Targeting Bioinformatics Predicted Biomarkers Associated with Cell Proliferation and Migration for Treating Gliomas: Preclinical Studies in a GL261 Mouse Model

Round 1

Reviewer 1 Report

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:   - I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.  - In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.  - How did the authors decide to split out the mice in the four groups with different numbers? Which is the rational and the statistical value of this sample size? - Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.  - In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance.  - Finally, a linguistic and syntactic English revision is advisable. 

Author Response

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 1:

The authors present an interesting preclinical study regarding the role of three specific antigens which could represent a target molecule for glioblastoma management.  The study is well designed with an accurate methodology; however, in my opinion, there are some issues that need to be addressed by the authors:  

I suggest to slight modify the Title to better highlight the focus of this manuscript and to specify that this preclinical study is performed on mice model.

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”.

In the Introduction, the authors should add a paragraph regarding the state of the art of HGG (current therapies and prognosis) as well as the experimental protocol studies.

We added a paragraph in the Introduction regarding the most recent WHO CNS5 classification for gliomas, as well as the current state-of-the art for high-grade gliomas.

How did the authors decide to split out the mice in the four groups with different numbers?

In the Methods section we added some explanation and rationale for the grouping of the mice into the four groups.

Which is the rational and the statistical value of this sample size?

We added in the statistical section of the Methods more information on the power analysis used and the statistical analyses.

Regarding the human investigation, what WHO CNS tumor classification are the authors referring to? Since the CNS5 recent update has upset the molecular tumor differentiation.

We have addressed at the end of the human IHC section in the Methods our rationale for separating our samples into low- and high-grade gliomas, as well as included additional information regarding their IDH1 status.

In the Discussion, the authors should add a paragraph aiming to discuss the limitations of this study and to purpose the future perspectives of this research enucleating also the possible translational relevance. 

In the Discussion, we have added several sections outlining the limitations of our study, and future studies that should be conducted to overcome the shortfalls.

Finally, a linguistic and syntactic English revision is advisable.

We have endeavored to correct any syntax errors in the manuscript, which has undergone editorial review by several English-speaking reviewers. We might add that English is the first language of most of the co-authors.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

I have read the manuscript entitled "Targeting in-silico predicted biomarkers for treating gliomas: 2 Preclinical studies" with interest.

I would appreciate if you could consider a few comments/quesions by me:

1) despite the emphasis in the title about "in-silico predicted biomarkers", the in-silico part of the work was presented in previous (duly cited) publications and the computational component presented in the manuscript at hand seems largely incidental

2) in facts, the discussion largely relies on assotiations between the target of the study and soe interesting families of proteins indicated by a bioinformatics search, unaccompanied (crucially) by experiments to validate their involvement in the animal models subjected to the study

3) I couldn't find any information about multiple testing correction being applied, however the control sample was shared among the several testing arms, and the sample sizes were "usual" for exploratory/pilot research but at the "small" end of the spectrum, and correcting might disperse dubious signal (e.g. the effect of SPON1 antibodies on survival)

4) the authors might want to detail a bit, in the methods section, what parameter settings have been employed when running GAMMA, and particularly when querying STRING since its results appear prominently in the discussion.

5) the limitations (e.g. sample size; speculative nature of the mechanistic explanation; non-randomization of the animals when attributing them to the treatments and control groups after the lesion implant surgery;...) of the study should be openly discussed by the authors, before reaching their conclusions

6) the conclusions could be made a bit less assetive, in considerations of the limitations of the study.

I look forward to the follow up of our exchange.

Best regards,

Author Response

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 2:

I have read the manuscript entitled "Targeting in-silico predicted biomarkers for treating gliomas: 2 Preclinical studies" with interest.

I would appreciate if you could consider a few comments/questions by me:

1) despite the emphasis in the title about "in-silico predicted biomarkers", the in-silico part of the work was presented in previous (duly cited) publications and the computational component presented in the manuscript at hand seems largely incidental

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”. WE also have emphasized in the manuscript that the study in the manuscript focuses on the validation of the selected biomarkers in their potential treatments by targeting them with antibodies. We still feel that it is necessary to point out that these biomarkers were identified by us to be highly expressed in high-grade gliomas as a result of bioinformatics GAMMA-predicted analyses. We have also indicated more clearly that we updated the bioinformatics analysis recently, in contrast to that conducted for the 2013 paper.

2) in facts, the discussion largely relies on associations between the target of the study and some interesting families of proteins indicated by a bioinformatics search, unaccompanied (crucially) by experiments to validate their involvement in the animal models subjected to the study

We have added more in the Discussion about our findings and the relevance. The bioinformatics STRING analyses is a crucial component of the study, as it indicates interesting gene/protein expressions associated with the biomarkers. From our updated bioinformatics GAMMA predictions, we have also included a table of mechanistic associations for each biomarker indicating some commonalities and differences. The full dataset is also added as Supplementary Data. This has resulted in additional MOA investigations that could be done for future studies. We totally agree that future studies require experiments to validate our MOA assertions.

3) I couldn't find any information about multiple testing correction being applied, however the control sample was shared among the several testing arms, and the sample sizes were "usual" for exploratory/pilot research but at the "small" end of the spectrum, and correcting might disperse dubious signal (e.g. the effect of SPON1 antibodies on survival)

We have added more information about the multiple testing corrections. We apologize for not including this in the original manuscript. We have also included statements in the figure legend for the SPON1 survival data, as well as in the Discussion regarding the relevance of SPON1 survival data regarding significance. Thank you for pointing this out to us.

4) the authors might want to detail a bit, in the methods section, what parameter settings have been employed when running GAMMA, and particularly when querying STRING since its results appear prominently in the discussion.

We have added more information in the bioinformatics section of the Methods regarding the parameter settings used for GAMMA (also included in Supplementary Data), and STRING.

5) the limitations (e.g. sample size; speculative nature of the mechanistic explanation; non-randomization of the animals when attributing them to the treatments and control groups after the lesion implant surgery;...) of the study should be openly discussed by the authors, before reaching their conclusions

We have included several sections in the Discussion regarding the limitations of the study, and suggested several future studies that should be conducted.

6) the conclusions could be made a bit less assertive, in considerations of the limitations of the study.

We have down-played the tone of our conclusions as suggested.

Reviewer 2:

I have read the manuscript entitled "Targeting in-silico predicted biomarkers for treating gliomas: 2 Preclinical studies" with interest.

I would appreciate if you could consider a few comments/questions by me:

1) despite the emphasis in the title about "in-silico predicted biomarkers", the in-silico part of the work was presented in previous (duly cited) publications and the computational component presented in the manuscript at hand seems largely incidental

We have changed the title to “Targeting bioinformatics predicted biomarkers associated with cell proliferation and migration for treating gliomas: Preclinical studies in a GL261 mouse model”. WE also have emphasized in the manuscript that the study in the manuscript focuses on the validation of the selected biomarkers in their potential treatments by targeting them with antibodies. We still feel that it is necessary to point out that these biomarkers were identified by us to be highly expressed in high-grade gliomas as a result of bioinformatics GAMMA-predicted analyses. We have also indicated more clearly that we updated the bioinformatics analysis recently, in contrast to that conducted for the 2013 paper.

2) in facts, the discussion largely relies on associations between the target of the study and some interesting families of proteins indicated by a bioinformatics search, unaccompanied (crucially) by experiments to validate their involvement in the animal models subjected to the study

We have added more in the Discussion about our findings and the relevance. The bioinformatics STRING analyses is a crucial component of the study, as it indicates interesting gene/protein expressions associated with the biomarkers. From our updated bioinformatics GAMMA predictions, we have also included a table of mechanistic associations for each biomarker indicating some commonalities and differences. The full dataset is also added as Supplementary Data. This has resulted in additional MOA investigations that could be done for future studies. We totally agree that future studies require experiments to validate our MOA assertions.

3) I couldn't find any information about multiple testing correction being applied, however the control sample was shared among the several testing arms, and the sample sizes were "usual" for exploratory/pilot research but at the "small" end of the spectrum, and correcting might disperse dubious signal (e.g. the effect of SPON1 antibodies on survival)

We have added more information about the multiple testing corrections. We apologize for not including this in the original manuscript. We have also included statements in the figure legend for the SPON1 survival data, as well as in the Discussion regarding the relevance of SPON1 survival data regarding significance. Thank you for pointing this out to us.

4) the authors might want to detail a bit, in the methods section, what parameter settings have been employed when running GAMMA, and particularly when querying STRING since its results appear prominently in the discussion.

We have added more information in the bioinformatics section of the Methods regarding the parameter settings used for GAMMA (also included in Supplementary Data), and STRING.

5) the limitations (e.g. sample size; speculative nature of the mechanistic explanation; non-randomization of the animals when attributing them to the treatments and control groups after the lesion implant surgery;...) of the study should be openly discussed by the authors, before reaching their conclusions

We have included several sections in the Discussion regarding the limitations of the study, and suggested several future studies that should be conducted.

6) the conclusions could be made a bit less assertive, in considerations of the limitations of the study.

We have down-played the tone of our conclusions as suggested.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors tried to validate the therapeutic significance of 3 predicted genes, including PLXNB2, SLIT3, and SPON1. The in vivo study is great, especially with molecular MR. However, the whole manuscript is not well organized. The authors could separate the whole study into 3 projects, and each could focused on one gene. There are also some other concerns. 

1. The diagnosis of glioma has progressed to WHO CNS5 in 2021, and high- and low- grade gliomas are seldom mentioned anymore. 

2. The bioinformatics have been improved a lot now. PLXNB2 and SLIT3 were reported in your previous paper (Neuro Oncol 2013). But SPON1 is new here, and it would be better to show some general silico analysis on these genes in gliomas with updated dataset and classification. 

3. The IHC staining is poor and confusing here. For the staining of FFPE samples, the quality needs to be improved. For the mouse model, if the whole general section of brain containing gliomas were shown, that would be great. The interested part of the borderline could be magnified then. 

4. The relationship between the 3 genes were not well demonstrated, and the function of these 3 genes were not mentioned too much. That is why I would suggest the authors to focus on one gene in one study. If the authors want to show the 3 genes, there needs a better explanation. 

So, in brief, some parts of the results are good. But, the whole manuscript and figures definitely need to be revised professionally. 

Author Response

We would like to thank the Reviewers for their insightful reviews providing helpful suggestions that can improve our manuscript.  Please find our responses to each separate criticism below.

Reviewer 3:

The authors tried to validate the therapeutic significance of 3 predicted genes, including PLXNB2, SLIT3, and SPON1. The in vivo study is great, especially with molecular MR. However, the whole manuscript is not well organized. The authors could separate the whole study into 3 projects, and each could focused on one gene. There are also some other concerns.

We have included more rationale, backed by our updated bioinformatics GAMMA-predicted data (summary table added to the results, and complete data included in a Supplementary Data file) as to our rationale for including the 3 biomarkers that we studied into one manuscript. Thank you for pointing this out, as it forced us to explain our combined study much better.

1. The diagnosis of glioma has progressed to WHO CNS5 in 2021, and high- and low- grade gliomas are seldom mentioned anymore.

We have updated information regarding the WHO 2021 CNS5 classifications in the Introduction and Discussion, included more information regarding the IDH1 status of our human tissue sample in the Methods, and provided a rational as to why we classified our human samples into low- and high-grade glioma categories.

2. The bioinformatics have been improved a lot now. PLXNB2 and SLIT3 were reported in your previous paper (Neuro Oncol 2013). But SPON1 is new here, and it would be better to show some general silico analysis on these genes in gliomas with updated dataset and classification.

Spondin 1 was included in our initial 2013 paper. Regardless, we have included more information regarding the parameters used for our bioinformatics GAMMA and STRING analyses, as well as the updated GAMMA prediction data in both a summary table of common and uncommon mechanism associations for the 3 biomarkers, as well as the complete GAMMA data set in Supplementary material.

3. The IHC staining is poor and confusing here. For the staining of FFPE samples, the quality needs to be improved. For the mouse model, if the whole general section of brain containing gliomas were shown, that would be great. The interested part of the borderline could be magnified then.

We have included new image datasets for the human IHC data regarding the expressions of the 3 biomarkers in human gliomas. Hopefully this has reduced any confusion.

We agree that whole general sections of the mouse brains would have been ideal, as we have done this in previous publications. Unfortunately, the PI no longer has access to any of the tissues used for the histological analyses, as he has moved to a different institution in a different country. The original tissue samples were unfortunately discarded by the PI’s previous institution a few months after he had left.

4. The relationship between the 3 genes were not well demonstrated, and the function of these 3 genes were not mentioned too much. That is why I would suggest the authors to focus on one gene in one study. If the authors want to show the 3 genes, there needs a better explanation.

We have included updated data and rationale for our reasoning for combining the 3 biomarkers into one manuscript. Thank you for pointing this out and allowing us to demonstrate this better in our revised manuscript.

So, in brief, some parts of the results are good. But, the whole manuscript and figures definitely need to be revised professionally.

We have endeavored to improve the text within the manuscript as suggested by the Reviewer. Regarding the figures, the graphical data was either obtained via Excel or GraphPad software programs, images obtained from state-of-the art microscopes or MRI systems, and the figures compiled by Acrobat Illustrator. I don’t think that we could be any more professional in the compilation of our figures. We have however changed Figure 1 as suggested by the Reviewer. Hopefully we have made sufficient changes that are agreeable to the Reviewer.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have addressed most of the points raised in the first revision. However, introduction should still be improved. I suggest to start from the beginning with the paragraph on the state of the art of gliomas, both from diagnostic and therapeutic point of view. In addition, this paragraph (that is now the second one but should be at the beginning), is not entirely correct. No grade differentiation has been described within glioma classification. In addition, chemotherapy is not always an immediate required therapy, as for patients with grade 2 gliomas, submitted to a complete total removal, particularly if younger than 40 yo, adjuvant treatment could be posponed at tumor recurrence. 

Author Response

The authors have addressed most of the points raised in the first revision.

However, introduction should still be improved. I suggest to start from the beginning with the paragraph on the state of the art of gliomas, both from diagnostic and therapeutic point of view.

We have added more relevant information to the Introduction regarding diagnosis and treatments.

In addition, this paragraph (that is now the second one but should be at the beginning), is not entirely correct. No grade differentiation has been described within glioma classification. In addition, chemotherapy is not always an immediate required therapy, as for patients with grade 2 gliomas, submitted to a complete total removal, particularly if younger than 40 yo, adjuvant treatment could be postponed at tumor recurrence.

We have reorganized the Introduction to include a modified version of the previous 2^nd paragraph upfront. We have also addressed the Reviewer’s concern regarding clarification on the new classification, as well as more recent treatment options based on the new classification. Hopefully the modifications meet the Reviewer’s concerns about our previous wording. Thank you for your suggestions, which have helped improve the quality of our manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

thank you for revising your manuscript in response to my questions/comments.

I hope you agree this has led to an improved presentation of your research, at least that is how I feel.

I will recommend the manuscript for acceptance.

Best regards and good luck with the continuation of your research

Author Response

Thank you for revising your manuscript in response to my questions/comments.

I hope you agree this has led to an improved presentation of your research, at least that is how I feel.

I will recommend the manuscript for acceptance.

Thank you for your suggestions, which have helped improve the quality of our manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

After revision, the whole manuscript and figures are much better. 

Author Response

After revision, the whole manuscript and figures are much better.

Thank you for your suggestions, which have helped improve the quality of our manuscript.

Author Response File: Author Response.docx