PONE-D-24-01608Extracellular ATP/P2X7 receptor, a regulatory axis of migration in ovarian carcinoma-derived cells.PLOS ONE

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“José David Núñez Ríos is a doctoral student from the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and has received CONAHCYT fellowship CVU #1044618. We are grateful to Jessica Gonzalez Norris for proofreading this manuscript. We are also grateful to Ing. Nydia Hernández Ríos, Biol Maria Eugenia Ramos Aguilar and Nuri Aranda López for technical assistance. This study was supported by the grant IN205223 from PAPIIT-UNAM; FONDECYT Regular Grant 1161490, FONDEQUIP EQM140100, intramural grant “Núcleo para el estudio del cáncer a nivel básico, aplicado y clínico”, VRIDT, UCN and Millennium Nucleus for the Study of Pain. MiNuSPain is a Millennium Nucleus supported by the Millennium Science Initiative of the Ministry of Science, Technology, Knowledge and Innovation (Chile). Funders had no role in study design, data collection and analysis, decision to publish, or preparation of this article.”

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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

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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: Authors elucidated pro-metastatic role of P2X7R in ovarian carcinoma cell line. Authors observed a decrease in cell migration and an increase in transepithelial electrical resistance and cell markers, suggesting P2X7R expression maintains a mesenchymal phenotype. Transcript levels analysis of some P2 receptors lead to three-fold higher P2RX7 levels in SKOV-3 cells than in a healthy cell line, namely HOSE6-3. Through bioinformatic analysis, authors identified a higher expression of the P2RX7 transcript in metastatic tissues than in primary tumors; thus, P2X7 seems to be a promising effector for the malignant phenotype. Subsequently, authors were demonstrated that presence and functionality of the P2X7 receptor in SKOV-3 cells and showed through pharmacological approaches, CRISPR-based knock-out system and in vivo Zebrafish embryonic assays role of P2X7 receptor is a regulator for cancer cell migration/invasion and thus a potential drug target. Overall, authors conclusions were drawn by experimental results on a metastatic ovarian cell line.

Comments:

1. Previously, many in vitro studies with different organ-site cancer cell lines shown the role of P2X7R in cell migration and invasion (eg. Molecular Cancer volume 14, Article number: 203, 2015; Mol Biol Cell. 2005;16:3260–72; FASEB J. 2010;24:3393–404). In several of published studies suggest P2X7R isoform A involvement in migration. Does authors tested whether Ovarian cancer SKOV-3 P2X7R over-expressed transcript is specific Isoform? Discuss in the text.

2. Figure 3C protein atlas data – Figure show very small deviation for survival (PFS) with Low: 18.83 months vs High: 18.65 months, just few days difference with p=0.044. Does authors analyze overall survival (OS) of patients with different stages with low and high P2X7R expressions. Please include these data and discuss significance PFS vs OS.

3. Although, authors Figure 8 experiments with Zebrafish interesting, how authors selected oxATP or P2X7R antagonist concentrations? It should have better used two or three doses!!

4. Even though, in vitro and some in vivo studies shown promoting effect of P2X7R over-expression; whereas in well-established experimental animal models either pharmacological targeting (Oncotarget. 2017 Nov 17; 8(58): 97822–97834) and genetic ablation (Cancer Res (2015) 75 (5): 835–845) of P2X7R lead to enhanced tumor growth, thus clearly suggesting pro-tumorigenic role. Authors should cite these articles and discuss in the discussion of part of text.

Reviewer #2: PONE-D-24-01608

Manuscript Title: Title: Extracellular ATP/P2X7 receptor, a regulatory axis of migration in ovarian carcinoma derived cells.

General comments: The authors of this study reported on the role of the P2X7 receptor in metastasis of ovarian cancer. Although this receptor's involvement in metastasis has been proven in other cancers, such as breast, pancreas, lung and melanoma, the authors suggest that it also has the same role in ovarian cancer. To prove their hypothesis, the authors used a single ovarian cancer cell line and studied a few human tissue samples. However, to provide more clarity on the role of P2X7 in ovarian cancer, they need to provide additional information, such as testing other types of ovarian cancer cell lines (PA-1, SW626, Caov-3), studying more human samples (a human samples panel), and performing additional experiments.

Specific questions:

It is unclear which specific gene mutations the tested ovarian cancer cell line has, as the authors did not mention it. Furthermore, the authors need to compare the data obtained from this cell line with other primary tumors or adenocarcinomas such as SW626 and Caov-3. They also need to provide data on the tissue samples collected from the metastatic site.

The authors need to provide details on whether the biopsies tested for P2X7 receptor expression were collected from the primary tumor site or the metastatic site. Additionally, the authors need to perform experiments with other primary tumor-derived ovarian cancer cell lines to show the difference in the data obtained using more than one cell line. The authors also need to address whether the P2X7 receptor is responsible for the aggressive nature of the serous histology type of ovarian cancer or if the serous type at the adenocarcinoma stage also expresses high levels of this receptor. In either case, they need to include experimental evidence for their explanation.

They also need to provide experimental evidence to show if this receptor expression causes the serous type to metastasize.

The authors need to explain how they optimized the doses of P2X7 antagonists A438079 125 nM (aA43) and non-reversible antagonist OxATP (200 μM).

The presence of the P2X7 receptor on immune cells such as neurons, DCs, macrophages, and B and T cells, etc., could influence the metastatic behavior of the tumor cells. Therefore, the authors need to discuss how the tumor cells interact with other cells that express this receptor and provide any data from human tissue samples.

Additionally, preclinical immune-competent animal experiments are required to understand how this receptor interacts with tumor cells in causing metastasis.

Finally, the authors need to provide data on the expression of P2X7 from lesions, adenomas, adenocarcinoma to metastatic tissues. This will give a clear indication of when this receptor is expressed during which stage of cancer development. They also need to explain the clinical significance of their observations reported in this paper.

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Reviewer #1: Yes: Chinthalapally V. Rao

Reviewer #2: No

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Extracellular ATP/P2X7 receptor, a regulatory axis of migration in ovarian carcinoma-derived cells.

PONE-D-24-01608R1

Dear Dr. Vázquez-Cuevas,

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,

Altaf Mohammed

Academic Editor

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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

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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: Yes

Reviewer #2: Yes

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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

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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 #2: 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: Authors addressed all the concerns of reviewers. No additional comments. Authors provided detailed and valid explanation reviewer comments.

Reviewer #2: (No Response)

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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: Chinthalapally V Rao

Reviewer #2: No

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