From the journal RSC Chemical Biology Peer review history

31-Dec-2023

Dear Dr Park:

Manuscript ID: CB-REV-10-2023-000200
TITLE: Recent advances in chemical biology tools for protein and RNA profiling of extracellular vesicles

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary.

Please submit a revised manuscript which addresses all of the reviewers’ comments. Further peer review of your revised manuscript may be needed. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

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Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Andrea Rentmeister

************

Reviewer 1

The review discusses the recent advancements in chemical biology tools for protein and RNA profiling of extracellular vesicles (EVs). EVs, nano-sized vesicles secreted by cells, contain various cellular components and can serve as circulating biomarkers for various diseases. Despite their potential, detecting and isolating targeted EVs in complex body fluids remains challenging. However, recent advances in chemical biology provide new opportunities for efficient profiling of the molecular contents of EVs. Various chemical biology tools have been used to enhance the analytical performance of conventional assays for a better understanding of EV biology. The review also discusses the improvements achieved by these tools.

This comprehensive review is commendable for its in-depth exploration of the advancements in chemical biology tools for protein and RNA profiling of EVs. The authors have done an excellent job in discussing the challenges and potential solutions in the field, providing valuable insights for future research. The interdisciplinary approach suggested in the review, combining chemical biology and biomedical research, is particularly noteworthy and holds great promise for further understanding of EV biology.

1. The review could benefit from a more structured layout, with clear subheadings for each section to guide the reader through the content.

2. The introduction could provide more background information on the importance and potential applications of extracellular vesicles (EVs) in biomedical research.

3. The authors could provide more specific examples of the diseases that can be detected or monitored through EVs, to illustrate their significance in practical applications.

4. The review could include more visual aids, such as diagrams or flowcharts, to help readers understand the complex processes involved in EV profiling.

5. The authors could elaborate more on the limitations and challenges of current technologies for EV detection and isolation, to provide a balanced view of the field.

6. The review could benefit from a more detailed discussion on the future directions and potential advancements in the field of EV profiling.

7. The conclusion could be more concise and focused, summarizing the key points of the review and highlighting the main takeaways for the reader.

8. The authors could consider including a section on the ethical considerations and potential risks associated with the use of EVs in biomedical research, to provide a comprehensive overview of the field.

Reviewer 2

This manuscript reviews studies of the chemical biology tools in the applications of protein and RNA profiling of extracellular vesicles. Generally, the manuscript is comprehensive. However, there are several areas that require further improvements. I recommend that the paper undergo significant revisions before being considered for publication.

1. In the review, some research groups have done a series of professional work. It is suggested that the author link up these works more logically to make the development and research of chemical biology tools more hierarchical.
2. The author needs to insert a detailed table to make the reader understand the manuscript more clearly.
3. It seems to be a problem with the layout of figure 9. Please revise.
4. Some important parts of the manuscript lack literature citations, for example, in the presentation of EV-based applications. There is also a lack of citations in each section where some background or methods are presented. Additionally, the authors should also summarize latest works reported recently.
5. The overview of the conclusions is novel but not sufficient, the author did not mention that the biggest challenge of chemical biology tools in EV research. And we hope the authors can provide more ideas.

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters
Reviewer #1

The review discusses the recent advancements in chemical biology tools for protein and RNA profiling of extracellular vesicles (EVs). EVs, nano-sized vesicles secreted by cells, contain various cellular components and can serve as circulating biomarkers for various diseases. Despite their potential, detecting and isolating targeted EVs in complex body fluids remains challenging. However, recent advances in chemical biology provide new opportunities for efficient profiling of the molecular contents of EVs. Various chemical biology tools have been used to enhance the analytical performance of conventional assays for a better understanding of EV biology. The review also discusses the improvements achieved by these tools.
This comprehensive review is commendable for its in-depth exploration of the advancements in chemical biology tools for protein and RNA profiling of EVs. The authors have done an excellent job in discussing the challenges and potential solutions in the field, providing valuable insights for future research. The interdisciplinary approach suggested in the review, combining chemical biology and biomedical research, is particularly noteworthy and holds great promise for further understanding of EV biology.

We deeply appreciate reviewer #1 for his/her strong support and encouraging comments to our work.


Q1. The review could benefit from a more structured layout, with clear subheadings for each section to guide the reader through the content.

We thank Reviewer #1 for this point. We revised our manuscript with more structure layout as suggested. We added new Table 1 to discuss about current limitation of EV profiling and new approaches to tackle those issues using chemical biology tools. We re-organized manuscript according to the listed challenges of EV profiling in table 1 as follows.


Please also check the following new subheadings in the revised manuscript.

2. EV enrichment
2.1. Immunoaffinity isolation
3. Analysis of EV RNA and protein
3.1. Enzyme reaction
3.2. Hybridization chain reaction
3.3. Rolling circle amplification
3.4. DNA nanostructure
3.5. Stimuli-responsive hydrogel
4. Analysis of EV heterogeneity
4.1. Cyclic imaging
4.2. Immuno-PCR and immuno-sequencing
5. EV glycoprotein analysis
5.1. Metabolic glycoengineering
6. Treatment response monitoring
6.1. EV drug occupancy analysis


Q2. The introduction could provide more background information on the importance and potential applications of extracellular vesicles (EVs) in biomedical research.

It is a good point. We deeply appreciate Reviewer #1 for this helpful comment. We now revised introduction part as follows. Now, we believe that importance and potential application of EV analysis toward disease diagnosis and prognosis are fully discussed in the revised manuscript.

Among the biological fluids, EVs in plasma or urine from patients are easily accessible and well-studied recently for various biomedical research.38 The comprehensive analysis of EV biomarkers in plasma and urine holds immense potential in enabling the diagnosis of various medical conditions, including cancers,17, 30, 32 kidney disease,39 and neurodegenerative diseases.40, 41 Along with diagnosis, treatment monitoring and prognosis by EV analysis have been also recently spotlighted.42 Plasma or urinal EV analysis is now expected to suggest a fast and personalized disease treatment strategy.

More importantly, EVs show strong potential in early diagnosis as the alterations in the EV molecular cargos may occur prior to changes in traditional serum markers or the size of lesion reaches the detection limit of imaging modalities.50


Q3. The authors could provide more specific examples of the diseases that can be detected or monitored through EVs, to illustrate their significance in practical applications.

We thank Reviewer #1 again for this helpful comment to improve our manuscript. We revised our manuscript accordingly as follows.

Among the biological fluids, EVs in plasma or urine from patients are easily accessible and well-studied recently for various biomedical research.38 The comprehensive analysis of EV biomarkers in plasma and urine holds immense potential in enabling the diagnosis of various medical conditions, including cancers,17, 30, 32 kidney disease,39 and neurodegenerative diseases.40, 41 Along with diagnosis, treatment monitoring and prognosis by EV analysis have been also recently spotlighted.42 Plasma or urinal EV analysis is now expected to suggest a fast and personalized disease treatment strategy.

Q4. The review could include more visual aids, such as diagrams or flowcharts, to help readers understand the complex processes involved in EV profiling.

We appreciate Reviewer #1 for this critical point to help readers to understand EV profiling more easily. We now updated Figure 1 with an EV profiling flowchart. We described an overall EV profiling process: Sampling -> EV enrichment -> Sample processing -> Detection.

Q5. The authors could elaborate more on the limitations and challenges of current technologies for EV detection and isolation, to provide a balanced view of the field.
We deeply appreciate reviewer #1 for the point. We now updated the conclusion part in the revised manuscript. We also added new Table 1 to show current limitation of conventional approaches and novel chemical biology approaches to overcome the limitations of EV research.

