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  • Fahad A. Alhumaydhi 1 and
  • Shehwaz Anwar 2,*

Reviewer 1: Anonymous Reviewer 2: Anonymous Reviewer 3: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Al-Humaydhi and Anwar submit a review article entitled „Extracellular vesicles in cancer: from intracellular communication to clinical applications”. In 2025 alone, at least 2,153 reviews on exosomes and 2,562 on extracellular vesicles were published. In view of this, the authors deserve credit for covering all aspects related to extracellular vesicles, rather than focusing on one or two. However, covering all aspects makes the manuscript lengthy as it necessitates repeating the same information from different perspectives. Without suffering a loss of information the manuscript could be shortened considerably, e.g.:

  • 100,101 and ll.113-115: repetitive/redundant.
  • 518-521, involvement of EVs in intercellular communications has been mentioned before (several times) and is mentioned again later (ll 664-666).
  • 817-820: “EVs help cells communicate and transport important materials like proteins, fats (lipids), and genetic information, making them strong options….disease diagnostics, prognosis, and therapy”. This has been said before already.
  • Parts of section 13 are redundant and could be included in/incorporated into previous chapters, e.g. 13.1. could be moved to section 5., 13.3. could be moved to sections 3. and 4., ll. 915-921 could be moved to section 12. .
  • 903-905: “Exosomes include certain subsets of cytosolic proteins, lipids, membrane proteins, nucleic acids, and other…”. This has been mentioned before already.
  • 910, 911: “…EV cargos, which include proteins, lipids and nuclear acids”; again repetitive.
  • 953-973: the part on the storage temperature can be shortened drastically.

Now and then, the citation praxis is questionable. For instance, in the first two paragraphs of the Introduction (ll. 45-64) the authors cite six (6/9) of their own papers. There is no problem with self citations per se, but when checking the cited reference one does not find the cited information but another citation/reference which then does provide the facts. For example, the statement (ll. 47, 48) “Cancer is associated with high rates of mortality and morbidity” refers to references 2 and 3. When checking reference 2 no direct information supporting this statement can be found. Instead, the first sentence of refence #2 (Introduction) mentions numbers based on/referring to “Siegel, Miller, Fuchs, Jemal  2022. Cancer statistics, 2022. CA Cancer J Clin 72, 7-33.” This latter reference instead of the authors’ “Hesperidin, a bioflavanoid in cancer therapy-paper” should be cited in the present manuscript.  In the same manner, reference 3 in the submitted manuscript, which is a review, should be replaced by the paper cited therein: “Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021, 71, 209–249.”

The two tables are great and really helpful.

  1. 183: “Typically, early endosomes are found outside of the cytoplasm.” This sentence is confusing. Are early endosomes not located inside the cell but extracellularly?
  2. 198,199: “The redistribution of phosphatidylserine to the extracellular-facing layer of the plasma membrane generates local curvatures, initiating vesicle-budding.” Phosphatidylserine in the outer layer of the membrane stimulates macrophages to phagocytose. Wouldn’t this result in a quick phagocytosis of EVs, i.e. a very short half life or survival of EVs?

Line 276: “There are seven major techniques…”. Both Figure and legend show and describe only six techniques, not seven.

Ll. 311, 312: “Exosomes produce short RNAs…”. Do exosomes produce short RNAs? Or do they “carry” or “contain” short RNA?

  1. 372-374: “Interestingly, miR-125 improves tip cell specification by lowering DLL4, whereas (while) miR-31 works by blocking the component that inhibits HIF-1-alpha.” This could be further explained in more detail: blocking HIF1alpha inhibition means that HIF1alpha can be activated which would result in the expression and secretion of VEGF and PDGF and thus stimulate (neo)angiogenesis.
  2. 621,622 and 627-629: I wonder whether these two sentences belong together. 627-29 could be shifted upwards so that it would read: “Researchers in one study discovered that EVs are more successful than liposomal delivery systems when comparing their biological effects. To decrease pancreatic cancer in mice models, exosomes have been utilized as delivery vehicles for small interfering RNA to target KrasG12D, and this method works better than liposome administration (reference).”

Ll. 947-949: Here, storage of EVs is described as a challenge, whereas in line 730, the authors state that EV vaccines have several benefits including the convenience of storage and transportation. How to reconcile these contrastive statements?

  1. 959 ff.: What about storing EVs in liquid nitrogen?

The list of abbreviation on page 28 should be kept in alphabetical order. It is by far not complete, and the following abbreviations should be added and additionally be defined at first use:

- MVs (line 158), please also define as micro vesicles (?) at first use,  

- line 368: EC (endothelial cells ???),

- line 503, entire section 8.8. : What does “T-EVs” mean?  It is not defined. I assume, T-EVs means tumor derived extracellular vesicles. Later on, the authors use tdEVs instead, but define it too late (l. 877). tdEVs is listed, T-EVs is not listed.

-  l. 573: RCC, probably “renal cell carcinoma”, should be defined an added to the list of abbreviations.

- l. 581: SACC, not defined, not in the list.

- l. 584: PMN, not defined, not in the list.

- l. 652: SCLC, not defined, not in the list.

- l. 711: A2780/DDP and A2780 cells, not defined, not in the list.

- l. 746: what does EVX-M+P mean? What does it stand for? Please define and add to the list of abbreviations.

- l. 750: memory CTLs; please define as cytotoxic T-cells and add to the list.

- l. 752: OVA-pulsed DEXs, please define/add to the list (Ovalbumin and DEXs)-

 

Typos, Errors:

  • 86-88, suggestion: “This review outlines the evolving significance of EVs in cancer diagnosis and therapy, emphasizing the_ biological characteristics of EVs, their roles in tumor progression, and….”
  • 98,99: “…mediators of various intracellular communications, affecting…”. Did the authors mean to write “intercellular” instead of “intracellular”?
  • 100, suggestion: “All bodily fluids, “such” as plasma, urine….” (“such” instead of “including”)
  • 220: “Gag”. It may be helpful to the reader to define Gag as “group-specific antigen”. Biochemists and ECM experts may associate Gag with glycosaminoglycan.
  • 221: remove “one” space between “.” and “Therefore,…”.
  • 294, 295: “Additionally, varios RNAs, including lncRNA, play in influencing the growth of cancer cells”. Something missing here? “…play a role in influencing the growth?
  • 314: suggestion: “A previous study…” instead of “Previous study…”.
  • 327-330: The content/purpose of (or the rationale behind) this sentence, particularly the second part (contrasting with…) does not get clear. Rephrasing is recommended.
  • 366: “were encouraged” or “are encouraged”?
  • 373: “whereas”. Do the authors want to express a “contrast” or the “opposite”? Or could “while” be used instead of “whereas”?
  • 376: suggestion: within “the” TME that promote…”.
  • 421; “The epithelial to EMT may also be facilitated…”. Suggestion: remove “The epithelial to” => “EMT may also be facilitated…”.
  • 487, 488: Suggestion: Connect the two sentences: “EVs are important regulators of immune response against cancer, while dendritic cell-derived…”.
  • 501, 502: “However”. Is “however” the word to use in this context? Maybe omit?
  • 600-602: “EVs are reported as promising therapeutic agents for solid tumor management, as both independent therapies as (?) supplements to improve the effectiveness of established approaches”. Syntax? Sentence not clear.
  • 676, 677: “the maturation of mature cells”. What does maturation of already mature cells mean? Pleonasm.
  • 707: “…by Liu et al.” Please add a second dot/period.
  • 764-768: Tenses not consistent?? “These T-EVs exert_ immonuadjuvant effects…, increase”d” secretion of, and improve”d”….”?
  • 785, 786: “…have been developed to EV performance…”. Something missing here? Sentence incomplete? …have been developed to “improve, optimize, enable, ensure…EV performance”?
  • 788 and 791: “Click chemistry” mentioned twice. On purpose?
  • 793: suggestion: “More work needs to be done….”.
  • 805: “Current research emphasize”s”….”.
  • 839: “…has emerged as a noble approach…” or “…as a novel approach...”?

Author Response

We sincerely thank the Editor and the reviewers for their insightful and constructive comments, which have significantly helped us to improve the quality and clarity of the manuscript. In response to the reviewers’ suggestions, we have carefully revised the manuscript.

  1. Comment- 100,101 and ll.113-115: repetitive/redundant.

Response- We are very grateful to our reviewer team for pointing it. The redundant description has been rewritten for removing repeated description as suggested.

  1. Comment- 518-521, involvement of EVs in intercellular communications has been mentioned before (several times) and is mentioned again later (ll 664-666).

Response- Done. We thank our respected reviewer for highlighting this redundancy. The repetitive content has been removed as suggested.

  1. Comment- 817-820: “EVs help cells communicate and transport important materials like proteins, fats (lipids), and genetic information, making them strong options….disease diagnostics, prognosis, and therapy”. This has been said before already.

Response- We are very grateful to our reviewer team for pointing it. The redundant description has been rewritten for removing repeated description as suggested.

  1. Comment 3- Parts of section 13 are redundant and could be included in/incorporated into previous chapters, e.g. 13.1. could be moved to section 5., 13.3. could be moved to sections 3. and 4., ll. 915-921 could be moved to section 12. .

Response- We sincerely thank the reviewer for this thoughtful comment. Although some aspects discussed in Section 13 relate to earlier sections, we felt it was important to keep this part as a dedicated Challenges section, as it brings together the practical, technical, and regulatory hurdles that emerge specifically at the stage of clinical translation. To address the reviewer’s concern, we have carefully edited the section to reduce repetition and ensure it complements, rather than reiterates, the preceding chapters.

  1. Comment 4- 903-905: “Exosomes include certain subsets of cytosolic proteins, lipids, membrane proteins, nucleic acids, and other…”. This has been mentioned before already.

Response- We are sincerely thankful to our respected reviewer for pointing out it. The repetitive content has been removed.

  1. Comment- 910, 911: “…EV cargos, which include proteins, lipids and nuclear acids”; again repetitive.

Response- We are sincerely thankful to our respected reviewer for pointing out it. The repetitive content has been removed.

  1. Comment- 953-973: the part on the storage temperature can be shortened drastically.

Response- Thanks for noticing it. This section has been substantially edited .

  1. Comment- Now and then, the citation praxis is questionable. For instance, in the first two paragraphs of the Introduction (ll. 45-64) the authors cite six (6/9) of their own papers. There is no problem with self citations per se, but when checking the cited reference one does not find the cited information but another citation/reference which then does provide the facts. For example, the statement (ll. 47, 48) “Cancer is associated with high rates of mortality and morbidity” refers to references 2 and 3. When checking reference 2 no direct information supporting this statement can be found. Instead, the first sentence of refence #2 (Introduction) mentions numbers based on/referring to “Siegel, Miller, Fuchs, Jemal Cancer statistics, 2022. CA Cancer J Clin 72, 7-33.” This latter reference instead of the authors’ “Hesperidin, a bioflavanoid in cancer therapy-paper” should be cited in the present manuscript.  In the same manner, reference 3 in the submitted manuscript, which is a review, should be replaced by the paper cited therein: “Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021, 71, 209–249.”

 

Response. We sincerely thank the reviewer for carefully pointing out this issue and for the constructive guidance regarding citation practices. We apologize for the inadvertent use of secondary citations in the Introduction. In response, we have revised the manuscript to replace the cited review and self-citations with the original primary sources that directly support the stated epidemiological information. These changes have been implemented to improve accuracy, transparency, and scholarly rigor. We appreciate the reviewer’s careful attention, which has helped strengthen the quality of the manuscript.

  1. Comment- The two tables are great and really helpful.

             Response- We sincerely thank the reviewer for this positive and encouraging comment.   We are pleased to hear that the tables were found to be clear and helpful, and we appreciate the reviewer’s thoughtful evaluation of our work.

  1. Comment- 183: “Typically, early endosomes are found outside of the cytoplasm.” This sentence is confusing. Are early endosomes not located inside the cell but extracellularly?

Response- We thank the reviewer for this helpful observation and apologize for the unclear wording in the original text. The sentence has been revised to accurately describe the intracellular localization of early endosomes. We appreciate the reviewer’s careful attention to this detail.

  1. Comment- 198,199: “The redistribution of phosphatidylserine to the extracellular-facing layer of the plasma membrane generates local curvatures, initiating vesicle-budding.” Phosphatidylserine in the outer layer of the membrane stimulates macrophages to phagocytose. Wouldn’t this result in a quick phagocytosis of EVs, i.e. a very short half life or survival of EVs?

Response- We sincerely thank the reviewer for this valuable comment. We have added a sentence clarifying that, although phosphatidylserine is exposed on extracellular vesicles, they are not necessarily cleared immediately, as their fate is influenced by size, surface proteins, and selective uptake by target cells.

  1. Comment- Line 276: “There are seven major techniques…”. Both Figure and legend show and describe only six techniques, not seven.

Response- We sincerely thank the reviewer for pointing out this oversight. We regret the error and have corrected the text to accurately reflect that six major techniques are shown and described in Figure 2 and its legend. We appreciate the reviewer’s careful attention to detail.

  1. Comment- 311, 312: “Exosomes produce short RNAs…”. Do exosomes produce short RNAs? Or do they “carry” or “contain” short RNA?

Response- We sincerely thank the reviewer for pointing out this oversight. We regret the error and have corrected the text to clarify that exosomes carry short RNA.

  1. Comment- 372-374: “Interestingly, miR-125 improves tip cell specification by lowering DLL4, whereas (while) miR-31 works by blocking the component that inhibits HIF-1-alpha.” This could be further explained in more detail: blocking HIF1alpha inhibition means that HIF1alpha can be activated which would result in the expression and secretion of VEGF and PDGF and thus stimulate (neo)angiogenesis.

Response. We sincerely thank the reviewer for this insightful comment. We have revised the text. We appreciate the reviewer’s thoughtful suggestion for improving the mechanistic clarity of this section.

  1. Comment - 621,622 and 627-629: I wonder whether these two sentences belong together. 627-29 could be shifted upwards so that it would read: “Researchers in one study discovered that EVs are more successful than liposomal delivery systems when comparing their biological effects. To decrease pancreatic cancer in mice models, exosomes have been utilized as delivery vehicles for small interfering RNA to target KrasG12D, and this method works better than liposome administration (reference).”

Response- We sincerely thank the reviewer for this helpful suggestion. We have revised the text. We appreciate the reviewer’s careful reading and valuable input.

  1. Comment- 947-949: Here, storage of EVs is described as a challenge, whereas in line 730, the authors state that EV vaccines have several benefits including the convenience of storage and transportation. How to reconcile these contrastive statements?

Response- We sincerely thank the reviewer for highlighting this important point. We revised the text and added following statement.

Even though EV vaccines are relatively easier to store and transport than cell-based therapies, this advantage is relative, as standardized long-term preservation strategies to maintain stability and bioactivity of EVs remain a significant challenge.  

  1. Comment- 959 ff.: What about storing EVs in liquid nitrogen?

Response- We thank the reviewer for this valuable suggestion. A new paragraph has been added to the manuscript addressing liquid nitrogen storage of EVs, summarizing its advantages, limitations, and the importance of optimized freezing protocols and cryoprotectants to preserve EV stability and functionality.

  1. Comment- The list of abbreviation on page 28 should be kept in alphabetical order. It is by far not complete, and the following abbreviations should be added and additionally be defined at first use:

      Response- The list has been corrected, completed, and reordered alphabetically.

  1. Comment- MVs (line 158), please also define as micro vesicles (?) at first use.

Response- Done. Defined as microvesicles at first mention.

  1. Comment- line 368: EC (endothelial cells ???),

Response. Done. Defined at first mention

  1. Comment- line 503, entire section 8.8. : What does “T-EVs” mean? It is not defined. I assume, T-EVs means tumor derived extracellular vesicles. Later on, the authors use tdEVs instead, but define it too late (l. 877). tdEVs is listed, T-EVs is not listed.

Response- Thanks to our respected reviewer. Defined at first mention. T-dEVs is replaced by T-EVs.

  1. Comment- 573: RCC, probably “renal cell carcinoma”, should be defined an added to the list of abbreviations.

Response- Done. Defined at first mention and added to the list.

  1. Comment- 581: SACC, not defined, not in the list.

Response- Done. Defined at first mention and added to the list.

  1. Comment- 584: PMN, not defined, not in the list

       Response. Added. Defined at first mention and added to the list

  1. Comment- 652: SCLC, not defined, not in the list.

Response- Done. Defined at first mention and added to the list.

  1. Comment- l. 711: A2780/DDP and A2780 cells, not defined, not in the list.

Response- Done. Defined at first mention and added to the list.

 

  1. Comment- 746: what does EVX-M+P mean? What does it stand for? Please define and add to the list of abbreviations.

Response- Done. Defined at first mention and added to the list.

  1. Comment- 750: memory CTLs; please define as cytotoxic T-cells and add to the list.

Response- Done. Defined at first mention and added to the list.

  1. Comment- 752: OVA-pulsed DEXs, please define/add to the list (Ovalbumin and DEXs)-

Response- Done. Defined at first mention and added to the list.

 

  1. Comment- Typos, Errors:

             Response- All typographical and grammatical errors have been corrected.

  1. Comment- 86-88, suggestion: “This review outlines the evolving significance of EVs in cancer diagnosis and therapy, emphasizing the_ biological characteristics of EVs, their roles in tumor progression, and….”

 

Response- Done. We revised the text.

  1. Comment- 98,99: “…mediators of various intracellular communications, affecting…”. Did the authors mean to write “intercellular” instead of “intracellular”?

Response- Done. We revised the text.

  1. Comment- 100, suggestion: “All bodily fluids, “such” as plasma, urine….” (“such” instead of “including”)

Response- Done. We revised the text.

  1. Comment- 220: “Gag”. It may be helpful to the reader to define Gag as “group-specific antigen”. Biochemists and ECM experts may associate Gag with glycosaminoglycan.

Response- Done. We revised the text.

  1. Comment- 221: remove “one” space between “.” and “Therefore,…”.

Response- Done. We revised the text.

  1. Comment- 294, 295: “Additionally, varios RNAs, including lncRNA, play in influencing the growth of cancer cells”. Something missing here? “…play a role in influencing the growth?

Response- Done. We revised the text.

  1. Comment- 314: suggestion: “A previous study…” instead of “Previous study…”.

Response- Done. We revised the text.

  1. Comment- 327-330: The content/purpose of (or the rationale behind) this sentence, particularly the second part (contrasting with…) does not get clear. Rephrasing is recommended.

Response- We agree with the reviewer that the sentence lacked clarity. It has been rephrased to clearly distinguish the reported finding from earlier studies and to improve logical flow and readability.

  1. Comment- 366: “were encouraged” or “are encouraged”?

Response- We have corrected the sentence to improve clarity.

  1. Comment- 373: “whereas”. Do the authors want to express a “contrast” or the “opposite”? Or could “while” be used instead of “whereas”?

Response- We agree with the reviewer. As the two microRNAs act through distinct but not opposing mechanisms, “whereas” was replaced with “while” to improve clarity.

  1. Comment- 376: suggestion: within “the” TME that promote…”.

Response- We have revised this sentence to correct minor grammatical issues, improve clarity, and ensure consistency with standard terminology.

  1. Comment- 421; “The epithelial to EMT may also be facilitated…”. Suggestion: remove “The epithelial to” => “EMT may also be facilitated…”.

Response- We agree with the reviewer and have revised the sentence by removing the redundant phrase “The epithelial to,” resulting in a clearer and grammatically correct statement. In revised manuscript, we revised the sentence

  1. Comment- 487, 488: Suggestion: Connect the two sentences: “EVs are important regulators of immune response against cancer, while dendritic cell-derived…”.

Response- We have corrected the sentence to improve clarity.

  1. Comment- 501, 502: “However”. Is “however” the word to use in this context? Maybe omit?

Response- We are thankful to our respected reviewer for figuring out the errors. All suggested edits have been implemented to improve clarity, grammar, consistency, and scientific precision. We omitted this word.

  1. Comment- 600-602: “EVs are reported as promising therapeutic agents for solid tumor management, as both independent therapies as (?) supplements to improve the effectiveness of established approaches”. Syntax? Sentence not clear.

Response- We are thankful to our respected reviewer for figuring out the errors. All suggested edits have been implemented to improve clarity, grammar, consistency, and scientific precision.

  1. Comment- 676, 677: “the maturation of mature cells”. What does maturation of already mature cells mean? Pleonasm.

Response- We are thankful to our respected reviewer for figuring out the errors. All suggested edits have been implemented to improve clarity, grammar, consistency, and scientific precision.

  1. Comment- 707: “…by Liu et al.” Please add a second dot/period.

Response- We revised the text and a second dot is added.

  1. Comment- 764-768: Tenses not consistent?? “These T-EVs exert_ immonuadjuvant effects…, increase”d” secretion of, and improve”d”….”?

Response. We are very thankful to our respected reviewer. We have corrected the sentence to improve clarity.

  1. Comment- 785, 786: “…have been developed to EV performance…”. Something missing here? Sentence incomplete? …have been developed to “improve, optimize, enable, ensure…EV performance”?

Response- We are very thankful to our respected reviewer. We have revised the text.

  1. Comment- 788 and 791: “Click chemistry” mentioned twice. On purpose?

Response- We are very thankful to our respected reviewer. We have corrected the sentence to improve clarity.”

  1. Comment- 793: suggestion: “More work needs to be done….”.

Response- We have corrected the sentence to improve clarity.

  1. Comment- 805: “Current research emphasize”s”….”.

Response- We have corrected the sentence to improve clarity.

  1. Comment- 839: “…has emerged as a noble approach…” or “…as a novel approach...”?

Response- We have corrected the sentence to improve clarity.

Reviewer 2 Report

Comments and Suggestions for Authors

Authors summarize literature focused on the role of EVs in cancer, in my opinion, the content of the review does not fully align with its title. While the authors extensively discuss the role of extracellular vesicles, they do not sufficiently focus on what is currently reported for different tumor types, which would have provided a broader and more comprehensive perspective. 

Several sections appear redundant and could be merged to improve clarity and conciseness. For example, paragraph 5, which describes methods for EV isolation and purification, is revisited in sections 13.1 and 13.2 when discussing related challenges. This repetition could be streamlined. 

The figures could be significantly improved, as they are currently too schematic and lack depth. More detailed and informative illustrations would enhance the overall quality of the review. 

Finally, the discussion of EVs in cancer seems largely confined to several types of tumors and their role in metastasis. Expanding this to include other relevant aspects—such as tumor initiation, progression, and therapy resistance—would make the review more comprehensive and better aligned with its stated scope. There are interesting papers which describe these aspects: doi.org/10.1002/jev2.70038, 10.1186/s12943-024-02083-y, doi.org/10.1186/s12943-020-01199-1, 0.1152/physrev.00019.2024.

I believe these issues need to be clarified before publication. 

 

Author Response

We sincerely thank Reviewer 2 for their thoughtful and constructive evaluation of our manuscript. We greatly appreciate their positive assessment of the scope, relevance, and review value of the article. We have carefully addressed all comments and suggestions, and revised the manuscript accordingly to improve clarity, depth, and overall quality. We hope that the revised version meets the reviewer’s expectations.

  1. Comment- Authors summarize literature focused on the role of EVs in cancer, in my opinion, the content of the review does not fully align with its title. While the authors extensively discuss the role of extracellular vesicles, they do not sufficiently focus on what is currently reported for different tumor types, which would have provided a broader and more comprehensive perspective.

Response- In response to our respected reviewer’s valuable comment, we have added a new section before the Conclusion that explicitly discusses tumor-type–specific roles of extracellular vesicles. This section highlights representative examples from solid and hematological malignancies, emphasizing cancer-specific EV cargo, mechanisms, and functional outcomes.

  1. Comment- Several sections appear redundant and could be merged to improve clarity and conciseness. For example, paragraph 5, which describes methods for EV isolation and purification, is revisited in sections 13.1 and 13.2 when discussing related challenges. This repetition could be streamlined.

Response- We sincerely thank our respected reviewer for this thoughtful comment. We have    streamlined the manuscript so that EV isolation methods are described only in Section 5, while challenges, reproducibility issues, and standardization are now clearly discussed in Section 13.1, and biological heterogeneity in Section 13.2. This improves clarity and avoids repetition.

  1. Comment- The figures could be significantly improved, as they are currently too schematic and lack depth. More detailed and informative illustrations would enhance the overall quality of the review.

Response- We sincerely thank our respected reviewer for this thoughtful comment. We have addressed this comment by adding two additional detailed and informative illustrations to enhance the depth and overall quality of the figures

  1. Comment- Finally, the discussion of EVs in cancer seems largely confined to several types of tumors and their role in metastasis. Expanding this to include other relevant aspects—such as tumor initiation, progression, and therapy resistance—would make the review more comprehensive and better aligned with its stated scope. There are interesting papers which describe these aspects: doi.org/10.1002/jev2.70038, 10.1186/s12943-024-02083-y, doi.org/10.1186/s12943-020-01199-1, 0.1152/physrev.00019.2024.

Response- We thank the reviewer for this insightful comment. In response, we have added a new subsection entitled “Role of EVs Beyond Metastasis” to expand the discussion on EV functions beyond metastatic dissemination. This section now addresses the roles of EVs in tumor initiation, progression, microenvironment remodeling, and therapy resistance, incorporating the suggested references. We believe this addition strengthens the scope and comprehensiveness of the review.

Reviewer 3 Report

Comments and Suggestions for Authors

This article reviews the multifaceted roles of extracellular vesicles (EVs) in cancer biology, including their involvement in tumorigenesis, development, immune regulation, pre-metastatic niche formation, and their potential as liquid biopsy biomarkers and drug delivery systems. The article has considerable review value. We recommend considering acceptance after revisions.

1. Some paragraphs are too general; for example, Section 5, "Methods for Isolation and Purification of EVs," could be supplemented with more recently developed technologies (such as microfluidic chips) and a comparison of their advantages and disadvantages.

2. Some references are outdated; we suggest updating them with high-impact research from the past 3–5 years, especially in the clinical translation section.

3. A brief discussion of whether different EV subtypes have specific functions in cancer would be beneficial.

4. Section 13 could briefly mention the ethical and regulatory challenges faced by EVs as therapeutic products.

In summary, the manuscript can be accepted after revisions.

Author Response

This article reviews the multifaceted roles of extracellular vesicles (EVs) in cancer biology, including their involvement in tumorigenesis, development, immune regulation, pre-metastatic niche formation, and their potential as liquid biopsy biomarkers and drug delivery systems. The article has considerable review value. We recommend considering acceptance after revisions.

Response- We sincerely thank the reviewer for this positive and encouraging assessment of our manuscript. We greatly appreciate the recognition of the review’s scope and relevance. In response to the valuable suggestions provided, we have carefully revised the manuscript to further improve its clarity, depth, and overall quality. We hope that the revised version meets the reviewer’s expectations and is now suitable for acceptance.

  1. Comment- Some paragraphs are too general; for example, Section 5, "Methods for Isolation and Purification of EVs," could be supplemented with more recently developed technologies (such as microfluidic chips) and a comparison of their advantages and disadvantages.

Response- We sincerely thank our respected reviewer for this constructive comment. Although microfluidic-based technologies were briefly mentioned in the original manuscript, we agree that the discussion was too general. We have therefore expanded Section 5 by adding a dedicated paragraph detailing microfluidic EV isolation strategies, including immunoaffinity- and size-based platforms, and explicitly comparing their advantages and limitations relative to conventional methods.

  1. Comment- Some references are outdated; we suggest updating them with high-impact research from the past 3–5 years, especially in the clinical translation section.

Response- We have updated the references by adding many recent high-impact studies from the past 3–5 years and replacing several outdated references, particularly in the clinical translation section.

  1. Comment- A brief discussion of whether different EV subtypes have specific functions in cancer would be beneficial.

Response- We sincerely thank our reviewer for this valuable and constructive feedback. In response, we have added a new dedicated section discussing the distinct biogenesis, molecular cargo, and cancer-specific functions of different EV subtypes, including exosomes, microvesicles, and apoptotic bodies, with emphasis on their differential roles in tumor progression and metastasis.

  1. Comment- Section 13 could briefly mention the ethical and regulatory challenges faced by EVs as therapeutic products.

Response- We thank the reviewer for this important comment. Section 13 has been expanded to include a concise discussion of ethical and regulatory challenges associated with EV-based therapeutics, including standardization, safety, scalability, and regulatory approval considerations.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

o.k.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have revised the manuscript in accordance with my suggestions, resulting in significant improvement.

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

accept

Comments on the Quality of English Language

accept