PONE-D-23-21611Changes in cell morphology and function induced by NRAS Q61R mutation in lymphatic endothelial cellsPLOS ONE

Dear Dr. Yasue,

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Avaniyapuram Kannan Murugan, M.Phil., Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

1. Though manuscript has merit, reviewers raised many concerns about the article and related experiments and is important to be addressed before consideration for publication.

2. The following articles are potentially important in RAS related research and hence can be cited them in the revised version (PMID: 31255772; PMID: 27102293; PMID: 26620726).

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: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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: The manuscript, “Changes in cell morphology and function induced by NRAS Q61R mutation in lymphatic endothelial cells” presents the morphological characterization and functional profiling of human dermal lymphocyte endothelial cells (LECs) upon transfection with lentivirus vector carrying specific NRAS mutation. The work includes protein expression analysis, and metabolomic profiling upon such transfection. According to this in vitro investigation NRAS Q61R human dermal LECs have poor tube formation and high cell proliferation and migration ability with increasing ratios of mutated cells. They demonstrate the inactivation of the PIK3/AKT/mTOR pathway and hyperactivation of the RAS/MAPK/ER pathway in the transfected cells. The latter is inhibited by trametinib, which targets MAP kinase. The study provides a possible mechanism for Kaposis lymphangiomatosis pathology and suggests the potential treatment strategies.

The study is well designed and presented in a coherent manner. There are a few presentation and language concerns:

Presentation Concerns:

i. In the Materials and Methods section under the heading “Gene transfection of HDLEC cell line using lentiviruses”, the authors have written “NRASQ61R expression in these cells was checked by polymerase chain reaction (PCR).” Probably they mean insertion rather than expression. In case of expression profiling “real time PCR” must have been performed. The authors may provide details as supplementary material.

ii. Figure 1: The magnification scale bar needs to be provided.

iii. Images (a) and (b) in Figure 1 need to be more clear to draw conclusions in the text.

iv. Under Metabolomics of HDLECs heading on page no. 26, last paragraph, Fig. 5. Is written instead of Fig. 6.

Language suggestions:

The manuscript may be revised for some minor language corrections for the purpose of clarity and precision. For instance:

i. In the abstract, “MEK” abbreviation is not explained.

ii. In the Introduction, it’s unclear what authors mean by “KLA is a subtype of GLA … [and presents with] poorer outcomes than GLA patients”. Do they actually mean “worse outcomes than GLA patients with other subtypes”?

iii. In the Methods section under the heading “Gene transfection of HDLEC cell line using lentiviruses”, the authors have written “The recombinant lentiviral solution was diluted with EBM2 to give solutions with initial concentrations of 1:4 to 1:10.” May be the authors should consider “prepare solutions with initial concentrations of 1:4 to 1:10”.

Reviewer #2: The manuscript by Yasue et al. describes the morphological and functional alterations observed in juvenile foreskin-derived human dermal lymphatic endothelial cells (HDLECs) after stable lentiviral insertion of a mutated form of the NRAS gene (Q61R variant). The Authors argue that somatic low-level mutation of the NRAS gene in HDLECs could explain the lymphatic abnormalities observed in patients with kaposiform lymphangiomatosis (KLA).

However, several concerns weaken the reliability of this work. The main concerns relate to the consistency and presentation of the data. Furthermore, the analysis presented is strictly descriptive, functional experiments are required to support the claimed correlation and causality. Previously published papers report the NRASQ61R variant found in KLA samples, with no evidence as to which cell type is mutated. The Authors propose that the NRASQ61R mutation of HDLECs is capable of inducing KLA-like aberrations based on similar molecular and morphological abnormalities observed in vitro. At the same time, they suggest that low-frequency mutated cells could affect the phenotype of distant cells even regardless of their mutational status, thus raising questions about the ability to induce the same phenotype on HDLECs by RAS mutation in other cell types in the microenvironment. To clarify this point, single cell RNA sequencing of KLA samples or coculture of WT HDLECs with NRAS mutant stromal cells should be performed.

MAJOR

- Intensive improvement of the image quality in the figures is required to appreciate the morphology of the cells and structures

- The WT cells in most of the figures are not visible as they are not colored. This is especially inconvenient when mixed cultures are being analyzed and NRASQ61R cells are the only observable, with no WT cell information in the dark areas. Red staining should be performed on WT cells to better understand the cell-cell interactions and morphological changes.

- The time-lapse photography in supplementary files a, b, allows observation of WT and mutant cells, however cell density at baseline appears to be different. Can you explain this discrepancy?

- The results of the proliferation assay should be better clarified: an increase in proliferation in NRASQ61R cells is reported in the abstract and discussion, while the opposite phenotype is described in the results, in figure 3C (decreased in NRASQ61R cells) and in the conclusion

- The same consideration for the number of nets formed in NRASQ61R cells with respect to WT: the result described in the text is opposite with respect to the graph of figure 2B. Please clarify this point.

- In figure 3C the amount of live cells after trametinib treatment is the same in all samples, regardless of the percentage of NRASQ61R cells; how can this apparent independence of drug efficacy from ERK MAPK activation be explained?

- Analysis of the metabolome of NRASQ61R cells revealed increased secretion of ANG-2 and decreased VEGFR3 compared with WT cells. The authors suggested that hypersecretion of ANG-2 by NRASQ61R HDLECs may cause decrease in VEGFR-3 levels, thereby imparing tube formation. Functional experiments based on loss or gain of function are needed to strenghten this claim.

