PONE-D-23-36221Examination of Wnt signaling as a therapeutic target for pancreatic ductal adenocarcinoma (PDAC) using a pancreatic tumor organoid library (PTOL).PLOS ONE

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Additional Editor Comments:

Reviewers have raised critical concerns regarding experiment design, result interpretations and statistical analysis. Authors need to point wise address the expert comments. Statistics may need major revisions.

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: No

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: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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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: The manuscript ‘Examination of Wnt signaling as a therapeutic target for pancreatic ductal adenocarcinoma (PDAC) using a pancreatic tumor organoid library (PTOL)’ by Hawkins et al, studied the Wnt dependent and independent phenotypes of pancreatic tumors using organoids. This reviewer provides the following comments/suggestions to be addressed by the Authors.

1. Did the authors observed any correlation between the mutations identified in the organoids and niche factor dependency? Please explain in detail regarding the same in the manuscript.

2. No much difference in viability was observed between 1μM or 10 μM concentration of ETC-159 and C59. And for combination analysis, ETC-159 was used at concentrations 0.313 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM. Since there is no much effect on viability beyond 1 μM concentration, authors should consider using lower concentrations and a maximum of 1 μM. The concept of analyzing combinatorial drug concentrations is to check if we could decrease the drug concentration and increase the efficacy of treatment by using two different mode of actions. Authors have not mentioned at what combination dose the tumor growth rate was determined or for the gene expression analysis. Figure 4D is missing. On what basis authors have chosen paclitaxel and gemcitabine for combination with ETC-159? May be these two drugs do not have synergistic effect with ETC-159 or the doses used for combination are not appropriate.

3. If combination of paclitaxel and gemcitabine is already known and reported, then authors should have focused on identifying if ETC-159 and combination of paclitaxel and gemcitabine can have increased efficacy. Authors can make an attempt of this combination.

4. Colony formation can be represented as images

5. Authors should carefully go through the figures and tables provided, the supplement figure 2 contains supplement table 2.

6. Statistical analysis for all the graphs needs to be provided

Reviewer #2: Hayley J Hawkins et al

Here are some constructive comments and observations you might consider for the provided based on the provided information about the research involving Examination of Wnt signaling as a therapeutic target for pancreatic ductal adenocarcinoma (PDAC) using a pancreatic tumor organoid library (PTOL), here are some questions you might consider:

I. The text provides a detailed and structured overview of the methods and procedures employed in the study.

II. The division into sections like "Human Specimens," "PDAC Patient-Derived Xenografts," and "Pancreatic Tumor Organoid Library (PTOL) Generation and Characterization" helps organize the information logically.

III. The process of generating PDAC organoid lines is clearly outlined, including details on tissue preparation, digestion, culture conditions, and media components.

IV. Assessment of Wnt Dependency:

a. the methodology for assessing the Wnt (in)dependency of PDAC tumor organoids is thoroughly explained, involving the manipulation of HPSC media and testing the effects on viability.

V. Wnt Inhibitor Studies:

a. The steps involving the use of Porcupine and Wnt inhibitors to confirm Wnt (in)dependency are well-documented, including the criteria used to define dependency.

VI. Minor Comments:

a. In certain sections, consider breaking down longer sentences for enhanced readability.

b. For instance, consider using bullet points or subheadings for better organization in sections with multiple steps.

c. The section on "Statistical Analysis of In vitro organoid drug treatment assays" is comprehensive but complex. Consider breaking down the information into subsections for clarity, separating details about individual drugs and combinations.

d. Clarify the notation "SMAD4(2, 3)." If these numbers represent specific mutations or isoforms, provide a brief explanation. Page 12 results section.

e. For better clarity, consider breaking the long sentence into two sentences: "Viability of Panc320 still required the presence of Noggin and A83-01. In contrast, Panc272 grew independently of all exogenous niche factors." Page 12

f. Clarify the significance or implications of the observed changes in gene expression in Panc320 and Panc269.

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Reviewer #1: Yes: Priya Arumugam

Reviewer #2: Yes: Manish Shukla

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Submitted filename: Review Comments.docx

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Submitted filename: Hayley J Hawkins et al.docx

Examination of Wnt signaling as a therapeutic target for pancreatic ductal adenocarcinoma (PDAC) using a pancreatic tumor organoid library (PTOL).

PONE-D-23-36221R1

Dear Dr. Hawkins,

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,

Yash Gupta, Ph.D.

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

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: (No Response)

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.

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 #2: Yes: Manish Shukla

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