Critical evaluation of an autologous peripheral blood mononuclear cell-based humanized cancer model

Harinarayanan Janakiraman; Scott A. Becker; Alexandra Bradshaw; Mark P. Rubinstein; Ernest Ramsay Camp

doi:10.1371/journal.pone.0273076

Peer Review History

Original SubmissionOctober 30, 2021
14 Feb 2022 Decision Letter - Anilkumar Gopalakrishnapillai, Editor PONE-D-21-34701Critical evaluation of an autologous peripheral blood mononuclear cell-based humanized cancer modelPLOS ONE Dear Dr. Janakiraman, 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 Mar 31 2022 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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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. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. 3. Thank you for stating in your Funding Statement: Supported in part by the Translational Science Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313). Supported in part by the Biorepository & Tissue Analysis Shared Resource, Hollings Cancer Center, Medical University of South Carolina. Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. 4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Additional Editor Comments: I would like authors go through the comments of the reviewers and include all the responses in the revised manuscript. I specifically would like to see authors including an immunotherapy modality in this model as a proof of principle to validate the work. [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: The article entitled "Critical evaluation of an autologous peripheral blood mononuclear cell-based humanized cancer model". Authors have established an autologous humanized PDX model for reproducible immunotherapy investigation. They have aimed to determine the technical aspects required to establish an autologous humanized cancer model system and demonstrated the ability to successfully engraft patient PBMCs that infiltrate the xenograft. The paper is well-written. Authors have highlighted the potential of using PDX models to evaluate the interaction between the human immune system and a heterogeneous tumour model, in addition to the identification of the limitations of an autologous PBMC-based humanized PDX approach. The article falls within the scope of PLOS ONE and is recommended to be considered for publication. Reviewer #2: This is a well written manuscript describing the development of an autologous PBMC derived PDX models in two different types of solid tumors for preclinical evaluation of immunotherapy. Comparison between the patient and mouse model is a bonus. Major Use of an immunotherapy modality in this model at least as proof-of-principle is lacking. Minor Fig. 3F How can the % of both CD4 and CD8 T cells be close to 80%? It is confusing as these markers are mutually exclusive. Figures 1 and 2 should be put in the supplement. ****** 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. https://doi.org/10.1371/journal.pone.0273076.r001
Revision 1
8 Mar 2022 Author Response Editor Comments: 1. specifically would like to see authors including an immunotherapy modality in this model as a proof of principle to validate the work. We agree that it would be ideal to test an immunotherapy modality in our model to broaden our conclusions. In fact, this was the overarching goal as we stated in the introduction section. However, for the reasons outlined below, we concluded that a patient-derived autologous PBMC method for humanization may not be the optimal strategy to study cancer immunotherapy in a rigorous and reproducible manner. Thus, this manuscript is focused on our model development approach and the barriers that we encountered in optimizing experimental conditions. Our goal was to develop a patient-derived humanized cancer mouse model based on the widely cited reference (Sanmamed et al. Cancer Research, 2015) that used a single gastric cancer patient tumor and PBMCs to study the use of Nivolumab and Urelumab. In retrospect, this elegant experiment only reported using a single patient sample and we could not find that it was repeated with the same patient samples or with another patient tissue samples. Our attempt to reproduce the published humanized model for immunotherapy testing with both colorectal cancer and pancreatic cancer tissue samples identified critical barriers that challenged the feasibility of the approach including: • Early evidence of xGVHD in the xenograft prior to clinical xGVHD in the mouse. • Significant intra- and inter model immune cell engraftment variability. These unexpected observations ultimately led us to conclude that a PBMC-based humanized model would not result in a reproducible model system that reflects the human condition for testing cancer immunotherapy rigorously. The degree of variability was particularly concerning and challenged the feasibility of the model for immunotherapy testing. In addition, the limited PBMC supply obtained from cancer patients decreased our ability to repeatedly test models. To further emphasize the valuable points our investigations uncovered, we have revised the manuscript. We have now emphasized these barriers in the manuscript conclusions. On page 24 line 373, we revised the manuscript as “We ultimately concluded that the intra- and inter-model variability is a critical limitation towards developing a reproducible experimental system that can rigorously test immunotherapy strategies. Using larger sample sizes and selecting mouse models based on engraftment might be an option to overcome the variability.” In the abstract, we revised the final statements as “In summary, the iPDX models can reproduce key features of the corresponding human tumor. However, the observed variability in T cell engraftment and high PD-1 expression are important considerations that need to be addressed in order to develop a reproducible model system.” While we were not able to achieve the original goal of our studies with the resources available for this effort, we believe that our manuscript will be very informative to future investigators who attempt a patient-derived autologous PBMC-humanized approach. On the surface, the model system is very appealing due to the relative ease of obtaining the needed patient samples. The issues we outline can provide future investigators with a realistic appreciation of the limitations and we expect the manuscript will serve as a vital reference for future investigators seeking to develop humanized mouse models for assessing immunotherapy. Reviewer’s comments and response. Reviewer #1: Thank you for the comments that you believe our paper is “well written” and that you recommend for publication. Reviewer #2 1. Use of an immunotherapy modality in this model at least as proof-of-principle is lacking. Please refer to our comments to the Editor above. We completely agree and we were sincerely disappointed in the significant limitations that we encountered that were previously not reported or discussed. We hope that you appreciate our effort to reproducibly create an effective model and that our rigorous testing demonstrated critical limitations that we decided were important to report in order to inform future investigators. 2. Fig. 3F How can the % of both CD4 and CD8 T cells be close to 80%? It is confusing as these markers are mutually exclusive. Thank you for your comment. We intended to demonstrate the % of cell populations that were PD-1 positive in the xenografts. The total CD4 and CD8 T cell populations are demonstrated in 3D. To make figure 3F less confusing, we have bolded and enlarged the Y-axis title to make this clearer that it is only the %PD-1 positive cell populations in the analysis. 3. Figures 1 and 2 should be put in the supplement. We agree and now have moved these figures to the supplemental section and revised the figure legend accordingly. Other requirements: 4. We have revised the Methods section to now include (1) methods of sacrifice (line 126), (2) methods of anesthesia and/or analgesia (line 130), and (3) efforts to alleviate suffering (line 137-138). 5. Funding statement has been updated and now includes the statement “There was no additional external funding received for this study.” 6. Funding Information’ and ‘Financial Disclosure’ sections now match. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0273076.r002
15 Jul 2022 Decision Letter - Anilkumar Gopalakrishnapillai, Editor PONE-D-21-34701R1 Critical evaluation of an autologous peripheral blood mononuclear cell-based humanized cancer model PLOS ONE Dear Dr. Janakiraman, 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 within 30 days. 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. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Anilkumar Gopalakrishnapillai Academic Editor PLOS ONE Additional Editor Comments: One of the major comments of the reviewer was to test the feasibility of this model for immunotherapy purpose. Please address this major comment along with others. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: [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.0273076.r003
Revision 2
25 Jul 2022 Author Response Editor Comments: 1. specifically would like to see authors including an immunotherapy modality in this model as a proof of principle to validate the work. We agree that it would be ideal to test an immunotherapy modality in our model to broaden our conclusions. In fact, this was the overarching goal as we stated in the introduction section. However, for the reasons outlined below, we concluded that a patient-derived autologous PBMC method for humanization may not be the optimal strategy to study cancer immunotherapy in a rigorous and reproducible manner. Thus, this manuscript is focused on our model development approach and the barriers that we encountered in optimizing experimental conditions. Our goal was to develop a patient-derived humanized cancer mouse model based on the widely cited reference (Sanmamed et al. Cancer Research, 2015) that used a single gastric cancer patient tumor and PBMCs to study the use of Nivolumab and Urelumab. In retrospect, this elegant experiment only reported using a single patient sample and we could not find that it was repeated with the same patient samples or with another patient tissue samples. Our attempt to reproduce the published humanized model for immunotherapy testing with both colorectal cancer and pancreatic cancer tissue samples identified critical barriers that challenged the feasibility of the approach including: • Early evidence of xGVHD in the xenograft prior to clinical xGVHD in the mouse. • Significant intra- and inter model immune cell engraftment variability. These unexpected observations ultimately led us to conclude that a PBMC-based humanized model would not result in a reproducible model system that reflects the human condition for testing cancer immunotherapy rigorously. The degree of variability was particularly concerning and challenged the feasibility of the model for immunotherapy testing. In addition, the limited PBMC supply obtained from cancer patients decreased our ability to repeatedly test models. To further emphasize the valuable points our investigations uncovered, we have revised the manuscript. We have now emphasized these barriers in the manuscript conclusions. On page 24 line 373, we revised the manuscript as “We ultimately concluded that the intra- and inter-model variability is a critical limitation towards developing a reproducible experimental system that can rigorously test immunotherapy strategies. Using larger sample sizes and selecting mouse models based on engraftment might be an option to overcome the variability.” In the abstract, we revised the final statements as “In summary, the iPDX models can reproduce key features of the corresponding human tumor. However, the observed variability in T cell engraftment and high PD-1 expression are important considerations that need to be addressed in order to develop a reproducible model system.” While we were not able to achieve the original goal of our studies with the resources available for this effort, we believe that our manuscript will be very informative to future investigators who attempt a patient-derived autologous PBMC-humanized approach. On the surface, the model system is very appealing due to the relative ease of obtaining the needed patient samples. The issues we outline can provide future investigators with a realistic appreciation of the limitations and we expect the manuscript will serve as a vital reference for future investigators seeking to develop humanized mouse models for assessing immunotherapy. Reviewer’s comments and response. Reviewer #1: Thank you for the comments that you believe our paper is “well written” and that you recommend for publication. Reviewer #2 1. Use of an immunotherapy modality in this model at least as proof-of-principle is lacking. Please refer to our comments to the Editor above. We completely agree and we were sincerely disappointed in the significant limitations that we encountered that were previously not reported or discussed. We hope that you appreciate our effort to reproducibly create an effective model and that our rigorous testing demonstrated critical limitations that we decided were important to report in order to inform future investigators. 2. Fig. 3F How can the % of both CD4 and CD8 T cells be close to 80%? It is confusing as these markers are mutually exclusive. Thank you for your comment. We intended to demonstrate the % of cell populations that were PD-1 positive in the xenografts. The total CD4 and CD8 T cell populations are demonstrated in 3D. To make figure 3F less confusing, we have bolded and enlarged the Y-axis title to make this clearer that it is only the %PD-1 positive cell populations in the analysis. 3. Figures 1 and 2 should be put in the supplement. We agree and now have moved these figures to the supplemental section and revised the figure legend accordingly. Other requirements: 4. We have revised the Methods section to now include (1) methods of sacrifice (line 126), (2) methods of anesthesia and/or analgesia (line 130), and (3) efforts to alleviate suffering (line 137-138). 5. Funding statement has been updated and now includes the statement “There was no additional external funding received for this study.” 6. Funding Information’ and ‘Financial Disclosure’ sections now match. Overall, we are grateful for the possible opportunity to publish our manuscript in PLOS ONE. We believe that our critical evaluation of a PBMC humanized cancer model system will provide important reference points for future investigators. Please do not hesitate to contact me should any questions arise. Thank you for your consideration in this matter. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0273076.r004
3 Aug 2022 Decision Letter - Anilkumar Gopalakrishnapillai, Editor Critical evaluation of an autologous peripheral blood mononuclear cell-based humanized cancer model PONE-D-21-34701R2 Dear Dr. Janakiraman, 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, Anilkumar Gopalakrishnapillai Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0273076.r005
Formally Accepted
2 Sep 2022 Acceptance Letter - Anilkumar Gopalakrishnapillai, Editor PONE-D-21-34701R2 Critical evaluation of an autologous peripheral blood mononuclear cell-based humanized cancer model Dear Dr. Janakiraman: 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. Anilkumar Gopalakrishnapillai Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0273076.r006

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