Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors The manuscript examines whether EV uptake is determined mainly by EV origin or recipent cell characteristics. Using EVs from HEK293T, SKBR-3 and Yummer cells, the author shows clear cell type-, dose- and time-dependent differences in EV uptake. The study is interesting, well presented and needs only minor revision:

1. Please expand the Introduction with a brief reference to the historical development and standardization of EV research: Int J Mol Sci. 2026;27:2470.
   
   
2. Please clarify the statistical analysis; Methods mention one-way ANOVA, whereas fig legends report two way ANOVA.
3. Please double check EV concentrations reported in Methods and Results, as different ranges are given.
4. Claims that EV uptake is determined by membrane properties are too strong, since these mechanisms werent directly investigated.
5. In conclusions, “unequivocally” should be replaced with “strongly supports”.

Author Response

I sincerely thank the editor and reviewers for their thorough and careful evaluation of our manuscript. I greatly appreciate the time and effort devoted to reviewing our work, as well as the insightful and constructive comments and suggestions, which have helped us improve the clarity and quality of the manuscript. I have carefully addressed all comments and revised the manuscript accordingly. The point-by-point responses are provided below.

Reviewer 1

The manuscript examines whether EV uptake is determined mainly by EV origin or recipent cell characteristics. Using EVs from HEK293T, SKBR-3 and Yummer cells, the author shows clear cell type-, dose- and time-dependent differences in EV uptake. The study is interesting, well presented and needs only minor revision:

1.Please expand the Introduction with a brief reference to the historical development and standardization of EV research: Int J Mol Sci. 2026;27:2470.

Response: I thank the reviewer for this valuable suggestion. I have expanded the Introduction to briefly describe the historical development of extracellular vesicle research, from their initial recognition as cellular particles to their established roles in intercellular communication. I have also highlighted the importance of standardisation through the ISEV and MISEV guidelines and incorporated the recommended reference (Guzowska et al., 2026). These additions provide historical and methodological context for the present study.

2.Please clarify the statistical analysis; Methods mention one-way ANOVA, whereas fig legends report two-way ANOVA.

Response: I thank the reviewer for highlighting this important point. Now the method section was corrected accordingly. I have used two-way ANOVA and Tukey's multiple comparisons test

3.Please double check EV concentrations reported in Methods and Results, as different ranges are given.

Response: I appreciate the reviewer’s carefully check my manuscript. Now I added the dose that was missed in line 203 and highlighted in yellow.

4.Claims that EV uptake is determined by membrane properties are too strong, since these mechanisms werent directly investigated.

Response: I appreciate the reviewer for pointing this out. I agree with the reveivwer however, I mentioned in the discussion part ‘’ the molecular mechanisms underlying differential EV uptake were not directly investigated, and no inhibition studies were performed to identify specific internalization pathways’’.

5.In conclusions, “unequivocally” should be replaced with “strongly supports”.

Response: I thank the reviewer for commenting on that now the word unequivocally has been replaced by strongly supports.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses an interesting question and presents a useful comparative dataset on the dependence of EV-associated fluorescence on recipient-cell identity. The experimental sequence is logical, and the comparative approach has potential value for the EV field.

However, the central conclusions currently extend beyond what the experimental design and uptake assay can establish. In particular, the distinction between cell association and internalization, the attribution of differences to recipient-cell membrane properties, the contribution of donor-cell origin, and the consistency of the statistical analysis require further attention.

Addressing these issues and moderating the mechanistic interpretations would substantially improve the clarity, rigor, and impact of the manuscript.

Comments for author File: Comments.pdf

Author Response

I sincerely thank the editor and reviewers for their thorough and careful evaluation of our manuscript. I greatly appreciate the time and effort devoted to reviewing our work, as well as the insightful and constructive comments and suggestions, which have helped us improve the clarity and quality of the manuscript. I have carefully addressed all comments and revised the manuscript accordingly. The point-by-point responses are provided below.

Reviewer 2

The manuscript addresses an interesting question and presents a useful comparative dataset on the dependence of EV-associated fluorescence on recipient-cell identity. The experimental sequence is logical, and the comparative approach has potential value for the EV field. However, the central conclusions currently extend beyond what the experimental design and uptake assay can establish. In particular, the distinction between cell association and internalization, the attribution of differences to recipient-cell membrane properties, the contribution of donor-cell origin, and the consistency of the statistical analysis require further attention.  Addressing these issues and moderating the mechanistic interpretations would substantially improve the clarity, rigor, and impact of the manuscript.

Major comments

Thank you for this interesting and well-integrated study entitled “Cell Type–Dependent Uptake of Extracellular Vesicles Independent of Cellular Origin”. The research question is highly relevant: To what extent is EV uptake determined by the recipient cell type, independently of the EV donor cell? The comparison across epithelial, immune, and melanoma cell lines, using EVs derived from different donor cells, is an attractive aspect of the study.

The main strength of the manuscript lies in its systematic comparative approach rather than in the identification of a novel molecular mechanism. The manuscript is logically organized, following a clear progression from the introduction and methods to EV characterization, the analysis of uptake across different recipient cell types, comparisons involving different EV donors, and finally the discussion and conclusions.

The experimental sequence is also logical and supports the progressive development of the central argument. The author first generate fluorescently labelled EVs and characterize them, then investigate HEK293T-derived EVs in epithelial cells, immune cells, and melanoma cells, subsequently change the EV donor to SKBR-3, and finally examine Yummer-derived EVs. Figure 1 is particularly helpful in illustrating the experimental design. However, several issues regarding the interpretation of the results, the validation of EV internalization, the strength of the conclusions, and the statistical analysis should be addressed before the manuscript can be considered for acceptance. My specific comments are detailed below.

1. The role of recipient-cell membrane properties requires further clarification

The manuscript attributes differences in EV uptake primarily to recipient-cell identity. However, these differences do not, by themselves, demonstrate that recipient-cell membrane properties are the underlying determinants. Other factors, including receptor expression, metabolic activity, endocytic pathways, and intracellular trafficking, may also contribute.

The author should clarify what they mean by “recipient-cell membrane properties” and which specific properties they consider relevant. Potential factors include surface charge, glycocalyx composition, lipid composition, membrane fluidity, and the presence of cell-surface or extracellular-matrix components such as collagen. At present, these properties are discussed as possible explanations, but their contribution has not been directly investigated.

A stronger approach would be to modify a specific membrane property in the same recipient-cell line and determine whether this alters EV uptake. An even more compelling experiment would be a rescue approach in which the modification changes uptake and restoration of the original membrane property reverses the effect.

The author should also clarify whether the sizes of the recipient cells are comparable across the cell lines examined. Biophysical models based on reaction–diffusion kinetics have demonstrated a linear relationship between recipient-cell radius and nanoparticle or vesicle uptake. This factor could therefore contribute to the observed differences and should be considered in the interpretation of the results (Biophysical Journal, 2019; 116(2):347–359. DOI: 10.1016/j.bpj.2018.11.3134).

I sincerely thank the reviewer for this valuable comment. I agree that differences in EV uptake cannot be attributed solely to recipient-cell membrane properties, as other cellular factors may also contribute. I have revised the Discussion to clarify that membrane properties and recipient-cell size are potential factors influencing EV uptake but were not directly investigated in this study. These limitations and the need for further mechanistic investigations have now been addressed in the revised Discussion.

 

2. The hypothesis should be explicitly stated at the end of the Introduction

The overall rationale of the study is understandable, but the central hypothesis would benefit from being stated explicitly at the end of the Introduction. I suggest the following formulation:

“We hypothesized that EV uptake is predominantly determined by recipient-cell properties rather than by EV donor-cell origin. We therefore aimed to systematically compare the uptake of EVs derived from different donor cell types across diverse recipient cells, while evaluating the effects of EV dose and exposure time.”

This would help establish a clearer link between the rationale, the experimental design, and the conclusions.

Response: I thank the reviewer for this helpful suggestion. I have revised the final paragraph of the Introduction to explicitly state our central hypothesis that recipient-cell characteristics play a greater role than EV donor-cell origin in determining EV association. We have also clarified the study objectives, including comparisons across different donor–recipient combinations and evaluation of EV dose and incubation time. The terminology has been adjusted to reflect the measurement of EV-associated fluorescence rather than confirmed internalization.

3. The scope of the study should be described more precisely

The overall objective—to compare the uptake of EVs derived from different donor cells across different recipient cell types—is clear. However, the description of the study as “conducting a comprehensive analysis of EV uptake across diverse cellular systems” seems overly broad in relation to the experiments performed.

The study examines three EV donor types, several recipient cell lines, two incubation times, two or three EV doses, and uptake-related fluorescence measured by flow cytometry. This represents a valuable and systematic in vitro comparison, but it does not constitute a comprehensive characterization of the determinants of EV uptake.

The author should consider replacing terms such as “comprehensive analysis” with more precise wording that reflects the actual scope of the work. For example, the following formulation would be more appropriate:

“A systematic experimental comparison demonstrating substantial recipient-cell-dependent differences in EV-associated fluorescence.” This wording better reflects the evidence generated without overstating the breadth of the study.

Response: I thank the reviewer for this valuable suggestion. I agree that describing the study as a “comprehensive analysis” overstated the scope of the experiments. We have therefore revised the final paragraph of the Introduction to describe the work more precisely as a “systematic in vitro comparison of EV-associated fluorescence”. The revised text specifies the EV donor-cell sources, recipient-cell types, flow cytometry measurements, and evaluation of EV dose and incubation time. We have also consistently used the term “EV association” rather than “EV uptake” to avoid implying that internalization was directly demonstrated.

4. The potential effects of CD81-mNG expression on EV properties should be addressed

The use of stable CD81-mNG expression is an interesting strategy for tracking EVs. However, it raises an important methodological question: could the expression of this fluorescently tagged tetraspanin alter EV biogenesis, membrane composition, EV number or size distribution, tetraspanin abundance, or interactions with recipient cells? The author should provide evidence that CD81-mNG-labelled EVs behave comparably to unmodified EVs. It may not be necessary to repeat the entire experimental series, but an appropriate structural or functional validation would strengthen confidence in the approach. One possibility would be to compare EV production in at least one donor-cell type using EVs labelled with a lipophilic fluorescent dye and EVs obtained from cells stably expressing CD81- mNG. Such a comparison could help establish whether the labelling strategy affects EV yield or other relevant characteristics. The limitations of each labelling approach should also be considered when interpreting the comparison.

Response: I thank the reviewer for this important comment. I acknowledge that stable expression of mNG-CD81 may affect EV biogenesis, membrane composition, and interactions with recipient cells. In this study, fluorescently labelled EVs were used to assess EV-associated fluorescence by flow cytometry. Although the EVs were characterised using nanoparticle tracking analysis, transmission electron microscopy, and EV marker analysis, their equivalence to unmodified EVs was not directly evaluated. We have therefore added this point to the limitations section of the Discussion. Future studies will compare labelled and unmodified EVs to assess potential effects on EV properties and recipient-cell interactions.

5. The characterization of EV preparations should be interpreted more cautiously

The statement “Collectively, these findings confirm the successful isolation of structurally intact and marker-positive EVs” is somewhat too categorical.

Although nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and the detection of CD81 and TSG101 provide useful complementary evidence, these results alone do not establish that the preparations consist of a highly pure EV population.

The author should consider whether additional positive EV-associated markers and negative markers for potential contaminants could strengthen the characterization. MISEV2023 recommends a multiparametric approach to EV characterization, including complementary categories of markers, rather than relying exclusively on CD81 and TSG101.

In addition, for Yummer-derived EVs, which marker would be suitable for Western blot analysis? A corresponding marker analysis, comparable to the TSG101 data presented for HEK293T- and SKBR- 3-derived EVs in Figure 2M, would improve consistency across the three donor types.

The author should also ensure that the conclusions regarding EV purity and integrity are proportionate to the characterization actually performed.

Response: I thank the reviewer for this valuable comment. I would like to clarify that EVs were purified using SEC and subsequently characterised by NTA, TEM, and detection of EV-associated markers, including CD81 and TSG101. Together, these complementary approaches provide evidence supporting the successful isolation and identification of EVs. However, I acknowledge that these analyses do not establish the absolute purity of the preparations. I have therefore revised the statement to avoid overinterpretation and acknowledge that additional positive and negative markers, including Western blot analysis of Yummer-derived EVs, would further strengthen EV characterisation in future studies.

6. The distinction between EV uptake and internalization needs to be established

The manuscript uses the terms “uptake” and “internalization” interchangeably. However, the assay appears to consist primarily of the incubation of cells with EVs labelled through CD81-mNG expression, followed by washing and flow cytometric analysis of fluorescence-positive cells.

This approach provides evidence of cell-associated EV-derived fluorescence, but it does not necessarily demonstrate that the EVs have been internalized.

The detected fluorescence could originate from several sources:

- EVs that have been internalized.

- EVs bound to the cell surface.

- EVs associated with the plasma membrane without being internalized.

- EVs trapped in aggregates associated with cells.

- EVs undergoing partial internalization.

PBS washing alone does not necessarily distinguish between these possibilities. This distinction is particularly important for recipient cells such as Huh7, for which the reported uptake reaches approximately 99%.

The author should therefore provide additional evidence to validate internalization or, alternatively, consistently use more cautious terminology that reflects what the assay directly measures.

Several complementary approaches could strengthen the evidence for internalization, including confocal microscopy, colocalization with endosomal markers, imaging flow cytometry demonstrating intracellular fluorescence, or experiments performed at 4 °C to assess the contribution of energy-dependent uptake processes. Endocytosis inhibitors could also be informative, provided their specificity and potential effects on cell viability are appropriately controlled.

This issue also affects the central conclusion that “EV uptake is predominantly determined by recipient-cell characteristics rather than the cellular source.” If the assay does not adequately distinguish cell-surface binding or association from internalization, the conclusion should be revised accordingly.

Response: I sincerely thank the reviewer for this important comment. I agree that flow cytometric detection of fluorescence following incubation with CD81-mNG-labelled EVs and subsequent washing does not definitively distinguish internalized EVs from EVs associated with the cell surface. Therefore, I acknowledge that our assay primarily measures cell-associated EV-derived fluorescence rather than confirmed intracellular EV uptake. Accordingly, I have revised the manuscript to use more precise terminology, including “EV-associated fluorescence” and “EV association,” where appropriate, and have avoided interpreting fluorescence-positive cells as direct evidence of EV internalization. I have also clarified that the high percentages of fluorescence-positive cells, including those observed in Huh7 cells, indicate EV-associated fluorescence rather than confirmed internalization. Furthermore, I have revised the Discussion and Conclusions to reflect this distinction and acknowledged the need for future studies using confocal microscopy, imaging flow cytometry, and intracellular localisation assays to distinguish cell-surface association from internalization.

 

7. The conclusion that recipient-cell properties outweigh donor-cell origin is too strong for the current experimental design

The conclusion that recipient-cell properties are more important than donor-cell origin is a central claim of the manuscript. However, the donor–recipient combinations are not fully balanced or systematically crossed.

For example:

- HEK293T-derived EVs are tested across several recipient cell types.

- SKBR-3-derived EVs are examined in a different subset of recipient cells.

- Yummer-derived EVs are tested in only two murine cell lines.

Consequently, donor-cell origin and recipient-cell identity cannot be compared robustly as independent factors across the entire experimental design. A more balanced factorial design, in which the different EV donors are tested across the same recipient-cell panel, would be required to estimate their respective contributions more rigorously.

Based on the current data, a more defensible conclusion would be: “The recipient cell type is a strong determinant of EV-associated fluorescence, and similar patterns are observed for EVs derived from two different human donor cell lines”. This formulation preserves the main finding while avoiding the unsupported assertion that recipient-cell properties predominantly determine uptake independently of donor-cell origin.

Response: I sincerely thank the reviewer for this important comment. I agree that the donor–recipient combinations were not fully balanced, limiting our ability to directly compare the relative contributions of EV donor-cell origin and recipient-cell identity. I would like to clarify that Yummer-derived EVs were tested not only in two murine melanoma cell lines but also in four human recipient-cell lines (HEK293T, PDAK, HepG2, and Huh7), as shown in Supplementary Figure 8. Similar patterns of EV-associated fluorescence were observed across these human recipient cells compared with HEK293T- and SKBR-3-derived EVs. Nevertheless, I acknowledge the limitations of the experimental design and have revised the Results, Discussion, and Conclusions to avoid suggesting that recipient-cell properties are the dominant determinant of EV uptake. I now emphasise that recipient-cell type is associated with substantial differences in EV-associated fluorescence, while the relative contributions of donor-cell origin and recipient-cell identity require further investigation using a balanced factorial experimental design.

8. The results obtained with Yummer-derived EVs should be reconciled with the central conclusion

The results involving Yummer-derived EVs appear to introduce an important qualification to the central argument. According to the relevant figure, Yummer-derived EVs are preferentially associated with Yummer cells, suggesting that donor–recipient identity may also influence the observed pattern.

The manuscript subsequently interprets these findings in terms of the biological membrane properties of recipient cells. However, this is an alternative hypothesis rather than a mechanism demonstrated by the experiments.

The author should explicitly acknowledge that donor-cell origin may contribute to the observed differences and discuss how the Yummer results fit with the overall conclusion. This would make the discussion more balanced and help distinguish experimental observations from mechanistic interpretations.

Response: I sincerely thank the reviewer for this important observation. I agree that the results obtained with Yummer-derived EVs provide an important qualification to our findings. Unlike the relatively low uptake of HEK293T- and SKBR-3-derived EVs by their respective parental cells, Yummer cells exhibited significantly higher EV-associated fluorescence following exposure to Yummer-derived EVs than B16F10 cells. This indicates that low uptake of EVs by their cells of origin is not a universal phenomenon. I also acknowledge that EV donor-cell origin, recipient-cell identity, and their interactions may contribute to the observed uptake patterns. Accordingly, I have revised the Results and Discussion to clarify that the underlying mechanisms, including potential contributions from recipient-cell membrane properties, were not directly investigated. I have also modified the Conclusions to avoid suggesting that EV uptake is determined independently of donor-cell origin.

9. The Discussion should be more cautious when proposing mechanisms and biological implications

The Discussion is generally well written, but several interpretations extend beyond the evidence directly provided by the experiments. For example, the author suggest that the higher fluorescence observed in Huh7 and HepG2 cells may reflect the naturally high endocytic and metabolic activity of hepatic cells. This is a plausible explanation, but neither endocytic capacity nor metabolic activity was directly assessed in the study. Similarly, the author discuss receptor availability, membrane composition, endocytic capacity, intracellular trafficking pathways, heparan sulfate, integrins, tetraspanin-binding partners, and macropinocytosis as potential determinants of EV uptake. These are reasonable hypotheses, but they remain speculative in the absence of experiments directly addressing their contributions.

I recommend clearly distinguishing the findings demonstrated by the data from possible mechanistic explanations. The Discussion would be stronger if these hypotheses were presented more explicitly as future directions rather than as explanations supported by the current results.

In particular, the statement that “liver-derived tumors may be particularly responsive to EV-based drug delivery systems” appears to go beyond the scope of the study. The experiments assess fluorescent EV association and/or internalization in cultured cell lines over a relatively short period. They do not directly investigate drug delivery, functional cargo delivery, therapeutic responses, or in vivo targeting. This statement should therefore be removed or substantially qualified. The results may provide a basis for future investigations into EV-based delivery systems, but they do not yet establish the suitability of these systems for tumor-targeted applications.

Response: I thank the reviewer for this valuable comment. I agree that the proposed mechanisms underlying differences in EV association should be interpreted cautiously. Accordingly, we have revised the Discussion to distinguish our experimental findings from potential mechanistic explanations. Factors such as membrane composition, receptor expression, metabolic activity, and endocytic pathways are now presented as hypotheses requiring further investigation rather than established explanations for the observed differences. I have also revised statements concerning the potential therapeutic applications of EVs to avoid implying that EV-associated fluorescence demonstrates functional cargo delivery or therapeutic efficacy. Furthermore, we have explicitly acknowledged these limitations and highlighted the need for mechanistic studies, direct internalization assays, and functional validation in future research.

10. The statistical analysis requires clarification and consistency

The statistical methods are not consistently described throughout the manuscript. The Methods section refers to “one-way ANOVA followed by Tukey’s and Dunnett’s multiple-comparisons tests,” whereas the figure legends state that statistical analysis was performed using two-way ANOVA. This discrepancy should be resolved, and the statistical methods should be reported consistently in the Methods section and figure legends.

Furthermore, the experimental design involves multiple factors, including EV dose, incubation time, and recipient-cell type. The statistical approach should reflect the specific design of each experiment and clearly identify the factors included in each analysis, as well as any interaction terms that were evaluated.

Given the use of n = 3 biological replicates, the author should also clarify the following points:

- What constitutes a biological replicate?

- Were the three EV preparations generated independently?

- Did each biological replicate involve a new, independently generated EV preparation?

- Were the measurements independent, or were repeated measurements obtained from the same biological sample?

- Were the assumptions of the selected statistical tests assessed, and how were potential violations addressed?

- Which comparisons were prespecified?

- Which multiple-comparison correction was applied in each analysis, and why was it appropriate for the corresponding experimental design?

These details are important for evaluating the robustness and reproducibility of the reported differences.

Response: I thank the reviewer for this valuable comment. We have clarified that statistical analyses were performed using two-way ANOVA followed by Tukey’s multiple-comparisons test, comparing all group means within each row. Three independent EV preparations were produced and isolated at different time points, representing three biological replicates (n = 3). For each biological replicate, three technical replicate wells were analyzed, and their measurements were averaged to obtain one value per biological replicate. The Methods section and figure legends have been revised accordingly.

11. The controls should be explicitly defined

The term “control” is used in several figures and should be defined more clearly. Does “control” refer to recipient cells incubated without EVs, EV preparations analyzed in the absence of cells, or another condition? These conditions address different experimental questions and should not be conflated. The author should specify the precise control condition in each relevant figure legend and, where necessary, in the Methods section.

Response: I thank the reviewer for this valuable comment. I have clarified that the control replaced with “untreated control” refers to recipient cells that were not exposed to mNG-labeled EVs. This definition has now been added to the Methods section and the relevant figure legends to avoid ambiguity.

Minor comments:

1. Line 41: Please write “extracellular vesicles” in full and introduce the abbreviation “EVs” at its first occurrence.

Response: I thank the reviewer for pointing this out. The term “Extracellular Vesicles (EVs)” has now been introduced at its first occurrence and highlighted in yellow in the revised manuscript.

2. Line 70: A period is missing at the end of the sentence.

Response: I thank the reviewer for carefully reviewing the manuscript. The missing period has now been added at the end of the sentence.

3. Line 147: Please clarify the terminology. NanoSight refers to a commercial instrument platform, whereas nanoparticle tracking analysis (NTA) is the analytical technique used to characterize the particles.

Response: I thank the reviewer for this clarification. The term “NanoSight” has been replaced with “Nanoparticle” to accurately describe the analytical technique. This correction has been highlighted in yellow in the revised manuscript.

4. Line 253: The TEM images appear to show EVs of different sizes. How do these observations correspond to the size distribution obtained by NTA? Please clarify whether the two techniques provide consistent results and discuss any apparent discrepancies.

Response: I thank the reviewer for this valuable comment. The TEM images show EVs of varying sizes, consistent with the heterogeneous size distribution observed by NTA. Minor differences between the two techniques may arise from their distinct measurement principles, as TEM examines dried EVs, whereas NTA measures particles in suspension. This clarification has been added to the revised manuscript and highlights in yellow.

5. Section 3.2, Line 281: Please include an explicit reference to Figure 3 at the appropriate point in the text.

Response: I sincerely thank the reviewer for carefully reviewing the manuscript. As suggested, an explicit reference to Figure 3 has now been added at the appropriate location in Section 3.2. This modification has been highlighted in yellow in the revised manuscript for ease of identification.

6. Figures 3–7: Please specify the meaning of “control” in each figure legend and ensure that the term consistently refers to the appropriate experimental condition.

Response: I appreciated the reviewer’s comment. The Control indicates cells that are not treated with EVs. Now it is defined in figure legends of supplementary figures.

7. Line 315: Please clarify the meaning of “reference control” and explain how this condition differs from the other controls used in the study.

Response: I thank the reviewer for this valuable comment. I have clarified that the term “reference control” refers to parental HEK293T cells treated with HEK293T-derived EVs to assess EV uptake. Unlike the untreated control, which consists of cells not exposed to EVs, the reference control was used to evaluate EV uptake by the parental cells. This clarification has been added to the revised manuscript and highlighted in yellow.

8. Figure 4A and 4C: Please correct “Jurcat” to “Jurkat.”

Response: I thank the reviewer for carefully checking the manuscript. The figure now has been updated.

9. Figures 3–7: The representative flow cytometry plots are difficult to interpret in their current form. Please improve their visibility and presentation, including axis labels, gate boundaries, and fluorescence distributions, as appropriate. The plots should allow readers to evaluate the data and understand how the reported percentages were determined. Maybe could be presented in supplementary information.

Response: I sincerely thank the reviewer for this constructive suggestion. We agree that clear presentation of the flow cytometry data is essential for accurate interpretation of the results. Accordingly, we have moved the representative flow cytometry plots from Figures 3–7 to the Supplementary Information to improve the overall presentation of the main figures. The plots have been presented at an improved resolution, allowing the fluorescence distributions, axis labels, and corresponding percentages to be clearly visualized when enlarged. I believe that these revisions have substantially improved the clarity and accessibility of the data.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for your careful revision of the manuscript and for providing clear and detailed responses to the reviewers’ comments. The authors have adequately addressed the concerns raised, and the revised manuscript is substantially improved in terms of clarity, rigor, and interpretation of the results. I consider that the revisions satisfactorily resolve the issues identified in my previous review.