Extracellular Vesicles in Cancer Diagnosis and Therapy: Advances, Challenges, and Prospects for Clinical Translation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review article focuses on the potential applications of extracellular vesicles (EVs, including exosomes and other subtypes) in oncology. However, the overall structure of the manuscript is highly disorganized, with unclear concepts and severe conflation of terms, indicating a profound lack of familiarity with the field on the part of the authors. A clear distinction exists between extracellular vesicles (EVs) and exosomes—the latter being a specific subtype of EVs—yet the review consistently conflates the two throughout.

The most prominent errors and deficiencies are as follows:

1. Figure 1 features redundant image descriptions with no clear emphasis or focus, rendering it ineffective and uninformative.
2. The discussion of traditional isolation methods (e.g., ultracentrifugation [UC], ultrafiltration [UF], size-exclusion chromatography [SEC], polymer-based precipitation, and immunoaffinity capture) and emerging approaches (including immunoaffinity enrichment, physical feature-based separation such as microfluidic chips, lipid-mediated isolation, and thermophoretic enrichment) relies exclusively on references from before 2022, with no incorporation of more recent literature.
3. Table 3 ("Gene and Drug Delivery Technologies and Their Clinical Applications") is entirely inconsistent with the article's main theme and appears unrelated to EVs.
4. Section 4.4 ("Application Strategies of RNA Therapy") is completely mismatched with the core topic of the review.
5. Subsection 2.2.4 ("Hot swimming rich collection") exhibits obvious translation artifacts and is highly unprofessional in phrasing (likely a mistranslation of "thermophoretic enrichment").

In summary, the manuscript suffers from fundamental conceptual inaccuracies, structural disarray, outdated citations, and thematic inconsistencies that undermine its scientific credibility. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled "Extracellular Vesicles in Cancer Diagnosis and Therapy: Advances, Challenges, and Prospects for Clinical Translation" is a comprehensive narrative review dedicated to summarizing recent advances in extracellilar vesicles (EVs) research. The article contains incredibly detailed information on the topic, so that it could serve as a chapter of an encyclopedia. This narrative review is strengthened by several quite informative and professionally created figures, as well as by tables. 
It is worth noting that there are numerous reviews on the topic of EVs in cancer available in the literature, and the reviewed manuscript does not bring any novelty (it does not contain a new perspective on any previously discussed problems, it does not focus on any underexplored aspects of EVs, it does not include latest research articles, results of which were not discussed previously, etc.). However, this manuscript is written so well that it might still find its reader (personally, I would advise scientists who only begin to work in this field to read it). Overall, in my opinion, it is written better and more comprehensively than most other reviews on the same topic.

Below is a list of my concerns which may require addressing to improve the manuscript.

1) The text is written in good English, yet there are several typos, which require correction (mainly absence of spaces after punctuation marks).

2) Please make sure that all abbreviations are explained in the text, there is no need to introduce abbreviations if they are not used later (for example, tEVs).

3) Lines 203-204. It is written that EVs containing several miRNAs in combination with other biomarkers are highly sensitive and specific in early detection of PDAC. This disease is usually diagnosed at advanced stages, making the research on early diagnosis quite difficult. In the provided reference [56] there is no such data, actually it is a literature review, which cites a different review. Please reconsider this statement and/or provide appropriate references.

4) Tables in this manuscript are quite comprehensive and the effort put to create them is commendable. However, with the present formatting their readability is poor. There is almost no transition between neighbouring cells of tables (especially, in table 3), making it hard to spot a beggining of a new sentence.

5) Table 3 is missing references.

6) There might be a typo in Figure 5 "Equatorow Cqments".

7) The conclusion is written in an unusual way: "This article elaborates... This articles delves... We elaborate on... This review focuses... etc.". This phrasing is more appropriate for the abstract or introduction, where the content of the article might be presented in a brief "teaser" form. Therefore, this section might require some correction (overall, the content of the conclusion is fine).

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. Insufficient focus on practical limitations: The manuscript describes EV isolation and detection methods well, but lacks critical discussion of practical clinical limitations (e.g., cost, time, need for specialized equipment, reproducibility across centers). For instance, in the “Emerging separation methods” section, the methods are described as efficient, but no real comparative data in clinical samples are presented.
2. Redundancy and information dispersion: Sections 2 and 3 show redundancy. For example, the advantages of EVs in liquid biopsy are repeated multiple times in slightly different wording. This reduces the logical flow and can fatigue the reader.
3. Limited critical discussion of challenges and clinical translation: While clinical challenges are mentioned, there is limited critical discussion on real-world translational barriers (e.g., large-scale EV production, long-term stability, safety issues, inter-cancer variability). A separate section with actual clinical trial examples would strengthen the manuscript.
4. The exercise and nutrition context is underdeveloped: The section on exercise and nutrition affecting EVs is brief and non-critical. The direct link to diagnostic or therapeutic applications in cancer is unclear and requires more in-depth analysis and supporting evidence.
5. Insufficient focus on practical limitations in EV applications: The manuscript describes EV applications in gene, drug, and RNA delivery and immunotherapy well, but offers limited critical discussion of practical challenges, such as production and purification complexity, high cost, scalability, and specialized infrastructure requirements. For instance, in the “EV-mediated gene/drug delivery” section, the advantages of EVs are described, but no real comparative clinical data are presented.
6. Redundancy and information dispersion: Certain information is repeated across sections, especially in “Gene Delivery” and “Drug Delivery.” For example, the benefits of EVs for precise drug targeting and reduced toxicity to healthy cells are repeated multiple times in similar wording, disrupting the logical flow and potentially fatiguing the reader.
7. Limited critical discussion of challenges and clinical translation: While clinical and safety challenges are mentioned, there is little critical discussion of real-world translational barriers. Examples include long-term stability, adverse immune responses, complexity of combining EVs with other immunotherapies, and individual patient limitations. A separate section with actual clinical examples would strengthen the manuscript.
8. Limited critical analysis in RNA therapy and immunotherapy: The RNA therapy and immunotherapy sections describe advantages and mechanisms, but practical limitations and potential risks, such as low mRNA/siRNA stability, adverse immune responses, production complexity, and limited target selection, are underexplored. Comparative data or real clinical examples would strengthen these sections.
9. Insufficient focus on the practical and manufacturing limitations of EVs: The manuscript highlights the potential of EVs in diagnostics and therapy but offers limited critical discussion of the practical challenges of production, purification, and standardization. For example, the complexity of controlling flow and electric fields in microfluidic chips and the difficulty of producing homogeneous, impurity-free EVs are only discussed in general terms, without practical data or comparative solutions.
10. Limited critical discussion of EV heterogeneity and marker selection: The manuscript notes EV heterogeneity and limitations of single markers, but lacks in-depth analysis or practical recommendations for selecting or standardizing a multi-molecule detection panel. The pancreatic cancer example highlights the need for combined markers to improve sensitivity and specificity, yet practical implementation strategies are not detailed.
11. Need for extensive clinical validation and standardization: The manuscript emphasizes that clinical translation of EVs requires large-scale clinical trials and standardization of production, but it does not provide a plan or an evaluation of the steps needed to achieve these goals. A lack of practical frameworks for multicenter studies and quality control may limit real-world clinical applicability.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The figure in Figure 2 is not correctly cited in the main text and was mistakenly written as Figure 3. The sources of the images in Figures 3-5 need to be credited.

Author Response

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Author Response File: Author Response.pdf