PONE-D-23-20515Cadherin-11 contributes to the heterogenous and dynamic Wnt-Wnt-β-catenin pathway activation in Ewing sarcoma.PLOS ONE

Dear Dr. Hayashi,

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==============================Both reviewers find your study interesting , but have raised a number of relevant points that needs to be addressed point by point. I would be happy to grant extra time should this be needed.

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Additional Editor Comments:

Both reviewers find your study interesting , but have raised a number of relevant points that needs to be addressed point by point. I would be happy to grant extra time should this be needed.

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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: No

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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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: The study by Shirai et al. aimed to investigate the contribution of CDH11 and b-catenin in Ewing sarcoma. This work could have been interesting; however, it lacks rigor and several points need to be clarified:

1. The English and scientific writing could be much improved.

In addition, the figure legends lack information that is important to understand the figures.

References need to be reformatted (e.g., #39 taking half a page!).

2. In general, blots and immunostaining should be quantified.

3. Fig 1: Good expression of b-catenin is still observed in TC71 cells after knockdown (A). So, how the authors can explain a such dramatic effect in the following experiments (like in B&C)? Especially given that basic b-catenin signaling is very low in these cells.

4. Fig 2: heterogeneity of Wnt/b-catenin activation in Ewing sarcoma is already published (reference #20 cited by the authors), and this figure does not bring any new information and should be removed. Moreover, following the methods described, the conclusions driven from this figure are biased: a) The whole A4573 xenograft has been used for the scRNAseq, so this includes mouse cells that have infiltrated the tumor. b) How Wnt signaling can be enriched when it is activated in only 4.3% of the A4573 population in vivo? c) It was performed on A4573 only, this is not representative.

It would have been more interesting to perform scRNAseq on CDH11 KO/WT cells.

5.Fig 4: IP (C) should be repeated in the other way, i.e., with CDH11 IP. The authors mentioned in the text that CDH11 interacted with "non-phosphorylated" b-catenin, but without showing it.

The immunostaining (B) is not convincing, several cells are negative; CDH11 antibody does not look very good.

6. Fig 5: Same comment as for Fig 4, immunostainings are not convincing (F): A4573 showed no expression of b-catenin in the control; we do not see any differences in nuclear localization of b-catenin between the different conditions in TC71 and A4573 cells (quantification is necessary). In addition, WB did not show any b-catenin translocation to nucleus in A4573 after Wnt stimulation (E) in contrast to what it should.

7. Fig 6: In C, counting cells with neurite outgrowth is a bit fishy (on pictures we can clearly see some in all conditions), it would have been better to quantify cell area.

In the cell migration assay (D), It is tricky to associate CDH11 to b-catenin signaling, since in basal condition this pathway is activated in only few cells (and here the control showed good migration). What is the status of b-catenin signaling during cell migration? The assay should be repeated including stimulation with Wnt.

Another type of cell migration like a wound assay could be considered.

8. Fig 7: Using CRISPR/Cas9, the authors have knock-out CDH11 (so the cells do not express it anymore, as seen in the blot 5C; knock-out is different than knock-down using shRNA, where in the latest some expression can still be observed). So why is there huge discrepancies between KO#1 and 2 in 7A and B (in the two cell type??)? In this context, the results does not look reliable.

To what correspond the control? Has flow cytometry analysis been performed for CDH11 expression at the cell surface?

Reviewer #2: The manuscript examines the role of Cadherin-11 in Wnt/β-Catenin signalling in Ewing sarcoma (ES) cells. The authors suggest that cadherin-11 contributes to Wnt/β-Catenin signalling in ES cells by controlling the expression of β-catenin. Using a xenograft model, the authors suggest that loss of cadherin-11 in ES cells can lead to a less metastatic phenotype. In general, the manuscript is technically sound. Although, some of the claims are supported with the experimental data, some major issues remain to be addressed.

Major comments:

1. The results showing the reduction in the protein levels of β-catenin expression in Cadherin-11 KO cells in interesting. In this context, mRNA levels of β-catenin and Cadherin-11 would help to know if the reduction was due to degradation or at transcription level. Authors could also use proteasomal inhibitors MG-132 to demonstrate the role of Cadherin-11 in the regulation or stabilization of β-catenin expression.

2. The data set in figure 5 could improve with quantification and statistical analysis. Importantly, the nuclear-cytoplasmic ratio of β-catenin in the western blots would help to understand if the reduced nuclear β-catenin in cadherin-11 KO cells are due to reduced total expression of β-catenin or due to changes in its localization.

3. The immunofluorescence staining of Cadherin-11 throughout the article is not very convincing to evaluate the expression level. Protein levels of Cadherin-11 in CRISPR-based Cadherin-11 KO cells would be better than immunofluorescence.

Minor comments:

1. The authors state that β-catenin is predominantly localized in the cell membrane. The fractions used for western blotting are cytoplasmic and not purely membrane fractions. Addition of Wnt ligands in experiments related to 3C would be helpful to know if β-catenin is predominantly localized in the cytoplasmic fractions despite activation of Wnt/β-Catenin signalling. Based on figure 2A, it seems there is very little activation of Wnt/β-Catenin signalling in the absence of Wnt ligands and this could explain the reduced nuclear localization of β-Catenin

2. For immunofluorescence the combination of 488 and 647 would be better instead of 488 and 555 to avoid cross-excitation issues mainly for cadherin-11 staining.

3. Figure legends are not completely clear enough to understand the data. For example, it is not clear what is Lcell and RPMI. Also, in figure 5C its mentioned as CDH11 KO but mentioned as CDH11 KD in 5E.

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Reviewer #1: No

Reviewer #2: No

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Cadherin-11 contributes to the heterogenous and dynamic Wnt-Wnt-β-catenin pathway activation in Ewing sarcoma.

PONE-D-23-20515R1

Dear Dr. Hayashi,

"We have had problems to reach one of the original reviewers, hence the delay.  Instead one editor has looked at your response to the non-responding reviewer and together with the positive response from the second reviewer we are glad to accept your revised version.

Donald Gullberg"

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,

Donald Gullberg, PhD

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

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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: (No Response)

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

Reviewer #1: (No Response)

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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 #1: (No Response)

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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: (No Response)

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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: (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: No

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