Mesenchymal Stem Cell-Derived Exosomes Reprogram Chemosensitivity Pathways in Cervical Cancer Spheroids

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.This is a valuable study aimed at investigating the effects of mesenchymal stem cell-derived exosome (MSC-exosome) pretreatment on chemotherapy sensitivity using three dimensional (3D) spheroid models generated from HeLa and SiHa CC cell lines.

2.It is recommended to adjust the order of the content in the introduction section. Specifically, first describe the rationale behind conducting this study, then explain why three-dimensional (3D) spheroid models generated from HeLa and SiHa CC cell lines were employed for the research, and finally outline the planned approach for executing this study.

3.Some figures do not match the content of the paper.For example:

Line177-203:  2.3. Proteomic profiling and enrichment analysis of MSC-exosome proteins(Figure 4a-c) ,but, Figure 4. Uptake of MSC-exosomes by cervical cancer cells.

 

LIne210-221: 2.4. STRING Enrichment Network of MSC-exosome Proteins: This network visualization underscores the complexity of the MSC-exosome proteome and identifies central “hub” proteins that may play critical roles in drug resistance and chemosensitivity, representing potential therapeutic targets (Figure 5). but, Figure 5. Assessment of cell viability and caspase activity in CC spheroids following MSC-exosome pretreatment and combination chemotherapy.

 

Line318-319:Exosomes are typically taken up by recipient cells through endocytosis, followed by intracellular cargo release. As shown in Figure 6.  but, Figure 6. Western blot analysis of DNA damage, NF-κB, and apoptosis pathway proteins in HeLa spheroids following MSC-exosome pretreatment and combination chemotherapy.

 

Line332-379: 2.7. Effect of MSC-Exo pretreatment on combination chemotherapy in cervical cancer spheroids(Figure 7a-c). but, Figure 7. Western blot analysis of DNA damage, NF-κB, and apoptosis pathway proteins in SiHa spheroids following MSC-exosome pretreatment and combination chemotherapy.

 

4.Caspase activity does not represent apoptosis. As the main focus of this study, it is recommended to provide results of apoptosis.

5.English requires professional editing.

6.Supplement references for 2025.

7.It is recommended that the paper be rewritten before resubmission.

Comments on the Quality of English Language

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

Author Response

Comment 1. This is a valuable study aimed at investigating the effects of mesenchymal stem cell-derived exosome (MSC-exosome) pretreatment on chemotherapy sensitivity using three dimensional (3D) spheroid models generated from HeLa and SiHa CC cell lines.

Response: Thank you for recognizing the value of our study. We sincerely appreciate your positive assessment. The investigation of MSC-exosome pretreatment on chemotherapy sensitivity in 3D spheroid models derived from HeLa and SiHa cervical cancer cell lines is indeed central to our work. Accordingly, we have carefully designed the experimental framework to capture both the mechanistic insights and the therapeutic implications of this approach.

Comment 2. It is recommended to adjust the order of the content in the introduction section. Specifically, first describe the rationale behind conducting this study, then explain why three-dimensional (3D) spheroid models generated from HeLa and SiHa CC cell lines were employed for the research, and finally outline the planned approach for executing this study.

Response: Thank you for the constructive suggestion. We have revised the introduction to follow the recommended sequence by first presenting the rationale for this study, including the clinical challenges associated with cervical cancer, the limitations of current treatment options, and the potential modulatory roles of MSC-derived exosomes in chemotherapy response. Thereafter, we include an explanation justifying the employing 3D spheroid models generated from HeLa and SiHa cervical cancer cell lines, while emphasizing their ability to better replicate in vivo tumor architecture, drug-penetration barriers, and the distinct biological characteristics of these two CC subtypes. Finally, we outline the planned study approach, which includes profiling MSC-exosome protein cargo, establishing HeLa and SiHa spheroids, and evaluating their chemotherapy response following exosome pretreatment (Lines 36-92).

Comment 3. Some figures do not match the content of the paper. For example:

Line177-203:  2.3. Proteomic profiling and enrichment analysis of MSC-exosome proteins (Figure 4a-c), but, Figure 4. Uptake of MSC-exosomes by cervical cancer cells.

LIne210-221: 2.4. STRING Enrichment Network of MSC-exosome Proteins: This network visualization underscores the complexity of the MSC-exosome proteome and identifies central “hub” proteins that may play critical roles in drug resistance and chemosensitivity, representing potential therapeutic targets (Figure 5). but, Figure 5. Assessment of cell viability and caspase activity in CC spheroids following MSC-exosome pretreatment and combination chemotherapy 

Line318-319: Exosomes are typically taken up by recipient cells through endocytosis, followed by intracellular cargo release. As shown in Figure 6.  but, Figure 6. Western blot analysis of DNA damage, NF-κB, and apoptosis pathway proteins in HeLa spheroids following MSC-exosome pretreatment and combination chemotherapy.

 

Line332-379: 2.7. Effect of MSC-Exo pretreatment on combination chemotherapy in cervical cancer spheroids (Figure 7a-c). but, Figure 7. Western blot analysis of DNA damage, NF-κB, and apoptosis pathway proteins in SiHa spheroids following MSC-exosome pretreatment and combination chemotherapy. 

 Response: Thank you very much for carefully reviewing our manuscript and for highlighting the inconsistencies between the figure numbers and the corresponding content. We sincerely apologize for these inadvertent and regrettable errors. These occurred due to missing figures and incorrect figure placements during the manuscript assembly process.

We have now thoroughly reviewed all figure assignments and made the necessary corrections. Specifically:

1. Lines 177–203 – The section “2.3. Proteomic profiling and enrichment analysis of MSC-exosome proteins (Figure 4a–c)” was incorrectly linked to a figure showing exosome uptake. Correction: We have inserted the correct proteomic profiling figures as Figure 4a–c and updated the caption accordingly.
2. Lines 210–221 – The description of the STRING enrichment network (Figure 5) was mistakenly paired with a figure on cell viability and caspase activity. Correction: The appropriate STRING protein–protein interaction network has now been added as Figure 5 with the correct caption.
3. Lines 318–319 – The text refers to exosome uptake pathways and intracellular cargo release (Figure 6), but the manuscript erroneously displayed a western blot panel. Correction: The exosome uptake image has been inserted as Figure 6, and the western blot figure has been moved to its correct section.
4. Lines 332–379 – The section “2.7. Effect of MSC-Exo pretreatment on combination chemotherapy (Figure 7a–c)” was linked to an unrelated western blot figure. Correction: The appropriate results illustrating the effects of MSC-exosome pretreatment on chemotherapy response have now been correctly inserted as Figure 7a–c. The western blot data have been reorganized and are presented as Figures 8 and 9 for the HeLaand SiHa cell lines, respectively.

These corrections ensure that all figures now match the narrative and accurately support the corresponding results. We appreciate the reviewer’s careful attention to detail, and we regret any confusion caused by these formatting errors. The revised manuscript now includes consistently correct figures, captions, and numbering.

 

Comment 4. Caspase activity does not represent apoptosis. As the main focus of this study, it is recommended to provide results of apoptosis.

Response: We thank the reviewer for this valuable comment. We agree that caspase activity alone does not fully represent apoptosis. In this study, apoptosis was evaluated using the ApoLive-Glo™ multiplex assay, which is a widely accepted method that simultaneously measures caspase-3/7 activity and cell viability. While we acknowledge that apoptosis is a complex, multistep process, activation of executioner caspases-3/7 represents a defining and irreversible event during apoptotic cell death, and is therefore, commonly used as an operational marker of apoptosis.

Importantly, in 3D spheroid models, caspase-based luminescence assays are particularly appropriate and frequently recommended, as membrane-dependent assays (e.g., Annexin V/PI staining or TUNEL assays) are often limited by restricted probe penetration, heterogeneous diffusion, and spheroid architecture. Additionally, recent evidence supports this approach: Rogmans et al. [1] demonstrated that caspase-3/7 activation provides a robust and reproducible readout of apoptosis in ovarian cancer spheroids and used caspase-3/7 luminescence as a primary indicator of treatment-induced apoptotic responses in 3D tumor models. This study exemplifies the accepted use of caspase-3/7 activity to represent apoptosis in spheroid-based drug-response evaluations. Accordingly, we consider caspase-3/7 activity measured using a validated multiplex assay to be an appropriate and biologically relevant representation of apoptosis in our experimental system. To further clarify this point and address the reviewer’s concern, we have revised the Methods section (Lines 935–936) to explicitly define apoptosis operationally as caspase-3/7 activation, and to emphasize the suitability of 3D spheroid models for evaluating treatment-induced apoptotic responses.

Reference

1. Rogmans, C.; Dittrich, J.; Hamm, E.; Weimer, J.P.; Holthaus, D.; Arnold, N.; Flörkemeier, I.; Maass, N.; Jansen, P.; Dempfle, A.; et al. Inhibiting ADAM17 enhances the efficacy of olaparib in ovarian cancer spheroids. Sci. Rep. 2024, 14, 26926.

 

Comment 5. English requires professional editing.

Response: Thank you for this valuable comment. The manuscript has been professionally edited for English language and style by Editage, and the clarity and readability have been improved throughout the revised version.

Comment 6. Supplement references for 2025.

Response: We thank the reviewer for this valuable suggestion. To strengthen the Discussion with recent literature, we have now incorporated relevant studies from 2025 that align closely with our experimental design and findings. Specifically, we cited Nittayaboon et al. (2025) to support the use of MSC-derived exosomes in cervical cancer spheroid models and to contextualize the observed differential responses between HeLa and SiHa spheroids. We also included Campora and Cicero (2025) to reinforce the rationale for using three-dimensional spheroid models as physiologically relevant systems for studying extracellular vesicle-mediated communication and treatment response. In addition, Shokati et al. (2025) was incorporated to place our findings within the broader context of emerging exosome-based therapeutic strategies, and establish their dual roles in apoptosis modulation and treatment resistance. These additions have been integrated into the Discussion to enhance the timeliness and relevance of the manuscript.

Comment 7. It is recommended that the paper be rewritten before resubmission.

Response: Thank you for the useful recommendation. We acknowledge the need for substantial revision, and we have now carefully rewritten the manuscript to improve clarity, coherence, and alignment between the text, figures, and methodology. The introduction has been reorganized according to the suggested structure, the Results section has been revised with corrected figure placements, and the Discussion has been expanded to better interpret the findings within the context of existing literature. Additionally, grammatical issues, formatting inconsistencies, and missing elements have been addressed throughout the manuscript. We appreciate your feedback and believe that these revisions have significantly strengthened the quality and presentation of the work.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors conducted a study on the role of exosomes in modulating chemosensitivity in cervical cancer. Some comments are provided below.

Line 51. It is important to mention the concept of 3D cell cultures.

Line 59. The role of exosomes is unclear. Exosomes can be considered both as therapeutic agents (as they can contain various proteins and nucleic acids) and as delivery agents for cargo (including chemotherapeutic drugs). Please clarify.

Line 78. There is no reference to the work on proteomic profiling.

Figure 2 c, d, e, f is very small. Please enlarge.

Line 158. Please briefly describe the steps involved in obtaining exosomes.

Figure 3a. The size scale is unclear.

Sections 2.3-2.5. 787 proteins were identified by LC–MS/MS. Can such a large number of proteins fit into an exosome with a diameter of ~200 nm? This is doubtful. The large number of proteins is likely due to insufficient purification of the exosomes. The authors should critically review this section. Proteomic analysis performed on insufficiently purified samples calls into question all subsequent bioinformatic conclusions regarding the functional composition of exosomes. Evidence of the high purity of the exosome preparation (absence of cytoplasmic and organelle markers) must be provided, or the data interpretation must be reconsidered, recognizing the possible contribution of co-isolated contaminants.

The reference to Figures 4 and 5 does not correspond to the figures provided.

Line 642. Since this study did not analyze microRNAs, discussion of microRNAs is beyond the scope of this paper and should be removed.

Author Response

Comment 1. The authors conducted a study on the role of exosomes in modulating chemosensitivity in cervical cancer. Some comments are provided below.

Line 51. It is important to mention the concept of 3D cell cultures.

Response: We thank the reviewer for this comment. The Introduction has been revised to include a description of the concept and relevance of 3D cell cultures in Lines 45-74.

 

Comment 2. Line 59. The role of exosomes is unclear. Exosomes can be considered both as therapeutic agents (as they can contain various proteins and nucleic acids) and as delivery agents for cargo (including chemotherapeutic drugs). Please clarify.

Response: We thank the reviewer for this important comment. We have now clarified the role of exosomes in the Introduction section of the revised manuscript. This study primarily considers exosomes to be biological modulators/therapeutic mediators, rather than drug delivery vehicles. Specifically, we investigated the effects of MSC-derived exosomes themselves, which contain endogenous proteins and nucleic acids, on modulating chemotherapy sensitivity in cervical cancer 3D spheroid models. The exosomes were not loaded with chemotherapeutic agents, nor were they used as carriers for drug delivery.

 

Comment 3. Line 78. There is no reference to the work on proteomic profiling.

Response: We appreciate the reviewer’s comment. The statement in Line 78 is intended to describe the proteomic profiling findings obtained in the present study. These data are reported and analyzed within this manuscript; therefore, an external reference was not originally included. We have now revised the sentence to clearly indicate that the statement refers to the current results.

 

Comment 4. Figure 2 c, d, e, f is very small. Please enlarge.

Response: We appreciate the reviewer’s suggestion. Figures 2c, 2d, 2e, and 2f have been resized and rearranged to increase their size and enhance clarity. The updated Figure 2 has been included in the revised manuscript.

 

Comment 5. Line 158. Please briefly describe the steps involved in obtaining exosomes.

Response: Thank you for this constructive comment. A brief description of the exosome isolation procedure has now been added in Lines 161–166. MSC-derived exosomes were obtained from conditioned medium by initial concentration using a 10 kDa Amicon Ultra centrifugal filter, followed by purification with qEV size-exclusion chromatography columns. The exosome-enriched fractions were subsequently subjected to ultracentrifugation, resuspended in PBS, and stored for downstream analyses. Detailed experimental procedures are provided in Section 4.2 of the Methods.

 

Comment 6. Figure 3a. The size scale is unclear.

Response: We appreciate the reviewer’s useful comment. A clear scale bar has now been added to Figure 3a, and the size scale has been adjusted to improve clarity and readability in the revised manuscript.

 

Comment 7. Sections 2.3-2.5. 787 proteins were identified by LC–MS/MS. Can such a large number of proteins fit into an exosome with a diameter of ~200 nm? This is doubtful. The large number of proteins is likely due to insufficient purification of the exosomes. The authors should critically review this section. Proteomic analysis performed on insufficiently purified samples calls into question all subsequent bioinformatic conclusions regarding the functional composition of exosomes. Evidence of the high purity of the exosome preparation (absence of cytoplasmic and organelle markers) must be provided, or the data interpretation must be reconsidered, recognizing the possible contribution of co-isolated contaminants.

Response: We fully acknowledge the reviewer’s concern that identification of a large number of proteins in extracellular vesicle (EV) preparations may reflect not only the intrinsic complexity of EV-associated proteins—including loosely associated protein corona and secreted proteins—but also the possible presence of co-isolated non-EV components resulting from incomplete purification. In our study, a total of 787 proteins were identified by LC–MS/MS. Importantly, comparable proteomic studies of MSC-derived EVs have reported protein numbers ranging from several hundred to over one thousand, depending on the cell source, EV isolation strategy, and the depth of mass spectrometry analysis. For example, Anderson et al. reported approximately 1,927 proteins in MSC-derived exosomes, Wang et al. identified 1,014 proteins, and Li et al. reported 807 proteins in human umbilical cord MSC-derived EVs. Furthermore, Abyadeh et al. demonstrated that different EV isolation methods substantially influence both the number and composition of identified proteins, thereby emphasizing that high protein counts may arise from sensitive MS detection as well as methodological differences in EV purification. Consistent with these reports, the number of proteins identified in our MSC EV–enriched preparations falls within the range previously described for MSC-EV proteomic studies.

In this study, exosomes were isolated using a combined approach of size-exclusion chromatography and ultracentrifugation, which is widely regarded as an effective strategy to reduce soluble protein contamination while preserving vesicle integrity. Exosome identity and quality were validated by nanoparticle tracking analysis (size distribution), transmission electron microscopy (morphology), and western blot detection of established exosomal markers, as shown in Figure 3. Notably, the mitochondrial marker cytochrome c, indicative of intracellular contamination, was not detected; however, other negative markers were not assessed.

Furthermore, the enrichment of canonical exosome-associated pathways and extracellular vesicle–related proteins, including those identified in STRING Cluster 1 (“extracellular exosome”), supports the overall validity of the dataset as vesicle-enriched rather than reflecting random cellular contamination. Nevertheless, we agree with the reviewer that complete exclusion of co-isolated proteins cannot be guaranteed. We have revised Sections 2.3–2.4 accordingly to include a more critical interpretation of the proteomic data. The revised manuscript explicitly acknowledges the potential contribution of vesicle-associated and extracellular proteins and avoids overinterpretation of the dataset as representing exclusively intraluminal exosomal cargo. We now emphasize that the bioinformatic analyses reflect the functional landscape of MSC exosome-enriched preparations rather than the proteomic content of individual purified vesicles.

Collectively, these revisions ensure a more balanced and cautious interpretation of the proteomic findings and strengthen the rigor and transparency of the study.

 

Comment 8. The reference to Figures 4 and 5 does not correspond to the figures provided.

Response: We appreciate the reviewer’s careful observation. The figure numbering and in-text citations for Figures 4 and 5 have been revised to ensure consistency between the text and the corresponding figures.

 

Comment 9. Line 642. Since this study did not analyze microRNAs, discussion of microRNAs is beyond the scope of this paper and should be removed.

Response: We agree with the reviewer’s insightful comment. Accordingly, since microRNAs were not analyzed in this study, the discussion related to microRNAs at Line 642 has been removed to ensure that the Discussion remains within the scope of the presented data.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1. The paper modified by the author has a clearer logic and the Figures and Tables are consistent with the content. However, there are still some issues that need to be.
2. What information did the results of the exosomal proteomics analysis (2.3, 2.4, 2.5, 26) provide for the “Effect of MSC-exosome pretreatment on CC spheroids (2.7, 2.8, 2.9)”?The authors need to explain this clearly in an appropriate place. Why did the authors choose to target key regulators of the DNA damage response, apoptosis, and NF-κB pathways in section “7, 2.8, 2.9” to prove the Effect of MSC-exosome pretreatment on CC spheroids? Information on NF-κB signaling pathways was not seen in the results of the exosomal proteomics analysis (2.3, 2.4, 2.5 2.6).
3. "2.5. Pathway enrichment of MSC-exosome proteins relevant to chemotherapy": Which database was used to obtain the intersection results?
4. Figure 7. (c) : It should be clear that caspase activation is caspase-3/7 activation.
5. Figure 9. (f) is empty.
6. Figure 8. (a), figure 9. (a): why are the WB result with or without MSCexosome pretreatment on two different membranes? Is it a simultaneous test? Is it comparable? The author needs to explain.

 

  Comments on the Quality of English Language

no

 

 

Author Response

1. The paper modified by the author has a clearer logic and the Figures and Tables are consistent with the content. However, there are still some issues that need to be.

Response We thank the reviewer for acknowledging that the revised manuscript has improved in clarity and consistency. The remaining concerns are addressed individually below.

1. What information did the results of the exosomal proteomics analysis (2.3, 2.4, 2.5, 26) provide for the “Effect of MSC-exosome pretreatment on CC spheroids (2.7, 2.8, 2.9)”? The authors need to explain this clearly in an appropriate place. Why did the authors choose to target key regulators of the DNA damage response, apoptosis, and NF-κB pathways in section “7, 2.8, 2.9” to prove the Effect of MSC-exosome pretreatment on CC spheroids? Information on NF-κB signaling pathways was not seen in the results of the exosomal proteomics analysis (2.3, 2.4, 2.5 2.6).

Response We thank the reviewer for raising this important point. In the revised manuscript, we have clarified how the exosomal proteomics results (Sections 2.3–2.6) informed the design and interpretation of the functional experiments in Sections 2.7–2.9. The exosomal proteomics analyses identified major functional themes within the MSC-exosome cargo, including extracellular matrix remodeling, integrin-mediated signaling, vesicle trafficking, metabolic reprogramming, redox regulation, proteostasis, and apoptosis-related proteins. Pathway enrichment and STRING network analyses demonstrated that these proteins form interconnected functional modules associated with stress adaptation, survival signaling, and chemotherapy-related processes such as drug efflux and detoxification. These functional categories are well known to regulate spheroid architecture, cellular stress responses, survival signaling, and drug sensitivity in 3D models.

Although classical DNA damage response (DDR) proteins were not directly identified as exosomal cargo, several enriched functional categories—particularly redox regulation, metabolic reprogramming, and proteostasis—are well known to influence cellular responses to chemotherapy-induced genomic stress. Platinum agents (cisplatin and carboplatin) primarily exert cytotoxicity through DNA crosslinking and double-strand breaks, while paclitaxel induces mitotic stress that ultimately converges on apoptosis. Therefore, if MSC-exosome cargo modulates oxidative stress balance, metabolic state, or survival signaling, it is mechanistically plausible that downstream DDR activation and apoptotic execution could be modulated at the recipient cell level.

For this reason, in Sections 2.7–2.9 we selected key regulators of the DNA damage response (γH2AX, ATR, pChk1) and apoptosis (Bax, caspase-3/7) as functional readouts. These pathways represent central decision nodes that determine whether chemotherapy-induced stress leads to DNA repair and survival or to apoptotic cell death. Thus, they served as mechanistic endpoints to test whether MSC-exosome pretreatment modifies chemotherapy sensitivity in CC spheroids.

Regarding NF-κB signaling, we acknowledge that canonical NF-κB pathway components were not directly enriched in the exosomal proteomics dataset. However, NF-κB is a well-established master regulator of chemoresistance, stress tolerance, inflammation, and survival in cervical cancer. Importantly, NF-κB signaling functionally intersects with redox balance, integrin/FAK signaling, and metabolic pathways—categories that were strongly represented in the proteomics analysis. Moreover, NF-κB can suppress apoptosis and enhance DNA damage tolerance, thereby influencing responses to platinum and taxane-based therapies.

Therefore, NF-κB pathway markers (IKKα and IκBα) were included not because they were directly identified as exosomal cargo proteins, but to determine whether MSC-exosome pretreatment indirectly modulates survival signaling downstream of the stress- and metabolism-related pathways defined by the proteomics data. In other words, we investigated whether exosome-induced alterations in the cellular microenvironment and stress responses converge on NF-κB–dependent survival regulation.

We have now added a clarifying transition paragraph at the beginning of Section 2.7 to explicitly state that the proteomic findings provided a mechanistic framework, and that DDR, apoptosis, and NF-κB pathways were selected as integrative downstream signaling axes that determine chemotherapy sensitivity. This revision would make clear that the functional experiments were designed to test the biological consequences of the proteome-defined stress, metabolic, and adhesion-related signatures in CC spheroids.

1. "2.5. Pathway enrichment of MSC-exosome proteins relevant to chemotherapy": Which database was used to obtain the intersection results?

Response The pathway enrichment and intersection results presented in Section 2.5 and summarized in Table 2 were obtained using both the PANTHER and Reactome databases. Gene Ontology (GO) biological process and molecular function enrichment analyses were performed using the PANTHER database, whereas chemotherapy-related pathway enrichment and intersection mapping were conducted using the Reactome database. Only pathways meeting the significance threshold (FDR < 0.05) were considered. Table 2 summarizes the proteins identified within these significantly enriched functional categories and chemotherapy-relevant pathways. The listed protein groups were organized based on their annotation in PANTHER (GO classification) and Reactome (pathway mapping), thereby providing a structured overview of MSC-exosome cargo associated with drug response mechanisms. To better reflect the scope of this analysis, we have revised the subheading to:

“2.5. Functional annotation and chemotherapy-related pathway analysis of MSC-exosome proteins.”

This revision clarifies both the databases used and the relationship between the enrichment results and Table 2.

 

4. Figure 7. (c) : It should be clear that caspase activation is caspase-3/7 activation.

Response Figure 7(c) has been revised to clearly indicate that the measured caspase activation corresponds to caspase-3/7 activity. Specifically, the Y-axis label has been changed to “Caspase-3/7 activity,” and the figure caption has been updated accordingly to avoid ambiguity.

1. Figure 9. (f) is empty.

Response Figure 9 have now been edited in the revised manuscript.

1. Figure 8. (a), figure 9. (a): why are the WB result with or without MSC exosome pretreatment on two different membranes? Is it a simultaneous test? Is it comparable? The author needs to explain.

Response We apologize for the lack of clarity in the original presentation. Due to a technical limitation of the western blot apparatus, which allows a maximum of 10 sample lanes per gel, it was not possible to load all treatment conditions on a single membrane. Consequently, samples with and without MSC-exosome pretreatment were resolved on separate gels. Importantly, all western blot experiments were performed simultaneously and processed in parallel under strictly identical experimental conditions, including protein extraction, gel electrophoresis, transfer, antibody incubation, exposure parameters, and signal detection. Each membrane contained the appropriate internal loading control (GAPDH), and protein expression levels were normalized accordingly.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The implemented revisions have substantially strengthened the manuscript. All issues I previously identified have been resolved, resulting in a marked improvement in the text's overall quality. I have no further feedback.

Author Response

We thank the reviewer for the positive feedback and are pleased that all concerns have been satisfactorily addressed.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The author explained and revised. There are no more questions. 

  Comments on the Quality of English Language

no