Identification of Potential Protein Targets in Extracellular Vesicles Isolated from Chemotherapy-Treated Ovarian Cancer Cells
Round 1
Reviewer 1 Report
Chan et al. described the effects on EV protein changes induced by different chemotherapeutic drugs treatment of ovarian cancer cells. In particular, they have identified that the expression levels of ANXA5, CD81, and RAB5C were correlated with drug sensitivity in cisplatin combined with paclitaxel-treated ovarian cancer patients.
The results sound interesting, the method used is appropriate, the coverage of the material is good and the collected data have been discussed in an informative manner. The manuscript should be accepted for publication in the journal after minor revision, on the following section:
- The authors should provide a graphical abstract.
- As stated by the authors, in cancer, the dysregulated secretion and altered cargoes of EVs (i.e., proteins, lipids and nucleic acids) can promote tumor growth and lead to metastasis, suggesting that targeting EVs may prevent metastasis. However, the knowledge about EV cargos’ subcellular/molecular sites of action in recipient cells is still fragmented. This is specifically true for proteins and nucleic acids associated with EVs. So, the collected data by the authors are very interesting and have Scientific Soundness.
However, in the introduction, the readers may benefit from some more background and context. For example, a short introduction on the ongoing preclinical efforts to elect EVs as a potential target to develop novel strategies to treat cancer would provide some orientation to readers and set the scene. Targeting EV biogenesis and secretion may have potential clinical implications for cancer metastatic therapy. For this purpose, I suggest seeing the following paper and adding the following references:
1. Int J Mol Sci. 2020 Jun 23;21(12):4463. doi: 10.3390/ijms21124463.
2. J Extracell Vesicles. 2021 Aug;10(10):e12132. doi: 10.1002/jev2.12132.
3. Nat Commun. 2023 Aug 10;14(1):4588. doi: 10.1038/s41467-023-40227-8.
- It could be of interest to investigate the effect of different chemotherapeutic drugs treatment on EV protein changes in vivo models.
Minor editing of English language required
Author Response
Manuscript ID: cimb-2560064
Type of manuscript: Article
Title: Identification of potential targets from extracellular vesicles of
chemotherapy-treated ovarian cancer cells
Reviewer Comments
Reviewer 1
We appreciate the comments of this reviewer and believe that our manuscript has been improved by attention to him or her. The following are our responses to the specific issues raised by this reviewer:
Point 1: The authors should provide a graphical abstract.
Response 1: Thanks for the reviewer's valuable comments. The graphical abstract is shown below (see in attachment):
Point 2: As stated by the authors, in cancer, the dysregulated secretion and altered cargoes of EVs (i.e., proteins, lipids and nucleic acids) can promote tumor growth and lead to metastasis, suggesting that targeting EVs may prevent metastasis. However, the knowledge about EV cargos’ subcellular/molecular sites of action in recipient cells is still fragmented. This is specifically true for proteins and nucleic acids associated with EVs. So, the collected data by the authors are very interesting and have Scientific Soundness.
However, in the introduction, the readers may benefit from some more background and context. For example, a short introduction on the ongoing preclinical efforts to elect EVs as a potential target to develop novel strategies to treat cancer would provide some orientation to readers and set the scene. Targeting EV biogenesis and secretion may have potential clinical implications for cancer metastatic therapy. For this purpose, I suggest seeing the following paper and adding the following references:
- Int J Mol Sci. 2020 Jun 23;21(12):4463. doi: 10.3390/ijms21124463.
- J Extracell Vesicles. 2021 Aug;10(10):e12132. doi: 10.1002/jev2.12132.
- Nat Commun. 2023 Aug 10;14(1):4588. doi: 10.1038/s41467-023-40227-8.
Response 2: Thanks for the reviewer's valuable comments. We have revised the introduction of the manuscript according to the reviewer's suggestions, as detailed in lines 86-127 of the revised manuscript. We also added the two references suggested by the reviewer: Reference#1&2 to our revised manuscript (references 22&23).
Point 3: It could be of interest to investigate the effect of different chemotherapeutic drugs treatment on EV protein changes in vivo models.
Response 3: Thanks for the reviewer's valuable comments. Regarding chemotherapeutic drugs treatment on EV protein changes in vivo models, we can not plan this part of the experiment because of the revision time. We are looking for some literature on how to detect EV in in vivo models. We will learn these methods and use them to plan this part of the experiment in the future.
- J Extracell Vesicles. 2020 Aug 21;9(1):1800222
- Bioengineering (Basel). 2023 Mar 3;10(3):325.
- Mol Imaging Biol. 2020 Dec;22(6):1501-1510.
Point 4: Minor editing of English language required
Response 4: Thanks for the reviewer's valuable comments. We have completed the English editing with the certificate as below (see in attachment):
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Diagnostic and therapeutic challenges of ovarian cancer are substantial, and research which advances this field is welcome. The manuscript offers information about Rx-induced changes in extracellular vesicles measured in ES2 cells, and the following issues should be addressed before its publication:
Major points
Malignancy cell type has tremendous relevance for both treatment and prognosis. In this investigation, ES2 was used as the substrate to examine EV features. However, ES2 is a cell line sourced from clear-cell (CC) ovarian carcinoma. Because CC carcinoma is a rare subset of epithelial ovarian cancer (see Gynecol Oncol 2021;162:741) this research would be strengthened by explaining why ES2 was selected? There are at least 100 other ovarian cancer cell lines commercially available, so why pick this one?
Could these data have relevance only to an elite band of clinical ovarian cancer cases? Not only are the dangers of inappropriately generalizing not discussed, the paper mentions no limitations whatsoever. Why?
The tendency to overreach also diminishes the title, where ‘potential targets’ are noted. Presumably, this refers to cargo proteins/upregulated genes recorded in EV efflux after Rx exposure. But this observation is not new – Am J Cancer Res 2015;5:629. More importantly, the Oklahoma group examined one application of ANXA5 (annexin V) with mutated cystathionine lyase to generate ROS against tumor cells – Cancer Lett 2018;425:174. Thus in 2023, just documenting another set of curious EV releasate features is not the same as translating that response into a meaningful therapeutic intervention. If it has already been done by others, then why not cite them?
Minor problems
Line 36-38: Worldwide rates are given, then USA data are inserted. Is there something special about American cases? USA doesn’t even have a national cancer registry. Consider reworking/simplifying this section.
Line 42-44: If effectiveness of Pt-based therapy is highlighted, but then a 70% recurrence rate is noted, it would be helpful to provide background on this apparent contradiction.
Subject-verb grammar disagreements occur with alarming frequency in this paper, beginning with the Abstract at line 25 (i.e., EV protein, not EVs protein).
Basic (English) spell-check should identify punctuation and S/V errors.
Author Response
Manuscript ID: cimb-2560064
Type of manuscript: Article
Title: Identification of potential targets from extracellular vesicles of
chemotherapy-treated ovarian cancer cells
Reviewer Comments
Reviewer 2
We appreciate the comments of this reviewer and believe that our manuscript has been improved by attention to him or her. The following are our responses to the specific issues raised by this reviewer:
Main problems:
Point 1: Malignancy cell type has tremendous relevance for both treatment and prognosis. In this investigation, ES2 was used as the substrate to examine EV features. However, ES2 is a cell line sourced from clear-cell (CC) ovarian carcinoma. Because CC carcinoma is a rare subset of epithelial ovarian cancer (see Gynecol Oncol 2021;162:741) this research would be strengthened by explaining why ES2 was selected? There are at least 100 other ovarian cancer cell lines commercially available, so why pick this one?
Response 1: Thanks for the reviewer's valuable comments. As for why we chose ES2 cells in the experiment, the main reason is that we refer to the study published by Prof. Takahiro Ochiya in Nat Commun. 2017 Mar 6;8:14470: Malignant extracellular vesicles carrying MMP1 mRNA facilitating peritoneal dissemination in ovarian cancer1. The author found that ES-2 can indeed secrete exosomes which can promote the metastasis of cancer cells through the secreted exosomes. This result confirms that ES-2 has the property of secreting exosomes and can affect neighboring cells through the secreted exosomes. According to this result, let us choose ES-2 cells to prove that ES-2 cells treated with therapeutic drugs can affect drug resistance by secreting EV.
Reference:
- Yokoi A, Yoshioka Y, Yamamoto Y, Ishikawa M, Ikeda SI, Kato T, Kiyono T, Takeshita F, Kajiyama H, Kikkawa F, Ochiya T. Malignant extracellular vesicles carrying MMP1 mRNA facilitate peritoneal dissemination in ovarian cancer. Nat Commun. 2017 Mar 6;8:14470.
Point 2: Could these data have relevance only to an elite band of clinical ovarian cancer cases? Not only are the dangers of inappropriately generalizing not discussed, the paper mentions no limitations whatsoever. Why?
Response 2: Thanks for the reviewer's valuable comments. In order to understand the correlations between the expression of deregulated proteins (genes) identified from the ES2 cells and drug sensitivity in clinical ovarian cancer patients, we chose the GSE30161 database for analysis. The main reason is that we used The Cancer Treatment Response gene signature DataBase (CTR-DB) (http://ctrdb.ncpsb.org.cn/) for analysis, CTR-DB is a unique tool for reusing clinical transcriptome data pertaining to cancer drug response. In the CRT-DB database, only GSE30161 and GSE14764 collect correlation between ovarian cancer patients and drug sensitivity of chemotherapy. However, the ovarian cancer patients collected by GSE14764 are treated with Carboplatin+Paclitaxel, and only GSE30161 collects the ovarian cancer patients treated with Cisplatin+Paclitaxel, in addition, the number of responder (sensitive) and non-responder (resistant) patients is close, so we selected this database for analysis.
|
Database |
Drug name |
Responders (sensitive) |
Non-responders (resistance) |
|
GSE30161 |
Cisplatin+Paclitaxel |
28 |
22 |
|
GSE14764 |
Carboplatin+Paclitaxel |
42 |
22 |
Point 3: The tendency to overreach also diminishes the title, where ‘potential targets’ are noted. Presumably, this refers to cargo proteins/upregulated genes recorded in EV efflux after Rx exposure. But this observation is not new – Am J Cancer Res 2015;5:629. More importantly, the Oklahoma group examined one application of ANXA5 (annexin V) with mutated cystathionine lyase to generate ROS against tumor cells – Cancer Lett 2018;425:174. Thus in 2023, just documenting another set of curious EV releasate features is not the same as translating that response into a meaningful therapeutic intervention. If it has already been done by others, then why not cite them?
Response 3: Thanks for the reviewer's valuable comments. We have revised the discussion of the manuscript according to the reviewer's suggestions, as detailed in lines 328-346 of the revised manuscript. We also added the two references suggested by the reviewer in our revised manuscript (references 63&65).
Minor problems:
Point 1: Line 36-38: Worldwide rates are given, then USA data are inserted. Is there something special about American cases? USA doesn’t even have a national cancer registry. Consider reworking/simplifying this section.
Response 1: Thanks for the reviewer's valuable comments. We have deleted the statement of the USA data and revised the introduction of the manuscript in the revised manuscript line 34-40.
Point 2: Line 42-44: If effectiveness of Pt-based therapy is highlighted, but then a 70% recurrence rate is noted, it would be helpful to provide background on this apparent contradiction.
Response 2: Thanks for the reviewer's valuable comments. We have revised paragraph 2 of the introduction and explained the function of cisplatin, which can explain the cause of drug resistance. The content has been revised in the revised introduction lines 53-83.
Point 3: Subject-verb grammar disagreements occur with alarming frequency in this paper, beginning with the Abstract at line 25 (i.e., EV protein, not EVs protein).
Response 3: Thanks for the reviewer's valuable comments. We have completed the English editing with the certificate as below (see in attachment):
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Thank you for this modified version.
Check punctuation throughout.
Author Response
