PONE-D-23-37839Monocyte-cancer cell fusion is mediated by phosphatidylserine - CD36 receptor interaction and induced by ionizing radiationPLOS ONE

Dear Dr. Shabo,

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,

Ajay Kumar, PhD

Academic Editor

PLOS ONE

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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: Partly

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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: In the present investigation “Monocyte-cancer cell fusion is mediated by phosphatidylserine - CD36 receptor interaction and induced by ionizing radiation”, Shabo et al have reported ionizing radiation resulted in the fusion of monocyte and tumor cells through the interaction between phosphatidylserine and CD36. The authors have co-cultured the tumor cell line and monocyte cell line and were subjected to ionizing radiation in the presence and absence of anti-CD36 antibodies to evaluate the interaction between phosphatidylserine and CD36 in the fusion of tumor cells and monocyte.

There are many studies studies regarding the fusion of cancer cells among themselves or/and with macrophages. However, to make this study more interesting and impactful, following comments can substantiate their findings.

1. The authors have presented and have explained the findings suitably. However, the language of the manuscript requires more improvement.

2. The authors have used the flow cytometry to show the fusion of the cells. However, the fusion of the cells should also be confirmed through microscopy. Since macrophages and monocytes are the scavenging and are involved in the phagocytosis, hence, microscopic images will confirm that the tumor cells are being phagocytosed not the apoptotic cell bodies.

3. The author should also measure the oxidative stress in the irradiated tumor and monocyte cell lines and fused tumor cells and cancer cell lines.

4. CD-36 have a prominent role in the scavenging potential of macrophages and phagocytosis. Therefore, the authors should also show the scavenging potential of the monocyte cell line in the presence of anti-CD36 antibodies.

5. The authors should confirm their findings with monocytes isolated from the human blood which will cement their findings.

6. The authors should show viability of the tumor cells and monocytes before and after the irradiation.

Reviewer #2: This study represents a significant leap forward in unraveling the intricate mechanisms of leukocyte-cancer cell fusion. By highlighting the role of the CD36-phosphatidylserine interaction and its modulation by ionizing radiation, the research not only deepens our understanding of tumor progression but also opens avenues for targeted therapeutic strategies in combating metastatic cancer. However, there are some potential drawbacks and limitations that should be considered.

1. The investigation is exclusively conducted in vitro, posing a potential limitation in fully replicating the intricate dynamics of the in vivo microenvironment. Cellular interactions within the controlled laboratory conditions may diverge from those occurring within living organisms, possibly constraining the generalizability of the study's outcomes.

2. The utilization of specific cell lines (THP-1 and MCF-7) may not comprehensively represent the diverse array of cancer and immune cells present in authentic tumors. Different responses among various cancer types and subtypes may exist, and relying solely on a single breast cancer cell line might overlook this inherent variability.

3. While the study underscores the significance of CD36 and phosphatidylserine in the fusion of leukocytes with cancer cells, it is imperative to acknowledge that additional molecular factors and signaling pathways could also play contributory roles in this intricate process. A more all-encompassing analysis of these involved molecules could yield a more comprehensive understanding.

4. The primary focus of the study is on unraveling mechanisms at the cellular and molecular levels. Translating these discoveries into clinical applications necessitates further exploration to ascertain the clinical relevance and potential therapeutic implications of targeting CD36-phosphatidylserine interactions.

5. Despite the compelling nature of the in vitro findings, the absence of in vivo validation imposes constraints on directly applying the results to the complexities of biological systems. Subsequent studies incorporating animal models or clinical samples hold promise for providing a more precise representation of the physiological relevance of these findings.

6. The study predominantly delves into the impact of ionizing radiation on leukocyte-cancer cell fusion. While this aspect is pivotal, an exclusive focus on a single treatment modality may fall short in capturing the diverse conditions encountered by cancer patients undergoing various therapeutic interventions.

7. The study overlooks the heterogeneity prevalent in cancer patients, encompassing variations in tumor microenvironments, patient responses to treatment, and the dynamic nature of cancer progression. Acknowledging and understanding these diverse factors are crucial for extending the applicability of the findings to a broader clinical context.

Reviewer #3: Reviewer Comments:

The manuscript entitled " Monocyte-cancer cell fusion is mediated by phosphatidylserine - CD36 receptor interaction and induced by ionizing radiation” Current study highlights irradiation leads to a significant dose-dependent increase in the number of cancer cells expressing PS, while it does not affect the expression of CD36 in either THP-1 or MCF cells. Moreover, cell fusion generates new metastatic cancer cell/Tumor hybrid cells (THC) that exhibits the unique properties like abilities in colony formation, cell migration, invasion and over all acquired growth promoting properties. This is very an interesting study that can provide clue of targeted therapy for high grade tumor. However, I still have some queries and suggestion for the improvement of work qualities. Authors are advice to deal with the comments and adapt the manuscript accordingly and submit a revised manuscript. At this stage manuscript does not hold merit of acceptance. I would like to review the manuscript again.

Major concerns:

1. As a results of Monocyte-cancer cell fusion a tumor hybrid cells (THC) get formed which exhibits a unique features like abilities of colony formation, cell migration, and invasion. In this study author has simply analyzed all data by FACS ARIA III. Since data is very unique and important so it should be validated by multiple parameters like morphological assessment by Immunofluorescence/ high contrast laser scanning confocal microscopy (LSCM ) images and some physiological assessment like colony formation assay, cell migration and invasion assay is very essential for further validation.

2. In the study, a single MCF7 luminal breast cancer cells have been selected for the study, which is more prone to form hybrids tumor cells than any other cells. Is there any specific reason of choosing this cell? Though cell fusion is a rare phenomenon, and the proportion of hybrid cells may be small compared to the total number of cells. Therefore, it is advice to use other cell lines too for validation of the results along with normal cells.

Manor concerns:

1. It is better to use term tumor hybrid cells (THC) which is more specific and technical instead of cancer cell fusion in running text.

2. Almost 80 to 90% references given to the article is quite old, kindly updates with some recent articles related to fields. There are some references related to the field may be included i.e. PMID: 30339548, PMID: 32612123, PMID: 28870843.

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Reviewer #1: Yes: Dr. VISHAL KUMAR GUPTA

Reviewer #2: No

Reviewer #3: Yes: Dr. Shyam Babu Prasad

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Attachments

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Submitted filename: PlosOne.pdf

Attachment

Submitted filename: Reviewer Comments_ PONE-D-23-37839_reviewer.doc

PONE-D-23-37839R1Monocyte-cancer cell fusion is mediated by phosphatidylserine - CD36 receptor interaction and induced by ionizing radiationPLOS ONE

Dear Dr. Shabo,

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 Aug 29 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. 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,

Ajay Kumar, PhD

Academic Editor

PLOS ONE

Journal Requirements:

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 #1: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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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 #1: Partly

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

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 #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 #1: Yes

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 #1: The study's findings are very interesting; however, they lack a mechanistic approach. The authors must have included the experiments and findings with this work during revision rather than keeping them for a future goal. The addition of these experiments would have increased the impact of the present finding and made this work more interesting to the readers.

Reviewer #3: The authors have addressed almost all the issues raised by the reviewer, but there is still some query:

Comments 1# Authors has added fluorescence cell images in a new figure (Fig 2), which is simple MCF7, THP1 cells, and THP-1 cells stained with CD36 images using cellTraceTM staining. Authors should provide the cells images showing CD36, PS expression, and images of ionizing radiation showing their impact on CD36 and PS expression in cancer cells. In each case merge images should be provided. Further if possible try to capture the tumor hybrid cells images.

Reviewer #4: Minor point

1. Statistical analysis shown in figure 5, 6 and 7 needs to be elaborated by including the error bar, significance or level of significance etc. Also include the number of replicates used in experiment.

2. Add the methodology of fluorescence microscope imaging in manuscript and include the magnification, scale bar etc. in figure 2.

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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 #3: Yes: Dr. Shyam Babu Prasad

Reviewer #4: No

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[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.

Attachments

Attachment

Submitted filename: Reviewer Comments_ PONE-D-23-37839_29042024.doc

Monocyte-cancer cell fusion is mediated by phosphatidylserine - CD36 receptor interaction and induced by ionizing radiation

PONE-D-23-37839R2

Dear Dr. Shabo

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.

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Kind regards,

Ajay Kumar, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have addressed all the comments of the reviewers, so, it can be accepted for the publication.

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