Reviewer ’s report  
 
Title: Development of a novel mouse model of hepatocellular carcinoma with nonalcoholic 
steatohepatitis using a high -fat, choline -deficient diet and intraperitoneal injection of 
diethylnitrosamine  
 
 
Version: 0 Date: 01 Oct 2015  
 
 
Reviewer: Hayato  Nakagawa  
 
 
Reviewer's report:  
 
This manuscript described a new mouse model of NASH -HCC induced by choline deficient, 
high-fat diet (HFCD) in combination with chemical carcinogen DEN. This mouse model may be 
potentially useful for analyzing the mechanism of NASH -HCC, and the greatest advantage of 
this model is ability to develop HCC quic kly. However, there are several important concerns as 
below.  
 
1. This study is very descriptive and does not include any experiments to clarify the mechanisms 
why HFCD induces NASH -like pathology and promotes HCC development. Experiments to 
assess the effi cacy of some drugs targeting insulin resistance, inflammation, and so on, may be 
helpful.  
 
2. Experiments of DEN drinking model (Figure 7) are very rough and immature at present. 
Assessment of effects of chronic DEN administration on oxidative stress and N ASH severity 
may be potentially interesting, because oxidative stress induced by single DEN injection is 
basically transient. In Figure 7, all of DEN dinking mice rapidly died even under standard diet. 
The authors should explore the appropriate DEN concent ration for mouse study. For example, 
Matsuda et al. reported that 50 μg/l DEN in drinking water could induce HCC in mice (Lab 
Invest. 2005 May;85(5):655 -63.) 
 
3. Park et al. have reported that feeding HFD to DEN -treated mice significantly promote HCC 
devel opment (Cell. 2010 Jan 22;140(2):197 -208.). So, the authors had better compare 
HFCD+DEN model with HFD+DEN model, and assess how much and why choline deficiency 
promotes HCC and inflammation.  
 
4. Wolf et al. recently reported that long -term feeding HFCD d iet to mice can induce HCC which 
depends on intrahepatic CD8+ T cells and NKT cells (Cancer Cell. 2014 Oct 13;26(4):549 -64.). 
In addition, as stated above, Park et al. have already reported that feeding HFD to DEN -treated 
mice promotes HCC development by T NF and IL -6 dependent manner. HFCD+DEN model is 
based on these models. The authors should discuss these previous studies.  
5. In Abstract, Introduction, and Discussion sections, the authors stated that "none" of previously 
reported mouse models of NASH ful ly recapitulate all of aspects of human NASH such as 
inflammation, insulin resistance, fibrosis, and HCC. But "none" is misleading, because several 
recent studies reported new mouse models with all of these features, such as high -fat and 
fructose diet mode l (Am J Pathol. 2014 May;184(5):1550 -61.), MUP -uPA transgenic mice with 
HFD (Cancer Cell. 2014 Sep 8;26(3):331 -43.), and MC4R knockout mice with HFD (Am J 
Pathol. 2011 Nov;179(5):2454 -63.).Therefore, "few" is better than "none", and the authors 
should make  reference to these studies.  
 
In HFCD+DEN model, tumor initiation is basically dependent on chemical carcinogen which is 
artificial compared with above spontaneous HCC models. So, HFCD+DEN model cannot assess 
the initiation step of NASH -HCC. On the other h and, the time to HCC development in 
HFCD+DEN model is the shortest among them. The authors can emphasize this point. 
HFCD+DEN model may be appropriate to assess how NASH environment promotes HCC.  
 
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.  
 
No 
 
Are the conclusions drawn adequately supported  by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please spec ify 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:  
 
Acceptable  
 
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 you r reply is yes to any, please give details below.  
 
I declare that I have no competing interes ts 
 
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 n amed report  
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 understand that any comments  
which I do not wish to be included in my named report can be i ncluded 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: Development of a novel mouse model of hepatocellular carcinoma with nonalcoholic 
steatohepatitis using a high -fat, choline -deficient diet and intraperitoneal injection of 
diethylnitrosamine  
 
 
Version: 0 Date: 01 Oct 2015  
 
 
Reviewer: Hayato  Nakagawa  
 
 
Reviewer's report:  
 
This manuscript described a new mouse model of NASH -HCC induced by choline deficient, 
high-fat diet (HFCD) in combination with chemical carcinogen DEN. This mouse model may be 
potentially useful for analyzing the mechanism of NASH -HCC, and the greatest advantage of 
this model is ability to develop HCC quic kly. However, there are several important concerns as 
below.  
 
1. This study is very descriptive and does not include any experiments to clarify the mechanisms 
why HFCD induces NASH -like pathology and promotes HCC development. Experiments to 
assess the effi cacy of some drugs targeting insulin resistance, inflammation, and so on, may be 
helpful.  
 
2. Experiments of DEN drinking model (Figure 7) are very rough and immature at present. 
Assessment of effects of chronic DEN administration on oxidative stress and N ASH severity 
may be potentially interesting, because oxidative stress induced by single DEN injection is 
basically transient. In Figure 7, all of DEN dinking mice rapidly died even under standard diet. 
The authors should explore the appropriate DEN concent ration for mouse study. For example, 
Matsuda et al. reported that 50 μg/l DEN in drinking water could induce HCC in mice (Lab 
Invest. 2005 May;85(5):655 -63.) 
 
3. Park et al. have reported that feeding HFD to DEN -treated mice significantly promote HCC 
devel opment (Cell. 2010 Jan 22;140(2):197 -208.). So, the authors had better compare 
HFCD+DEN model with HFD+DEN model, and assess how much and why choline deficiency 
promotes HCC and inflammation.  
 
4. Wolf et al. recently reported that long -term feeding HFCD d iet to mice can induce HCC which 
depends on intrahepatic CD8+ T cells and NKT cells (Cancer Cell. 2014 Oct 13;26(4):549 -64.). 
In addition, as stated above, Park et al. have already reported that feeding HFD to DEN -treated 
mice promotes HCC development by T NF and IL -6 dependent manner. HFCD+DEN model is 
based on these models. The authors should discuss these previous studies.  
5. In Abstract, Introduction, and Discussion sections, the authors stated that "none" of previously 
reported mouse models of NASH ful ly recapitulate all of aspects of human NASH such as 
inflammation, insulin resistance, fibrosis, and HCC. But "none" is misleading, because several 
recent studies reported new mouse models with all of these features, such as high -fat and 
fructose diet mode l (Am J Pathol. 2014 May;184(5):1550 -61.), MUP -uPA transgenic mice with 
HFD (Cancer Cell. 2014 Sep 8;26(3):331 -43.), and MC4R knockout mice with HFD (Am J 
Pathol. 2011 Nov;179(5):2454 -63.).Therefore, "few" is better than "none", and the authors 
should make  reference to these studies.  
 
In HFCD+DEN model, tumor initiation is basically dependent on chemical carcinogen which is 
artificial compared with above spontaneous HCC models. So, HFCD+DEN model cannot assess 
the initiation step of NASH -HCC. On the other h and, the time to HCC development in 
HFCD+DEN model is the shortest among them. The authors can emphasize this point. 
HFCD+DEN model may be appropriate to assess how NASH environment promotes HCC.  
 
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.  
 
No 
 
Are the conclusions drawn adequately supported  by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please spec ify 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:  
 
Acceptable  
 
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 you r reply is yes to any, please give details below.  
 
I declare that I have no competing interes ts 
 
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 n amed report  
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 understand that any comments  
which I do not wish to be included in my named report can be i ncluded 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: Development of a novel mouse model of hepatocellular carcinoma with nonalcoholic 
steatohepatitis using a high -fat, choline -deficient diet and intraperitoneal injection of 
diethylnitrosamine  
 
 
Version: 0 Date: 01 Oct 2015  
 
 
Reviewer: Hayato  Nakagawa  
 
 
Reviewer's report:  
 
This manuscript described a new mouse model of NASH -HCC induced by choline deficient, 
high-fat diet (HFCD) in combination with chemical carcinogen DEN. This mouse model may be 
potentially useful for analyzing the mechanism of NASH -HCC, and the greatest advantage of 
this model is ability to develop HCC quic kly. However, there are several important concerns as 
below.  
 
1. This study is very descriptive and does not include any experiments to clarify the mechanisms 
why HFCD induces NASH -like pathology and promotes HCC development. Experiments to 
assess the effi cacy of some drugs targeting insulin resistance, inflammation, and so on, may be 
helpful.  
 
2. Experiments of DEN drinking model (Figure 7) are very rough and immature at present. 
Assessment of effects of chronic DEN administration on oxidative stress and N ASH severity 
may be potentially interesting, because oxidative stress induced by single DEN injection is 
basically transient. In Figure 7, all of DEN dinking mice rapidly died even under standard diet. 
The authors should explore the appropriate DEN concent ration for mouse study. For example, 
Matsuda et al. reported that 50 μg/l DEN in drinking water could induce HCC in mice (Lab 
Invest. 2005 May;85(5):655 -63.) 
 
3. Park et al. have reported that feeding HFD to DEN -treated mice significantly promote HCC 
devel opment (Cell. 2010 Jan 22;140(2):197 -208.). So, the authors had better compare 
HFCD+DEN model with HFD+DEN model, and assess how much and why choline deficiency 
promotes HCC and inflammation.  
 
4. Wolf et al. recently reported that long -term feeding HFCD d iet to mice can induce HCC which 
depends on intrahepatic CD8+ T cells and NKT cells (Cancer Cell. 2014 Oct 13;26(4):549 -64.). 
In addition, as stated above, Park et al. have already reported that feeding HFD to DEN -treated 
mice promotes HCC development by T NF and IL -6 dependent manner. HFCD+DEN model is 
based on these models. The authors should discuss these previous studies.  
5. In Abstract, Introduction, and Discussion sections, the authors stated that "none" of previously 
reported mouse models of NASH ful ly recapitulate all of aspects of human NASH such as 
inflammation, insulin resistance, fibrosis, and HCC. But "none" is misleading, because several 
recent studies reported new mouse models with all of these features, such as high -fat and 
fructose diet mode l (Am J Pathol. 2014 May;184(5):1550 -61.), MUP -uPA transgenic mice with 
HFD (Cancer Cell. 2014 Sep 8;26(3):331 -43.), and MC4R knockout mice with HFD (Am J 
Pathol. 2011 Nov;179(5):2454 -63.).Therefore, "few" is better than "none", and the authors 
should make  reference to these studies.  
 
In HFCD+DEN model, tumor initiation is basically dependent on chemical carcinogen which is 
artificial compared with above spontaneous HCC models. So, HFCD+DEN model cannot assess 
the initiation step of NASH -HCC. On the other h and, the time to HCC development in 
HFCD+DEN model is the shortest among them. The authors can emphasize this point. 
HFCD+DEN model may be appropriate to assess how NASH environment promotes HCC.  
 
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.  
 
No 
 
Are the conclusions drawn adequately supported  by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please spec ify 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:  
 
Acceptable  
 
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 you r reply is yes to any, please give details below.  
 
I declare that I have no competing interes ts 
 
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 n amed report  
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 understand that any comments  
which I do not wish to be included in my named report can be i ncluded as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant