Treatment of Mesenchymal Stem Cell-Derived Extracellular Vesicles with Extract of Cultured Lentinula edodes Modulates Breast Cancer Stem Cells and MicroRNA Reprogramming

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study addresses a highly relevant clinical problem by investigating an innovative and biologically meaningful approach to check the modulation of chemoresistance using AHCC-treated MSC-derived extracellular vesicles. The following comments should be addressed

1. In the results, "miRNA analysis revealed that AHCC remarkably affected the expression of several microRNAs, amongst which are miR-155, miR-34a, miR-Let7 and miR-200c."  Not clear whether upregulated or downregulated 
2. Some latest references can be added in the introduction 
3. Was a cell viability assay performed before any cell-based assay experiments to distinguish viable and non-viable cells?
4. Use the correct unit of volume. (ml use mL)
5. I recommend using the same font style for the graph on the y-axis  
6. Why was the positive control not used?
7. The control cell line should be used to check the modulation in the normal cell line
8. Why was 24 hours selected for expression?

Author Response

Comment 1

In the results, "miRNA analysis revealed that AHCC remarkably affected the expression of several microRNAs, amongst which are miR-155, miR-34a, miR-Let7 and miR-200c." Not clear whether upregulated or downregulated.

Response:
We thank the reviewer for pointing out this lack of clarity. We would like to clarify that the direction of microRNA regulation was already described in the original version of the manuscript using the terms “decrease” and “increase.” However, to further improve clarity and avoid any possible ambiguity, we have revised the figure 1 and Abstract sections to state the direction of regulation more explicitly. The revised text now clearly indicates that AHCC decreased the expression of the oncogenic miR-155, while increased the expression of the tumor-suppressive microRNAs miR-34a, miR-Let7a, and miR-200c in both wild-type and doxorubicin-resistant MCF-7 cell.

 

 

Comment 2

Some latest references can be added in the introduction.

Response:
We agree with this suggestion. Recent and relevant references (2023–2025) have now been added to the Introduction, particularly in sections discussing cancer stem cells, miRNA-mediated chemoresistance, and the role of natural compounds and nutritional interventions in cancer therapy. These additions strengthen the clinical and translational relevance of the study.

 

Comment 3

Was a cell viability assay performed before any cell-based assay experiments to distinguish viable and non-viable cells?

Response:
We appreciate this important question. In this study, the AHCC concentration (4 mg/mL) and exposure time (24 h) were selected based on our previously published work, in which cell viability assays confirmed the absence of cytotoxic effects at these conditions. Therefore, the observed effects reflect biological modulation rather than nonspecific cytotoxicity. This clarification has now been added to the Materials and Methods and Discussion sections.

 

Comment 4

Use the correct unit of volume. (ml use mL)

Response:
Thank you for noting this formatting issue. All volume units throughout the manuscript have been corrected to SI-compliant notation (mL).

 

Comment 5

I recommend using the same font style for the graph on the y-axis.

Response:
We agree. All figures have been revised to ensure consistent font type and size across axes, labels, and legends, in accordance with MDPI figure guidelines.

Comment 6

Why was the positive control not used?

Response:
We acknowledge this comment. The study was designed to compare AHCC-treated MSC-derived EVs with naïve MSC-derived EVs, which served as the biologically relevant control to isolate the specific effect of AHCC modification. A classical pharmacological positive control was not included, as the objective of this study was to evaluate EV-mediated modulation of microRNA expression and cancer stem cell behavior, rather than direct cytotoxic drug activity. This rationale has now been clarified in the Methods and Discussion sections.

.

 

Comment 7

The control cell line should be used to check the modulation in the normal cell line.

Response:
We thank the reviewer for this valuable suggestion. While the present study focused on chemoresistant versus sensitive breast cancer models, we fully agree that inclusion of a normal epithelial control would provide additional insight. This has been acknowledged as a limitation of the current study, and we have explicitly stated in the Future Investigations section that normal mammary epithelial cells will be incorporated in subsequent studies.

 

Comment 8

Why was 24 hours selected for expression?

Response:
The 24-hour time point was selected based on our prior studies and existing literature demonstrating that miRNA transcriptional changes occur rapidly and are reliably detectable within this timeframe, particularly for epigenetic and signaling modulation. Moreover, this time point minimizes secondary effects such as differentiation or nutrient depletion, allowing assessment of direct regulatory effects. This justification has now been added to the Methods and Discussion sections.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigates the effects of AHCC (Lentinula edodes extract) on mesenchymal stromal cells (MSC) and the extracellular vesicles (EVs) derived from them, focusing on modulation of selected microRNAs and functional consequences on breast cancer cell lines (MCF-7 and MCF-7/DOX). The topic is timely and scientifically relevant, and the model selection is appropriate. However, the study suffers from major methodological inconsistencies, insufficient EV characterization, unclear quantification procedures, and several critical gaps in experimental design. The manuscript requires substantial revision before it can be considered for publication.

 

1. Different sections list incompatible doses (40 mg/mL, 4 mg/mL, 3 mg/mL, 4 µg/mL). These contradictions undermine reproducibility and must be corrected with a clear description of preparation, dilution, and justification of selected concentrations.
2. EV identity is not convincingly demonstrated. The study lacks required MISEV-compliant validation:

• No Western blots for EV-positive markers (CD9, CD63, CD81, TSG101) and a negative marker (calnexin).
• NTA results are incomplete: no size distribution graphs, no modal/median size, no replicate details, and no instrument parameters.
  Without these, the particles cannot be confirmed as EVs.

1. The authors inconsistently use protein content (µg/mL) and particle number (particles/mL). It must be clarified which metric was used for dosing, how it was normalized to cell number, and whether particles and protein correlate across conditions.
2. Inadequate controls to demonstrate miRNA transfer via EVs
   Conclusions that EVs deliver miRNA to cancer cells are premature. Missing essential controls include:

• EV-depleted conditioned medium,
• heat-inactivated/refolded EV controls,
• RNase ± detergent assays to confirm membrane-protected RNA,
• fluorescently labeled EV uptake experiments.

1. Incorrect reference gene for miRNA qPCR
   The use of SNORD65 (mmu) is inappropriate for human cell lines and may invalidate normalization. Authors must use a validated human small RNA reference (e.g., U6, RNU48) and demonstrate stability.
2. Biological replicate numbers are unclear. Multiple t-tests are used where ANOVA is required. Exact p-values, effect sizes, and distribution checks are missing. All figures must include n, error bar type, and statistical tests.
3. Insufficient methodological detail
   Key steps are missing: MSC culture conditions, RNA quality control (RIN), RT parameters, qPCR primer identifiers, EV storage cycles, sphere quantification criteria, blind analysis, etc. These are required for reproducibility.
4. Figures
• Unit errors (µg vs mg) appear in legends.
• Images lack scale bars and adequate resolution.
• NTA data show only particle concentration without size distribution.
• Sphere/colony images, if included, require much clearer presentation.
• Figure legends must include n, statistical tests, and full descriptions.

Abstract

Clear but overly vague: should provide actual fold-changes and specific p-values. Avoid subjective phrasing (“remarkably affected”). State exact AHCC concentrations. Focus on aim of the study. Include the purpose of the research in the abstract.

Introduction

The background on miRNAs and CSC biology is adequate but should better justify the selection of miR-155, miR-34a, let-7a, and miR-200c. The introduction needs to be shortened.

 

Methods

• Correct AHCC dosing scheme,
• Detailed EV isolation workflow (pre-clearing, filtration, kit volumes),
• Full NTA protocol and data reporting standards,
• Information about biological vs technical replicates,
• Detailed qPCR workflow (RNA input, primer IDs),
• Check upper and down index in whole manuscript

 

Comments on the Quality of English Language

The manuscript contains numerous stylistic inconsistencies and repetitive phrasing that reduce the clarity of the text. Sentence structure is often uneven, with several overly long constructions that should be divided into shorter and more precise statements to improve readability. Grammar and punctuation require careful revision, particularly regarding article usage, verb tenses, and comma placement. The English language is occasionally imprecise, and some terminology is used inconsistently, which should be standardized throughout. It is also important to carefully verify the correct formatting of superscripts and subscripts, as several instances appear inconsistent or unclear and may affect the interpretation of biological data and numerical values. Overall, the manuscript would significantly benefit from thorough language editing by a native English speaker or a professional scientific proofreading service to ensure clarity and consistency.

Author Response

1. Different sections list incompatible doses (40 mg/mL, 4 mg/mL, 3 mg/mL, 4 µg/mL). These contradictions undermine reproducibility and must be corrected with a clear description of preparation, dilution, and justification of selected concentrations.

 

 

We thank the reviewer for identifying the inconsistent AHCC doses reported across sections. We have now standardized and corrected all AHCC concentration units and values throughout the manuscript and added a clear description of stock preparation, dilution, and final working concentrations to ensure full reproducibility.

Specifically, AHCC was prepared as a 40 mg/mL sterile-filtered stock solution and diluted to a final working concentration of 4 mg/mL for cell exposure experiments. This selection is consistent with prior breast cancer stem cell/mammosphere studies from our group, where 2 and 4 mg/mL AHCC were used and biological effects were assessed at defined time points (including 24 h) (Graham et al., 2017). All instances of “4 µg/mL” were identified as unit/typographical errors and have been corrected to 4 mg/mL. Any remaining concentration values (e.g., 3 mg/mL) that appeared in earlier drafts have been reconciled to match the finalized dosing scheme and are now presented consistently across the Abstract, Methods, Results, figure legends, and text.

In the revised manuscript, we also explicitly state where AHCC is applied (directly to breast cancer cells vs. MSC conditioning, where applicable) and provide the corresponding final concentrations and exposure durations in each subsection.

 

 

2. EV identity is not convincingly demonstrated. The study lacks required MISEV-compliant validation:

• No Western blots for EV-positive markers (CD9, CD63, CD81, TSG101) and a negative marker (calnexin).
• NTA results are incomplete: no size distribution graphs, no modal/median size, no replicate details, and no instrument parameters.
  Without these, the particles cannot be confirmed as EVs.

 

We thank the reviewer for highlighting the importance of MISEV-compliant characterization. We acknowledge that comprehensive EV validation, including Western blot analysis of canonical EV-positive markers (CD9, CD63, CD81, TSG101) and negative markers (e.g., calnexin), was not performed in the present study.

EV-enriched fractions were isolated using the exoEasy Maxi kit and characterized by nanoparticle tracking analysis (NTA) and protein quantification. We have revised the manuscript to clarify that the isolated preparations represent EV-enriched fractions within the expected size range, rather than fully MISEV-validated extracellular vesicles.

This limitation has now been explicitly acknowledged in the Discussion section, and future studies will include comprehensive marker validation to comply with updated MISEV recommendations.

 

3. Inadequate controls to demonstrate miRNA transfer via EVs
   Conclusions that EVs deliver miRNA to cancer cells are premature. Missing essential controls include:

• EV-depleted conditioned medium,
• heat-inactivated/refolded EV controls,
• RNase ± detergent assays to confirm membrane-protected RNA,
• fluorescently labeled EV uptake experiments.

 

We thank the reviewer for this important comment. We agree that definitive demonstration of EV-mediated microRNA transfer would require additional mechanistic controls such as EV-depleted conditioned medium, RNase protection assays, heat-inactivation experiments, or uptake tracking studies. These experiments were not performed in the present study.

The primary objective of this work was to evaluate whether AHCC-treated MSC-derived EV-enriched fractions are associated with modulation of microRNA expression and cancer stem cell behavior in recipient breast cancer cells. Therefore, we have revised the manuscript to avoid definitive statements regarding direct miRNA delivery and instead describe the findings as modulation of microRNA expression following exposure to AHCC-treated EV-enriched fractions.

This limitation has now been explicitly acknowledged in the Discussion section, and future studies will aim to investigate the mechanistic aspects of EV-mediated RNA transfer.

 

Incorrect reference gene for miRNA qPCR
The use of SNORD65 (mmu) is inappropriate for human cell lines and may invalidate normalization. Authors must use a validated human small RNA reference (e.g., U6, RNU48) and demonstrate stability.

We thank the reviewer for this important observation. We confirm that SNORD65 was used as the endogenous control for normalization in our human cell line experiments; the “(mmu)” designation was a typographical error and has been corrected throughout the manuscript. In addition, consistent with QIAGEN guidance for reference controls, we verified stable expression of the endogenous control across our experimental conditions.
We agree that U6/RNU48 are also commonly used human endogenous controls; future studies will include additional reference controls to further strengthen normalization robustness.

 

Biological replicate numbers are unclear. Multiple t-tests are used where ANOVA is required. Exact p-values, effect sizes, and distribution checks are missing. All figures must include n, error bar type, and statistical tests.

We thank the reviewer for this important comment. We have revised the manuscript to clearly distinguish biological replicates from technical replicates, and we now report the exact number of independent biological replicates (n = 3) for each experiment in the Methods and in all figure legends.

We would like to clarify that the study design involved only two-group comparisons within each experiment (e.g., control vs AHCC; naïve MSC-derived EV-enriched fractions vs AHCC-treated MSC-derived EV-enriched fractions). Therefore, an unpaired two-tailed Student’s t-test was used as the appropriate statistical method.

In the revised figures and legends, we now explicitly indicate the error bar type (mean ± SEM), the statistical test applied, and the corresponding p-values. These revisions improve clarity and transparency of the statistical analysis.

Insufficient methodological detail
Key steps are missing: MSC culture conditions, RNA quality control (RIN), RT parameters, qPCR primer identifiers, EV storage cycles, sphere quantification criteria, blind analysis, etc. These are required for reproducibility.

We thank the reviewer for highlighting the need for additional methodological clarity. We have substantially expanded the Methods section to improve reproducibility. Specifically, we now provide detailed information regarding MSC culture conditions, EV isolation workflow, RNA extraction procedures, qPCR parameters (including primer identifiers), and statistical analysis. Where applicable, we clarified sample handling procedures, storage conditions, and experimental criteria (e.g., sphere quantification). These revisions enhance transparency and reproducibility of the stu

Figures

• Unit errors (µg vs mg) appear in legends.
• Images lack scale bars and adequate resolution.
• NTA data show only particle concentration without size distribution.
• Sphere/colony images, if included, require much clearer presentation.
• Figure legends must include n, statistical tests, and full descriptions.

We thank the reviewer for the valuable comments regarding figure presentation. We have carefully revised all figures and legends to improve clarity, consistency, and methodological transparency.

First, all unit inconsistencies (µg, mg, and mL) have been thoroughly checked and corrected throughout the manuscript and figure legends to ensure accuracy and reproducibility.

Second, scale bars have been added to representative microscopy images where available, and image resolution has been improved to the extent permitted by the original raw data.

Third, we clarified the NTA data presentation by specifying that particle concentration was measured using nanoparticle tracking analysis, and we have added additional methodological details regarding measurement conditions in the Methods section.

Fourth, sphere images are now described as representative morphological observations rather than quantitative measurements, and the quantification criteria have been clearly defined in the Methods section.

Finally, all figure legends have been revised to explicitly include the number of biological replicates (n = 3), error bar definitions (mean ± SEM), and the statistical tests used for each experiment. These revisions improve the overall clarity and reproducibility of the figures.

Reviewer 3 Report

Comments and Suggestions for Authors

1. I noticed that the word "god strategy" appeared in the Abstract. I think the author meant "good strategy" but misspelled it. This needs to be corrected.
2. The article does not have line numbers. The author should add them.
3. In the Introduction, there was a spelling mistake: "re-newal". It should be "renewal".
4. In the text, sometimes it says "miR-Let7a" and other times "miR-Let7". This is not consistent.
5. In the Introduction, I saw the phrase "an good candidate", which is grammatically wrong. It should be "a good candidate".
6. Also in the Introduction, "lot of evidence" is incorrect. It should be "a lot of evidence".
7. In the Introduction, "MSCs-CSCs" has an extra space. The author should check the whole paper carefully for similar spacing issues.
8. The materials used in the study should include details like the company's location, model number, and brand name.
9. In section 2.7, the author mentioned "SNORD65 (mmu)" where "mmu" means mouse. However, the study used human cells. This was confusing to me.
10. In section 2.8, "value < 0.05" should be written as "p < 0.05". This is basic for statistical reporting.
11. In the caption for Figure 1, "4 ug/mL" is not the standard unit. It should be "4 μg/mL".
12. In the Discussion, there is a grammar mistake: "Our data indicates". Since "data" is plural, it should be "Our data indicate".
13. I think there are too many references for this paper. It doesn't need so many.
14. The format of the references is messy and not consistent.
15. In the Introduction, the author talked about data from Canada and then data from the USA. The logic here seemed unclear and jumped around.
16. In the Abstract, "Extracellular vehicles" is a basic term error. The correct term is "Extracellular vesicles".
17. The Abstract does not clearly state what is new about this research. After reading it, I still did not understand its main contribution.
18. In the Introduction, the author cited 2024 data saying 310,720 women in the USA would be diagnosed with invasive breast cancer. Since it is now late 2025, nearly 2026, I believe the author should use the latest and most authoritative statistics.
19. The author should explain why they chose the MCF-7 and MCF-7/DOX cell lines, and why they did not use other breast cancer cell lines.
20. The English in the paper needs significant improvement. For example, phrases in the Introduction like "first in cancer related to female" and "plasticity’s" sound awkward and have grammar problems. The author should consider professional English editing.
21. All the Figures are very hard to read. I could not see the details clearly in any of them.
22. In section 2.3, the author wrote "0.3x105 cells" without using superscript for the exponent.
23. In section 2.4, the description of how EVs were extracted is too vague. There are not enough details for someone else to repeat the experiment.
24. In the text, sometimes it says "Figure", other times "Figures" or "figures". The way figures are referred to should be consistent.
25. In section 3.3, the author mentioned "colony formation". However, there is no description at all in the Materials and Methods section about how the colony formation assay was done. This is a major omission that hurts the paper's credibility.
26. In the Discussion, the author referred to "figures (1,2,4,5)". But the paper has no Figure 4 or Figure 5. This kind of mistake is unacceptable and greatly reduces credibility.
27. In the Discussion, the author made an important claim about a "dose-dependent" mechanism. However, I could not find any data in the Results section to support this. This is a major problem for the data's completeness and the conclusion's soundness.
28. In section 2.8, the description of the statistical methods is very brief. The author should provide more specific details about the methods used.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

We sincerely thank Reviewer 3 for the thorough and constructive editorial review. Many of the comments relate to language clarity, terminology consistency, formatting, and methodological transparency. We have carefully revised the manuscript throughout to correct typographical errors, improve English language quality, standardize terminology, and expand methodological details where require

 

 

1. I noticed that the word "god strategy" appeared in the Abstract. I think the author meant "good strategy" but misspelled it. This needs to be corrected.

The typographical error “god strategy” in the Abstract has been corrected to “good strategy.

 

2. The article does not have line numbers. The author should add them.

Line numbers have been added to the revised manuscript as requested.

3. In the Introduction, there was a spelling mistake: "re-newal". It should be "renewal".

The spelling error “re-newal” has been corrected to “renewal.”

4. In the text, sometimes it says "miR-Let7a" and other times "miR-Let7". This is not consistent.

The miRNA nomenclature has been standardized throughout the manuscript (e.g., consistently using miR-Let7a).

 

5. In the Introduction, I saw the phrase "an good candidate", which is grammatically wrong. It should be "a good candidate".

The grammatical error “an good candidate” has been corrected to “a good candidate.”

6. Also in the Introduction, "lot of evidence" is incorrect. It should be "a lot of evidence".

The phrase “lot of evidence” has been revised to “a lot of evidence.”

 

7. In the Introduction, "MSCs-CSCs" has an extra space. The author should check the whole paper carefully for similar spacing issues.

Spacing inconsistencies (including “MSCs-CSCs”) have been carefully checked and corrected across the entire manuscript.

8. The materials used in the study should include details like the company's location, model number, and brand name.

We thank the reviewer for this important suggestion. We have revised the Materials and Methods section to include detailed information on reagents, instruments, manufacturers, and locations (company name, city, and country) to improve reproducibility. In addition, the entire manuscript has undergone comprehensive language editing to ensure clarity, consistency, and grammatical accuracy.

9. In section 2.7, the author mentioned "SNORD65 (mmu)" where "mmu" means mouse. However, the study used human cells. This was confusing to me.

We thank the reviewer for this important observation. We confirm that SNORD65 was used as the endogenous control for normalization in our human cell line experiments; the “(mmu)” designation was a typographical error and has been corrected throughout the manuscript. In addition, consistent with QIAGEN guidance for reference controls, we verified stable expression of the endogenous control across our experimental conditions.
We agree that U6/RNU48 are also commonly used human endogenous controls; future studies will include additional reference controls to further strengthen normalization robustness.

 

10. In section 2.8, "value < 0.05" should be written as "p < 0.05". This is basic for statistical reporting. The statistical notation has been corrected throughout the manuscript. “value < 0.05” has been revised to the standard format “p < 0.05” in Section 2.8 and elsewhere where applicable.

 

11. In the caption for Figure 1, "4 ug/mL" is not the standard unit. It should be "4 μg/mL".

The unit formatting in the figure captions has been carefully reviewed and standardized. The AHCC concentration is now consistently reported using the correct SI unit format (μg/mL or mg/mL, as appropriate) across all figures and legends.

12. In the Discussion, there is a grammar mistake: "Our data indicates". Since "data" is plural, it should be "Our data indicate".

The grammatical error in the Discussion has been corrected, and the sentence now reads “Our data indicate” to reflect proper plural usage.

 

13. I think there are too many references for this paper. It doesn't need so many.

We appreciate the reviewer’s comment. We have carefully revised the reference list and removed redundant or less directly relevant citations, retaining only the most essential and up-to-date references to improve focus and readability.  

14. The format of the references is messy and not consistent.

The reference list has been thoroughly reformatted according to the journal (MDPI) guidelines to ensure consistency in citation style, punctuation, and formatting.

