Exosome Analysis in Prostate Cancer: How They Can Improve Biomarkers’ Performance

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The review article entitled “Exosomes analysis in prostate cancer: How they can improve biomarkers performance” by Salciccia et al., is comprehensive review that specifically analyzed and compared different methodologies available for the use of exosomes in prostate cancer. Authors have described methodologies that optimizes the exosome extraction form cancer patients and may serve as great non-invasive diagnostic tool for prostate cancer patients. Current biomarkers for prostate cancer diagnosis including tissue, urine and serum samples have been discussed very well. As the acidity of the microenvironment significantly increases the release of exosomes by cancer cells, authors have claim that exosomes may considered as tumor biomarkers, with potential use in screening, diagnosis and prognosis of the disease. This is the strength of the manuscript. The current review also demonstrated that not only the increased levels of CA IX in exosomes of prostate cancer patients but also the direct correlation between the acidic microenvironment, the release of exosomal particles in the extracellular compartment, and the upregulation of CA IX expression and activity. The authors have done amazing job in compiling the article references according to clinical relevance in oncology research.

Conceptual comments-

The manuscript is clear, comprehensive and of relevance to the oncology diagnostics. Important articles in the recent past have been included and justified in the manuscript. Although there are excessive self-citations in the review, it justified by the fact that authors have significantly contributed with respect to exosome quantification in the prostate cancer field. In last 10 years there has not been good enough compilation of exosome as biomarkers in oncology research in general and prostate cancer in particular. This information has been made available in the current manuscript. It is therefore relevant and a great addition to the existing knowledge of the scientific community.

The conclusions and future perspectives are consistent with the evidence and arguments presented in the manuscript. Authors did mention the limitation of using exosomes as biomarker in clinical practice due to a lack of standardization in the analytical process, high costs and prolonged time. Additionally, authors mention further development in exosome research may enable liquid biopsy through exosomes analysis to become a routine diagnostic tool in prostate cancer diagnosis.

In my opinion this manuscript will help to increase the knowledge in oncology research and in future may use exosome as biomarker for diagnosis of different types of cancers. 

Specific comments-

Please elaborate the full form of PSA at the beginning in the introduction.

It would be great if authors show different biomarkers information in a pictorial form. Tests for serum, tissue and urine markers could be clubbed together in a single figure thereby simplifying the types of tests to the readers.

Please correct the following typo errors-

Page 9, line number 256, miRNa-exosomes

Page 10, line 314, the main limit

Comments on the Quality of English Language

At a few places in the manuscript, it is difficult to follow what the authors are trying to say. Reframing the sentences would help in clear understanding of the context.

Author Response

We thank the reviewer for appreciating our work. All  your comments has been addressed so to improve the manuscript status.

 

Comment 1: As requested, the full form of prostate specific antigen (PSA) was reported correctly at the beginning of the introduction.

 

Comment 2: We agree with the reviewer that a figure with these characteristics would have been interesting from a clinical point of view. However we apologize but Due to editorial regulations on the number of tables and figures we were unable to make a new figure. Moreover, given the type of molecular biology journal, we thought it appropriate to focus on exosome extraction techniques rather than the clinical implications of these technologies.

 

Comment 3: As requested, “typo errors” have been corrected

 

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Stefano et al. reviewed the prospects of using extracellular vesicles as prostate cancer biomarkers in clinical applications and summarized the methods and advantages/disadvantages of extracellular vesicle isolation and detection techniques. The main focus of the review is to describe the methods used for extracellular vesicle analysis in prostate cancer, how extracellular vesicles can improve the specificity and sensitivity of the serum biomarker PSA in prostate cancer diagnosis, and the relationship between extracellular vesicles and carbonic anhydrase expression in prostate cancer. This is a meaningful review study that provides a pathway for clinical urologists to understand the latest molecular biomarkers. However, the authors need to address the following issues to improve the quality of this paper.

1. List the main extracellular vesicle biomarkers associated with prostate cancer, such as mRNA, proteins, ncRNA, etc.

2. Discuss whether prostate fluid can be used for extracellular vesicle extraction and applied in the diagnosis of clinical prostate cancer.

3. Discuss whether extracellular vesicle-related techniques can be applied to the study of metastasis and castration resistance in prostate cancer patients.

4. Provide definitions for abbreviations used in the text.

5. Carefully check for grammar and descriptive errors in the manuscript.

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

We thank the reviewer for appreciating our work. All  your comments has been addressed so to improve the manuscript status.

 

Comment 1: we thank the reviewer for his suggestion. As requested, the main extracellular biomarkers associated with prostate cancer have been reported discursively in the text (page 7-8-9). Due to editorial regulations on the number of tables and figures we were unable to make a summary table of the main biomarkers. moreover, given the type of molecular biology journal, we thought it appropriate to focus on exosome extraction techniques rather than developing the clinical implications of these technologies

 

Comment 2-3: we thank the reviewer for his interesting suggestion. As requested now in the conclusion section (page 10, Line 324-332) we have included comments as perspectives for future research on the role of exosomes in prostate cancer progression and metastasis. Moreover the role of semen fluid for extracellular vesicle extraction was also included

 

Comment 4: As requested, now at the end of the manuscript the list of abbreviations was included.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors of the study entitled "EXOSOMES ANALYSIS IN PROSTATE CANCER: HOW THEY CAN IMPROVE BIOMARKERS PERFORMANCE” are trying to establish exosomes as a biomarker and promising diagnostic tool for Prostate cancer. This is a very helpful study for researchers to explore new avenues for the management of prostate cancers in humans. This topic is gaining a lot of attention due to its large clinical and scientific importance. This study could be suitable for publication with significant major revision.

Comments :

1.    This topic has been addressed in a number of recently published comprehensive reviews and research articles in the last 2-3 years. (DOI: 10.3389/fonc.2022.873296; doi: 10.3389/fmed.2022.736110. eCollection 2022; DOI: 10.1016/j.biopha.2022.113093). Therefore, the introduction should give a clear answer to the question: What fields did not get proper attention in previous recent reviews, and why this current review is still relevant and of interest to the scientific community?

2.    The writing style is redundant, in some parts negligent, and should be improved throughout the paper. All irrelevant statements should be deleted.

3.    Abstract should be more focused based on the content of the manuscript. It looks like any original research article, not as a review. Please provide a more focused aim and significance of this study.

4. It would be recommended to provide a few lines of world statistics on the prevalence of PC and

5.    Please provide the symptoms of PC and the main issues faced during clinical studies in PC patients.

6. Is there any recent studies in the serum markers except PSA? If Yes, Please mention it.

7.    Section 2.4: As the main foundation of this review is exosomes, Please provide more precise and detailed information at the molecular level of exosomes as biomarkers.

8.     Line 133-138, Please make it a separate heading OR merge it with section 3.

9.    It looks like a few lines copy and paste. Authors should check the plagiarism.

 

 

Comments on the Quality of English Language

Need to improve throughout the manuscript.

Author Response

ANSWERS TO REVIEWER 1

 

The authors of the study entitled "EXOSOMES ANALYSIS IN PROSTATE CANCER: HOW THEY CAN IMPROVE BIOMARKERS PERFORMANCE” are trying to establish exosomes as a biomarker and promising diagnostic tool for Prostate cancer. This is a very helpful study for researchers to explore new avenues for the management of prostate cancers in humans. This topic is gaining a lot of attention due to its large clinical and scientific importance. This study could be suitable for publication with significant major revision.

Comments :

1. This topic has been addressed in a number of recently published comprehensive reviews and research articles in the last 2-3 years. (DOI: 10.3389/fonc.2022.873296; doi: 10.3389/fmed.2022.736110. eCollection 2022; DOI: 10.1016/j.biopha.2022.113093). Therefore, the introduction should give a clear answer to the question: What fields did not get proper attention in previous recent reviews, and why this current review is still relevant and of interest to the scientific community?

Answer: The topic of exosomes as biomarkers has been addressed in previous reviews, this is true. However our review analyzed different topics not present in previous reviews:

1. Methodology to be use for exosomes analysis in prostate cancer is more specifically described and this point follow the requests of Editors for this Special issue. We specifically describe, compare and comment on NTA, IC-ELISA e Flow Cytometry analysis
2. Differently to previous reviews we specifically analyze how exosome analysis can improve specificity and sensitivity of the major marker used in PC diagnosis, PSA
3. Differently to previous reviews we specifically analyze the relationship between exosome and Carbonic anhydrase expression in PC

As requested by the reviewer, this point is better underlined in the Introduction section line 63-69

 

2. The writing style is redundant, in some parts negligent, and should be improved throughout the paper. All irrelevant statements should be deleted.

Answer: Writing style has been revised and improved removing redundant

3. Abstract should be more focused based on the content of the manuscript. It looks like any original research article, not as a review. Please provide a more focused aim and significance of this study.

Answer: Abstract has been revised and modified (line 17-33) as requested with a style for review and more focused on aim and significance

4. It would be recommended to provide a few lines of world statistics on the prevalence of PC and

Answer: A few lines on world statistic on the prevalence of PC has been provided in Introduction lines: 46-47

 

5. Please provide the symptoms of PC and the main issues faced during clinical studies in PC patients.

Answer: PC is not related to specific symptoms. The main issue for clinical studies in the initial diagnosis of PC is provided in lines 48-50

 

 

6. Is there any recent studies in the serum markers except PSA? If Yes, Please mention it.

Answer:Recent studies and information on other serum markers except PSA are provided in Line 88-92. From line 93-126 other recent urine and tissue markers are described.

7. Section 2.4: As the main foundation of this review is exosomes, Please provide more precise and detailed information at the molecular level of exosomes as biomarkers.

Answer:Specific and precise detailed information at the molecular level of exosomes as biomarkers are provided in section 2.4 and following also in Table 1 and section 3.0

 

8. Line 133-138, Please make it a separate heading OR merge it with section 3.

 Answer:This part now is included in section 3.0 as requested

 

9. It looks like a few lines copy and paste. Authors should check the plagiarism.

Answer:We checked better. Data are all used from our previous clinical trials on this topic and well referenced.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors aim to review the potential of exosomes (should be referred to as small EVs) as PC biomarkers. However, the authors focus too much of the review on methodology to validate and characterize EVs in general, which is not relevant to their biomarker potential (entire section 2.4 and large part of section 3). Also, most of the review is focused on 2 exosomal biomarkers, one of which is a great PC biomarker outside of EVs (PSA) so not clear what value EV isolation are adding to classical PSA measurements. There is NO discussion of PC EV biomarkers in urine! There are numerous reviews out there on EVs as biomarkers in PC, reviews that are much more comprehensive so the present study does not distinguish itself or bring anything new in either depth or interpretation. Below is the PMC extract for such studies.

https://pubmed.ncbi.nlm.nih.gov/?term=prostate+cancer+extracellular+vesicles+and+biomarkers

Author Response

 

ANSWERS TO REVIEWERS 2

 

The authors aim to review the potential of exosomes (should be referred to as small EVs) as PC biomarkers. However, the authors focus too much of the review on methodology to validate and characterize EVs in general, which is not relevant to their biomarker potential (entire section 2.4 and large part of section 3).

Answer: Before submission to the Journal, the review was checked by two Editor. In particular Editor Dr Linnie Yin specifically asked to develop more detail on methodology used for our studies and a specific section (3.0) on methodology. This is the reason of our focus. We specifically described methods used, we compared them providing information on the advantages and disadvantages of each method. However, also the clinical results of exosomes as markers are well described in the other sections (4.0 and 5.0).

Also, most of the review is focused on 2 exosomal biomarkers, one of which is a great PC biomarker outside of EVs (PSA) so not clear what value EV isolation are adding to classical PSA measurements.

Answer: In section 4.0 we specifically analyze how exosome analysis can significantly improve the use of PSA as marker of PC. The major limit of PSA is its low specificity. An increase in PSA can be related to PC but also to benign conditions such as BPH. We demonstrated how the analysis of exosomes and in particular of PSA expressing exosomes, significantly improve specificity of this marker. This point is related to a potentially high clinical value. The isolation of PSA expressing PSA adds specificity for PC to the classical PSA measurement and this is well described in section 4.0

There are numerous reviews out there on EVs as biomarkers in PC, reviews that are much more comprehensive so the present study does not distinguish itself or bring anything new in either depth or interpretation. Below is the PMC extract for such studies.

Answer

The present review is well distinguishable from the previous reviews for the following aspects:

1. Methodology to be use for exosomes analysis in prostate cancer is more specifically described and this point follow the requests of Editors for this Special issue. We specifically describe, compare and comment on NTA, IC-ELISA e Flow Cytometry analysis
2. Differently to previous reviews we specifically analyze how exosome analysis can improve specificity and sensitivity of the major marker used in PC diagnosis, PSA
3. Differently to previous reviews we specifically analyze the relationship between exosome and Carbonic anhydrase expression in PC

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all the suggestions during the first round of revision. 

Recommendation: Accepted in present form

Comments on the Quality of English Language

Minor revision is still required

Author Response

Thank you for your positive evaluation

Reviewer 2 Report

Comments and Suggestions for Authors

We thank the authors for their response. While the authors state that the journal Editor had requested the extensive methodology description, this is not in any way specific for the prostate studies described here and instead describes standard approaches to extracellular vesicle characterization in general. In addition, as mentioned before, the correct terminology is extracellular vesicles or small EVs rather than exosomes.

The authors also fail to address EV cargo as broader biomarkers of prostate cancer to cover the variety of studies already published, as the title would lead a reader to believe. 

It does not seem that through this revision the authors have attempted to address the reviewer’s concerns.

Author Response

I do not agree with the comment of the Reviewer. As agreed with the Editor I firmly believe that the chapter on methodology used, strongly improved and personalized the Review article and is perfectly enclosed with the other sections on clinical results.

Related to the terminology exosomes or extracellular vesicles, exosomes is diffusely used in studies, and in particular from the studies considered in the present Review the term exosome is used and not extracellualr vesicle. We modified in some sections of the review article the terminology in EV but we maintain exosomes in several other parts as the original articles used.