-->PONE-D-26-13623-->-->Caerin 1.1 and 1.9 inhibit glioblastoma growth by regulating the ARHGAP26-β-catenin axis and enhancing intratumoral CD8+ T cell infiltration-->-->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: 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: Yes

Reviewer #2: 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

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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 titled, “Caerin 1.1 and 1.9 inhibit glioblastoma growth by regulating the ARHGAP26–β-catenin axis and enhancing intratumoral CD8⁺ T cell infiltration” investigates the anti-tumor effects of caerin peptides (1.1 and 1.9) in glioblastoma using in vitro assays, xenograft models, and a PBMC-humanized mouse model. The study addresses an important clinical challenge in glioblastoma and presents a multi-layered experimental approach. The integration of tumor-intrinsic signaling and immune modulation is a strength. However, several issues regarding mechanistic rigor, data interpretation, redundancy, and clarity need to be considered before the manuscript can be considered for publication.

Major Comments:

1.The central claim that ARHGAP26 mediates the anti-tumor effects is not fully supported. The study shows correlation, however, no causal experiments are conducted. There is no knockdown or overexpression validation of ARHGAP26.

2.The manuscript concludes that caerin peptides induce “atypical pyroptosis.” However, IL-1β and IL-18 secretion is minimal or absent, Key pyroptosis effectors (e.g., GSDMD) are downregulated and no direct evidence of inflammasome activation is provided. Hence, The data do not convincingly support pyroptosis as a major mechanism.

3.The manuscript states that apoptosis is the primary mechanism of cell death based on gene expression data. But there is no functional validation like caspase inhibitor assay or annexinV/PI quantification. Authors should perform additional validation experiments to confirm the mechanism of cell death

4.The PBMC-humanized mouse experiments use very small group sizes (n=3). This limits statistical power, weakens conclusions about immune mechanisms. Authors should increase the sample size or explicitly acknowledge this limitation in the Discussion.

5.The study shows increased CD8⁺ T cell infiltration but does not assess their functional state. Increased infiltration does not necessarily imply enhanced anti-tumor activity. Authors should include functional assays to support their claim

Minor Concerns

1.The description of PCR array results is excessively long and appears twice (Results and Discussion). Summarize key findings concisely in the main text.

2.Several grammatical errors and awkward phrases are present.

3.The number of replicates and independent experiments is not always clearly stated. Clarify whether data represent biological or technical replicates.

4.Ensure all figures include exact n values, statistical tests used and clear labeling of error bars

5.Use consistent terminology for pyroptosis-like death and immune micro-environment remodeling

Reviewer #2: This is a well-conducted and clinically relevant study that raises one of the most encountered problems in GBM: Resistance to all treatment modalities, including immune checkpoint inhibitors and CAR-T cell therapies. The authors investigate two host-defense peptides, caerin 1.1 and 1.9 (F1/F3), in U87 and U118 glioblastoma cells and propose ARHGAP26–β-catenin signaling as a mechanistic axis that links peptide treatment to the suppression of pro-invasive effectors, including MMP2, MMP7, and VEGFA. In immunodeficient hosts, F1/F3 has no measurable effect on tumor growth, but in the humanized group, it inhibits U87 growth, increases infiltration of CD8⁺ T cells, promotes M1 polarization, and reduces tumor-cell PD-L1. The paper is a meaningful addition to the field, and I recommend minor revisions.

The study design is appropriate. My main comment concerns about the interpretation of the humanized NSG model, which is the most important point. The reconstituted human T cells in this system are naive, they have not been chronically stimulated, and lack the exhausted phenotype that defines tumor-infiltrating lymphocytes in real GBM patients. It would be worth adding a brief paragraph discussing whether there is a feasible way to introduce a naive immune compartment into human GBM patients, whether prior literature has addressed this question, or at least acknowledging it as a limitation. The analyses are appropriate, consistent variance reporting, and standard significance thresholds. The supplementary tables and figures are available, The manuscript is well written.

Overall the central finding is well-supported and contributes meaningfully to a field where most candidate therapies fail. The most important revision is the framing of the humanized model.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Comments_PlosONE.docx

Caerin 1.1 and 1.9 inhibit glioblastoma growth associated with modulation of the ARHGAP26-β-catenin axis and enhancing intratumoral CD8+

T cell infiltration

PONE-D-26-13623R1

Dear Dr. Wang,

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,

Ryota Tamura

Academic Editor

PLOS One

Additional Editor Comments (optional):

This manuscript is in a nice condition. Thank you very much for your effort.

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: Manuscript has been revised appropriately and all the shortcomings have been addressed. It can be accepted for publication

Reviewer #2: The authors have satisfactorily addressed all the comments and recommendations raised in the previous review. The revised manuscript reflects the requested corrections, and I have no further concerns regarding the methodology, results, or interpretation presented. I recommend acceptance of the manuscript in its current form.

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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

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