 

15. In the Introduction, the author talked about data from Canada and then data from the USA. The logic here seemed unclear and jumped around.

 

We thank the reviewer for this insightful comment. The Introduction has been substantially revised to improve logical flow and clarity. Epidemiological data are now presented in a coherent structure (global to North American context, followed by Canada-specific statistics). In addition, the rationale for selecting MCF-7 and MCF-7/DOX cell lines has been explicitly clarified as a paired model of breast cancer chemoresistance. The novelty and study aim have also been rewritten for greater clarity.

 

16. In the Abstract, "Extracellular vehicles" is a basic term error. The correct term is "Extracellular vesicles".

We thank the reviewer for pointing out this terminology error. The term “Extracellular vehicles” in the Abstract has been corrected to the scientifically accurate term “Extracellular vesicles.” This correction has also been verified throughout the manuscript to ensure consistent and appropriate use of EV-related terminology.

17. The Abstract does not clearly state what is new about this research. After reading it, I still did not understand its main contribution.

We thank the reviewer for this valuable comment. We agree that the novelty of the study was not sufficiently highlighted in the original Abstract. We have now revised the Abstract to explicitly state the main contribution of this work. Specifically, this study is, to our knowledge, among the first to investigate the indirect anti-chemoresistance effect of AHCC through MSC-derived extracellular vesicles, rather than direct treatment, and to demonstrate their role in modulating key oncogenic and tumor-suppressive microRNAs (miR-155, miR-34a, miR-Let7a, and miR-200c) in both sensitive (MCF-7) and doxorubicin-resistant (MCF-7/DOX) breast cancer models, including sphere-forming cancer stem-like cells. The revised Abstract now clearly emphasizes the study’s novelty, mechanistic focus on EV-mediated microRNA modulation, and its relevance to chemoresistance and tumor microenvironment regulation.

18. In the Introduction, the author cited 2024 data saying 310,720 women in the USA would be diagnosed with invasive breast cancer. Since it is now late 2025, nearly 2026, I believe the author should use the latest and most authoritative statistics.

We thank the reviewer for this insightful comment. The Introduction has been substantially revised to improve logical flow and clarity. Epidemiological data are now presented in a coherent structure (global to North American context, followed by Canada-specific statistics). In addition, the rationale for selecting MCF-7 and MCF-7/DOX cell lines has been explicitly clarified as a paired model of breast cancer chemoresistance. The novelty and study aim have also been rewritten for greater clarit

 

19. The author should explain why they chose the MCF-7 and MCF-7/DOX cell lines, and why they did not use other breast cancer cell lines.

We thank the reviewer for this insightful comment. The Introduction has been substantially revised to improve logical flow and clarity. Epidemiological data are now presented in a coherent structure (global to North American context, followed by Canada-specific statistics). In addition, the rationale for selecting MCF-7 and MCF-7/DOX cell lines has been explicitly clarified as a paired model of breast cancer chemoresistance. The novelty and study aim have also been rewritten for greater clarity.

 

20. The English in the paper needs significant improvement. For example, phrases in the Introduction like "first in cancer related to female" and "plasticity’s" sound awkward and have grammar problems. The author should consider professional English editing.

We thank the reviewer for this important comment. The manuscript has undergone thorough language revision to improve grammar, clarity, and scientific tone. Awkward expressions such as “first in cancer related to female,” “plasticity’s,” and similar phrasing have been corrected throughout the Introduction and the entire manuscript. The text has been carefully edited for consistency and readabilit

 

21. All the Figures are very hard to read. I could not see the details clearly in any of them.

We appreciate the reviewer’s observation. All figures have been revised to improve readability by enhancing resolution, increasing font size, and standardizing labeling and contrast. In addition, figure legends were expanded to provide clearer descriptions of the experimental conditions and result

 

22. In section 2.3, the author wrote "0.3x105 cells" without using superscript for the exponent.

Thank you for noting this formatting issue. The exponent notation has been corrected to the appropriate scientific format (3 × 10⁴ cells) throughout the manuscrip

 

23. In section 2.4, the description of how EVs were extracted is too vague. There are not enough details for someone else to repeat the experiment.

We agree with the reviewer and have substantially expanded Section 2.4 to provide a detailed description of the EV isolation workflow, including conditioning steps, collection, centrifugation, storage conditions, and downstream handling to ensure reproducibilit

 

24. In the text, sometimes it says "Figure", other times "Figures" or "figures". The way figures are referred to should be consistent.

The manuscript has been carefully revised to ensure consistent formatting of figure citations. All figure references are now standardized according to journal guidelines

 

25. In section 3.3, the author mentioned "colony formation". However, there is no description at all in the Materials and Methods section about how the colony formation assay was done. This is a major omission that hurts the paper's credibility.

We thank the reviewer for identifying this inconsistency. The term “colony formation” was an imprecise wording and has been corrected to “sphere formation” throughout the manuscript to accurately reflect the experimental assay described in the Materials and Methods section

 

26. In the Discussion, the author referred to "figures (1,2,4,5)". But the paper has no Figure 4 or Figure 5. This kind of mistake is unacceptable and greatly reduces credibility.

We sincerely apologize for this oversight. All incorrect figure citations have been corrected, and references to non-existent figures have been removed. The Discussion section has been carefully revised to ensure accurate figure numberings.

 

27. In the Discussion, the author made an important claim about a "dose-dependent" mechanism. However, I could not find any data in the Results section to support this. This is a major problem for the data's completeness and the conclusion's soundness.

We appreciate the reviewer’s critical observation. The wording suggesting a “dose-dependent” mechanism has been revised to avoid overinterpretation. The Discussion and Conclusion sections now strictly reflect the experimental findings without implying dose-response effects beyond the presented data.

 

28. In section 2.8, the description of the statistical methods is very brief. The author should provide more specific details about the methods used.

The Statistical Analysis section (Section 2.8) has been expanded to include detailed information on the statistical tests used, definition of biological replicates (n = 3), error bar representation (mean ± SEM), and the specific analytical approaches applied (unpaired t-test and two-way ANOVA where appropriate).

 

Minor grammatical, formatting, and terminology issues (Comments 20–24) have been comprehensively corrected throughout the manuscript. Methodological descriptions (Sections 2.4 and 2.8) were expanded, figure readability and labeling were improved, incorrect terminology such as “colony formation” was replaced with “sphere formation,” and unsupported claims (e.g., dose-dependent effects) were revised to accurately reflect the presented data.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

no comments 

Author Response

Thank you for your valuable comment. The manuscript has undergone professional English language editing by MDPI Author Services to improve grammar, clarity, and overall readability.

     

Reviewer 2 Report

Comments and Suggestions for Authors

In the present manuscript, the following minor revisions are recommended:

– Harmonization and clarification of the NTA analysis description. The manuscript states that only limited size distribution data were retained; however, mean EV diameters are reported in the Results section. It is recommended to clearly specify which NTA parameters were prospectively analyzed and which were derived from archival data.

– Further clarification of the rationale for selecting the 4 mg/mL AHCC concentration, particularly in the context of potential clinical translation and with reference to physiologically achievable concentrations.

– Enrichment of the miRNA analysis by briefly discussing potential target genes of the investigated miRNAs (e.g., in a supplementary table), which would strengthen the biological interpretation of the findings without requiring additional experimental work.

– Additional language and editorial revision to eliminate minor inconsistencies, typographical errors, and variations in unit formatting and terminology.

– Standardization of terminology throughout the manuscript (e.g., MSC-derived EV vs MSC-EVs; miR-Let7a vs let-7a) to ensure terminological consistency.

Comments on the Quality of English Language

The English language of the manuscript is generally clear and understandable. However, minor linguistic and stylistic revisions would further improve clarity and readability. Occasional grammatical inaccuracies, awkward phrasing, and minor inconsistencies in terminology and unit formatting are present throughout the text.

A careful language polishing, preferably by a native English speaker or professional editing service, is recommended prior to publication.

Author Response

1– Harmonization and clarification of the NTA analysis description. The manuscript states that only limited size distribution data were retained; however, mean EV diameters are reported in the Results section. It is recommended to clearly specify which NTA parameters were prospectively analyzed and which were derived from archival data.

Response 1: Thank you for this important comment. We have revised the Methods and Results sections to clarify the NTA workflow. Particle concentration (particles/mL) was the primary prospectively recorded parameter. Mean and median EV diameters were derived from archival NanoSight output files that remained available, while full size-distribution profiles were not retained due to data archiving limitations. This clarification has been added to the revised manuscript.

2– Further clarification of the rationale for selecting the 4 mg/mL AHCC concentration, particularly in the context of potential clinical translation and with reference to physiologically achievable concentrations.

Response 2 :Thank you for this helpful suggestion. We have expanded the rationale for selecting the 4 mg/mL concentration in the Methods section. This dose was chosen based on our previously published work demonstrating biological modulation of breast cancer stem cells without cytotoxicity. We have also added discussion regarding translational considerations and physiologically achievable exposure ranges.

3– Enrichment of the miRNA analysis by briefly discussing potential target genes of the investigated miRNAs (e.g., in a supplementary table), which would strengthen the biological interpretation of the findings without requiring additional experimental work.

Response 3: Thank you for this insightful recommendation. Identification and validation of miRNA target genes would indeed provide additional mechanistic depth; however, this was beyond the predefined scope of the current study, which focused on miRNA expression modulation and functional cellular outcomes. This point has now been acknowledged in the Discussion as a study limitation and proposed for future research

4– Additional language and editorial revision to eliminate minor inconsistencies, typographical errors, and variations in unit formatting and terminology.

Response 4: Thank you for your comment. The manuscript has undergone professional English language editing by MDPI Author Services. Additional editorial revisions were also performed to correct minor inconsistencies, typographical errors, unit formatting, and terminology standardization

5– Standardization of terminology throughout the manuscript (e.g., MSC-derived EV vs MSC-EVs; miR-Let7a vs let-7a) to ensure terminological consistency.

Response 5: Thank you for noting this. Terminology has now been standardized throughout the manuscript. Consistent forms such as “MSC-derived EVs” and “let-7a” are used uniformly across the text, figures, and legends.

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

Overall Recommendation: Major Revision
The author’s response to my comments is highly positive and responsible, which I greatly appreciate. However, several points still need improvement:

1. Responses 18 and 19 are identical, which is confusing.
2. The author has pledged a thorough linguistic revision of the entire manuscript and corrected awkward phrasing in the examples. As this is an extensive and challenging task, I will need a few more days to carefully check whether the revised language meets publication standards.
3. Figure 2 is poorly legible. The text inside is barely readable.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

. Responses 18 and 19 are identical, which is confusing.

Response : We sincerely apologize for the oversight. The duplicated responses were unintentional. The response has now been corrected, and each comment is addressed separately and appropriately in the revised response document.
2. The author has pledged a thorough linguistic revision of the entire manuscript and corrected awkward phrasing in the examples. As this is an extensive and challenging task, I will need a few more days to carefully check whether the revised language meets publication standards.

Response : Thank you for your patience and for taking the time to evaluate the revised language. The manuscript has undergone comprehensive professional English language editing through MDPI Author Services, followed by additional careful proofreading by the authors to improve clarity, flow, and readability. We appreciate the reviewer’s additional time to assess the revision

3. 
   Figure 2 is poorly legible. The text inside is barely readable.

Response : Thank you for pointing this out. Figure 2 has been redesigned and exported at higher resolution. Font sizes, panel labels, and graphical elements have been enlarged to ensure readability in both print and digital formats.

 

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

Overall Recommendation: Accept with minor revisions.

The author has made targeted revisions in response to my comments, and I am very satisfied with the changes. I have no further content-related suggestions at this time. However, the author should note that the iThenticate similarity report for the revised manuscript shows a score of 65%, which may require the author’s careful consideration. Additionally, I suggest that the author cite the following reference at line 78 in the introduction. This reference is a recent multi-omics and experimental validation study targeting the TLR4 pathway in tumors. It provides cutting-edge experimental evidence for the mechanism by which AHCC/MSC-EVs regulate the tumor microenvironment through TLR4. Furthermore, its research approach offers a high-level methodological reference for this study while also extending the relevance of TLR4 as a target across different tumor types: DOI: 10.1016/j.jpet.2026.103836.

Author Response

 

The author has made targeted revisions in response to my comments, and I am very satisfied with the changes. I have no further content-related suggestions at this time. However, the author should note that the iThenticate similarity report for the revised manuscript shows a score of 65%, which may require the author’s careful consideration. Additionally, I suggest that the author cite the following reference at line 78 in the introduction. This reference is a recent multi-omics and experimental validation study targeting the TLR4 pathway in tumors. It provides cutting-edge experimental evidence for the mechanism by which AHCC/MSC-EVs regulate the tumor microenvironment through TLR4. Furthermore, its research approach offers a high-level methodological reference for this study while also extending the relevance of TLR4 as a target across different tumor types: DOI: 10.1016/j.jpet.2026.103836.: Thank you for your comment regarding the similarity report.
The elevated similarity score is primarily due to the publicly available preprint version of this manuscript. As the preprint represents an earlier version of the same work by the same authors, textual overlap is expected. All other sources have been properly cited and referenced.

We appreciate your careful review.