable high in both 2D and 3D culture.” in line 368, although the indication may require further investigation. 6. Previously conducted studies have also addressed the role of M1 and M2 macrophages and their activation, authors are suggested to cite the following publications at line 415. a. PMID: 36769180 b. PMID: 38461312 We thank the reviewer for pointing out these two important references and have cited them at line 415. 7. Authors should briefly highlight the pros and cons of fibroblast 2D and 3D culture from previous conducted cancer model studies in discussion section. We thank the reviewer of pointing out this very important point that requires further clarification and referencing so we have added “Numerous studies have highlighted the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models.” in the discussion section of line 458 and added two supporting references. Reviewer #2: This study examines how 2D and 3D culturing conditions affect cancer-associated fibroblasts (CAFs) in a tumor microenvironment (TME). In 2D cultures, CAFs displayed a myofibroblast (myCAF) subtype, while in 3D tumor spheroids, they shifted to a more inflammatory (iCAF) state. Using RNA sequencing and proteomics, researchers found that 3D CAFs enhanced tumor growth and shielded cancer cells from natural killer (NK) cell attacks. Additionally, 3D CAF-secreted proteins promoted monocyte migration and differentiation into immunosuppressive macrophages. The findings suggest that 3D TME models offer a more clinically relevant system for drug discovery in cancer treatment. To make this manuscript more informative, please consider to perform following experiments. For example, characteristic changes in CAFs in 3D culture need to be assessed in in vivo setting, using PDAC model mouse. Cytokine production, immune cell infiltration and their characteristics must be evaluated. In addition, authors are encouraged to confirm these findings also in human PDAC samples. For 3D culture, authors should use adequate scaffold such as collagen and other ECM to recapitulate cancer stroma. These settings will further reinforce authors’ claim. Above mentioned experiments seem indispensable to make this manuscript suitable for publication. Please consider to perform these experiments accordingly. The authors thank the reviewer for pointing out two important points of our research and for the suggested experiments to address those points. Our current manuscript focuses on comparison of two common in vitro methods for recapitulating human CAF tumor biology. The models proposed here are engineered to mimic human-specific tumor biology, which may not be represented in murine subcutaneous tumor models. Therefore, validating our findings through mouse models would not support our point of clinical translatability. That is the reason we have chosen to use clinically-derived snRNAseq data sets to support our findings. Given the new FDA 2.0 act which does not require rodent models for advancing clinical therapeutics, we feel the human tumor models proposed here are particularly relevant. Regarding the use of collagen or other type of ECM in our 3D models, the authors appreciate the suggestion. Typically, exogeneous ECM is incorporated into 3D tumor models that require these reagents to “stick” the various cell types together. Given that BxPC3 cells are a secretory ductal cell type, our 3D PDAC models did not require addition of exogeneous ECM. Further, snRNAseq data from the CAF cells show important ECM components are additionally secreted from the primary CAFs as well, consistent with clinical CAF function. Regarding the use of primary PDAC samples, we used BxPC3 human PDAC cells in our study due to their desirable oncogenic mutational profile of Braf, TP53 and ease of 3D culture. We were unable to locate a primary PDAC sample with a similar mutational profile. Additionally, we felt the incorporation of both primary human CAFs and PBMCs into our models offered a translational nature to our tumor model irrespective of tumor cell origin. Having said that, we agree with the reviewer that future studies incorporating primary patient-derived PDAC tumor samples may reveal novel tumor cell – CAF interactions. 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. Yes, the authors wish to publish the peer review history of our article. We again thank the reviewers for their thoughtful edits and suggestions and have revised and modified the text according to reviewers’critiques. We believe these changes have improved the manuscript considerably and we hope it can be published without delay. Sincerely， Jian-Ping Yang, PhD Attachments Attachment Submitted filename: Response to Reviewers_Final_1.docx https://doi.org/10.1371/journal.pone.0314227.r002
7 Nov 2024 Decision Letter - Zhiming Li, Editor Unveiling Immune Cell Response Disparities in Human Primary Cancer-Associated Fibroblasts between Two- and Three-Dimensional Cultures PONE-D-24-36264R1 Dear Dr. Yang, Thank you for the efforts in revising your manuscript. 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Zhiming Li, 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: Yes: NAMRATA ANAND ******** https://doi.org/10.1371/journal.pone.0314227.r003
Formally Accepted
8 Dec 2024 Acceptance Letter - Zhiming Li, Editor PONE-D-24-36264R1 PLOS ONE Dear Dr. Yang, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Zhiming Li Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0314227.r004

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