PONE-D-20-05598

SULF1 suppresses Wnt3A-driven growth of bone metastatic prostate cancer in perlecan-modified 3D cancer-stroma-macrophage triculture models

PLOS ONE

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3. Bone marrow tissue specimens containing PCa tumors were obtained with consent under IRB approved protocols at The University of Texas Health Science Center School of Dentistry.'

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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: Partly

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

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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5. Review Comments to the Author

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Reviewer #1: Authors of the manuscript examined the role of SULF1 in reactive bone marrow stroma within metastatic bone microenvironment using biomimetic hydrogel incorporating tumor, fibroblast and macrophage cell types. The function of SULF1 in Wnt3A-mediated prostate tumor growth was also examined. Authors concluded that SULF1 loss favors prostate cancer progression by inability to suppress Wnt3A-mediated growth signals in the stroma. Despite of an interesting model with potential relevance to bone metastasis field, there are several issues with this manuscript that need to be addressed by the authors:

1) The significance of studying SULF1 in a context of Wnt3A is not clear. Aside from stating that Wnt3A is a key HBGF implicated in PCa progression there is little rationale provided for investigating the SULF1-Wnt3A axis. Better background needs to be provided by the authors

2) Fig.1 – it is concerning that authors were not able to detect SULF1 protein with any of the available SULF1 antibodies

3) Fig.2 – RNA signal is very difficult to visualize; mock controls need to be shown for RISH

4) Fig.3 – the presence of macrophages in bone marrow tissue is not surprising and has been shown before; the statement that macrophages accumulate around tumors is too strong based on data provided; lack of CD163 expression in the biomimetic model is concerning. CD163 is an M2 marker known to be expressed by the macrophages in the metastatic microenvironment

5) How was experimental concentration of Wnt3A determined for the studies in Fig. 5?

6) If macrophage-derived TNFa is suspected to be a key-contributor to SULF1 expression, this needs to be shown. Do TNFa levels in macrophages change in response to interaction with tumor cells or in triculture?

7) Fig 5- why was not staining for macrophage markers performed rather than using sphericity filter? This is not an accurate way of identifying macrophages, and certainly an approach that does not allow to look at specific phenotypes of macrophages. Not all macrophages are vimentin-positive. What was the significance of only looking at vimentin-positive macrophages?

8) Overall contribution of macrophages to tumor cell growth in this model is overstated. The data, as provided, do not convincingly support the conclusions

9) The model is somewhat confusing. An increase of SULF1 expression in the stroma and loss in the tumor cells is an interesting concept that needs to be investigated. Since the analyses of human databases indicate that SULF1 is lost in metastatic tumors (which one would presume contain both tumor cells and the stroma) – how do the data presented here fil with these findings. This disconnect between SULF1 levels in the stroma vs the tumor needs to be better demonstrated by the authors. The experimental evidence provided here is not very strong.

Minor points:

1) The sentence in Discussion (page 7, line 192) is incomplete: “Here we report that the up-regulation of SULF1 and HSPG2 transcripts in CAFs by M2-like macrophages.”

2) The references to Supplementary Figures in text are not in agreement with Figures and Figure Legends provided. For example: Fig S5 referenced in text should actually be S6, Fig S6 should be S7, and so forth….. In addition, no reference at all is made to Fig S10 in text.

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Reviewer #1: No

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SULF1 suppresses Wnt3A-driven growth of bone metastatic prostate cancer in perlecan-modified 3D cancer-stroma-macrophage triculture models

PONE-D-20-05598R1

Dear Dr. Farach-Carson,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Daotai Nie, 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

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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

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

Reviewer #1: 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

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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: This reviewer has no additional concerns. Authors adequately responded to the comments and provided additional data to support their conclusions

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Reviewer #1: No