Reviewer’s report
Title:  Glucose metabolic upregulation via phosphorylation of S6 ribosomal protein affects tumor
progression in distal cholangiocarcinoma
Version: 1 Date: 16 Mar 2023
Reviewer's report:
In this work Fujinaga and collaborators evaluate the activity of the mTORC1 pathway by analyzing the
levels of S6 ribosomal protein phosphorylation (pS6) along with the expression of GLUT1 in dCCA
tissue samples. The authors observed a correlation between higher pS6 and GLUT1 tissue levels with
increased  FDG  tumor  uptake  (FDG-PET)  and  advanced  disease.  In  cultured  CCA  cells,  the
pharmacological inhibition of mTORC1 pathway resulted in reduced cell growth and lowered GLUT1
expression, along with reduced glycolysis and TCA pathways activity. This is a potentially interesting
study. Nevertheless, there are some issues for the authors to consider which if properly addressed may
increase the significance of this study.
1. The experiments in which the effects of PF are tested in CCA cells should be more complete. The 
GI50 doses of PF need to be calculated for all CCA cell lines. Moreover, these tretaments should be 
better described in the Methods section.
2. Regarding the data shown in Fig. 3d, the authors need to perform dose- and time-dependent studies 
on the effects of PF, and include all four cell lines.
3. Does PF affect the migratory and invasive capacity of CCA cells? Does PF induce apoptosis and/or 
cell cycle arrest in CCA cells?
4. It would be interesting to know if PF synergizes with chemotheraputics used for the management of 
CCA patients, such as platin compounds and gemcitabine, in the inhibition of CCA cells growth.
5. The metabolomic data need to be better and further discussed.
6. The anatomical origing of the CCA cell lines used needs to be clearly indicated. As fas as I know, 
HuCCT1 cells were not derived from a dCCA.
Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of this
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that 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
Reviewer Publication Consent . 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 ) if this manuscript is
accepted for publication. Any comments that I do not wish to be included in the published report have
been included as confidential comments to the editor, which will not be published. If you are not happy
for us to publish this report, please contact the editorial office before completing the review. If you
wish, you can include your name in your published report. Please note you must decide whether to
include your name at the start of the process and confirm this decision whenever prompted. If you
change your decision later, we will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)
Is the study design appropriate to answer the research question (including the use of appropriate
controls), and are the conclusions supported by the evidence presented?
Yes
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
Yes
Are  the  images  in  this  manuscript  (including  electrophoretic  gels  and  blots)  free  from  apparent
manipulation?
Yes
Reviewer’s report
Title:  Glucose metabolic upregulation via phosphorylation of S6 ribosomal protein affects tumor
progression in distal cholangiocarcinoma
Version: 1 Date: 16 Mar 2023
Reviewer's report:
In this work Fujinaga and collaborators evaluate the activity of the mTORC1 pathway by analyzing the
levels of S6 ribosomal protein phosphorylation (pS6) along with the expression of GLUT1 in dCCA
tissue samples. The authors observed a correlation between higher pS6 and GLUT1 tissue levels with
increased  FDG  tumor  uptake  (FDG-PET)  and  advanced  disease.  In  cultured  CCA  cells,  the
pharmacological inhibition of mTORC1 pathway resulted in reduced cell growth and lowered GLUT1
expression, along with reduced glycolysis and TCA pathways activity. This is a potentially interesting
study. Nevertheless, there are some issues for the authors to consider which if properly addressed may
increase the significance of this study.
1. The experiments in which the effects of PF are tested in CCA cells should be more complete. The 
GI50 doses of PF need to be calculated for all CCA cell lines. Moreover, these tretaments should be 
better described in the Methods section.
2. Regarding the data shown in Fig. 3d, the authors need to perform dose- and time-dependent studies 
on the effects of PF, and include all four cell lines.
3. Does PF affect the migratory and invasive capacity of CCA cells? Does PF induce apoptosis and/or 
cell cycle arrest in CCA cells?
4. It would be interesting to know if PF synergizes with chemotheraputics used for the management of 
CCA patients, such as platin compounds and gemcitabine, in the inhibition of CCA cells growth.
5. The metabolomic data need to be better and further discussed.
6. The anatomical origing of the CCA cell lines used needs to be clearly indicated. As fas as I know, 
HuCCT1 cells were not derived from a dCCA.
Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of this
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that 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
Reviewer Publication Consent . 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 ) if this manuscript is
accepted for publication. Any comments that I do not wish to be included in the published report have
been included as confidential comments to the editor, which will not be published. If you are not happy
for us to publish this report, please contact the editorial office before completing the review. If you
wish, you can include your name in your published report. Please note you must decide whether to
include your name at the start of the process and confirm this decision whenever prompted. If you
change your decision later, we will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)
Is the study design appropriate to answer the research question (including the use of appropriate
controls), and are the conclusions supported by the evidence presented?
Yes
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
Yes
Are  the  images  in  this  manuscript  (including  electrophoretic  gels  and  blots)  free  from  apparent
manipulation?
Yes
Please review this paper.
user
Reviewer’s report
Title:  Glucose metabolic upregulation via phosphorylation of S6 ribosomal protein affects tumor
progression in distal cholangiocarcinoma
Version: 1 Date: 16 Mar 2023
Reviewer's report:
In this work Fujinaga and collaborators evaluate the activity of the mTORC1 pathway by analyzing the
levels of S6 ribosomal protein phosphorylation (pS6) along with the expression of GLUT1 in dCCA
tissue samples. The authors observed a correlation between higher pS6 and GLUT1 tissue levels with
increased  FDG  tumor  uptake  (FDG-PET)  and  advanced  disease.  In  cultured  CCA  cells,  the
pharmacological inhibition of mTORC1 pathway resulted in reduced cell growth and lowered GLUT1
expression, along with reduced glycolysis and TCA pathways activity. This is a potentially interesting
study. Nevertheless, there are some issues for the authors to consider which if properly addressed may
increase the significance of this study.
1. The experiments in which the effects of PF are tested in CCA cells should be more complete. The 
GI50 doses of PF need to be calculated for all CCA cell lines. Moreover, these tretaments should be 
better described in the Methods section.
2. Regarding the data shown in Fig. 3d, the authors need to perform dose- and time-dependent studies 
on the effects of PF, and include all four cell lines.
3. Does PF affect the migratory and invasive capacity of CCA cells? Does PF induce apoptosis and/or 
cell cycle arrest in CCA cells?
4. It would be interesting to know if PF synergizes with chemotheraputics used for the management of 
CCA patients, such as platin compounds and gemcitabine, in the inhibition of CCA cells growth.
5. The metabolomic data need to be better and further discussed.
6. The anatomical origing of the CCA cell lines used needs to be clearly indicated. As fas as I know, 
HuCCT1 cells were not derived from a dCCA.
Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of this
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that 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
Reviewer Publication Consent . 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 ) if this manuscript is
accepted for publication. Any comments that I do not wish to be included in the published report have
been included as confidential comments to the editor, which will not be published. If you are not happy
for us to publish this report, please contact the editorial office before completing the review. If you
wish, you can include your name in your published report. Please note you must decide whether to
include your name at the start of the process and confirm this decision whenever prompted. If you
change your decision later, we will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)
Is the study design appropriate to answer the research question (including the use of appropriate
controls), and are the conclusions supported by the evidence presented?
Yes
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
Yes
Are  the  images  in  this  manuscript  (including  electrophoretic  gels  and  blots)  free  from  apparent
manipulation?
Yes
assistant