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  • Hiroko Kono,
  • Takanori Goi* and
  • Kenji Koneri
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous Reviewer 3: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript Kono et al presented their findings on the role of anti-prokineticin1 (anti-PROK1) antibody on colorectal cancer metastasis using a liver metastasis mouse model. Anti-PROK1 monoclonal antibody was shown to suppress cell proliferation ability of liver metastatic lesions, suggesting its potential use as an anticancer agent. Additionally, anti-PROK1 treatment was shown to significantly increase survival.

This laboratory has already published data on PROK1 involvement in CRC angiogenesis and metastasis, and they have developed the anti-PROK mAb, which warrants further investigations into therapeutic modalities for hematogenous metastases in colorectal cancer. The authors should consider adding their proposal for future studies in the Discussion section.

The manuscript is succinct, clear, and well written. The sentence in the lines 9-11 is not completely clear, and should be rephrased.

Comments on the Quality of English Language

The quality of English language is adequate.

Author Response

[CIMB] Manuscript ID: cimb-2717120

We greatly appreciate for your suggestions. We would like to re-submit our manuscript(Anti-Prokineticin1 Suppresses Liver Metastatic Tumors in a Mouse Model of Colorectal Cancer with Liver Metastasis : Kono H, Goi T, et.al.) to CIMB.

We modified the paper to each comment below.

 

Reviewer 1

  1.   This laboratory has already published data on PROK1 involvement in CRC angiogenesis and metastasis, and they have developed the anti-PROK mAb, which warrants further investigations into therapeutic modalities for hematogenous metastases in colorectal cancer. The authors should consider adding their proposal for future studies in the Discussion section.

Respose→ We rewritten the discussion as below. Please refer to the text.

We are working on creating the anti-PROK1 antibody that can be used in human. Ultimately, we hope to conduct clinical trials on human and lead to new treatments.

 

2.. The manuscript is succinct, clear, and well written. The sentence in the lines 9-11 is not completely clear, and should be rephrased.

Respose→We worked over the sentence in the lines 9-11.

However, one problem with current molecular targeted therapeutics is that they must be used in combination with anticancer agents. New molecular targeted therapies that can be used alone are needed.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Kono and colleagues evaluated the effects of PROK1 antibody on colorectal cancer metastasis using an in vivo mouse model. Overall, the authors were able to demonstrate that PROK1 antibody reduces the liver metastasis and improved the overall survival of the animals. Furthermore, authors evaluated a proliferation marker on liver metastatsis. Although the paper showed several interesting findings, there are some study design issues. These issues are a) sample size (too small) b) no. of liver metastasis for controls and Ab group are not the same time span (i.e. Ab group were treated substantially longer due to animals living significantly longer).

 

Specific comments:

In order to demonstrate the efficiency of the anti-PROK1 mAb. Please use cell lines to show the effectiveness of anti-PROK1 mAb (provide data that anti-PROK1 mAb actually targets PROK1 expression). Authors showed that IHC of PROK1 in cell lines but not with mAb. 

 

Since overall survival of two groups are significantly different, authors should show the overall survival data before liver metastasis data and highlight the fact that liver of control mouse where collected significantly before the mAb group. 

 

Show average survival time as graphs. 

 

Methods: Mann Whitney U test mentioned are not discussed in the statistical analysis. Please provide statistical information more comprehensively. 

 

Table 1: please include SE or SEM to show the errors between treatment groups. Please also show this data as graphs.

 

Considering that the sample size of the study is very small, but the trend of the experiments are similar across cell lines, please generate data for combining 3 cell lines. This will improve the statistical analysis by providing a stronger p-values. 

 

Discussion should include the limitations of the manuscripts. 

Author Response

We greatly appreciate for your suggestions.

 

1.In order to demonstrate the efficiency of the anti-PROK1 mAb. Please use cell lines to show

the effectiveness of anti-PROK1 mAb (provide data that anti-PROK1 mAb actually targets

PROK1 expression). Authors showed that IHC of PROK1 in cell lines but not with mAb. 

 Respose→In this paper (reference　No.16), we showed that anti-PROK1 mAb react with PROK1 protein.

 

 

2.Since overall survival of two groups are significantly different, authors should show the overall survival data before liver metastasis data and highlight the fact that liver of control mouse where collected significantly before the mAb group. 

Show average survival time as graphs. 

 

Respose→Figure 4 is added. Please refer to the text.

Figure 4. The average survival time. Mice injected with HCT116, HT29, and DLD-1 cells exhibited a significant increase in the average survival time when treated with the anti-PROK1 mAb(Ab group).

 

 Results Addition:　The average survival time of mice transfected with each cell line was 35.4 days compared to 27.6 days in control mice transfected with HCT116 cells, 48.2 days in mice transfected with antibody compared to 32.6 day in control mice transfected with HT29 cells, and 103.6 days in mice transfected with antibody compared to 53.6 days in control mice transfected with DLD-1 cells, indicating that the survival time of mice transfected with the anti-PROK1 mAb was significantly longer than that of control mice(Figure4).

 

 

3.Methods: Mann Whitney U test mentioned are not discussed in the statistical analysis. Please　provide statistical information more comprehensively. 

 　　Respose→We have changed it. Please refer to the text.

→Since we confirmed that the outcome data were not normally distributed with the Kolmogorov-Smirnov test, we tested them with the Monn Whitney U test.

 

 

 

4.Table 1: please include SE or SEM to show the errors between treatment groups. Please also

show this data as graphs.

Respose →I'm sorry, due to the median number of liver metastases, it was not possible to indicate SE or SEM.

 

 

5.Considering that the sample size of the study is very small, but the trend of the

experiments are similar across cell lines, please generate data for combining 3 cell

lines. This will improve the statistical analysis by providing a stronger p-values.

 

Respose →Figure 5 is added. Please refer to the text.

 

Figure 5. The median survival times of 15 mice from a total of three cell lines.

Mice injected with colon cancer cells exhibited a significant increase in median survival times when treated with the anti-PROK1 mAb. The survival curve was created via the Kaplan–Meier method. (control vs. the anti-PROK1 mAb group, 31 vs. 46 days; p = 0.00273)

 

 

 

Results Addition:　 And in a study of the survival period of 15 mice from a total of three cell lines, it was found that the median survival times of the mice using the anti-PROK1 mAb was sig-nificantly longer than that of the control mice. (control vs. the anti-PROK1 mAb group, 31 vs. 46 days; p = 0.00273). (Figure 5).

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, the authors investigated the effects of an anti-PROK1 monoclonal antibody (mAb) on inhibiting the metastasis of human colorectal cells using a liver metastasis mouse model. The authors concluded that the anti-PROK1 antibody suppressed liver metastatic lesions in a mouse model of colorectal cancer with liver metastasis.

Comments

The reviewer has some concerns as follows:

1.    The methods for preparation of anti-PROK1 mAb can be shortly mentioned in the Methods section. Moreover, how the quality of the anti-PROK1 mAb prepared by the authors themselves? The issues for mAb identification and quality control should be described in the Methods section.

2.     In line 70, for “SHO”, the full name or a trademark symbol ® can be shown.

3.     The reason for using single dose of 500 μg anti-PROK1 mAb can be described.

4.     In Figure 1, the size of DLD-1 cells seems too large compared to other cell sizes with same scale bars. Please check and confirm it.

5.     The scale bars for Figures 2 and 4 can be shown.

6.     The PROK1 expression in the tumors of liver metastases should be confirmed.

7.     The sample size (n=5) for this study is really small, making it difficult to judge the correctness of the conclusions.

Author Response

  1. [CIMB] Manuscript ID: cimb-2717120

    We greatly appreciate for your suggestions. We would like to re-submit our manuscript(Anti-Prokineticin1 Suppresses Liver Metastatic Tumors in a Mouse Model of Colorectal Cancer with Liver Metastasis : Kono H, Goi T, et.al.) to CIMB.

    We modified the paper to each comment below.

    1.The methods for preparation of anti-PROK1 mAb can be shortly mentioned in the Methods section. Moreover, how the quality of the anti-PROK1 mAb prepared by the authors themselves? The issues for mAb identification and quality control should be described in the Methods section.

Respose→We have investigated this in our laboratory. (Ex.Please refer to the text. as previously described [18])

 

  1. In line 70, for “SHO”, the full name or a trademark symbol® can be shown.

Respose→We rewritten and changed it as below.

five-week-old male Crlj:SHO-PrkdcscidHrhr mice (Charles River, Japan).

 

  1. The reason for using single dose of 500 μg anti-PROK1 mAb can be described.

Respose→We changed the text as below.

The dose of anti-PROK1 mAb was determined to be 500 μg (20 mg/kg of mouse body weight) by pre-experiment.

 

  1. In Figure 1, the size of DLD-1 cells seems too large compared to other cell sizes with same scale bars. Please check and confirm it.

Respose→We have confirmed and changed it. Please refer to the text.

 

  1. The scale bars for Figures 2 and 4 can be shown.

Respose→We have changed it. Please refer to the text.

 

  1. The PROK1 expression in the tumors of liver metastases should be confirmed.

Respose→We have confirmed that the PROK1 expression has decreased and am conducting further research. I would like to report the mechanism in the next issue of CIMB.

 

  1. The sample size (n=5) for this study is really small, making it difficult to judge the correctness of the conclusions.

Respose→I rewritten and changed it as below.

Although further studies are required, anti-PROK1 antibodies may be used to control and treat hematogenous metastasis of colorectal cancer.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

This revised manuscript has a great improvement and can be accepted.