Promising Therapeutic Impact of a Selective Estrogen Receptor Downregulator, Fulvestrant, as Demonstrated In Vitro upon Low-Grade Serous Ovarian Carcinoma Cell Lines
, Kentaro Nakayama 1,*, Mohammad Mahmud Hossain 1, Sultana Razia 1, Tomoka Ishibashi 1, Masako Ishikawa 1, Hitomi Yamashita 1, Kosuke Kanno 1, Seiya Sato 1, Satoru Nakayama 2, Yoshiro Otsuki 3 and Satoru Kyo 1
Round 1
Reviewer 1 Report
This study represents a good effort towards identifying more specific treatment of serous low grade ovarian carcinoma, serous borderline ovarian tumors and ovarian serous cystadenoma and promotes better tumor characterization.
But I would like to see a more appropriate title: 'Promising therapeutic impact of selective estrogen receptor down-regulator fulvestrant as demonstrated in vitro upon low-grade serous ovarian carcinoma cell lines'
At line 98 is described the ER expression grading but it does not permeate to the results section to describe fulvestrant effect according to grading.
I find this a very interesting research and maybe it could extended to associate pi3k inhibitors.
Author Response
Point-wise responses to the comments made by Reviewer 1
Comments and Suggestions for Authors
Thank you for your valuable comments and suggestions. We have hereinafter provided point-wise responses to all the comments and suggestions.
Comment 1
This study represents a good effort towards identifying more specific treatment of serous low grade ovarian carcinoma, serous borderline ovarian tumors and ovarian serous cystadenoma and promotes better tumor characterization.
But I would like to see a more appropriate title: 'Promising therapeutic impact of selective estrogen receptor down-regulator fulvestrant as demonstrated in vitro upon low-grade serous ovarian carcinoma cell lines'
Response 1
We have modified the title according to your suggestion.
Comment 2
At line 98 is described the ER expression grading but it does not permeate to the results section to describe fulvestrant effect according to grading.
Response 2
Although we described the ER expression grading in the Materials and Methods section (immunohistochemistry) we did not mention this description in the Results section, due to some limitations. Per your recommendation, we have mentioned the ER expression grading under study strength and limitation in the Discussion section of the revised manuscript.
Page 11, lines: 457-465 “This study has a major strength: it is the first molecular study demonstrating that the use of fulvestrant, a selective ER downregulator, is effective in LGSOC treatment. However, this study has a limitation. We did not observe the effect of fulvestrant on the ER expression grading of the clinical samples. Therefore, the use of three-dimensional cell cultures such as patient-derived cancer organoids, which closely mimic the in vivo conditions, is essential to confirming the current findings. Although we observed a significant association between positive ER expression and PIK3CA mutation, further research is required to analyze the sensitivity of PI3K inhibitors in ER-positive MPSC1 LGSOC cells, in order to clarify the therapeutic potential of PI3K inhibitors.”
Comment 3
I find this very interesting research and maybe it could extended to associate pi3k inhibitors.
Response 3
We have added a recommendation that future studies should evaluate LGSOC cell sensitivity to PI3K inhibitors in the Discussion section
Page 11, lines: 462-465 “Although we observed a significant association between positive ER expression and PIK3CA mutation, further research is required to analyze the sensitivity of PI3K inhibitors in ER-positive MPSC1 LGSOC cells, in order to clarify the therapeutic potential of PI3K inhibitors.”
Reviewer 2 Report
I read with great interest the enclosed Manuscript which falls within the aim of Current Oncology.
In my honest opinion, the topic is interesting enough to attract the readers’ attention. Methodology is accurate and conclusions are supported by the data analysis. Nevertheless, authors should clarify some point and improve the discussion citing relevant and novel key articles about the topic.
Authors should consider the following recommendations:
- Manuscript should be further revised by a native English speaker
- Inclusion/exclusion criteria should be better clarified.
- The Authors did not mention the sample size calculation for their study. It is essential to specify this data in order to guarantee an adequate significance of the results obtained by the Authors.
Author Response
Point-wise response to the comment made by Reviewer 2:
Comments and Suggestions for Authors
Thank you for your valuable comments and suggestions. We have hereinafter provided point-wise responses to all the comments and suggestions.
Comment 1
I read with great interest the enclosed Manuscript which falls within the aim of Current Oncology.
In my honest opinion, the topic is interesting enough to attract the readers’ attention. Methodology is accurate and conclusions are supported by the data analysis. Nevertheless, authors should clarify some point and improve the discussion citing relevant and novel key articles about the topic.
Authors should consider the following recommendations:
- Manuscript should be further revised by a native English speaker
Response 1
We have endeavored to get the revised manuscript edited by a native English speaker.
Comment 2
Inclusion/exclusion criteria should be better clarified. The Authors did not mention the sample size calculation for their study. It is essential to specify this data to guarantee an adequate significance of the results obtained by the Authors.
Response 2
In our experiment, we conducted a retrospective cohort study from 2007-2017 in our university hospital and related hospitals. Thus, we have not mentioned sample size calculation.
Comment 3
The authors have not adequately highlighted the strengths and limitations of their study. I suggest better specifying these points
Response 3
We have highlighted the study strength and limitation accordingly.
Page,11 lines: 457-465 “This study has a major strength: it is the first molecular study demonstrating that the use of fulvestrant, a selective ER downregulator, is effective in LGSOC treatment. However, this study has a limitation. We did not observe the effect of fulvestrant on the ER expression grading of the clinical samples. Therefore, the use of three-dimensional cell cultures such as patient-derived cancer organoids, which closely mimic the in vivo conditions, is essential to confirming the current findings.”
Comment 4
Discussion could benefit of a brief paragraph discussing the role of an integrated physiological and gynecological approach for ovarian cancer (see PMID: 32662343).
Response 4
Per your suggestion, we have added a brief paragraph wherein we discussed the role of an integrated physiological and gynecological approach for ovarian cancer in the Discussion section.
Page 9, line 338–356 “LGSOC is an atypical histologic subtype of ovarian carcinoma with distinct clinical features, and is characterized by a high rate of metastasis during diagnosis; moreover, the rate of resistance to conventional chemotherapy is high, with an extended overall survival of patients with LGSOCs. LGSOC accounts for more than 10% of serous carcinomas [6]. Women typically present LGSOC at a younger age and have an indolent clinical course compared with that of high-grade serous ovarian cancers. A previous study reported that ovarian malignancies in children and adolescents accounted for 10–20% of all ovarian masses [28]. Most ovarian cysts in children are benign and self-resolving. However, if malignancy is identified, it is usually necessary to perform surgery with a wide range of interventions [29]. The primary treatment of LGSOC is the same as that of other epithelial ovarian cancer subtypes and comprises debulking surgery and platinum/taxane-based chemotherapy; however, the use of platinum-based chemotherapy is debated due to low response rates in patients with LGSOCs. Several recommendations can be followed to preserve the fertility of young patients with LGSOCs: (1) removing the whole body of the tumor, (2) sparing the fallopian tube without filmy and dense adhesions, (3) collecting ascitic fluid for cytology, (4) removing areas with suspected tumor invasion, and (5) examining the iliac and aortocaval nodes and biopsying areas with suspected tumor invasion [29]. In cases of tumor recurrence, the use of hormonal therapies, bevacizumab, and targeted therapies such as MEK inhibitors may offer benefits in combination with cytotoxic chemotherapy.”
Comment 5
Discussion could also benefit from a small paragraph showing other new approaches for advanced ovarian cancer treatment, including HIPEC (PMID: 32953629) or PARP-i (PMID: 35290101)
Response 5
We have added statements pertaining to the use of other new approaches such as hyperthermic intraperitoneal chemotherapy and PARP inhibitors for advanced cancer treatment in the Discussion section of the revised manuscript.
Page 10, lines:390-394 “Hitherto, different treatment options such as bevacizumab, PARP inhibitors [44], or hyperthermic intraperitoneal chemotherapy [45] have been used to increase survival in patients with ovarian cancer (including LGSOCs). However, the results obtained are not completely satisfactory.”
Reviewer 3 Report
Dear Authors,
Please see the following comments to improve the manuscript.
- Give some brief introduction about Serous borderline tumors (SBT) and Serous cystadenomas (SCAs) in the introduction part.
- In figure-1, mark the area for immunostaining of ER, where it is expressed in the section. It is better to show the area of positivity with arrow in all the panels.
- How all the cell line used in this study plated at the same density for expansion. Every cell line has different doubling time/proliferation potential. If the authors have doubling time data of the cells, it will be better to include in this manuscript.
- 3 Cell culture and cell line condition: First sentence structure need to revise; it is not giving clear meaning.