From the perspective of clinical diagnostics, time-consuming process of EV purification presents obstacles to their effective utilization in robust clinical application. In addition, limited sensitivity for low abundant target EVs is one of the big hurdles for early diagnosis.144

Despite above mentioned recent advances in EV analysis, there are still multiple issues to be solved for bringing EV profiling to real clinic. i) Discovery of disease specific EV marker is urgently required.146 EV profiling-based diagnosis has been commonly demonstrated for a specifically targeted disease instead of differentiating multiple diseases due to the lack of disease-specific EV markers. Combination of previously reported disease markers in cell lines or tissues has been widely used to increase the accuracy of EV based disease diagnosis.121 Due to this limitation, differential diagnosis of multiple diseases was barely reported.
ii) Another limitation of EV profiling-based research is the size of patient cohort for validation.147 Mostly EV analysis for disease diagnosis is validated in small patient cohorts.148-150 To claim the potential clinical significance, EV based disease diagnosis should be demonstrated in the large size of patient cohorts. iii) Most of the current EV analysis technologies are high-cost device dependent assays, which needs well- trained technicians.57 Considering the situation of real clinic, development of easily accessible EV assays with low resource setting is highly demanding. iv) Ethical issues of EV research should be considered during the analysis and diagnosis processes. Personal information of each patient must be handled securely, and de-identification methods are needed to be employed for the protection of patient information. Moreover, false positives and negatives of EV profiling-based diagnosis can contribute to unnecessary stress, or treatments of patients. Enhancing the accuracy of EV profiling based diagnosis would address this ethical issue.151 Researchers are needed to be aware of these ethical issues and manage those concerns in clinical applications.

Q6. The review could benefit from a more detailed discussion on the future directions and potential advancements in the field of EV profiling.

It is another good point. We now revised our manuscript with the future directions and potential advancements in this field.

However, only limited number of chemical biology tools has been used to tackle those issues.82, 120, 121, 130 To shed light on the unexplored territory of EV biology, we believe that the biggest challenge of chemical biology in EV research is to develop new bioorthogonal chemical reactions with superior efficiency. We anticipate that these future advancements in chemical biology tools will offer promising avenues for innovating the field of EV profiling. Chemical biology tools would be further engineered for nanoscale targets, addressing the issue of sensitivity, specificity, and EV heterogeneity. Moreover, standardizing the analytical protocol will make EV profiling robust, facilitating the translational application of EV profiling in real-world clinical diagnostics

Q7. The conclusion could be more concise and focused, summarizing the key points of the review and highlighting the main takeaways for the reader.

We do agree with Reviewer #1 for this point. We now revised our manuscript with the key points of our review in the conclusion part. We also provided future perspectives of chemical biology research in EV profiling for successful translational application in the last part of the revised manuscript.

We introduced an efficient and robust EV enrichment strategy based on immunoaffinity isolation. In particular, we highlight the recent applications that integrate click chemistry with immunoaffinity isolation, facilitating the rapid and selective EV enrichment. Next, we review various chemical biology tools for signal amplification and multiplexed analysis to enhance the assay sensitivity and capture EV heterogeneity. Finally, we explore recent advancements in the field of EV profiling, such as EV visualization, biosensing platform and treatment response monitoring.

To shed light on the unexplored territory of EV biology, we believe that the biggest challenge of chemical biology in EV research is to develop new bioorthogonal chemical reactions with superior efficiency. We anticipate that these future advancements in chemical biology tools will offer promising avenues for innovating the field of EV profiling. Chemical biology tools would be further engineered for nanoscale targets, addressing the issue of sensitivity, specificity, and EV heterogeneity. Moreover, standardizing the analytical protocol will make EV profiling robust, facilitating the translational application of EV profiling in real-world clinical diagnostics.

Q8. The authors could consider including a section on the ethical considerations and potential risks associated with the use of EVs in biomedical research, to provide a comprehensive overview of the field.

We deeply appreciate Review #1 for this point, which we did not previously consider at all. We now revised the conclusion part of our manuscript to discuss current limitations and ethical issues in EV based liquid biopsy.

Despite above mentioned recent advances in EV analysis, there are still multiple issues to be solved for bringing EV profiling to real clinic. i) Discovery of disease specific EV marker is urgently required.146 EV profiling-based diagnosis has been commonly demonstrated for a specifically targeted disease instead of differentiating multiple diseases due to the lack of disease-specific EV markers. Combination of previously reported disease markers in cell lines or tissues has been widely used to increase the accuracy of EV based disease diagnosis.121 Due to this limitation, differential diagnosis of multiple diseases was barely reported.
ii) Another limitation of EV profiling-based research is the size of patient cohort for validation.147 Mostly EV analysis for disease diagnosis is validated in small patient cohorts.148-150 To claim the potential clinical significance, EV based disease diagnosis should be demonstrated in the large size of patient cohorts. iii) Most of the current EV analysis technologies are high-cost device dependent assays, which needs well- trained technicians.57 Considering the situation of real clinic, development of easily accessible EV assays with low resource setting is highly demanding. iv) Ethical issues of EV research should be considered during the analysis and diagnosis processes. Personal information of each patient must be handled securely, and de-identification methods are needed to be employed for the protection of patient information. Moreover, false positives and negatives of EV profiling-based diagnosis can contribute to unnecessary stress, or treatments of patients. Enhancing the accuracy of EV profiling based diagnosis would address this ethical issue.151 Researchers are needed to be aware of these ethical issues and manage those concerns in clinical applications.

Reviewer #2

This manuscript reviews studies of the chemical biology tools in the applications of protein and RNA profiling of extracellular vesicles. Generally, the manuscript is comprehensive. However, there are several areas that require further improvements. I recommend that the paper undergo significant revisions before being considered for publication.

We do appreciate reviewer #2 for his/her thoughtful ideas to improve our work. We now believe the critical comments of reviewer #2 are now fully address in the revised manuscript.

Q1. In the review, some research groups have done a series of professional work. It is suggested that the author link up these works more logically to make the development and research of chemical biology tools more hierarchical.

We deeply appreciate Review #2 for this helpful comment. We updated the conclusion part in the revised manuscript to describe how the chemical biology tools has been used hierarchically to explore new territories of EV biology.

Hierarchical step-by-step employment of chemical biology tools assisted to unveil above mentioned EV biology as follows. For example, Weissleder and Lee group has developed efficient isolation of biomolecules using Tz-TCO click chemistry,14, 62 which has been widely used for EV isolations. They harnessed the same Tz-TCO click reaction for image cycling to understand heterogeneity of EVs.106 Smart structural modification of TCO realized chemical bond cleavage with remarkable yield for successful multiplex imaging. They again utilized Tz-TCO for single EV analysis by combining droplet encapsulations and immunosequencing. Shao et al. further hired Tz-TCO click chemistry to monitor drug occupancy of target proteins in EVs for the first time.122 Another example is the utilization of enzyme based radical reactions for signal amplification. Lee and Weissleder group developed the SAViA and DEST assay using HRP based radical reaction for tyramide polymerization medicated signal amplification.58,68 Shao group used HRP based radical reaction for the deposition of metal[ref] or insoluble optical products122 to improve sensitivity of EV analysis. Shao group also achieved excellent EV detection sensitivity by mechanical transition of metamaterial using HRP based radical reaction.98 These various smart applications of existing chemical biology tools suggest new opportunities in EV biology.

Q2. The author needs to insert a detailed table to make the reader understand the manuscript more clearly.

We thank Reviewer #2 for this critical comment. We now added a new table 1 in the revised manuscript to compare current limitations of conventional approaches and new approaches to overcome the challenges. We also revised Figure 1 to elucidate EV profiling process with a flowchart to help readers to understand which process has been improved with chemical biology tools.

Q3. It seems to be a problem with the layout of figure 9. Please revise.

We thank reviewer #2 for his/her careful proof-reading of our manuscript. We now revised the figure (new Figure 7).

Q4. Some important parts of the manuscript lack literature citations, for example, in the presentation of EV-based applications. There is also a lack of citations in each section where some background or methods are presented. Additionally, the authors should also summarize latest works reported recently.

We appreciate Review #2 for this thoughtful comment. We now updated our manuscript with more citations. We added 29 new references in the revised version. We also summarized latest works in the perspective of hierarchical workflow to help readers to understand how chemical biological tools has been involved to EV research as we answered to Q1.

Q5. The overview of the conclusions is novel but not sufficient, the author did not mention that the biggest challenge of chemical biology tools in EV research. And we hope the authors can provide more ideas.

It is a very thoughtful idea. We thank Reviewer #2 for this comment. We now revised the conclusion part of our manuscript to suggest the current limitations and biggest challenge of chemical biology tools in EV research.

Despite above mentioned recent advances in EV analysis, there are still multiple issues to be solved for bringing EV profiling to real clinic. i) Discovery of disease specific EV marker is urgently required.146 EV profiling-based diagnosis has been commonly demonstrated for a specifically targeted disease instead of differentiating multiple diseases due to the lack of disease-specific EV markers. Combination of previously reported disease markers in cell lines or tissues has been widely used to increase the accuracy of EV based disease diagnosis.121 Due to this limitation, differential diagnosis of multiple diseases was barely reported.
ii) Another limitation of EV profiling-based research is the size of patient cohort for validation.147 Mostly EV analysis for disease diagnosis is validated in small patient cohorts.148-150 To claim the potential clinical significance, EV based disease diagnosis should be demonstrated in the large size of patient cohorts. iii) Most of the current EV analysis technologies are high-cost device dependent assays, which needs well- trained technicians.57 Considering the situation of real clinic, development of easily accessible EV assays with low resource setting is highly demanding. iv) Ethical issues of EV research should be considered during the analysis and diagnosis processes. Personal information of each patient must be handled securely, and de-identification methods are needed to be employed for the protection of patient information. Moreover, false positives and negatives of EV profiling-based diagnosis can contribute to unnecessary stress, or treatments of patients. Enhancing the accuracy of EV profiling based diagnosis would address this ethical issue.151 Researchers are needed to be aware of these ethical issues and manage those concerns in clinical applications.

However, only limited number of chemical biology tools has been used to tackle those issues.82, 120, 121, 130 To shed light on the unexplored territory of EV biology, we believe that the biggest challenge of chemical biology in EV research is to develop new bioorthogonal chemical reactions with superior efficiency. We anticipate that these future advancements in chemical biology tools will offer promising avenues for innovating the field of EV profiling. Chemical biology tools would be further engineered for nanoscale targets, addressing the issue of sensitivity, specificity, and EV heterogeneity. Moreover, standardizing the analytical protocol will make EV profiling robust, facilitating the translational application of EV profiling in real-world clinical diagnostics.

10-Apr-2024

Dear Dr Park:

Manuscript ID: CB-REV-10-2023-000200.R1
TITLE: Recent advances in chemical biology tools for protein and RNA profiling of extracellular vesicles

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link :

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The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Andrea Rentmeister

************

Reviewer 3

The authors provided a great overview of various chemical biology tools for extracellular vesicle (EV) isolation, EV biomarker analysis, and clinical applications. The strength of the review manuscript is that it provides a comprehensive, balanced overview of different technologies developed for EVs. Simple but clear illustrations of different methods are another strength of the manuscript, which will help readers better understand the working principle of various approaches and identify their differences. I have a couple of minor suggestions to make it more timely and address one of the main technical challenges for EV studies.

1. For EV RNA sensing, besides PCR and sequencing mentioned in the manuscript, recent studies demonstrated the use of CRISPR tools for sensitive and multiplexed analysis of EV protein and RNA biomarkers (e.g., Nature Nanotech, 2021;16:1039 with Cas12a for SARS-CoV-2, Adv Sci, 2023;10(24):e2301766 with Cas13a for amplification-free RNA detection, and others). These newer technologies would be worth noting to represent more recent developments in RNA sensing

2. Universal EV labeling is a key challenge in the field. The authors discussed it briefly with the metabolic labeling methods, but further elevation and discussions with other EV labeling methods would be really useful for many readers in the field.

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters

Point By Point Response to reviewer’s comments.

Reviewer #3

The authors provided a great overview of various chemical biology tools for extracellular vesicle (EV) isolation, EV biomarker analysis, and clinical applications. The strength of the review manuscript is that it provides a comprehensive, balanced overview of different technologies developed for EVs. Simple but clear illustrations of different methods are another strength of the manuscript, which will help readers better understand the working principle of various approaches and identify their differences.
We deeply appreciate reviewer #3 for his/her strong support and encouraging comments to our work.


Q1. For EV RNA sensing, besides PCR and sequencing mentioned in the manuscript, recent studies demonstrated the use of CRISPR tools for sensitive and multiplexed analysis of EV protein and RNA biomarkers (e.g., Nature Nanotech, 2021;16:1039 with Cas12a for SARS-CoV-2, Adv Sci, 2023;10(24):e2301766 with Cas13a for amplification-free RNA detection, and others). These newer technologies would be worth noting to represent more recent developments in RNA sensing.

We thank Reviewer #3 for this wonderful suggestion. We revised our manuscript with discussions about CRISPR tools for the analysis of EV cargos as suggested by Reviewer #3. Section 3.5 is now added to the revised manuscript. EV-liposome fusion technology is now added to Table 1. We also added new Figure 5 to help readers to understand the CRISPR tools.



3.5 EV-liposome fusion
EV-liposome fusion technologies have been developed for detecting intra-vesicular RNA cargos. These technologies incorporate RNA sensing components in liposomes (for example, lipid-polymer hybrid nanoparticles98, fusogenic vesicles99 and lipid nanoparticles100, 101) and delivered them directly to EVs through EV-liposome fusion. The strategy allows extraction-free detection of EV RNAs, without labor-intensive and time-consuming procedures of EV RNA extraction and amplification. For sensitive intra-vesicular RNA sensing, various sensing components including molecular beacons99 and catalyzed hairpin DNA circuit (CHDC)98 have been employed. Most recently, CRISPR-Cas sensing systems are loaded to liposomes and sensitively detect RNAs in EVs. Ning et al. developed Cas12a based assay for the detection of SARS-CoV-2 RNA-positive EVs in in monkey and human plasma.100 EVs in plasma were captured with CD81 antibody and subsequently fused with liposome containing reverse transcriptase (RT), a primer for N gene, Cas12a protein, and a fluorescence-quencher (FQ) probe. In the fused vesicles, RT mediated recombinase polymerase amplification (RPA) amplifies the target and triggers Cas12a-mediated fluorescent signal enhancement for SARS-CoV-2 diagnosis. Hong et al. showed the potential of multiplexed EV protein-miRNA analysis for more accurate cancer diagnosis (Fig. 5A).101 For EV miRNA detection, liposome having crRNA, Cas13a, and a FQ probe was designed. After EV and the liposome fusion, hybridization of target miRNA and crRNA activates Cas13a and subsequently cleaves the FQ probe, which generates fluorescent signals. Furthermore, they integrated a gold microdisk arrays to the liposome-based miRNA detection system for capturing protein marker-specific EVs and detect miR-21-5p in the captured EVs (Fig. 5B). Using this assay, the authors not only successfully differentiated ovarian cancer patients from healthy controls, but also probed that miR-21-5p-positive and EpCAM-positive subpopulation were significantly higher than the CD63-positive subpopulation in patient samples.




Q2. Universal EV labeling is a key challenge in the field. The authors discussed it briefly with the metabolic labeling methods, but further elevation and discussions with other EV labeling methods would be really useful for many readers in the field

It is a good point. We deeply appreciate Reviewer #3 for this helpful comment. We now revised section 5.1 and added discussions about other EV labeling methods as follows. Now, we believe that the revised manuscript would be very helpful for the readers in EV research field.

Lipid dyes (for example, DiO/DiI, PKH67, MemGlow, ExoBrite) are also often used for visualizing EVs.138, 139 The lipophilic nature of the dyes allows them to incorporate into the lipid bilayer of the EVs. However, the dyes are easy to be aggregated during the labeling process and form non-EV particles, which is hard to be separated from EVs and complicating the downstream applications.138, 140 For universal EV labeling, fluorescent proteins (FPs) also can be genetically encoded to EV cargos such as tetraspanin family members (CD63, CD9 and CD9).141, 142 This genetic labeling allows tracking and visualizing the EV’s biogenesis, secretion, biodistribution and uptake in living cells and animals. However, genetic incorporation of large reporter proteins may disturb cellular signaling pathways for EV biogenesis and trafficking.

25-Apr-2024

Dear Dr Park:

Manuscript ID: CB-REV-10-2023-000200.R2
TITLE: Recent advances in chemical biology tools for protein and RNA profiling of extracellular vesicles

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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Andrea Rentmeister

Reviewer 3

I appreciate the authors considering my comments and revising the manuscript. The scope of the review articles is timely with recent advances in the field of extracellular vesicle analysis. I therefore recommend the publication of the manuscript in RSC Chemical Biology.