PONE-D-22-01036Combining Ablative Radiotherapy and Anti CD47 Monoclonal Antibody Improves Infiltration of Immune Cells in Tumor MicroenvironmentsPLOS ONE

Dear Dr. Jalali,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Jian Jian Li, M.D., Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

Although radiation combined with anti-CD47 immunotherapy has been reported, this work demonstrates some interesting results in mouse cancer model treated by radiation and anti-CD47 immunotherapy. However, several major concerns are raised by both reviewers which should be carefully addressed with additional data supports.

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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: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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

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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

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study displayed some experiment results that, in comparison with radiotherapy alone, the single high dose of 16 Gy radiotherapy combined with αCD47 enhanced CT26 tumor growth delay and thus increased the survival of tumor-bearing mice, probably by increasing CD8+ T cells and M1 macrophages and decreasing M2 macrophages and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME) and increasing CD8+INF� in spleen. However, these phenomena have been reported by other literatures and there is no novel mechanistic investigation in this work, although these data may helpful to strengthen the experimental evidence for tumor radioimmunotherapy.

Comments:

1. Many similar studies applied male mice as an animal model. But this work applied a female mice model. Please explain.

2. The full names of abbreviations, TILs, DLNs, TTE, TGD and so on, should be given.

3. The calculation methods of TTE, TGD should be given.

4. Fig. 1B showed the value of CD47 MFI in tumor cells. The representative fluorescence images of this MFI should be given.

5. Fig. 2A was missing in the text.

6. Corresponding to the MFI in Fig. 2E, the representative tissue images should be given.

7. Fig. 4 showed that the percentage of CD45+CD8+ immune cells expressing IFNγ was increased in spleen of both �CD47 and RT+�CD47 groups. How about it in tumors? If this increase contributes to the anti-tumor effect, why didn’t �CD47 reduce tumor growth in Fig. 5? By the way, lacking of “%” in the Y-axis of Fig. 4.

8. In Fig. 5, the tumor sizes of control and �CD47 groups approached to a maximum value 1500 mm3 on day 45 and day 60. Why had they the same volume (1500 mm3) on these different days?

9. This reviewer suggest to perform more mechanistic studies, for example, to demonstrate how M1-macrophage contributes to the anti-tumor effect of RT+�CD47 treatment.

Reviewer #2: This study evaluated the effect of a single high dose radiotherapy combined with an anti-CD47 monoclonal

antibody (αCD47 mAb) in CT26 tumor‐bearing BALB/c mice. Also, immune cell changes were analyzed by flow cytometry in tumors, lymph nodes, and spleen. Combination therapy enhanced the anti-tumor response in treated mice by increasing CD8+ T cells and M1 macrophages and decreasing M2 macrophages and myeloidderived suppressor cells (MDSCs) in the tumor microenvironment (TME).

(1) The combination of Radiotherapy and CD47 blockade was investigated before. This work did not provide novel point and evidence. The novelty is poor.

(2) Most the data were presented based on three mice in each group, which is not enough. All the data were statistical results but without original picture of flow cytometry, which greatly impaired the scientific level of the work.

(3) All the data were based on one time mice experiment but without other experiments to verify the hypothesis.

(4) Figure 2 should be combined with Figure 5, and Fig 5B-5C should be deleted. Figure 4 should be combined with Figure 1.

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Reviewer #1: Yes: Chunlin Shao

Reviewer #2: No

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PONE-D-22-01036R1Combining Ablative Radiotherapy and Anti CD47 Monoclonal Antibody Improves Infiltration of Immune Cells in Tumor MicroenvironmentsPLOS ONE

Dear Dr. Seyed Amir Jalali,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 02 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.

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,

Norikatsu Miyoshi, M.D., Ph.D., FACS

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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 #3: All comments have been addressed

Reviewer #4: (No Response)

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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 #3: Partly

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: Yes

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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 #3: Yes

Reviewer #4: Yes

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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 #3: Yes

Reviewer #4: Yes

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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 #3: (No Response)

Reviewer #4: The article is in line with the current hot topics of radiotherapy activating immunity. Using radiotherapy and single-targeted immunotherapy in combination，it is very inspiring to draw the conclusion that this combination regimen is superior to radiotherapy or immunotherapy alone. But there are still some details to discuss in this article:

1. The author's intention of using high-dose irradiation alone as part of a combined regimen is based on previous published results, from high-dose alone, or hypofractionation, or multiple fractions. The one with the most enrichment of immune cells in the tumor microenvironment after radiation has been selected. But not all radiation regimens have been considered, such as low-dose 1-2 Gy radiation，which can induce more immune cells, or flash radiation，which has been reported to have a effect of activating abscopal effect.

2.The recruitment of CD8+ cells increases after the combination therapy, and the next immune mechanism has not been explored. Currently, it is only limited to the illustration of recruited immune cells.

3. Since we all know the effectiveness of PD-1 antibody combined with radiotherapy, why do the authors choose CD47 antibody instead of PD-1 antibody? Are there specific groups of people? or specific tumor subtypes? Is there any cooperation with concurrent clinical research? No experiment on toxicity caused by immunotherapy or combination therapy is described. Even if the program improves the tumor growth rate and survival time, it is not explained whether there are toxic adverse reactions.

4. It is recommended to conduct further validation in human xenograft vectors (PDX/mini-PDX/organoids）.

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Reviewer #3: No

Reviewer #4: No

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Combining Ablative Radiotherapy and Anti CD47 Monoclonal Antibody Improves Infiltration of Immune Cells in Tumor Microenvironments

PONE-D-22-01036R2

Dear Dr. Seyed Amir Jalali,

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.

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Kind regards,

Norikatsu Miyoshi, M.D., Ph.D., FACS

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: