Reviewer ’s report  
Title: Apatinib affect VEGF -mediated cell proliferation, m igration, invasion via blocking 
VEGFR2/MEK/ERK in cholan giocarcinoma cell of QBC939  
 
Version: 0 Date: 11 Jan 2018  
 
Reviewer: Darrell Borger  
 
Reviewer's report:  
The manuscript entitled "Apatinib affect VEGF -mediated cell proliferation, migra tion, invasion 
via blocking VEGFR2/MEK/ERK in cholangiocarcinoma cell of QBC939" by Manping Huang 
et al. provides compelling evidence that interference with VEGF signaling through VEGFR2 
targeting can attenuate proliferation, migration, and invasion of cho langiocarcinoma cells in 
vitro. The major limitation of this study is that evaluations were performed in only a single cell 
line. However, this study is still relevant and timely as there are many actively accruing clinical 
trials evaluating apatinib acros s a variety of cancer types, including advanced intrahepatic 
cholangiocarcinoma. With these factors in mind, this reviewer feels that this is an informative 
study that requires several issues to be addressed:  
 
Major Comments:  
1) While the authors should b e commended for writing a scientific manuscript that is not in 
their native language, there are many grammatical errors that need to be corrected 
throughout the entire manuscript. This reviewer urges the authors to have this manuscript 
edited by a colleagu e who is proficient in the English language. Plus, any new submission 
should include page numbers and figure numbers.  
 
2) The reported time course of apatinib treatment throughout this study was 24h. Please 
indicate why this time -point was chosen and inclu de any time -course data using your 
model.  
 
3) The MTT assay is described sometimes as a proliferation assay and sometimes as a cell 
viability assay throughout this manuscript. Please clarify and elaborate more on how these 
experiments used in Figure 2 were  conducted in regards to the cell confluence at the start of 
the experiment and how many replicates were performed. Please also include data on 
siKDR cells undergoing the same testing to show that VEGF cannot overcome the mRNA 
knockdown and that VEGFR leve ls are not inducible.  
 
4) When evaluating the effects of apatinib with and without VEGF addition on cell viability 
(Figure 3), the authors indicated that cytotoxicity could be seen at concentrations >1,000 
nM apatinib (page 6, line 7). However, when looking at the data in Figure  3, there seems to 
be a statistically significant decrease in cell viability starting at 1,000 nM apatinib 
treatment in the absence of VEGF addition. I think this may have been a typographical 
error.  
 
5) In Figure 4, the pictures from the cell culture woun d closure assay do not show adequate 
detail. Please retake images that clearly show the cells and the channel.  
 
6) The authors are encouraged to add a recent case report of apatinib treatment in a 
cholangiocarcinoma patient (PMID 29138576) who demonstrated  a favorable response.  
 
7) It should be added in the discussion that it would not be known whether the potential 
therapeutic effects of apatinib in vivo would be attributed to effects on endothelial cells 
(anti-angiogenic), tumor cells, or both.  
 
Minor C omments:  
1) On page 6, lines 22 -26 under Results, the authors state that there was "nearly no VEGFR2 
band" on western blotting. That is overstated and should be rewritten to indicated that the 
VEGFR2 protein level was reduced by siRNA. It is encouraged to  measure the magnitude of 
protein reduction, as was done in Figure 6.  
 
2) In the Figure Legends, only list the appropriate P values instead of a generic P value legend 
that may or may not pertain to that figure.  
 
3) For the Figure 1A legend, it should be s tated more clearly that siKDR mRNA levels are 
expressed relative to siControl levels.  
 
4) For Figure legend 2, the term "relative" should also be clearly defined. Additionally, 
"gradually increasing concentration of rhVEGF" should be changed to "increased 
concentrations…", but using proper sentence structure. Finally, there is a contradiction 
between the figure and the figure legend: was the reference protein used b -actin or GAPDH?  
 
5) In Figure 3, there was a clear decrease in viable cell number with incr easing apatinib 
concentrations. Did the authors determine whether this was attributed to apoptosis, as 
identified by Peng et al. (PMID 26967384)? If not, please add to this to the Discussion.  
 
6) In Figures 3, 4, 5 apatinib is misspelled.  
 
7) I found Figur e Legend 3 confusing. Please rewrite.  
 
8) Please indicate the amounts of rhVEGF and apatinib used in each figure legend.  
 
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 statistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Not suitable for publication unless extensively edited  
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, ple ase give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named r eport 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I unders tand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
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 cholan giocarcinoma cell of QBC939  
 
Version: 0 Date: 11 Jan 2018  
 
Reviewer: Darrell Borger  
 
Reviewer's report:  
The manuscript entitled "Apatinib affect VEGF -mediated cell proliferation, migra tion, invasion 
via blocking VEGFR2/MEK/ERK in cholangiocarcinoma cell of QBC939" by Manping Huang 
et al. provides compelling evidence that interference with VEGF signaling through VEGFR2 
targeting can attenuate proliferation, migration, and invasion of cho langiocarcinoma cells in 
vitro. The major limitation of this study is that evaluations were performed in only a single cell 
line. However, this study is still relevant and timely as there are many actively accruing clinical 
trials evaluating apatinib acros s a variety of cancer types, including advanced intrahepatic 
cholangiocarcinoma. With these factors in mind, this reviewer feels that this is an informative 
study that requires several issues to be addressed:  
 
Major Comments:  
1) While the authors should b e commended for writing a scientific manuscript that is not in 
their native language, there are many grammatical errors that need to be corrected 
throughout the entire manuscript. This reviewer urges the authors to have this manuscript 
edited by a colleagu e who is proficient in the English language. Plus, any new submission 
should include page numbers and figure numbers.  
 
2) The reported time course of apatinib treatment throughout this study was 24h. Please 
indicate why this time -point was chosen and inclu de any time -course data using your 
model.  
 
3) The MTT assay is described sometimes as a proliferation assay and sometimes as a cell 
viability assay throughout this manuscript. Please clarify and elaborate more on how these 
experiments used in Figure 2 were  conducted in regards to the cell confluence at the start of 
the experiment and how many replicates were performed. Please also include data on 
siKDR cells undergoing the same testing to show that VEGF cannot overcome the mRNA 
knockdown and that VEGFR leve ls are not inducible.  
 
4) When evaluating the effects of apatinib with and without VEGF addition on cell viability 
(Figure 3), the authors indicated that cytotoxicity could be seen at concentrations >1,000 
nM apatinib (page 6, line 7). However, when looking at the data in Figure  3, there seems to 
be a statistically significant decrease in cell viability starting at 1,000 nM apatinib 
treatment in the absence of VEGF addition. I think this may have been a typographical 
error.  
 
5) In Figure 4, the pictures from the cell culture woun d closure assay do not show adequate 
detail. Please retake images that clearly show the cells and the channel.  
 
6) The authors are encouraged to add a recent case report of apatinib treatment in a 
cholangiocarcinoma patient (PMID 29138576) who demonstrated  a favorable response.  
 
7) It should be added in the discussion that it would not be known whether the potential 
therapeutic effects of apatinib in vivo would be attributed to effects on endothelial cells 
(anti-angiogenic), tumor cells, or both.  
 
Minor C omments:  
1) On page 6, lines 22 -26 under Results, the authors state that there was "nearly no VEGFR2 
band" on western blotting. That is overstated and should be rewritten to indicated that the 
VEGFR2 protein level was reduced by siRNA. It is encouraged to  measure the magnitude of 
protein reduction, as was done in Figure 6.  
 
2) In the Figure Legends, only list the appropriate P values instead of a generic P value legend 
that may or may not pertain to that figure.  
 
3) For the Figure 1A legend, it should be s tated more clearly that siKDR mRNA levels are 
expressed relative to siControl levels.  
 
4) For Figure legend 2, the term "relative" should also be clearly defined. Additionally, 
"gradually increasing concentration of rhVEGF" should be changed to "increased 
concentrations…", but using proper sentence structure. Finally, there is a contradiction 
between the figure and the figure legend: was the reference protein used b -actin or GAPDH?  
 
5) In Figure 3, there was a clear decrease in viable cell number with incr easing apatinib 
concentrations. Did the authors determine whether this was attributed to apoptosis, as 
identified by Peng et al. (PMID 26967384)? If not, please add to this to the Discussion.  
 
6) In Figures 3, 4, 5 apatinib is misspelled.  
 
7) I found Figur e Legend 3 confusing. Please rewrite.  
 
8) Please indicate the amounts of rhVEGF and apatinib used in each figure legend.  
 
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 statistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Not suitable for publication unless extensively edited  
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, ple ase give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named r eport 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I unders tand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
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 cholan giocarcinoma cell of QBC939  
 
Version: 0 Date: 11 Jan 2018  
 
Reviewer: Darrell Borger  
 
Reviewer's report:  
The manuscript entitled "Apatinib affect VEGF -mediated cell proliferation, migra tion, invasion 
via blocking VEGFR2/MEK/ERK in cholangiocarcinoma cell of QBC939" by Manping Huang 
et al. provides compelling evidence that interference with VEGF signaling through VEGFR2 
targeting can attenuate proliferation, migration, and invasion of cho langiocarcinoma cells in 
vitro. The major limitation of this study is that evaluations were performed in only a single cell 
line. However, this study is still relevant and timely as there are many actively accruing clinical 
trials evaluating apatinib acros s a variety of cancer types, including advanced intrahepatic 
cholangiocarcinoma. With these factors in mind, this reviewer feels that this is an informative 
study that requires several issues to be addressed:  
 
Major Comments:  
1) While the authors should b e commended for writing a scientific manuscript that is not in 
their native language, there are many grammatical errors that need to be corrected 
throughout the entire manuscript. This reviewer urges the authors to have this manuscript 
edited by a colleagu e who is proficient in the English language. Plus, any new submission 
should include page numbers and figure numbers.  
 
2) The reported time course of apatinib treatment throughout this study was 24h. Please 
indicate why this time -point was chosen and inclu de any time -course data using your 
model.  
 
3) The MTT assay is described sometimes as a proliferation assay and sometimes as a cell 
viability assay throughout this manuscript. Please clarify and elaborate more on how these 
experiments used in Figure 2 were  conducted in regards to the cell confluence at the start of 
the experiment and how many replicates were performed. Please also include data on 
siKDR cells undergoing the same testing to show that VEGF cannot overcome the mRNA 
knockdown and that VEGFR leve ls are not inducible.  
 
4) When evaluating the effects of apatinib with and without VEGF addition on cell viability 
(Figure 3), the authors indicated that cytotoxicity could be seen at concentrations >1,000 
nM apatinib (page 6, line 7). However, when looking at the data in Figure  3, there seems to 
be a statistically significant decrease in cell viability starting at 1,000 nM apatinib 
treatment in the absence of VEGF addition. I think this may have been a typographical 
error.  
 
5) In Figure 4, the pictures from the cell culture woun d closure assay do not show adequate 
detail. Please retake images that clearly show the cells and the channel.  
 
6) The authors are encouraged to add a recent case report of apatinib treatment in a 
cholangiocarcinoma patient (PMID 29138576) who demonstrated  a favorable response.  
 
7) It should be added in the discussion that it would not be known whether the potential 
therapeutic effects of apatinib in vivo would be attributed to effects on endothelial cells 
(anti-angiogenic), tumor cells, or both.  
 
Minor C omments:  
1) On page 6, lines 22 -26 under Results, the authors state that there was "nearly no VEGFR2 
band" on western blotting. That is overstated and should be rewritten to indicated that the 
VEGFR2 protein level was reduced by siRNA. It is encouraged to  measure the magnitude of 
protein reduction, as was done in Figure 6.  
 
2) In the Figure Legends, only list the appropriate P values instead of a generic P value legend 
that may or may not pertain to that figure.  
 
3) For the Figure 1A legend, it should be s tated more clearly that siKDR mRNA levels are 
expressed relative to siControl levels.  
 
4) For Figure legend 2, the term "relative" should also be clearly defined. Additionally, 
"gradually increasing concentration of rhVEGF" should be changed to "increased 
concentrations…", but using proper sentence structure. Finally, there is a contradiction 
between the figure and the figure legend: was the reference protein used b -actin or GAPDH?  
 
5) In Figure 3, there was a clear decrease in viable cell number with incr easing apatinib 
concentrations. Did the authors determine whether this was attributed to apoptosis, as 
identified by Peng et al. (PMID 26967384)? If not, please add to this to the Discussion.  
 
6) In Figures 3, 4, 5 apatinib is misspelled.  
 
7) I found Figur e Legend 3 confusing. Please rewrite.  
 
8) Please indicate the amounts of rhVEGF and apatinib used in each figure legend.  
 
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 statistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
Not suitable for publication unless extensively edited  
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, ple ase give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named r eport 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I unders tand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant