Correlation between Cancer Stem Cells, Inflammation and Malignant Transformation in a DEN-Induced Model of Hepatic Carcinogenesis

Round 1

Reviewer 1 Report

cimb-1739610

The authors made a very clean and well-presented work. But still, they have to make some modifications to improve their manuscript.

Material and methods
 Why did the authors choose the times 7, 27, 33, and 50? (please inform in relation to references in the area)
Line 81: inform the cellular markers.
Line 84: the pathologist was no conflict of interest? were double-blind?
Please inform the reference where did you choose that score… (the same situation for 2.4)
Line 126: statistical software? Did you use the media or the average (and why?)

Result
 In Fig 1: please uniform the back color (there are not the same) and present all the images with a scale bar.

In legend, please inform the animal´s number for all independent time. And the X scale (do the same for all the microphotography presented in this manuscript)
Figures 1 and 2. The authors should present a representative image for all the times assayed (stadia reported in results). The present form is not correct and we can not elucidate if there exist changes thought the time. (Same for the immunohistochemical microphotography’s)

Figure 4… score of why????, where are the Figures that the authors analyzed. (Please describe all the statistical analyses).

Discussion
DEN at delimited doses and time, acts as a hepatotoxic and fibrotic (inflammatory) liver stimulus, for that reason you need to discuss these differences between fibrosis and HCC, and explain your result concerning that information.

The discussion is weak, and the authors need to make some assays related to inflammation (maybe TNF-a and IL-6´s ELISAs) to discuss it better and respond to the “inflammation progression informed in the abstract”.

Please discuss (profoundly) the role of OV-6 and how it increases is related to your findings (a molecular/cellular relationship).

Conclusion

The description of a “simple method” for inducing liver tumors is very well known (DEN induced) in the hepatologist community, you do not make a new advance in that area, please erase that and make a better conclusion

Author Response

The authors made a very clean and well-presented work. But still, they have to make some modifications to improve their manuscript.
Thank you for your affirmation and encouragement
Material and methods
 Why did the authors choose the times 7, 27, 33, and 50? (please inform in relation to references in the area)

Thanks for your valuable recommend. It is not any reference. We inject with DEN in all rats at the same time. We followed the guideline from “IACUC - American Association for Laboratory Animal Science”. When any animal need to euthanasia, we had sacrificed all animals in that time group.

Line 81: inform the cellular markers.

Thanks for your valuable recommend. It is an unnecessary section. I had deleted it.

Line 84: the pathologist was no conflict of interest? were double-blind?

Thanks for your valuable recommend. The histological scores were independently evaluated by two pathologists. If there was any discrepancy, the pathologists reanalyzed the slides together and came to made a consensus on the final score.

Please inform the reference where did you choose that score… (the same situation for 2.4)

Thanks for your valuable recommend. Inflammation scoring was modified following “Grading and staging systems for inflammation and fibrosis in chronic liver diseases” in 2007. And Reticulin staining scoring was modified from “Human colorectal cancer progression correlates with LOX-induced ECM stiffening” in 2017.

Line 126: statistical software? Did you use the media or the average (and why?)

Thanks for your valuable recommend. SPSS 24.0 (IBM, NY, USA) software was used for statistical analysis. We had added the statistical software in manuscript. We had presented the time-dependent of scores in figure 4, we choosed the average score to make this line graph.

Result
 In Fig 1: please uniform the back color (there are not the same) and present all the images with a scale bar.

Thanks for your valuable recommend. We had modified these figures.

In legend, please inform the animal´s number for all independent time. And the X scale (do the same for all the microphotography presented in this manuscript)

Thanks for your valuable recommend. We had modified these figure legends and figures in manuscript.

Figures 1 and 2. The authors should present a representative image for all the times assayed (stadia reported in results). The present form is not correct and we can not elucidate if there exist changes thought the time. (Same for the immunohistochemical microphotography’s)
Thanks for your valuable recommend. Because each week has different scores, it is difficult to present a representative image in every time, and this way maybe not provide the image from all scores. Therefore, we recommended to keep these data intact.

Figure 4… score of why????, where are the Figures that the authors analyzed. (Please describe all the statistical analyses).
Thanks for your valuable recommend. In 2.6 Data Analyses: SPSS 24.0 (IBM, NY, USA) software was used for statistical analysis. The relation-ships between the score of malignant transformation, inflammation and OV-6 were determined using Spearman’s correlation test. Statistical significance was considered when P < 0.05. In 3.4 Relationship between cancer stem cell, inflammation and malignant transformation: DEN-induced inflammation, malignant transformation and high-level OV-6 expression in hamster liver as shown above and applying Spearman’s correlation to the data showed that expression of OV-6 was significantly correlated to inflammation (p = 0.001) and malignant transformation (p < 0.001) (Figure 4)

Discussion
DEN at delimited doses and time, acts as a hepatotoxic and fibrotic (inflammatory) liver stimulus, for that reason you need to discuss these differences between fibrosis and HCC, and explain your result concerning that information.
Thanks for your valuable recommend. We had added the section “Previous studies showed that the progression of HCC was found through Wnt pathway, loss function of p53, Ras signalling, and ROS pathway. Chronic inflammation induced the proliferation of hepatocytes, shortening of telomeres and malignant transformation. Moreover, repeatedly and continuously injured in liver is significantly increased in ad-vanced cirrhosis leading to a propensity towards cancer and fibrosis. Therefore, inflammation lead to fibrosis and malignant transformation.”.

The discussion is weak, and the authors need to make some assays related to inflammation (maybe TNF-a and IL-6´s ELISAs) to discuss it better and respond to the “inflammation progression informed in the abstract”.
Thanks for your valuable recommend. In this study, we discussed the relationship between inflammation, malignant transformation and cancer stem cells in pathology. Therefore, we excluded these cytokines in this study. We discussed a theory “more and more inflammatiom induced malignant transformation, even to create the cancer stem cell.” This is a preliminary study to provide relationship between inflammation, malignant transformation and cancer stem cells.

Please discuss (profoundly) the role of OV-6 and how it increases is related to your findings (a molecular/cellular relationship).
Thanks for your valuable recommend. We had added the section “Oval cells played important role of development of HCC. OV-6 was a biomarker of oval cells and was found from hepatic stem cells. In addition, OV-6 was well-know as-sociation with prognosis in various tumors including HCC. However, no reports showed OV-6 was association with inflammation or malignant transformation. In our result, DEN-induced inflammation, malignant transformation and high-level OV-6 expression in hamster liver, and OV-6 expression was significantly correlated to inflammation and malignant transformation (Fig. 4).”

Conclusion

The description of a “simple method” for inducing liver tumors is very well known (DEN induced) in the hepatologist community, you do not make a new advance in that area, please erase that and make a better conclusion

Thanks for your valuable recommend. We had modified the section “This study describes that repeatedly inflammatory response induced malignant trans-formation and development of cancer stem cells to resistant chemotherapy and radio-therapy.”

Author Response File: Author Response.docx

Reviewer 2 Report

 The Authors conclude, "There was a significant correlation between number of cancer stem cells, inflammation and malignant transformation in a DEN-induced model of hepatic carcinogenesis"   Do the Authors have any data on CD44, CD90, CD133, and EpCAM in relation to Ov6 if yes discuss on it

 

Author Response

 The Authors conclude, "There was a significant correlation between number of cancer stem cells, inflammation and malignant transformation in a DEN-induced model of hepatic carcinogenesis"   Do the Authors have any data on CD44, CD90, CD133, and EpCAM in relation to Ov6 if yes discuss on it

Thanks for your valuable recommend. We had the IHC data from CD44 and CD133. Unfortunately, they are not significantly association with inflammation and transformation. Maybe CD44 and CD133 are the biomarker of cancer stem cells. At the same time, they are the biomarker of epithelial–mesenchymal transition. Therefore, CD44 and CD133 did not applied in this study. But, it is not confirmed. We can not discuss on this study.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors made all the requeired changes, but still it is necesary that they present representatives images related to the data presented (Figure 1, 2, 4).

Author Response

The authors made all the requeired changes, but still it is necessary that they present representatives images related to the data presented (Figure 1, 2, 4).

 

Thanks for your valuable recommend. We provided representatives images related to the data presented from 0 week to 50 weeks after DEN-induced.

 

Author Response File: Author Response.pdf