-->PONE-D-26-05376-->-->Optimization of Extracellular Vesicle Extraction from Hepatic Tissue Interstitial Fluid and Analysis of Their ncRNA Expression Profiles-->-->PLOS One

Dear Dr. Gao,

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PLOS One

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Reviewers' comments:

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

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Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: Shubo Liu and colleagues investigated extracellular vesicles (EVs) isolated from hepatic tissue interstitial fluid (TIF) and analyzed the expression patterns of several lncRNAs and miRNAs in hepatocellular carcinoma. The attempt to explore ncRNA signatures in EVs derived from TIF is interesting, and the study may provide useful information for identifying potential biomarkers related to HCC. However, several issues should be addressed to strengthen the manuscript.

1. The authors isolated hepatic tissue interstitial fluid using an enzymatic digestion method. While the Discussion compares this protocol with previously reported methods, enzymatic digestion may induce cellular disruption and artificial release of extracellular vesicles during tissue processing. Therefore, it remains unclear whether the isolated vesicles truly represent EVs present in the native interstitial space or vesicles released as a consequence of tissue digestion. The authors should clarify how they minimized potential contamination from intracellular components and discuss this methodological limitation more explicitly.

2. The current study mainly provides a descriptive analysis of ncRNA expression profiles in extracellular vesicles derived from hepatic tissue interstitial fluid. Although the authors discuss the potential diagnostic value of several ncRNAs based on previous literature, the biological significance of the findings in this study remains largely unexplored. Without functional validation or mechanistic investigation, it is difficult to determine the role of these ncRNAs in hepatocellular carcinoma biology. Additional biological interpretation or functional validation would strengthen the conclusions of the study.

3. The manuscript first identifies differentially expressed lncRNAs and subsequently analyzes miRNAs. However, the rationale for presenting the relative expression relationship between these ncRNAs is unclear. It may be more informative to first analyze the biological pathways or functional processes associated with the differentially expressed lncRNAs and miRNAs separately. Subsequently, investigating whether these ncRNAs are linked to shared pathways or regulatory mechanisms could provide a more biologically meaningful basis for comparing their relative expression patterns.

4. There appears to be an inconsistency between the text and the cited figure numbers in lines 118–146. The authors should carefully review and correct the figure references to ensure that they correspond appropriately to the described results.

5. The manuscript suggests that EVs isolated from TIF may originate from liver tissue. However, it is generally difficult to determine the precise cellular origin of EVs present in interstitial fluid, as vesicles from multiple cellular sources may coexist in this compartment. Therefore, interpretations implying a definitive hepatic origin should be made with caution.

6. Although the authors detected the hepatocyte-associated membrane protein ASGPR, additional characterization using other liver-associated EV markers could further strengthen the interpretation regarding the potential hepatic origin of the isolated vesicles.

7. The characterization of extracellular vesicles should also follow established guidelines such as the Minimal Information for Studies of Extracellular Vesicles (MISEV). The authors should clarify whether their EV characterization fully meets these criteria and consider including additional validation markers, particularly negative markers for non-vesicular contaminants.

Reviewer #2: Summary

This manuscript examines a method for isolating liver tissue–derived TIF-sEVs and explores the non-coding RNAs they contain, with the aim of identifying potential biomarkers for the early diagnosis and precision intervention of hepatocellular carcinoma. However, several points in the manuscript require further clarification.

Comment:

Introduction

1. Please clarify the potential of TIF-sEVs as biomarkers for early diagnosis in cancer and other diseases, and support this statement with additional recent references.

Materials and methods

1. Please provide more detailed methodological information on the ROC analysis.

Results

1. Please clarify why six lncRNAs (e.g., AL031985, TMCC1-AS1) and eleven miRNAs (e.g., miR-1224-5p, miR-483-5p) were examined. Were these non-coding RNAs selected from the array results or from the literature?

2. Please improve the figure quality, particularly the western blot bands and font, which appear blurry.

3. The ROC analysis should also present separate curves for the lncRNA GAS5 and the miRNA hsa-miR-194-5p. Please also report the corresponding sensitivity and specificity (at the selected cutoff).

4. ROC analyses for the other non-coding RNAs should be reported either in the Results section or in the Supplementary Materials.

Discussion

1. Because the study cohort included only HCC and non-HCC groups, without stratification by HCC stage, the current analysis may not adequately support the stated objective of “providing potential biomarkers for early diagnosis.” The authors should therefore discuss this limitation and clarify how their findings relate to early-stage HCC detection.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Reviewer comment.docx

-->PONE-D-26-05376R1-->-->Optimization of Extracellular Vesicle Extraction from Hepatic Tissue Interstitial Fluid and Analysis of Their ncRNA Expression Profiles-->-->PLOS One

Dear Dr. Gao,

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.-->

• Reviewers appreciated your revised manuscript, however to improve the quality of the future article, you need to introduce the changed asked by the second reviewer.

-->Please submit your revised manuscript by Jul 29 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

-->

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Boyan Grigorov

Academic Editor

PLOS One

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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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: Partly

**********

-->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 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

**********

-->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:  I appreciate the effort the authors have made to address the issues raised during the review process and to improve the manuscript. I hope that your future mechanistic and functional studies will build upon these interesting preliminary findings and provide further insights into the biological roles of TIF-derived extracellular vesicle ncRNAs.

Reviewer #2:  Summary

The authors have addressed the reviewer’s comment and made the appropriate revisions in the manuscript. However, several aspects of the manuscript require further clarification and improvement before it can be considered for publication.

Comment:

1. The source of the lncRNA and miRNA data derived from The Cancer Genome Atlas (TCGA) hepatocellular carcinoma dataset and the Gene Expression Omnibus (GEO) dataset under accession number GSE302990 should be clearly described in the Materials and Methods section.

2. The images in Fig. 1C–E are not sufficiently clear, and the graph labels and protein names in the western blot panels appear blurred. The figure should be improved to ensure that all labels are clearly legible and of publication quality.

3. In the ROC curve analysis, please clarify why hsa-miR-194-5p was included, given that it was not identified among the eleven miRNAs and does not appear to have been reported as significantly different in the prior study. The rationale for selecting this miRNA for ROC analysis should be clearly explained.

4. If hsa-miR-194-5p and GAS5 are proposed as potential biomarkers for hepatocellular carcinoma, the authors should expand the Discussion to include their previously reported biological roles and relevance to hepatocellular carcinoma. This additional context would strengthen the rationale for their biomarker potential and better support the interpretation of the findings.

5. Sections 4.9 and 4.10 appear to have duplicate headings, both titled “qRT-PCR Detection of miRNAs.” Please revise the section titles to avoid duplication and to clearly distinguish the methods described in lines 502 and 519.

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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

**********

[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

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-->

Attachments

Attachment

Submitted filename: summary R1.docx

Optimization of Extracellular Vesicle Extraction from Hepatic Tissue Interstitial Fluid and Analysis of Their ncRNA Expression Profiles

PONE-D-26-05376R2

Dear Dr. Gao,

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,

Boyan Grigorov

Academic Editor

PLOS One

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