Near-infrared photoimmunotherapy is effective treatment for colorectal cancer in orthotopic nude-mouse models

Hannah M. Hollandsworth; Siamak Amirfakhri; Filemoni Filemoni; Justin Molnar; Robert M. Hoffman; Paul Yazaki; Michael Bouvet

doi:10.1371/journal.pone.0234643

Peer Review History

Original SubmissionJanuary 31, 2020
19 Mar 2020 Decision Letter - Irina V. Lebedeva, Editor PONE-D-20-02950 Near-Infrared Photoimmunotherapy is Effective Treatment for Colorectal Cancer in Orthotopic Nude-Mouse Models PLOS ONE Dear Dr Bouvet, 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. Quantification of fluorescent intensity and tumor size need to be provided. Also histology images should be provided. What is the rationale for the cell line used? Have experiments been done with other cell lines? What is the rationale for treatment time? The limitations should be discussed for the mouse model and for technology application. Are there any considerations for the mechanism(s) involved? We would appreciate receiving your revised manuscript by May 03 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Irina V. Lebedeva, Ph.D. Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 3. We note that you have a patent relating to material pertinent to this article. Please provide an amended statement of Competing Interests to declare this patent (with details including name and number), along with any other relevant declarations relating to employment, consultancy, patents, products in development or modified products etc. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” If 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. * This information should be included in your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following in the Financial Disclosure section: "This study was funded by VA Merit Review grant numbers 1 I01 BX003856-01A1 and 1 I01 BX004494-01 (MB), NIH/NCI T32CA121938 (HH). " We note that one or more of the authors are employed by a commercial company: 'AntiCancer 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. 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. 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. * Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 5. In the Methods, please provide the formula by which tumour volume was calculated. [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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No 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: No 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: In this manuscript, Hollandsworth et al. describe the application of photoimmunotherapy (PIT) in the treatment of a human colon cancer cell line in vitro and in an orthotopic cecal transplantation model. The authors demonstrate a dose-dependent response to PIT in a cancer cell line. They then show that a single dose of PIT effectively treats the cecal transplantation model. However, a key weakness is that this manuscript fails to provide any mechanism whatsoever to support the conclusion that this technology may be useful for clinical application. Comments: 1. The authors state that PIT suppressed tumor growth in subcutaneous colon cancer mouse model but this conclusion is demonstrated only in a single mouse image in Figure 3. Quantification of fluorescence intensity and tumor size measurements need to be provided to support this claim. Figure 4 should also be accompanied with quantification and images of tumors. 2. What is the purpose of performing subcutaneous transplant studies if the authors also perform cecal transplants, which are presumably better? 3. Finally, they show inhibition of tumor growth in orthotopic nude mouse models with repeated exposure to PIT one week after initial treatment. Overall the results are promising as this is the first study of PIT in an orthotopic mouse model of colon. However, the conclusions drawn are largely hypothetical as the tumors were grown in athymic nude mice without a competent immune system. Although these mice have dendritic cells, they lack T cells. Thus, this model may have little relevance to humans. Studies in a mouse cell line or organoid transplant model or genetically engineered model would be more relevant. 4. The authors also failed to discuss why the tumors rapidly increased in size following PIT cessation in comparison to control tumors. 5. No experiments were performed to study the possible mechanism of action of PIT in colon cancer. Thus, these experiments add little value to the literature on PIT. 6. Why did the authors select this particular cell line, and why were studies not repeated in additional cell lines or organoid lines? 7. As the authors themselves note, the application of this technology is questionable since the tumor will need to be exposed to the light source. One possible application not discussed by the authors is ablation of positive margins of adenomas during colonoscopy. 8. Why was the treatment only provided for 1-2 weeks? This time scale is extremely short. 9. Histology of the tumors should be provided. Reviewer #2: The figure legends and results discussion appear to be out of order for several figures. This appears to be an issue of uploading but does not impact the content. The study is technically proficient and highlights an important potential approach to margin positive disease and potentially for peritoneal metastasis. ****** 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: Yes: Tony R Reid MD, Ph.D. [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Review PIT for colorectal cancer.docx https://doi.org/10.1371/journal.pone.0234643.r001
Revision 1
4 Apr 2020 Author Response Thank you for your email and reviewers’ comments regarding our manuscript entitled “Near-Infrared Photoimmunotherapy is Effective Treatment for Colorectal Cancer in Orthotopic Nude-Mouse Models.” Thank you also for your interest and the thoughtful comments provided by the Reviewers. For our revision submission, we are providing a revised manuscript that addresses all Reviewer comments and suggestions. Below, please find the Reviewer’s comments and how each was addressed in the revised manuscript. The Reviewer comments are numbered and our responses to each comment are below in bold. We thank you again for the opportunity to submit this revised manuscript and look forward to your comments. Reviewers' comments: Reviewer 1: 1. The authors state that PIT suppressed tumor growth in subcutaneous colon cancer mouse model but this conclusion is demonstrated only in a single mouse image in Figure 3. Quantification of fluorescence intensity and tumor size measurements need to be provided to support this claim. Figure 4 should also be accompanied with quantification and images of tumors. Figure 4 legend (now labelled Figure 3) and the Results now include quantification of fluorescence intensity in the orthotopic models. Please see answer to question 2 below. 2. What is the purpose of performing subcutaneous transplant studies if the authors also perform cecal transplants, which are presumably better? Data on subcutaneous models was removed from the revised manuscript. 3. Finally, they show inhibition of tumor growth in orthotopic nude mouse models with repeated exposure to PIT one week after initial treatment. Overall the results are promising as this is the first study of PIT in an orthotopic mouse model of colon. However, the conclusions drawn are largely hypothetical as the tumors were grown in athymic nude mice without a competent immune system. Although these mice have dendritic cells, they lack T cells. Thus, this model may have little relevance to humans. Studies in a mouse cell line or organoid transplant model or genetically engineered model would be more relevant. A section in the discussion has been included to discuss the limitation highlighted above: “In the present study, we utilized athymic nude mice that lack T cells, which are not representative of the complex conditions of the immune system in patients. However, even in the absence of T cells, PIT was shown to be effective, suggesting that T cells are not the major factor in immunogenic cell death. Prior to translation into clinical studies, we will perform PIT on orthotopic syngeneic models, humanized or genetically engineered mice, all with an intact immune system.” 4. The authors also failed to discuss why the tumors rapidly increased in size following PIT cessation in comparison to control tumors. This is an important question. We can only speculate that the first PIT caused the production of growth factors, such is sometimes the case in surgery, where residual tumors grow faster. Future experiments are needed to answer this question. This is stated in the revised manuscript. 5. No experiments were performed to study the possible mechanism of action of PIT in colon cancer. Thus, these experiments add little value to the literature on PIT. The present studies are a proof-of-principle that PIT is effective on orthotopic models of colon cancer, as previous studies were only on subcutaneous colon cancer models, which are artificial. This is stated in the revised manuscript. However, general mechanisms of PIT are discussed in the revised manuscript. 6. Why did the authors select this particular cell line, and why were studies not repeated in additional cell lines or organoid lines? We have done previous studies showing human colon cancer LS174T cell line is targeted well by anti-CEA antibodies (1) and would be appropriate for an orthotopic PIT study and proof-of-principle. This is stated in the revised manuscript. 7. As the authors themselves note, the application of this technology is questionable since the tumor will need to be exposed to the light source. One possible application not discussed by the authors is ablation of positive margins of adenomas during colonoscopy. PIT can be an intra-operative procedure; therefore, illuminating an intra-abdominal tumor is feasible. This is discussed in the revised version. The potential application for polyp margins during endoscopy is also now included in the Discussion. 8. Why was the treatment only provided for 1-2 weeks? This time scale is extremely short. The limitation of the time scale is included in the Discussion. This also includes the reason for short time period, which was tumor growth in the control group that exceeded the allowed tumor burden. It was discussed that survival and recurrence studies can increase clinical applicability of PIT in colorectal cancer. 9. Histology of the tumors should be provided. As the present study was a proof-of-principle study that PIT could arrest an orthotopic tumor, histology studies were not done. Detailed histology studies will be done in the future. This is discussed in the revised edition. As part of this submission, we have included a revised version of the main manuscript file that shows changes made by highlighting, as well as a clean version of the revised manuscript. Again, we thank the editors and reviewers for their efforts in improving our manuscript. We would be happy to answer any further questions and/or concerns. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0234643.r002
5 May 2020 Decision Letter - Irina V. Lebedeva, Editor PONE-D-20-02950R1 Near-Infrared Photoimmunotherapy is Effective Treatment for Colorectal Cancer in Orthotopic Nude-Mouse Models PLOS ONE Dear Dr Bouvet, 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. We would appreciate receiving your revised manuscript by Jun 19 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Irina V. Lebedeva, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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 Reviewer #2: 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 Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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: The author's has appropriately limited the scope of their conclusions and added appropriate discussion. Below please see our comments about specific points with minor revision requests. Reviewers' Response to comments: 1. The authors state that PIT suppressed tumor growth in subcutaneous colon cancer mouse model but this conclusion is demonstrated only in a single mouse image in Figure 3. Quantification of fluorescence intensity and tumor size measurements need to be provided to support this claim. Figure 4 should also be accompanied with quantification and images of tumors. Author’s Response: Figure 4 legend (now labelled Figure 3) and the Results now include quantification of fluorescence intensity in the orthotopic models. Please see answer to question 2 below. Reviewer’s Response: How many fluorescence measurements were calculated? It would strengthen the claim if this data was shared as a graph in updated Figure 3. 2. What is the purpose of performing subcutaneous transplant studies if the authors also perform cecal transplants, which are presumably better? Author’s Response: Data on subcutaneous models was removed from the revised manuscript. Reviewer’s Response: Author’s can include other relevant experiments as supplemental figures. 3. Finally, they show inhibition of tumor growth in orthotopic nude mouse models with repeated exposure to PIT one week after initial treatment. Overall the results are promising as this is the first study of PIT in an orthotopic mouse model of colon. However, the conclusions drawn are largely hypothetical as the tumors were grown in athymic nude mice without a competent immune system. Although these mice have dendritic cells, they lack T cells. Thus, this model may have little relevance to humans. Studies in a mouse cell line or organoid transplant model or genetically engineered model would be more relevant. Author’s Response: A section in the discussion has been included to discuss the limitation highlighted above: “In the present study, we utilized athymic nude mice that lack T cells, which are not representative of the complex conditions of the immune system in patients. However, even in the absence of T cells, PIT was shown to be effective, suggesting that T cells are not the major factor in immunogenic cell death. Prior to translation into clinical studies, we will perform PIT on orthotopic syngeneic models, humanized or genetically engineered mice, all with an intact immune system.” Reviewer’s Response: This is acceptable. 4. The authors also failed to discuss why the tumors rapidly increased in size following PIT cessation in comparison to control tumors. Author’s Response: This is an important question. We can only speculate that the first PIT caused the production of growth factors, such is sometimes the case in surgery, where residual tumors grow faster. Future experiments are needed to answer this question. This is stated in the revised manuscript. Reviewer’s Response: Histology of the tumors on future studies can also help identify characteristics of the residual tumor. 5. No experiments were performed to study the possible mechanism of action of PIT in colon cancer. Thus, these experiments add little value to the literature on PIT. Author’s Response: The present studies are a proof-of-principle that PIT is effective on orthotopic models of colon cancer, as previous studies were only on subcutaneous colon cancer models, which are artificial. This is stated in the revised manuscript. However, general mechanisms of PIT are discussed in the revised manuscript. Reviewer’s Response: The new information provided will help readers understand the possible future directions. 6. Why did the authors select this particular cell line, and why were studies not repeated in additional cell lines or organoid lines? Author’s Response: We have done previous studies showing human colon cancer LS174T cell line is targeted well by anti-CEA antibodies (1) and would be appropriate for an orthotopic PIT study and proof-of-principle. This is stated in the revised manuscript. 7. As the authors themselves note, the application of this technology is questionable since the tumor will need to be exposed to the light source. One possible application not discussed by the authors is ablation of positive margins of adenomas during colonoscopy. Author’s Response: PIT can be an intra-operative procedure; therefore, illuminating an intra-abdominal tumor is feasible. This is discussed in the revised version. The potential application for polyp margins during endoscopy is also now included in the Discussion. 8. Why was the treatment only provided for 1-2 weeks? This time scale is extremely short. Author’s Response: The limitation of the time scale is included in the Discussion. This also includes the reason for short time period, which was tumor growth in the control group that exceeded the allowed tumor burden. It was discussed that survival and recurrence studies can increase clinical applicability of PIT in colorectal cancer. 9. Histology of the tumors should be provided. Author’s Response: As the present study was a proof-of-principle study that PIT could arrest an orthotopic tumor, histology studies were not done. Detailed histology studies will be done in the future. This is discussed in the revised edition. As part of this submission, we have included a revised version of the main manuscript file that shows changes made by highlighting, as well as a clean version of the revised manuscript. Again, we thank the editors and reviewers for their efforts in improving our manuscript. We would be happy to answer any further questions and/or concerns. Reviewer #2: The study demonstrates dose dependent tumor cell killing with PIT and the potential application of this technology to surgical settings. The non-toxic aspect of PIT in the absence of the conjugated antibody offers the potential to treat otherwise surgically inoperable disease as well as debulk disease. The paper offers an important potential path to managing complex intra-abdominal metastatic disease. ****** 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 Reviewer #2: Yes: Tony Reid MD, Ph.D. [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0234643.r003
Revision 2
8 May 2020 Author Response May 7, 2020 To: Drs. Joreg Heber and Irina V. Lebedeva, Editor-in Chief and Academic Editor, PLOS One Manuscript – “Near-Infrared Photoimmunotherapy is Effective Treatment for Colorectal Cancer in Orthotopic Nude-Mouse Models” Drs. Heber and Lebedeva, Thank you for your email and reviewers’ comments regarding our manuscript entitled “Near-Infrared Photoimmunotherapy is Effective Treatment for Colorectal Cancer in Orthotopic Nude-Mouse Models.” Thank you also for your interest and the thoughtful comments provided by the Reviewers. For our revision submission, we are providing a revised manuscript that addresses all Reviewer comments and suggestions. Below, please find the Reviewer’s comments and how each was addressed in the revised manuscript. The most recent Reviewer comments are italicized and our responses to each comment are below in bold. We thank you again for the opportunity to submit this revised manuscript and look forward to your comments. Reviewers' Response to comments: 1. The authors state that PIT suppressed tumor growth in subcutaneous colon cancer mouse model but this conclusion is demonstrated only in a single mouse image in Figure 3. Quantification of fluorescence intensity and tumor size measurements need to be provided to support this claim. Figure 4 should also be accompanied with quantification and images of tumors. Author’s Response: Figure 4 legend (now labelled Figure 3) and the Results now include quantification of fluorescence intensity in the orthotopic models. Please see answer to question 2 below. Reviewer’s Response: How many fluorescence measurements were calculated? It would strengthen the claim if this data was shared as a graph in updated Figure 3. Figure 3 has been updated to include a graph depicting the difference in mean maximum tumor fluorescence signal before and after PIT. The figure legend provides description of how many fluorescence measurements were calculated and statistical significance. Materials and methods have been revised to include image analysis and statistical analysis of tumor fluorescence. 2. What is the purpose of performing subcutaneous transplant studies if the authors also perform cecal transplants, which are presumably better? Author’s Response: Data on subcutaneous models was removed from the revised manuscript. Reviewer’s Response: Author’s can include other relevant experiments as supplemental figures. All relevant experiments are presented in the main manuscript. 3. Finally, they show inhibition of tumor growth in orthotopic nude mouse models with repeated exposure to PIT one week after initial treatment. Overall the results are promising as this is the first study of PIT in an orthotopic mouse model of colon. However, the conclusions drawn are largely hypothetical as the tumors were grown in athymic nude mice without a competent immune system. Although these mice have dendritic cells, they lack T cells. Thus, this model may have little relevance to humans. Studies in a mouse cell line or organoid transplant model or genetically engineered model would be more relevant. Author’s Response: A section in the discussion has been included to discuss the limitation highlighted above: “In the present study, we utilized athymic nude mice that lack T cells, which are not representative of the complex conditions of the immune system in patients. However, even in the absence of T cells, PIT was shown to be effective, suggesting that T cells are not the major factor in immunogenic cell death. Prior to translation into clinical studies, we will perform PIT on orthotopic syngeneic models, humanized or genetically engineered mice, all with an intact immune system.” Reviewer’s Response: This is acceptable. 4. The authors also failed to discuss why the tumors rapidly increased in size following PIT cessation in comparison to control tumors. Author’s Response: This is an important question. We can only speculate that the first PIT caused the production of growth factors, such is sometimes the case in surgery, where residual tumors grow faster. Future experiments are needed to answer this question. This is stated in the revised manuscript. Reviewer’s Response: Histology of the tumors on future studies can also help identify characteristics of the residual tumor. This point was added to the revised discussion. 5. No experiments were performed to study the possible mechanism of action of PIT in colon cancer. Thus, these experiments add little value to the literature on PIT. Author’s Response: The present studies are a proof-of-principle that PIT is effective on orthotopic models of colon cancer, as previous studies were only on subcutaneous colon cancer models, which are artificial. This is stated in the revised manuscript. However, general mechanisms of PIT are discussed in the revised manuscript. Reviewer’s Response: The new information provided will help readers understand the possible future directions. 6. Why did the authors select this particular cell line, and why were studies not repeated in additional cell lines or organoid lines? Author’s Response: We have done previous studies showing human colon cancer LS174T cell line is targeted well by anti-CEA antibodies (1) and would be appropriate for an orthotopic PIT study and proof-of-principle. This is stated in the revised manuscript. 7. As the authors themselves note, the application of this technology is questionable since the tumor will need to be exposed to the light source. One possible application not discussed by the authors is ablation of positive margins of adenomas during colonoscopy. Author’s Response: PIT can be an intra-operative procedure; therefore, illuminating an intra-abdominal tumor is feasible. This is discussed in the revised version. The potential application for polyp margins during endoscopy is also now included in the Discussion. 8. Why was the treatment only provided for 1-2 weeks? This time scale is extremely short. Author’s Response: The limitation of the time scale is included in the Discussion. This also includes the reason for short time period, which was tumor growth in the control group that exceeded the allowed tumor burden. It was discussed that survival and recurrence studies can increase clinical applicability of PIT in colorectal cancer. 9. Histology of the tumors should be provided. Author’s Response: As the present study was a proof-of-principle study that PIT could arrest an orthotopic tumor, histology studies were not done. Detailed histology studies will be done in the future. This is discussed in the revised edition. As part of this submission, we have included a revised version of the main manuscript file that shows changes made by highlighting, as well as a clean version of the revised manuscript. Again, we thank the editors and reviewers for their efforts in improving our manuscript. We would be happy to answer any further questions and/or concerns. As part of this submission, we have included a revised version of the main manuscript file that shows changes made by highlighting, as well as a clean version of the revised manuscript. Again, we thank the editors and reviewers for their efforts in improving our manuscript. We would be happy to answer any further questions and/or concerns. Sincerely, Hannah Hollandsworth, MD Attachments Attachment Submitted filename: Response to Reviewers_2.docx https://doi.org/10.1371/journal.pone.0234643.r004
1 Jun 2020 Decision Letter - Irina V. Lebedeva, Editor Near-Infrared Photoimmunotherapy is Effective Treatment for Colorectal Cancer in Orthotopic Nude-Mouse Models PONE-D-20-02950R2 Dear Dr. Bouvet, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Irina V. Lebedeva, 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 review questions / comments have been addressed. I recommend proceeding with acceptance and publication. ****** 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: Jatin Roper https://doi.org/10.1371/journal.pone.0234643.r005
Formally Accepted
3 Jun 2020 Acceptance Letter - Irina V. Lebedeva, Editor PONE-D-20-02950R2 Near-Infrared Photoimmunotherapy is Effective Treatment for Colorectal Cancer in Orthotopic Nude-Mouse Models Dear Dr. Bouvet: 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. Irina V. Lebedeva Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0234643.r006

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