Reviewer ’s report  
Title: Apatinib affect VEGF -mediated cell proliferation, m igration, invasion via blocking 
VEGFR2/MEK/ERK in cholangiocarcinoma cell of QBC939  
 
Version: 1 Date: 15 Jun 2018  
 
Reviewer: Darrell Borger  
 
Reviewer's report:  
This updated manuscript submitted by Manping Hua ng et al. titled "Apatinib affect VEGF -
mediated cell proliferation, migration, invasion via blocking VEGFR2/RAF/MEK/ERK and 
PI3K/AKT pathways in cholangiocarcinoma cell" has been revised to address reviewers 
comments. Updates have included a second cell li ne TFK -1 in all analyses, which fully support 
original observations in the QBC939 cell line. This provides strong evidence that VEGF -
mediated cell invasion are at least partly mediated by VEGFR2 expression directly in 
cholangiocarcinoma cells and that this  can be largely reversed using the therapeutic agent 
apatinib. Support is further provided by additional analyses that identified modulated expression 
of invasion mediators SLUG and SNAIL in addition to downstream signaling to that now 
includes the AKT sig naling pathway. Significantly more details were also added to how 
experimentation was conducted. This reviewer feels that all comments from Reviewer #1 were 
adequately addressed and this manuscript provides important information that help identify 
potentia l therapeutic strategies in cholangiocarcinoma. However, a few minor points should be 
addressed:  
 
1. While the English was dramatically improved in this updated manuscript, further 
improvements are required.  
2. On page 9, lines 26 -32 in the Results section, I do not see the 3 -fold induction in cell viability 
by rhVEGF in Figure 4. Overall, I find this sentence confusing.  
3. The title of the the Results section on page 10, lines 20 -22 should be changed to som ething 
more appropriate to the actual experimentation performed, such as "Apatinib suppresses 
VEGF/KDR -Mediated Signaling Through the MAPK and AKT Signaling Pathways".  
4. On Figure 1, the labeling of the "C." panel was left off.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
Yes 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
Yes 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
Yes 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy what aspects require further 
assessment in your comments to the editors.  
I am able to assess the sta tistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Needs some language corrections before being published  
Decla ration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, f ees, funding, or salary from an 
organisation that may in any way gain or l ose financially from the publication of this  
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently applying for any patents  relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the j ournal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree f or my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as confidential commen ts 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
Title: Apatinib affect VEGF -mediated cell proliferation, m igration, invasion via blocking 
VEGFR2/MEK/ERK in cholangiocarcinoma cell of QBC939  
 
Version: 1 Date: 15 Jun 2018  
 
Reviewer: Darrell Borger  
 
Reviewer's report:  
This updated manuscript submitted by Manping Hua ng et al. titled "Apatinib affect VEGF -
mediated cell proliferation, migration, invasion via blocking VEGFR2/RAF/MEK/ERK and 
PI3K/AKT pathways in cholangiocarcinoma cell" has been revised to address reviewers 
comments. Updates have included a second cell li ne TFK -1 in all analyses, which fully support 
original observations in the QBC939 cell line. This provides strong evidence that VEGF -
mediated cell invasion are at least partly mediated by VEGFR2 expression directly in 
cholangiocarcinoma cells and that this  can be largely reversed using the therapeutic agent 
apatinib. Support is further provided by additional analyses that identified modulated expression 
of invasion mediators SLUG and SNAIL in addition to downstream signaling to that now 
includes the AKT sig naling pathway. Significantly more details were also added to how 
experimentation was conducted. This reviewer feels that all comments from Reviewer #1 were 
adequately addressed and this manuscript provides important information that help identify 
potentia l therapeutic strategies in cholangiocarcinoma. However, a few minor points should be 
addressed:  
 
1. While the English was dramatically improved in this updated manuscript, further 
improvements are required.  
2. On page 9, lines 26 -32 in the Results section, I do not see the 3 -fold induction in cell viability 
by rhVEGF in Figure 4. Overall, I find this sentence confusing.  
3. The title of the the Results section on page 10, lines 20 -22 should be changed to som ething 
more appropriate to the actual experimentation performed, such as "Apatinib suppresses 
VEGF/KDR -Mediated Signaling Through the MAPK and AKT Signaling Pathways".  
4. On Figure 1, the labeling of the "C." panel was left off.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
Yes 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
Yes 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
Yes 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy what aspects require further 
assessment in your comments to the editors.  
I am able to assess the sta tistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Needs some language corrections before being published  
Decla ration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, f ees, funding, or salary from an 
organisation that may in any way gain or l ose financially from the publication of this  
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently applying for any patents  relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the j ournal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree f or my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as confidential commen ts 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
Title: Apatinib affect VEGF -mediated cell proliferation, m igration, invasion via blocking 
VEGFR2/MEK/ERK in cholangiocarcinoma cell of QBC939  
 
Version: 1 Date: 15 Jun 2018  
 
Reviewer: Darrell Borger  
 
Reviewer's report:  
This updated manuscript submitted by Manping Hua ng et al. titled "Apatinib affect VEGF -
mediated cell proliferation, migration, invasion via blocking VEGFR2/RAF/MEK/ERK and 
PI3K/AKT pathways in cholangiocarcinoma cell" has been revised to address reviewers 
comments. Updates have included a second cell li ne TFK -1 in all analyses, which fully support 
original observations in the QBC939 cell line. This provides strong evidence that VEGF -
mediated cell invasion are at least partly mediated by VEGFR2 expression directly in 
cholangiocarcinoma cells and that this  can be largely reversed using the therapeutic agent 
apatinib. Support is further provided by additional analyses that identified modulated expression 
of invasion mediators SLUG and SNAIL in addition to downstream signaling to that now 
includes the AKT sig naling pathway. Significantly more details were also added to how 
experimentation was conducted. This reviewer feels that all comments from Reviewer #1 were 
adequately addressed and this manuscript provides important information that help identify 
potentia l therapeutic strategies in cholangiocarcinoma. However, a few minor points should be 
addressed:  
 
1. While the English was dramatically improved in this updated manuscript, further 
improvements are required.  
2. On page 9, lines 26 -32 in the Results section, I do not see the 3 -fold induction in cell viability 
by rhVEGF in Figure 4. Overall, I find this sentence confusing.  
3. The title of the the Results section on page 10, lines 20 -22 should be changed to som ething 
more appropriate to the actual experimentation performed, such as "Apatinib suppresses 
VEGF/KDR -Mediated Signaling Through the MAPK and AKT Signaling Pathways".  
4. On Figure 1, the labeling of the "C." panel was left off.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
Yes 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
Yes 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
Yes 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy what aspects require further 
assessment in your comments to the editors.  
I am able to assess the sta tistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Needs some language corrections before being published  
Decla ration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, f ees, funding, or salary from an 
organisation that may in any way gain or l ose financially from the publication of this  
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently applying for any patents  relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the j ournal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree f or my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as confidential commen ts 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant