PONE-D-19-19270

The Clinicopathological Significance of Thrombospondin-4 Expression in the Tumor Microenvironment of Gastric Cancer

PLOS ONE

Dear Dr. Yashiro,

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.

(1) Immunostaining of Podoplanin (PDPN) as a widely accepted marker of cancer associated fibroblasts (CAFs)

(2) Detailed methodological description of CAFs and normal-associated fibroblasts (NAFs) and presentation of their images

(3) Explanation about THBS4 expression in stroll cells but not cancer cells in primary gastric cancer, as well as presentation of more clear images of THBS4 immunostaining, and results of THBS4 immunostaining

(4) Presentation and statistical analysis related to Western blot analysis

(5) Refinement of Kaplan-Meier analysis

(6) Additional experiments or discussion on the functions of thrombospondin-4 (THBS4) in CAFs

(7) Other issues pointed out by Reviewers

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Masaru Katoh, M.D., Ph.D.

Academic Editor

PLOS ONE

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This study is partially founded by KAKENHI Grant-in-Aid for Scientific Research, Nos. 18H02883 (M.Y.).

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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: Partly

Reviewer #3: Yes

Reviewer #4: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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: No

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #4: 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: This manuscript is to evaluate that THBS4 expressed in CAFs is a prognostic marker to predict poor prognosis in gastric cancer. They proved that THBS4 was expressed in stromal cells expressing alpha-SMA in gastric cancer, and positive THBS4 expression is correlated with poor prognosis of gastric cancer patients. The results are clear, but for publication in Plos One, following points should be fixed.

1. Authors determined the expression of THBS4 in gastric cancer tissues according to the intensity and the percentage of stained cancer cells and stromal cells (Page 4, Line 71). However, they said that it was only expression in stromal cells. How did authors calculate the percentage of positive THBS cells? Was the percentage meaning the ratio of positive stromal cells in all cells including cancer cells and stromal cells?

2. In figure 1, the positive staining of THBS4 look like too diffuse in the entire tissues. Can authors show the clearer photo for THBS4 staining?

3. The gastric cancer generally showed histologic heterogeneity according to the classification. In figure 1, I recommend that authors show the representative photos for THBS4 expression in two different histological subtypes (diffuse and intestinal type).

4. In figure 1, the staining photo of cytokeratin, cancer cell specific maker, should be added to support that THBS4 was not stained in cancer cells.

5. In figure 3, authors just showed the expression of THBS4 in NAF and CAF. I recommend authors that the results should be added the expression of THBS of several gastric cancer cell lines.

6. Authors described in abstract and conclusion that “THBS4 from CAFs is associated with the metastasis of cancer cells” and “THBS4 from CAFs might be associated with the invasion of cancer cells”. However, they did not show the any data for proof of concept. Authors should add the experimental data to prove them, or the descriptions in abstract and conclusion should be toned down.

7. Authors emphasized that THBS4 is especially expression on CAFs of macroscopic type-4 gastric cancer. It would be better to add the results of subgroup analysis for type-4 or other type gastric cancer.

8. Authors insisted that non-significance of stage II would be explained as recurrent pattern and adjuvant chemotherapy. To supper this hypothesis, recurrent pattern or adjuvant regimen according to the stage should be added.

Reviewer #2: The authors investigated the clinicopathological significance of stromal thrombospondin-4 expression in the tumor microenvironment of gastric cancer, and found that THBS4 was expressed on CAFs. Stromal THBS4 was associated with the metastasis of cancer cells. The results are interesting and promising. However, this article suffers from several major flaws:

1. The authors used α-SMA to indicate the location of CAFs. However, α-SMA is a microfilament protein with contractile ability, mainly expressed at myoepithelial cells. Although α-SMA expression could also appear in the transformation of fibroblasts to myofibroblasts around cancer cells, the positive rate of α-SMA in CAFs is not high, and some of muscle tissues may be unexpectedly positive. I recommend the authors to add podoplanin (PDPN) in immunostaining assays, which is a widely accepted marker for CAFs with a relative high expression level.

2. Primary culture was performed to obtain CAFs and NAFs in this research. However, there was no demonstrations or photos to show the status of cells in order to verify the results. And the isolations and purifications of CAFs and NAFs were not mentioned either.

3. Inconsistent data were found between abstract and main text (e.g. positive rate of THBS4 expression).

4. For WB assay, the authors did not elucidate the sample size and necessary statistical analysis (t-test). A solitary result cannot clarify the real world.

5. According to the clinical data, THBS4 in stroma has obvious correlations with tumor invasion and lymph node metastasis. Since the primary CAFs have been cultured successfully, the authors should perform gene function experiments (e.g. migration & invasion) subsequently and explore the potential mechanisms to explain the phenomena they observed.

6. Are there any references to support the IHC evaluating method of THBS4 expression? If yes, please cite the articles, otherwise the authors should state the reason why score 3 was the watershed.

7. Terms were not used properly in this article. NAF was not common in the description of normal fibroblast. And definitions of positive and high were confused.

Reviewer #3: Dear editors,

In this study, the authors mainly investigated the clinicopathological value of THBS4

In gastric cancer based on the retrospective cohort from their own center. The research methods were statistical analyses of clinical data, including chi-square test, Cox proportional hazards regression models, as well as survival curves. The endpoint parameter was overall survival (OS). The article was well written, and focused on practical clinical aspects. While some problems still existed and should be improved.

1. The exact correlation of α-SMA or CAFs and THBS4 should be explained more apparently both in Introduction and Discussion.

2. The qualities of all K-M curves were poor. We believe this problem should be focused since key results of this study were reflected herein. Some normative forms could be referenced, for instance, ANNALS OF ONCOLOGY.

3. Information from Figure 3 was too simple to explain exact relation between the expression of THBS4 and α-SMA or CAFs.

4. References should be updated by using articles in recent 5 years.

Reviewer #4: The authors Kuroda et.al have identified THBS4 as a marker of CAFs in gastric cancer. IHC analysis on 584 GC patients specimen shows statistical significance when THBS4 expression is compared with the clinico-pathological characteristics of the patients. However, I have some comments with respect to the methodology of CAFs and NAFs isolation for western blot analysis. The authors have not elaborated on the specific markers that were used to identify these from the samples.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Kakoli Das

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The Clinicopathological Significance of Thrombospondin-4 Expression in the Tumor Microenvironment of Gastric Cancer

PONE-D-19-19270R1

Dear Dr. Yashiro,

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,

Masaru Katoh, M.D., 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

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 #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

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 #1: Yes

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 #1: Authors revised manuscript according to reviewers' comments.

It is acceptable to be published in PlosOne

Reviewer #2: The authors answered most of the questions I raised, and modified the mistakes. I am satisfied with the revised version.

Reviewer #3: (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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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No