- It will be better to show figures of all the different cells line used in this study make a composite of all the cell line used.
- 4 Western blot analysis: How extracted protein from the cells measured? Need to provide brief details about the method adopted. How much protein loaded per lane of gel?
- What is the primary or secondary antibody dilution used in the western blot analysis?
- How many times washing of the western blot membrane?? Need to revise the material method of western blot membrane in a descriptive way.
- Need to Provide full membrane of western blot as a supplementary figure, and also provide the bar graph for western blot analysis.
- Need to revise the figure 2A, what is the value of the Y axis, Axis should be labelled also.
- 5 Cell proliferation assay: line 139, it should be “drug only”, not all the drugs as author used only single drug Fulvestrant.
- What is the method used to check the ER expression in LGSOC/SBT/SCA cells?
- Need to provide more data to prove the hypothesis, either by knockdown the ER receptor in cell line and then treatment or no treatment with Fulvestrant to see the effect.
Author Response
Point-wise responses to the comments made by Reviewer 3
Comments and Suggestions for Authors
Thank you for your valuable comments and suggestions. We have hereinafter provided point-wise responses to all the comments and suggestions.
Comment 1
Give some brief introduction about Serous borderline tumors (SBT) and Serous cystadenomas (SCAs) in the introduction part.
Response 1
We have briefly mentioned serous borderline tumors (SBT) and serous cystadenomas (SCAs) in the Introduction section of the revised manuscript.
Page 1, line 42-44 “LGSOCs are thought to arise from serous cystadenomas or adenofibromas in a slow stepwise fashion; LGSOC development passes through serous borderline tumor (SBT) development.”
Comment 2
In figure 1, mark the area for immunostaining of ER, where it is expressed in the section. It is better to show the area of positivity with arrow in all the panels.
Response 2
Per your suggestion, we have added a new Figure 1.
Figure 1. Representative image of ER immunostaining in LGSOCs. A. Negative ER expression in LGSOCs; B. Weak ER expression in LGSOCs; C. Moderate ER expression in LGSOCs; D. Strong ER expression in LGSOCs. ER, estrogen receptor; LGSOCs, low-grade serous ovarian carcinomas.
Comment 3
How all the cell line used in this study plated at the same density for expansion. Every cell line has different doubling time/proliferation potential. If the authors have doubling time data of the cells, it will be better to include in this manuscript.
Response 3
In the revised manuscript, we have calculated and included the cell doubling time in the Materials and Methods section (Cell culture subsection).
Page 4, lines:-129-132“In our experiments, we cultured an equal number of cells for each cell line; the number of cells was determined using a cell counting machine, and the cell doubling times of the MPSC1, T47D, and TOV-21G cell lines were 54 h 55 min, 62 h 49 min, and 21 h 19 min, respectively.”
Comment 4
Cell culture and cell line condition: First sentence structure need to revise; it is not giving clear meaning.
Response 4
According to your suggestion, we have changed the first sentence structure.
Page,3 lines:– 120-122 “The human LGSOC cell line, MPSC1, was used in this study as an experimental cell line; the breast cancer cell line, T47D (ER-positive), was used as a positive control; and ovarian clear cell line, TOV-21G (ER-negative), was used as a negative control.”
Comment 5
It will be better to show figures of all the different cells line used in this study make a composite of all the cell line used.
Response 5
We have added figures of all the different cell lines in the Supplementary Materials section. (Supplementary Figure S1).
Comment 6
Western blot analysis: How extracted protein from the cells measured? Need to provide brief details about the method adopted. How much protein loaded per lane of gel?
Response 6
In our experiment, the protein concentration of the cell lysates was measured using the Bradford method, and 10 µg of denatured protein was loaded per well for gel electrophoresis. Page 4, lines: 140-141 “Protein concentration in the cell lysates was measured using the Bradford method, and 10 µg of denatured protein was loaded per well for gel electrophoresis.”
Comment 7
What is the primary or secondary antibody dilution used in the western blot analysis?
Response 7
In the western blot analysis, we used primary ERα and secondary goat anti-rabbit antibodies at dilutions of 1:100 and 1:10000, respectively.
Page 4, lines: - 148-154 “Thereafter, primary ERα antibodies were added (dilution- 1:100, abcam, CA, USA), and the membranes were incubated overnight at 4 C on a shaker. The next day, the primary antibody solution was discarded, and the membrane was washed with 0.1% PBS-T four times for 5 min each. The membrane was placed in secondary antibodies (goat anti-rabbit 1:10000, horseradish peroxidase conjugated IgG) for 1 h at room temperature (25 C) and subsequently washed with 0.1% PBS-T four times for 5 min each.”
Comment 8
How many times washing of the western blot membrane? Need to revise the material method of western blot membrane in a descriptive way.
Response 8
According to our laboratory protocol of western blot analysis, we washed the membrane with 0.1% PBS-T four times for 5 min each. We have revised the Materials and Methods section (Western blot analysis subsection) to include this description.
Page 4, lines: 148-154 “Thereafter, primary ERα antibodies were added (dilution- 1:100, abcam, CA, USA), and the membranes were incubated overnight at 4 C on a shaker. The next day, the primary antibody solution was discarded, and the membrane was washed with 0.1% PBS-T four times for 5 min each. The membrane was placed in secondary antibodies (goat anti-rabbit 1:10000, horseradish peroxidase conjugated IgG) for 1 h at room temperature (25 C) and subsequently washed with 0.1% PBS-T four times for 5 min each.”
Comment 9
Need to Provide full membrane of western blot as a supplementary figure, and also provide the bar graph for western blot analysis.
Response 9
Per your suggestion, we have provided a full western blot membrane as well as a bar graph for western blot analysis in the Supplementary Materials section. (Supplementary Figures 2A and 2B.
Comment 10
Need to revise the figure 2A, what is the value of the Y axis, Axis should be labelled also.
Response 10
We have revised Figure 2A; the Y-axis indicates ER expression levels (H-score).
Figure 2. ER expression levels in ovarian serous tumors and MPSC1 cells. (A) ER expression levels in SCAs, SBTs, and LGSOCs. ER expression levels in SBTs and LGSOCs are significantly higher than that in SCAs (*p< 0.05); however, ER expression variation was not significant between SBTs and LGSOCs. (B) ER expression by Western blotting in MPSC1 and T47D cells. SCAs, serous cystadenomas; SBTs, serous borderline tumors.
Comment 11
Cell proliferation assay: line 139, it should be “drug only”, not all the drugs as author used only single drug Fulvestrant.
Response 11
We apologize for this oversight; we have made the necessary correction in the Materials and Methods section (Cell proliferation assay subsection) of the revised manuscript.
Page 4, line: 160 “Fluvestrant was purchased from Sigma-Aldrich (St. Louis, MO, USA).”
Comment 12
What is the method used to check the ER expression in LGSOC/SBT/SCA cells?
Response 12
We used immunohistochemistry to assess ER expression in LGSOC, SBT, and SCA cells. This information is mentioned in the Materials and Methods section (Immunohistochemistry subsection).
Comment 13
Need to provide more data to prove the hypothesis, either by knockdown the ER receptor in cell line and then treatment or no treatment with Fulvestrant to see the effect.
Response 13
Per you comment, we have performed ER knockdown in the cell line, and no treatment with fulvestrant was performed MTT assay to observe the effect. (Supplementary Figure S4).
Page 9, lines: 324-329 “3.5. siRNA knockdown of ERα decreased ERα expression in MPSC1 cells as shown by RT-PCR
To confirm the effects of the ER on MPSC1 cells, the ERα underwent ERα siRNA knockdown. RT-PCR analysis revealed that ERα siRNA knockdown significantly suppressed mRNA ERα expression in the MPSC1 cell line (p<0.001) (Figure S3. The T47D cell line was used as a positive control (p<0.002). GAPDH expression was used as a control for the cDNA input.
3.6. ERα knockdown decreases cell proliferation in the MPSC1 cell line
To confirm the effects of ER expression on MPSC1 cell proliferation, we applied a complementary approach using a gene knockdown system to reduce ER expression in the MPSC1 cell line. Figure S4 shows that ERα siRNA knockdown significantly inhibited MPSC1 cell proliferation ability after 72 h in contrast to the control siRNA group (79.05 ± 0.0175% vs. 179.89 ± 0.025%, p <0.001).”
Author Response File: 
Author Response.docx
Round 2
Reviewer 2 Report
The authors have resolved all the issues highlighted in the previous version of the manuscript. Paper can be accepted for publication.
Reviewer 3 Report
Dear Authors,
Thanks for revising your manuscript as per the comments and suggestions.
All the best.
