PONE-D-19-24224

Necroptosis in pancreatic cancer promotes cancer cell migration and invasion by release of CXCL5

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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

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: 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: Pancreatic cancer is an aggressive disease often discovered at a late stage, and treatment options are limited. Better understanding of the determinants of PC progression may lead to more effective treatments. This research article suggests possible mechanisms by which necroptosis in PC cells may contribute to tumor invasiveness, with a focus on impacts to tumor epithelium itself and not on tumor-associated macrophages or neutrophils, development of immune responses, or angiogenesis. The topic is of general interest, and the writing is clear. Note that there are many published observations regarding CXCL5, CXCR2, and CXCL1 in PC, and the authors should be meticulous in positioning their work in the context of what has already been shown.

1) The authors begin with the observation of RIP3 and MLKL expression in human PC tissue (previously published by Seifert et al 2016 Nature) then extend the observation by stating the detection is at the leading, invasive edge of the tumor. Steele et al (Cancer Cell 2016) previously showed CXCR2 expression at the PC tumor border. What was the expression pattern for CXCR2, CXCL5, and CXCL1 in the PC tissue panel? Which patients received chemotherapy, and was there any relationship to the expression of RIP3, MLKL, or the chemokines and receptor of interest?

2) Li et al (AJ Pathol 2011 178:1342) and Matsuo (Int J Cancer 2009 125: 1032) show differences between PC cells in their baseline expression of CXCL5. Matsuo et al additionally show substantially stronger expression of CXCL1 compared with CXCL5 in untreated PC lines. Does the CXCL5 released into conditioned medium after death-inducing stimulus represent stored chemokine that is released when membrane integrity is lost? Or is CXCL5 expression induced in cells undergoing programmed cell death, in the bystanders not undergoing cell death, or both? A repeat of immunofluorescence staining as performed in Figure 2, with a time course that includes CXCL5 detection, would clarify this point. Is CXCL1 also released into conditioned medium? Is expression of CXCL2 impacted by necroptotic stimuli? Is the in vitro activity of conditioned medium dependent on CXCL1?

3) The in vitro analysis would be strengthened with the inclusion of a relevant, necroptosis-inducing chemotherapeutic, for comparison with the experimental TSZ necroptosis induction strategy chosen. Any changes in CXCR2 expression related to treatment in the chosen cell lines should be shown (immunoblot, mRNA).

4) The number of times in vitro experiments were performed is missing in places.

Reviewer #2: The authors examined roles of necroptosis of human pancreatic cancer cells by immunohistochemistry of cancer samples and in vitro experiments. They show that CXCL5 is a key mediator from necroptotic pancreatic cancer cells and promotes invasion and migration of cancer cells via the CXCR5/CXCR2 axis. This result suggests that, although necroptosis of cancer cells is an anti-cancer effect, this type of cell death could enhance cancer progression. The reviewer supposes that this information is useful for the readers of PLOS ONE.

Minor comments:

1) Page 22, line 354 and 355. Two different description, such as S1 Figs a-c and Supplementary Fig 1d.

2) Page 19, line 297-298. Cleaved caspase-8 was upregulated in TS-treated PANC-1 cells (Fig. 2d). However, it seems no change.

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Reviewer #1: No

Reviewer #2: No

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Necroptosis in pancreatic cancer promotes cancer cell migration and invasion by release of CXCL5

PONE-D-19-24224R1

Dear Dr. Ohuchida,

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,

Yi-Hsien Hsieh, 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

**********

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

**********

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 made a good faith effort to address the comments provided to them. Additional references cited for context and considerable additional data have been incorporated into the manuscript. The manuscript provides a more complete picture of CXCL5, CCR2, and CXCL1 expression in tumor tissues and in PC lines challenged with necroptosis-inducing stimuli. Additional clarity has been provided in figure legends. The current version of the manuscript meets publication criteria.

Reviewer #2: MS# PLOS-D-19-24224R1

The authors respond to my comments properly. The reviewer supposes that this study is worth publishing in the PLOS ONE.

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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: No

Reviewer #2: No