Dear Dr. Noh,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Amr Ahmed El-Arabey

Academic Editor

PLOS ONE

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7. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Scientific Information

1. Inadequate Research Gap Justification

o The introduction is overly broad and fails to specify the precise knowledge gap this study fills or why it is necessary.

It requires more solid placement in the body of current literature.

The second is methodological transparency.

o A completely replicable process is required by PLOS ONE. A few details are lacking: the recruitment process and sample size calculation are imprecise, and the precise inclusion/exclusion criteria are not properly specified.

The way statistical approaches handle missing data and correct for confounding is not explained.

3. Results Presentation: Some findings are descriptive and lack critical analysis.

o According to PLOS ONE statistical reporting guidelines, p-values should be accompanied with confidence ranges.

4. Discussion o Excessively detailed and devoid of critical comparison with the most recent research.

Given the cross-sectional/observational study methodology, a number of statements are exaggerated.

o The limitations section is inadequate and needs to address potential biases (generalisability, reporting, and selection) in greater detail.

5. Conclusion: Overly conclusive; has to be careful and closely linked to the results of the study.

Formatting and Journal Style Issues

1. Abstract: Structured abstracts (Background, Methods, Results, Conclusion) are required by PLOS ONE. The abstract as it stands now is repetitious and narrative.

2. References: The formatting is not in accordance with PLOS ONE policy, which requires a numbered citation style based in Vancouver.

o A number of the references are out of date; there are no fresh sources from 2022–2024.

3. Tables and Figures: o A few figure legends lack details and make it impossible to understand them without consulting the main text.

o There is excessive narrative material in the tables, which deviates from journal formatting guidelines.

4. Language and Grammar o Several typographical and grammatical mistakes.

o Uncomfortable wording (such as "patients was," "can able to") makes it harder to read.

o For clarity and flow, extensive English editing is required.

Reviewer #2: This manuscript entitled “The Role of exosomal miRNA-125b derived from colon cancer-associated fibroblasts in skeletal muscle cachexia” explores the contribution of CAF-derived exosomal miR-125b to skeletal muscle atrophy. The study is interesting and addresses an important gap in understanding how stromal components of the tumor microenvironment influence cancer-associated cachexia. Overall, the manuscript is clearly written and logically structured, but several issues related to experimental robustness, quantitative analysis, and mechanistic depth should be addressed before acceptance.

1. miRNA sequencing appears to have been performed on a single CAF-derived exosome sample. This significantly limits the robustness of the findings. At minimum, please validate the enrichment of miR-125b across all CAF isolates using qRT-PCR and acknowledge this limitation explicitly in the Methods and Discussion.

2. The conclusions are primarily based on morphological observations (myosin diameter). Without functional markers (e.g., atrogin-1, MuRF-1, MyoD, Myogenin, or IGF-1 pathway readouts), it remains uncertain whether the observed changes truly represent cachexia-related atrophy rather than a general cytoskeletal alteration. Incorporating at least one molecular validation experiment or a more nuanced discussion of this limitation is strongly encouraged.

3. The statistical analysis relies exclusively on Student’s t-tests, even in cases where more than two groups were compared (e.g., Figs 4, 7, 8, 9, 10). Please confirm that all data met the assumptions of parametric testing or use one-way ANOVA with post-hoc correction. Figure legends should include n values, the type of error bars (SD or SEM), and exact p-values.

4. The reported mean particle size of ~28 nm is notably smaller than typical exosomes (50–150 nm). This discrepancy raises concern about the accuracy of NTA measurements or sample purity. Please re-examine the data or provide an explanation (e.g., detection threshold differences or aggregation).

5. The manuscript occasionally overstates causality. For example, asserting that miR-125b “directly induces muscle atrophy.” Given the correlative nature of the current data, the statement should be tempered and rephrased to reflect a potential association or preliminary mechanistic indication.

Other comments:

- The discussion is generally well-written but repetitive. It should focus more on how CAF-derived miR-125b differs from tumor-cell-derived or serum exosomal miRNAs.

- The limitations section is appreciated but should include the lack of in vivo validation and absence of protein-level mechanistic studies.

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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 #1: Yes: Omer Al-lela

Reviewer #2: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

The Role of exosomal miRNA-125b derived from colon cancer-associated fibroblasts in skeletal muscle cachexia

PONE-D-25-43356R1

Dear Dr. Noh,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Amr Ahmed El-Arabey

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The text was meticulously edited after peer review to take into account the feedback and recommendations from each reviewer. Clarifying the study's goals and reasoning in the Introduction, adding more methodological information to promote repeatability and transparency, and improving the results' presentation with better tables and figures when necessary were among the changes made. Expanding the interpretation of the results, enhancing the comparison with pertinent literature, and more explicitly recognising the limits of the study all boosted the Discussion section. To enhance the manuscript's coherence, clarity, and scientific readability, linguistic and stylistic changes were also made. The manuscript's overall quality and rigour have been much enhanced by these modifications.

Reviewer #2: The authors have adequately addressed all comments raised in the previous round of review. The revised manuscript is clearer, better structured, and the experimental findings are appropriately supported by the presented data. The study provides novel and relevant insights into the role of cancer-associated fibroblast–derived exosomes in cancer cachexia. The experimental design is sound, the data are well presented, and the conclusions are justified within the scope of the study. Limitations have been transparently acknowledged and appropriately discussed.

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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 #1: Yes: Omer Qutaiba Al-lela

Reviewer #2: No

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