MINOR

- The titles in the results section are more suitable for materials and methods; please replace them with short sentences that briefly describe the result obtained

- Figures 4B-d and 4B-e are mentioned in the text but missing from the figure

- Please specify in figure 3B what it refers to: the legend of the figure refers to the proliferation data, while the text shows the space ratio result and the title on the y axis is missing

- in the last paragraph of the results chapter “Analysis of signaling pathways in HDLECs by western blotting and multiplex protein assay” there are some refuses to correct: AKT was described in the previous paragraph, so it shoud be something different; moreover, only the efficacy of trametinib is described while two P-values are reported which raise doubts about the described target.

- Please check the captions in the graphs of Figures 4B and 4C, they look confusing

- In the M&M section, the chapter “Analysis of NRAS mutational status” appears to be a refuse of the previous paper by the group, as NRAS mutational status was not analyzed in this study

Reviewer #3: In this manuscript, Yasue et al, aimed to explore a low-level somatic mutation in NRAS gene (c.182 A > G, Q61R) in LA patients. The authors generated stable lymphatic endothelial cells (LECs) with expressing NRASQ61R mutation using Lentiviral system to understand morphological and functional characterization, protein expression profiling, and metabolome analysis of those mutated cells compared to WT cells. They found out that NRASQ61R human dermal LECs showed poor tube formation and high cell proliferation and migration ability with increasing ratios of mutated cells. Moreover, signaling pathways analysis showed inactivation of the PIK3/AKT/mTOR pathway and hyperactivation of the RAS/MAPK/ERK pathway, which was improved by MEK inhibitor treatment. Overall, the findings they present are interesting, technically sound, and could be valuable to the field. Their claims are convincing and supported by the experimental data with comprehensive experimental controls. The manuscript provides relevant, up-to-date information and it is well written and supported with proper figures. All the data appears to be available within the manuscript and supplementary data.

I have minor suggestions that may be helpful for the reader to better understand the current study.

1. Figure 1: NRASQ61R HDLECs showed larger and irregular-shaped morphology compared with NRASWT HDLECs microscopically (Fig.1 a–c). Please define the image name which one is WT or Q61R? If there is no WT cell images, please include it as well.

2. Figure 2B: It is better to include X-axis name as (e.g. WT: Q61R cell ratio) in the figure for better understanding

3. Figure 2D: It is better to include “DMSO treated control cells” to show the changing of the poor tube-forming ability.

4. Figure 3B: Please define the Y-axis name in the graph (e.g. space ratio)

5. Figure 5: What is the control group?

6. Page 26: “Among other amino acids, aspartic acid in NRASWT HDLECs was significantly higher than in NRASQ61R (p=0.008) (Fig.5)” Figure number should be 6

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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PONE-D-23-21611R1Changes in cell morphology and function induced by the NRAS Q61R mutation in lymphatic endothelial cellsPLOS ONE

Dear Dr. Ozeki,

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 Mar 23 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,

Avaniyapuram Kannan Murugan, M.Phil., Ph.D.

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.

Additional Editor Comments:

Authors have partially addressed the comments: The following points to be taken care before considering the article for publication.

Comments;

1. The Reviewer #2 still has some points which is critical.

2. The following important RAS-related articles to be cited in the manuscript.

Importance of RAS in human cancer: PMID: 31255772; PMID: 27102293

How RAS can be modulated by miRNAs: PMID: 26620726

RAS mutations in other cancers: PMID: 22240207; PMID: 19628422

3. The spell, grammar and expressions to be checked as PLoS one does not edit for Grammar.

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 #2: (No Response)

Reviewer #3: 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 #2: Partly

Reviewer #3: Yes

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

Reviewer #2: I Don't Know

Reviewer #3: 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 #2: Yes

Reviewer #3: 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 #2: Yes

Reviewer #3: 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 #2: The Authors have provided a revised version of the manuscript entitled “Changes in cell morphology and function induced by the NRAS Q61R mutation in lymphatic endothelial cells”. Although the Authors have addressed all the minor concerns and most of the major concerns that have been raised, some unaddressed points still persist:

- The mixed cultures in figure 1c are still difficult to evaluate as only NRASQ61R cells are visible. Since enhanced images of the NRASWT and NRASQ61R HDLECs with fluorescence have been added, consider deleting figure 1c.

- Since no functional experiments have been performed demonstrating that hypersecretion of ANG-2 by NRASQ61R HDLECs leads to decreased VEGFR-3 levels, the work is still descriptive and provides only speculative conclusions.

- the same perplexity concerns the effect of inhibitors of the MEK and mTOR pathways which is not clearly linked to the NRAS mutation. It is unclear why mTOR inhibition has the same effects as MEK inhibition in cells without hyperactivation of the PI3K/AKT/mTOR pathway.

Reviewer #3: All my comments have been addressed by the author. I have no more suggestions. The paper is ready for publishing.

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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 #2: No

Reviewer #3: No

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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.

Changes in cell morphology and function induced by the NRAS Q61R mutation in lymphatic endothelial cells

PONE-D-23-21611R2

Dear Dr. Ozeki,

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,

Avaniyapuram Kannan Murugan, M.Phil., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: