Patient-Derived Exosomes as siRNA Carriers in Ovarian Cancer Treatment

Round 1

Reviewer 1 Report

In this study, the authors explored the potential therapeutic applications of exosomes derived from patients with OC as carriers for delivering siRNA. The exosomes were isolated from the culture medium of primary fibroblasts obtained from the omentum of OC patients during surgery. Specifically, the authors focused on targeting the c-Met gene for silencing using synthesized c-Met siRNAs loaded into the exosomes through electroporation. The investigation involved assessing the efficacy of these engineered exosomes, referred to as Met-siExosomes, both in vitro and in vivo. Results demonstrated that Met-siExosomes effectively reduced c-Met protein levels, leading to the inhibition of ovarian cancer cell proliferation, migration, and invasion. In xenograft experiments with SKOV3-13 and ES-2 cells, Met-siExosomes were selectively taken up by peritoneally disseminated tumors. Intraperitoneal treatment with Met-siExosomes not only suppressed downstream targets of c-Met in cancer cells but also extended the survival of mice. In summary, the findings suggest that exosomes derived from OC patients hold promise as a viable and patient-specific carrier for therapeutic siRNA, offering a potential avenue for the treatment of OC. 

While going through the manuscript, I could not find anything new that can be taken home. A ton of similar studies/reviews can be found in the literature, so I regret that I could not provide any positive comments this time.

Minor editing is required

Author Response

We partly understand the comments from this reviewer. As this reviewer mentioned, a number of reports have been published on the potential of exosomes as drug delivery carriers [new ref. 13 and 14]. However, in most of these preclinical reports, the sources of exosomes were cell lines or cells collected from mice. To promote the clinical application of exosomes, a method of collecting ample volumes of exosomes from patients must be developed to establish exosome‑based delivery strategies for cancer treatment. Thus, the strength of our study is that we showed that it is possible to collect an ample volume of exosomes from patient-derived tissues, which is clinically meaningful. Furthermore, omentum contains fibroblasts as well as adipose cells and macrophages [new ref. 32], indicating the possibility of obtaining a variety of exosome types. If exosome‑based delivery strategies using patient-derived omentum are established, laparoscopic omentectomy can be applied not only for OC but also for various other types of cancers, such as colorectal cancer or gastric cancer, because of the less-invasive nature of this surgery. We would ask the reviewer to reconsider this contribution as a strength of the manuscript.

Reviewer 2 Report

Dear Authors,
The manuscript under review “Patient-derived exosomes as a novel siRNA carrier in ovarian  cancer treatment”

1.     In over-all, I think the knowledge of this article is interesting and the authors' fascinating observations on this timely topic may be of interest to the readers of Cancer. However, some comments, as well as some crucial evidence that should be included to support the authors' argumentation, need to be addressed to improve the quality of the article, its adequacy, and its readability prior to the publication in the present form. My overall judgment is to publish this article after the authors have carefully considered my suggestions below.

2.     In general, I recommend authors to use more evidence to back their claims, especially in the Introduction of the article. Thus, I recommend the authors to attempt to deepen the subject of their manuscript, as the bibliography is too concise: nonetheless, in my opinion, some recent references of the year 2023-24 are highly recommended. Therefore, I suggest the authors focus their efforts on researching the most recent and relevant literature: I believe that adding few more studies will help to provide better and more accurate background to this study.

3.     Zhang, Yixin, et al. "Research progress of extracellular vesicles in the treatment of ovarian diseases." Experimental and Therapeutic Medicine 27.1 (2024): 1-14.

4.     Liu, Yang, et al. "The Roles of Exosomes in Ovarian Cancer Chemo-resistance." Journal of Cancer 14.11 (2023): 2128.

5.     Sharma, Vriti, and Chitrangada Das Mukhopadhyay. "Exosome as drug delivery system: Current advancements." Extracellular Vesicle 3 (2024): 100032

6.     Amina, Sundus Jabeen, et al. "A review on the use of extracellular vesicles for the delivery of drugs and biological therapeutics." Expert Opinion on Drug Delivery j(2024).

7.     Guo, Zun Y., Yue Tang, and Yi C. Cheng. "Exosomes as targeted delivery drug system: advances in exosome loading, surface functionalization and potential for clinical application." Current Drug Delivery (2024)

8.     Tendulkar, Reshma, and Mugdha Tendulkar. "Current Update of Research on Exosomes in Cancer." Current Molecular Medicine 24.1 (2024): 26-39.

9.     As the reviewer desired to see the overall study design before going into detail of manuscript. Hence the author is suggested to incorporate comprehensive flow sheet abstract (colored Scheme), which must be part of the manuscript, including all information that is ranging from material experiment parts, results and other key points mentioned in the text. This part is very important, as the reader will understand the whole manuscript without going into detail. See the following for example:   https://doi.org/10.1080/21655979.2020.1865607

10. One page: Similarly infographic abstract should also part of the manuscript, covering all the detail mentioned in the manuscript, especially pictures, where it is required, for ready reference, https://doi.org/10.1080/21655979.2020.1867405

11. Why was this study done? (Separate paragraph)

12. What did the author and co-authors do and find? (Separate paragraph)

13. What do these findings mean? (Separate paragraph)

14. What is the impact of this research on society? (Separate paragraph)

15. Limitation / short coming of the study must be included as a separate heading. (Separate paragraph)

16. Key highlights/ Future direction must also be part of the manuscript. (Separate paragraph)

17. c-Met, SKOV3-13 and ES-2 use time as abbreviations, it is recommended to write first time when used in the manuscript. (Abstract).

18. Material, well written with cat numbers.

19. Cell culture BJ cells, SKOV3, CLEA, GEPIA  and CAOV3 use as abbreviations, it is recommended to write first time when used in the manuscript.

20. Please upload the letter as supplementary file (The Institutional Animal Care and Use Committee of Osaka University approved all animal experiments (No. J006461-010).)

21. Figure 1D Explanation please?

22. Refer to the paragraph: Met expression was transiently knocked down in SKOV3, CAOV3, and ES-2 cells, but whereas in Figure ES 2 data has been not shared? When compared to paragraph: Met-siExosomes were constructed using 317 two siRNA sets (3270 and 404); these Met-siExosomes successfully inhibited c-Met expression in ES-2, SKOV3, and CAOV-3 cells (Figure 3B).

23. Refer to Figure 4D,4E, 4F, 4G, 4H, COAV3, data has not been shared? In refer to Figure 4B observation which quite noticeable from the gel. Met-siExosomes were constructed using 317 two siRNA sets (3270 and 404); these Met-siExosomes successfully inhibited c-Met expres-318 sion in ES-2, SKOV3, and CAOV-3 cells. VS Although c-Met expression was not affected by treatment with exosomes or siRNAs alone in SKOV3 and ES-2 cells, Met-siEx-342 osomes inhibited c-Met expression in these cells (Figure 3C), CAOV-3 data is missing?

24. One million 407 SKOV3-13 cells were inoculated into female BALB/c mice. Why not CAOV 3?

Recommended to review the language and include orcid numbers 

Author Response

Comments from Reviewer 2

Dear Authors,

The manuscript under review “Patient-derived exosomes as a novel siRNA carrier in ovarian cancer treatment”

1. In over-all, I think the knowledge of this article is interesting and the authors' fascinating observations on this timely topic may be of interest to the readers of Cancer. However, some comments, as well as some crucial evidence that should be included to support the authors' argumentation, need to be addressed to improve the quality of the article, its adequacy, and its readability prior to the publication in the present form. My overall judgment is to publish this article after the authors have carefully considered my suggestions below.

2. In general, I recommend authors to use more evidence to back their claims, especially in the Introduction of the article. Thus, I recommend the authors to attempt to deepen the subject of their manuscript, as the bibliography is too concise: nonetheless, in my opinion, some recent references of the year 2023-24 are highly recommended. Therefore, I suggest the authors focus their efforts on researching the most recent and relevant literature: I believe that adding few more studies will help to provide better and more accurate background to this study.
3. Zhang, Yixin, et al. "Research progress of extracellular vesicles in the treatment of ovarian diseases." Experimental and Therapeutic Medicine 27.1 (2024): 1-14.
4. Liu, Yang, et al. "The Roles of Exosomes in Ovarian Cancer Chemo-resistance." Journal of Cancer 14.11 (2023): 2128.
5. Sharma, Vriti, and Chitrangada Das Mukhopadhyay. "Exosome as drug delivery system: Current advancements." Extracellular Vesicle 3 (2024): 100032
6. Amina, Sundus Jabeen, et al. "A review on the use of extracellular vesicles for the delivery of drugs and biological therapeutics." Expert Opinion on Drug Delivery (2024).
7. Guo, Zun Y., Yue Tang, and Yi C. Cheng. "Exosomes as targeted delivery drug system: advances in exosome loading, surface functionalization and potential for clinical application." Current Drug Delivery (2024)
8. Tendulkar, Reshma, and Mugdha Tendulkar. "Current Update of Research on Exosomes in Cancer." Current Molecular Medicine 24.1 (2024): 26-39.

We appreciate these comments from the reviewer. As suggested, four of the above manuscripts were added as new references. (line 62-69)

9. As the reviewer desired to see the overall study design before going into detail of manuscript. Hence the author is suggested to incorporate comprehensive flow sheet abstract (colored Scheme), which must be part of the manuscript, including all information that is ranging from material experiment parts, results and other key points mentioned in the text. This part is very important, as the reader will understand the whole manuscript without going into detail. See the following for example: https://doi.org/10.1080/21655979.2020.1865607

According to the requirements of Cancers listed in the author guidelines, a flow sheet graphic is only required for systematic reviews and meta-analyses and not for articles. However, since the reviewer requested it, we have produced this flow sheet and added it to the revised version. We kindly ask the editor and editorial office to judge whether such a flow sheet should be included or not.

10. One page: Similarly infographic abstract should also part of the manuscript, covering all the detail mentioned in the manuscript, especially pictures, where it is required, for ready reference, https://doi.org/10.1080/21655979.2020.1867405

As this reviewer asked us to include a graphical abstract that describes the overall study, Figure 5H was removed and presented as a graphic abstract. Again, we kindly ask the editor and editorial office whether this graphic is more suitable as a graphical abstract or figure.

11. Why was this study done? (Separate paragraph)

The purpose of this study was included in the Introduction as follows:

“The key to the effective clinical application of exosomes for siRNA replacement therapy is the collection of sufficient amounts of patient-derived exosomes. Most patients with OC undergo surgery, including partial omentectomy, as the primary treatment modality. As the omentum contains fibroblasts that secrete abundant exosomes, we attempted to use primary fibroblasts from the omentum as an exosome source for delivering siRNAs.

In this study, we elucidated the potential use of exosomes as siRNA carriers, investigated whether siRNA replacement therapy could be performed with patient-derived exosomes from the omentum, and developed a novel precision therapy for OC.” (line 71-78).

12. What did the author and co-authors do and find? (Separate paragraph)

To address this comment, we added the following text to the Discussion:

“In this study, we utilized patient-derived omental fibroblasts as the source of exosomes. Omentectomy is a common procedure for patients with OC that provides readily accessible omental tissue from which fibroblasts can be isolated. These fibroblasts secrete exosomes in ample quantities suitable for siRNA encapsulation, thereby enabling their use as a novel treatment modality. Our in vitro results demonstrated that these siRNA-loaded exosomes are effectively taken up by cancer cells, leading to the silencing of the target gene.” (line 541-7).

13. What do these findings mean? (Separate paragraph)

The following sentence was added to the Discussion:

“Furthermore, our in vivo experiments revealed that these patient-derived exosomes are efficiently accumulated at tumor sites and significantly inhibit tumor growth, thus offering a promising direction for OC treatment strategies.” (547-50).

14. What is the impact of this research on society? (Separate paragraph)

The following sentences were added to the Discussion:

“The most important requirement for the establishment of exosome‑based delivery strategies for cancer treatment is the development of a method of collecting ample volumes of exosomes from patients. Although recent reviews have introduced a variety of exosomes delivery methods, the sources of exosomes in these preclinical studies were mostly cell lines and bone marrow cells collected from mice. Therefore, one of the strengths of this study is that we revealed the possibility of collecting ample volumes of exosomes from patient-derived tissues, which is clinically meaningful. Furthermore, omentum contains fibroblasts as well as adipose cells and macrophages, indicating that a variety of exosome types may be obtained. Moreover, considering the less-invasive nature of this surgery, the establishment of exosome‑based delivery strategies using patient-derived omentum could lead to the application of laparoscopic omentectomy for OC as well as other types of cancers, such as colorectal cancer or gastric cancer.” (line 560-71)

15. Limitation / short coming of the study must be included as a separate heading. (Separate paragraph)

Thank you for this comment. We listed several limitations of this paper in the Discussion.

“This study has several limitations. First, exosomes were collected using differential ultracentrifugation, which is a complicated method that cannot remove the debris inevitably produced during the collection process. Thus, for applications in clinical settings, easier methods of collecting high-quality exosomes are warranted. Second, although the omentum was used as the exosome source, laparotomy must be performed to utilize the omentum. However, in many cases of recurrent OS, surgery is not a treatment option. Third, because the median PFS of patients with advanced OC is 16–21 months [33], it is impractical to store exosomes collected from the omentum during primary surgery to prevent future recurrence.” (line 572-583)

16. Key highlights/ Future direction must also be part of the manuscript. (Separate paragraph)

Thank you for this comment. The following sentence was added to the Discussion:

“However, ample amounts of exosomes are difficult to collect from immune cells in the peripheral blood; therefore, future studies should focus on developing innovative methods of collecting exosomes to promote their clinical use.” (line 583-7)

17. c-Met, SKOV3-13 and ES-2 use time as abbreviations, it is recommended to write first time when used in the manuscript. (Abstract).

SKOV3, SKOV-13, and CAOV3 are the full names of cell lines and do not represent abbreviations.

18. Material, well written with cat numbers.

Thank you for this comment.

19. Cell culture BJ cells, SKOV3, CLEA, GEPIA and CAOV3 use as abbreviations, it is recommended to write first time when used in the manuscript.

BJ cells, SKOV3, and CAOV3 are the full names of cell lines and do not represent abbreviations. CLEA is the accurate company name. GEPIA is the abbreviation of “Gene Expression Profiling Interactive Analysis,” and the full name has been provided in line 122 at the first appearance (yellow in highlighted).

20. Please upload the letter as supplementary file (The Institutional Animal Care and Use Committee of Osaka University approved all animal experiments (No. J006461-010).)

As requested, the approval letter was uploaded as a supplementary file; however, it is written in Japanese.

21. Figure 1D Explanation please?

Green fluorescent spots seen in cells indicate FAM-labeled Met siRNAs (si-Met-3270 and si-Met-404). We added an explanation in the figure legend.

22. Refer to the paragraph: Met expression was transiently knocked down in SKOV3, CAOV3, and ES-2 cells, but whereas in Figure ES 2 data has been not shared? When compared to paragraph: Met-siExosomes were constructed using 317 two siRNA sets (3270 and 404); these Met-siExosomes successfully inhibited c-Met expression in ES-2, SKOV3, and CAOV-3 cells (Figure 3B).

In this manuscript, we included the in vitro data using at least two cell lines as requested by the journal. Thus, in this figure, the data for SKOV3 and ES-2 cells were included. Experiments with CAOV3 cells were not performed. If the editors require us to include such information, we will add these data. However, the experiments cannot be performed within the 10-day resubmission period indicated by the editorial office.

23. Refer to Figure 4D,4E, 4F, 4G, 4H, COAV3, data has not been shared? In refer to Figure 4B observation which quite noticeable from the gel. Met-siExosomes were constructed using two siRNA sets (3270 and 404); these Met-siExosomes successfully inhibited c-Met expression in ES-2, SKOV3, and CAOV-3 cells. VS Although c-Met expression was not affected by treatment with exosomes or siRNAs alone in SKOV3 and ES-2 cells, Met-siExosomes inhibited c-Met expression in these cells (Figure 3C), CAOV-3 data is missing?

CAOV3 cells grow slowly; thus, in vivo experiments could not be performed with CAOV3. Please refer to the following previous manuscript that compared in vivo tumor growth among high-grade serous ovarian cancer cell lines: Gynecol Oncol. 2015 Aug;138(2):372-7.

24. One million 407 SKOV3-13 cells were inoculated into female BALB/c mice. Why not CAOV 3?

As mentioned above, it was impossible to perform in vivo experiments with CAOV3 cells.

Round 2

Reviewer 1 Report

The authors' response will not sway my view. Nonetheless, whether to proceed lies within the editor's discretion.

Author Response

As we mentioned, the strength of our study is that we showed that it is possible to collect an ample volume of exosomes from patient-derived tissues. However, we understand this reviewer will not change his/her decision. Therefore, we sincerely ask the editor and editorial office to decide how to handle our manuscript. Please consider asking another reviewer to review it, if needed. 

Reviewer 2 Report

Thanks addressing the comments.

Author Response

We appreciate careful review of the reviewer for improvement.