PONE-D-20-03388

Establishment and Characterization of VOA1066 cells: an Undifferentiated Endometrial Carcinoma Cell Line

PLOS ONE

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Reviewers' comments:

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Wang et al. describe the generation of an undifferentiated human endometrial carcinoma cell line VOA1066, which harbours characteristics such as intact MMR, ER negativity, genomic stability and is capable of being grown as xenografts. This work is important given the highly aggressive nature of this type of endometrial cancer and the lack of models to investigate the disease. The cell line displays a truncated form of ARID1A, the loss of ARID1B and a p.D1810Y mutation in DICER1, which the authors determine likely retains wild type function. Given the model recapitulates the features of the original tumour from which it was derived, overall the study is well done and establishes a new experimental model that will facilitate future investigation of this rare endometrial cancer type. I have some comments for clarification and suggestions.

Specific points:

1. Figure 4D. Please provide details about the HEC50 cell line in the methods. Please explain the shift in molecular weight for SMARCA4 in the VOA1066 sample. As defects in ARID1A are often associated with constitutive PI3K/AKT pathway activation, the authors should assess pathway activation in addition to PTEN status.

2. The authors speculate that the truncated ARID1A protein will likely result in impaired incorporation into SWI/SNF complexes, but the claim that this model could be used as a model of both defective ARID1A and ARID1B would be improved if this were demonstrated.

3. In addition to the genomic characterisation of the VOA1066 cell line, RNA sequencing analysis would provide important insight into potential pathways that could be targeted clinically and bolster the utility of this line for mechanistic studies.

Typographical errors:

Ethics statement and page 5 - “eithics” should be “ethics.”

Page 9 – “alternations” should be “alterations.”

Reviewer #2: DDEC has recently been recognised as a distinct EC subtype for its aggressive behaviour. The mechanism associated with pathogenesis remains largely unknown. No matter how great proportion the lower-grade endometrioid adenocarcinoma accounted for the DDEC, the component of UEC contributed largely to a highly aggressive manner. Therefore, understating of DDEC starts with the knowledge of UEC. This paper established a UEC cell line and validate through specific genomic features. The study is of translational important but needs to address a couple of major points:

1. Molecular signature specific to UEC remains unclear. The cell line established in this paper could be useful to study to role of ARID1A and ARID1B mutations in UEC, but not quite useful for other inactivating mutations such as SMARCA4. Recent studies suggest a role for inactivation of SMARCA4 in UEC (Stewart et al, 2015; Karnezis et al, 2015) but according to the western blotting result of this study, SMARCA4 showed expression in the UEC cell line. It would also be useful to include IHC for SMARCA4.

2. Grade 2 or 3 endometrioid adenocarcinoma are the 2 most misdiagnoses of DDEC. More markers such as Mullerian epithelial markers: keratin, EMA and PAX8 should be included to validate the portion of UEC in the primary tumor as well as cell line to discriminate from grade 2 or 3 endometrioid adenocarcinoma.

3. The VOA1066 cell line had more than 30% protein altering mutations compared to the primary tumour, which could be problematic when understanding related mechanisms. It would be helpful to include a functional summary of the cell line specific mutations.

4. For the IHC of VOA1066 primary tumor, it would be reasonable to include positive controls for ER staining (such as from adjacent endometrioid portions).

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Reviewer #1: Yes: Keefe Chan

Reviewer #2: No

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Establishment and Characterization of VOA1066 cells: an Undifferentiated Endometrial Carcinoma Cell Line

PONE-D-20-03388R1

Dear Dr. Wang,

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,

Elizabeth Christie

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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

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

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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: 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: The authors have addressed all of my comments from the initial review of the manuscript, which has been strengthened. The new model for UEC will be useful for researchers investigating this disease.

Reviewer #2: (No Response)

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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.

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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: Keefe T Chan

Reviewer #2: No