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Academic Editor

PLOS One

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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?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: This is an interesting pan-cancer plus mechanism paper with real experimental effort, but it still feel overextended and under-contextualized in places, so a major revision make sense to tighten the logic and strengthen the translational angle.

In the introduction, the background read solid but also a bit generic, and it would really benefit from stepping back and framing PRDX3 within the broader cancer biomarker landscape instead of jumping so fast into oxidative stress biology. Biomarker discovery is not just about association but about eventual utility, and this is where a short conceptual bridge help a lot. I strongly suggest anchoring the rationale by briefly discussing how discovery-stage biomarkers move toward prognosis, stratification, and eventually intervention, for example by citing “Bridging Discovery and Treatment: Cancer Biomarker,” which fit naturally here and give the reader a clearer sense of why PRDX3 matter beyond being another differentially expressed gene. That kind of framing would also make the pan-cancer scope feel more intentional rather than simply exhaustive.

For the results sections that rely heavily on TCGA analyses, the overall workflow is acceptable and quite standard, but the manuscript treat TCGA almost as a neutral truth source, which is risky. Many readers already know this, but it still need to be said explicitly. It would help to situate the approach within prior TCGA-based efforts, such as TCGA-Based Analysis of Oncogenic Signaling Pathways Underlying Oral Squamous Cell Carcinoma or Immune prognostic model for glioblastoma based on the ssGSEA enrichment score, just to show that the strategy follow an established analytical tradition. At the same time, this is exactly where a more critical tone is needed. Bulk transcriptomic resources come with built-in biases, in sample composition, stromal contamination, batch effects, and clinical heterogeneity, and ignoring this weaken the interpretation of differential expression, immune infiltration, and pathway enrichment. A brief but explicit acknowledgement, supported by “Genetic Expression in Cancer Research: Challenges and Complexity” (2024) and “Technical and Biological Biases in Bulk Transcriptomic Data Mining for Cancer Research” (2025), would go a long way toward making the conclusions feel more measured and trustworthy.

When moving into the functional and immune-related analyses, the story become interesting but also a bit scattered. The immune microenvironment results are extensive, yet the biological implications are not always clear, and the paper sometimes describe correlation without really slowing down to explain what that might mean in a tumor context. I think the authors should lean more into interpretation here, especially where PRDX3 appear linked to immune modulation, checkpoint markers, or stromal features. Even a few sentences per subsection that ask what this could imply for therapy response or resistance would make the data feel less like a catalogue and more like a hypothesis-generating framework. here you should mention review paper "Identification of new immune target and signaling for cancer immunotherapy".

The experimental validation in KIRC is a strength, but the discussion stop short of translating this into a broader drug development perspective. Biomarkers do not live in isolation, and the manuscript would benefit from explicitly addressing how PRDX3-associated pathways, particularly the PPAR axis, could inform therapeutic targeting, patient selection, or combination strategies. In this context, citing and briefly discussing “Fast, tracking drug development with biomarkers and companion diagnostics.” would fit very naturally and help bridge the gap between molecular mechanism and clinical application. This does not require overclaiming, just a clearer statement of how such a biomarker could realistically be used.

Finally, the discussion and future outlook feel a bit conservative given how much data is presented. With deeper insight into tumor biology, cancer treatment is clearly moving toward personalized and precision approaches, especially those targeting the tumor immune microenvironment and specific molecular dependencies. I encourage the authors to end on a more forward-looking note, connecting their findings to emerging therapeutic paradigms and the idea that PRDX3-related vulnerabilities could eventually support tailored intervention strategies. Referencing “Overview of perspectives on cancer, newer therapies, and future directions” would help frame this vision and signal that the work is aligned with where the field is heading, even if much validation still remain to be done.

Reviewer #2: This study presents an interesting multi-omics investigation into the role of PRDX3 across 33 cancer types, with a specific focus on Kidney Renal Clear Cell Carcinoma (KIRC). While the bioinformatic integration is a clear strength, the manuscript requires additional experimental depth and technical clarification to support the authors’ mechanistic claims.

- Please add recent statistics regarding kidney cancer prevalence and survival rates in the Introduction to provide a clearer background on the disease’s impact.

- Correlation between PRDX3 expression and PPAR pathway markers via Western Blot is insufficient to claim a regulatory relationship.

- Clarify if the reduced migration/invasion observed in KIRC cells is a direct effect on motility or a secondary effect of decreased cell proliferation.

- The study notes that PRDX3 exhibits "dual roles" in tumor biology across different cancers. Please provide a table or supplementary figure clarifying which of the 33 cancers show PRDX3 as an oncogene/ an antioncogene.

- The authors should provide data on the intracellular ROS levels in KIRC cell models after PRDX3 manipulation.

- Please justify the choice of KIRC cell lines used. Are these lines representative of the "high-methylation" or "low-methylation" groups identified in your TCGA analysis?

- While the in vitro data supports the role of PRDX3 in KIRC, the lack of animal models makes it difficult to assess the tumor microenvironment and immune response mentioned in the conclusion.

- The authors state that 50 pg of protein was loaded for Western blot analysis. Standard protocols typically employ 20 - 50 µg. Could the authors clarify if this is a typographical error? If 50 pg was indeed the amount used, please provide details on the high-sensitivity detection system or ultra-pure recombinant protein used to achieve a signal at this level.

- The English needs improvement, and there are some errors.

- Be careful of abbreviations.

Reviewer #3: 1. Why do authors particularly chose KIRC not any other cancer type cell lines that are more significant in tumor microenvirnoment? Justify.

2. How the findings can be clinically applied?

3. Fig.2 represent the same information so except Fig.2A others can be moved to supplement file.

4. Clarify the term TBM whether it is TMB or TBM

5. Fig. 4, Fig. 6, Fig.7 results does have much correlation with KIRC. Justify their importance or move them to supplement file.

6. Give a brief description of peroxisome proliferator-activated receptor signaling pathway in KIRC.

7. Give a pictorial representation of the identified pathway mechanism.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Yingcai Hong

Reviewer #2: No

Reviewer #3: Yes: Priyadharshini Annadurai

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Multiple omics analyses and experiments validation identify PRDX3 as a biomarker of prognosis and antioncogene in kidney clear cell carcinoma

PONE-D-25-58601R1

Dear Dr. Chen,

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,

Xiaoen Wei

Academic Editor

PLOS One

Additional Editor Comments (optional):

The authors have satisfactorily addressed the major concerns raised in the previous round of review

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

Reviewer #2: (No Response)

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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 #2: No

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