Infliximab Inhibits Colitis Associated Cancer in Model Mice by Downregulating Genes Associated with Mast Cells and Decreasing Their Accumulation

Round 1

Reviewer 1 Report

The review is of interest in my area, however there are few citations to be considered recent. Without taking this observation into account, the publication has a lot of informative value.

Author Response

1. Comments and Suggestions for Authors from Reviewer 1

The review is of interest in my area, however there are few citations to be considered recent. Without taking this observation into account, the publication has a lot of informative value.

Thank you for the suggestion. We added more recent citations on IBDs to the Introduction section and changed the text. The modified parts are highlighted in yellow in the manuscript (L37-42, 47-53).

Reviewer 2 Report

In this article, the authors (Wang et.al) aimed to elucidate the relationship between colon carcinogenesis and gene expression in a drug-induced colitis mouse model, with or without the anti-TNF-a antibody (infliximab) treatment.

Although I find the topic very interesting, I have several concerns regarding this article. Please find detailed comments below:

Minor:

1. Table 1 - could the authors please define the criteria of "offset"? A significant change at p< 0.05? Or at p < 0.01? To help the readers better understand, I suggest adding the data collected from the anti-TNF-a Ab group as a separate column in this table.

2. Lines 103-104 should be moved to the "Materials and methods" section.

3. Figure 4c and 4e need titles and labels for the y-axis.

4. Figure 4b and 4d - I see that some areas in the picture are highlighted by red circles, assuming those are tumor-like lesions or colon adenocarcinoma. The authors may consider adding an explanation of those red marks in the figure legend. 

5. Line 307 - could the authors please define the area? For example, 50 um * 50 um? And what is the sample size used? How many of these areas were counted? Based on how many animals? 

Major:

1. The microscopic figures presented in this article (Figures 2a, 3a, 4d, and 5a) do not have scale bars. This is not acceptable and very unprofessional. Please consider adding scale bars to these figures.

2. Figure 4b-e: could the authors explain why the control group data is omitted in these figures?

3. Figure 5c - according to lines 152-154, the data should look like this: "...the mRNA expression of these genes (Mcpt1, Mcpt2, and Cma1) was 100-, 125-, and 60- fold higher, respectively, in cancer tissues from AOM/DSS than control mice". This part matches what is presented in Figure 5c. However, it also suggests that "infliximab significantly inhibited their expression 20-, 20-, and 5-fold, respectively". These fold-changes are compared to which group? Control? Or AOM/DSS tumor? The authors need to clarify here. Also, no matter which group, this part does not match Figure 5c. Could the authors please explain how their data is interpreted? 

4. I found the article's conclusion rather disappointing - first, the authors concluded that anti-TNF-a Ab infliximab significantly inhibited colon carcinogenesis in AOM/DSS mice. This was discovered a long time ago. Please see this article, which was published in 2016, as an example - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5124977/

Then, the authors concluded that mast cells play pivotal roles in the development of colitis-associated cancer, yet, failed to prove any concrete evidence besides mRNA expressions, which is rather weak support. I think a mast cell-deficient mouse model (https://www.sciencedirect.com/science/article/abs/pii/S0006291X21004083) may be used to prove the roles of mast cells during colitis with or without infliximab treatment. 

Third, in the introduction, the authors aimed to elucidate the relationship between colon carcinogenesis and gene expression in a drug-induced colitis mouse model. Yet, I fail to see the connection.

Overall, I think the topic of this article has been shifted to a different direction in the middle of the study - whatever was concluded at the end of this article is not what the authors claimed to focus on at the beginning of the article. Hence, either the authors should consider revising their introduction section or adding more data to support the aims originally brought up in the introduction. 

Author Response

1. Comments and Suggestions for Authors from Reviewer 2

In this article, the authors (Wang et.al) aimed to elucidate the relationship between colon carcinogenesis and gene expression in a drug-induced colitis mouse model, with or without the anti-TNF-a antibody (infliximab) treatment. Although I find the topic very interesting, I have several concerns regarding this article. Please find detailed comments below:

Minor:

• Table 1 - could the authors please define the criteria of "offset"? A significant change at p< 0.05? Or at p < 0.01? To help the readers better understand, I suggest adding the data collected from the anti-TNF-a Ab group as a separate column in this table.

Thank you for the comments. “Offset” means that TNF-α mediated inhibition of proliferation was recovered by adding of human anti TNF-α Ab. To help the readers better understand, the column of “Offset by anti TNF-a Ab (2 mg/mL)” was removed. Instead, we added the data collected from the anti-TNF-a Ab group as a separate column, named “Recovery effect by anti TNF-α Ab (2 µg/mL)” in Table 1. Furthermore, we put a significant change at P < 0.01 or P < 0.05 for Table 1.

• Lines 103-104 should be moved to the “Materials and methods” section.

Lines 103-104 were moved to the “Materials and methods” section. (line 138-140)

• Figure 4c and 4e need titles and labels for the y-axis.

The labels for the y-axis were inserted.　Titles of Figure 4c and 4e were inserted into Figure legends.

• Figure 4b and 4d – I see that some areas in the picture are highlighted by red circles, assuming those are tumor-like lesions or colon adenocarcinoma. The authors may consider adding an explanation of those red marks in the figure legend. 

Thank you for suggestion. The areas highlighted by red circles indicate tumor-like (protruding part) lesions in macroscopic view and adenocarcinoma in microscopic view. We added an explanation of those red marks in the figure legends (Because the images of Figure 4b and 4d were distorted, those were replaced with the correct one).

• Line 307 – could the authors please define the area? For example, 50 um * 50 um? And what is the sample size used? How many of these areas were counted? Based on how many animals? 

Thank you for critical questions. The positively stained mast cells in the sample tissues were detected by selecting seven random fields in high-power microscope (x400) after microscopic examination scanning the entire colon tissues in three mice. The mast cell counts are the mean of seven individual high-power fields. We put above sentences into the Materials and Methods section (L158-161).

Major:

1. The microscopic figures presented in this article (Figures 2a, 3a, 4d, and 5a) do not have scale bars. This is not acceptable and very unprofessional. Please consider adding scale bars to these figures.

Thank you for pointing out our mistake. We added scale bars in the figures.

1. Figure 4b-e: could the authors explain why the control group data is omitted in these figures?

Thank you for the comment. Because the control group　(no administration of AOM and DSS) does not develop tumors, so it was omitted in Figure 4b-e.

2. Figure 5c - according to lines 152-154, the data should look like this: "...the mRNA expression of these genes (Mcpt1, Mcpt2, and Cma1) was 100-, 125-, and 60- fold higher, respectively, in cancer tissues from AOM/DSS than control mice". This part matches what is presented in Figure 5c. However, it also suggests that "infliximab significantly inhibited their expression 20-, 20-, and 5-fold, respectively". These fold-changes are compared to which group? Control? Or AOM/DSS tumor? The authors need to clarify here. Also, no matter which group, this part does not match Figure 5c. Could the authors please explain how their data is interpreted? 

Thank you for the critical comments. Sorry for the confusing explanation. The numbers of 20, 20, and 5 were derived by the following calculations: the actual expression levels of Mcpt1, Mcpt2, and Cma1 of cancer tissue in the AOM/DSS group were divided by those in the AOM/DSS/Ab group. As the reviewer pointed out, those numbers do not match Figure 5c. To avoid confusion for the reader, the text has been revised based on the data just presented in Figure 5c. Infliximab significantly inhibited their expression compared to control, 5-, 5-, and 21-fold, respectively. We corrected the text in the Result section. (L273-274)

4. I found the article's conclusion rather disappointing - first, the authors concluded that anti-TNF-a Ab infliximab significantly inhibited colon carcinogenesis in AOM/DSS mice. This was discovered a long time ago. Please see this article, which was published in 2016, as an example - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5124977/
   Then, the authors concluded that mast cells play pivotal roles in the development of colitis-associated cancer, yet, failed to prove any concrete evidence besides mRNA expressions, which is rather weak support. I think a mast cell-deficient mouse model (https://www.sciencedirect.com/science/article/abs/pii/S0006291X21004083) may be used to prove the roles of mast cells during colitis with or without infliximab treatment.
   Third, in the introduction, the authors aimed to elucidate the relationship between colon carcinogenesis and gene expression in a drug-induced colitis mouse model. Yet, I fail to see the connection.
   Overall, I think the topic of this article has been shifted to a different direction in the middle of the study - whatever was concluded at the end of this article is not what the authors claimed to focus on at the beginning of the article. Hence, either the authors should consider revising their introduction section or adding more data to support the aims originally brought up in the introduction. 

Thank you for critical comments and suggestions. Yes, we had known that infliximab inhibited colon carcinogenesis in AOM/DSS mice as the reference paper was cited [26]. After we validated that infliximab inhibited colon carcinogenesis in AOM/DSS mice, we tried to elucidate the mechanism of colon carcinogenesis in AOM/DSS mice in this study. Then, microarray analysis and RT-PCR confirmed that mast cell related genes were upregulated in cancer tissues of AOM/DSS mice and those genes were downregulated by administration of infliximab. Although the detailed mechanism has not yet been investigated, it was suggested that mast cells play an important role in the colon carcinogenesis. Following the reviewer's suggestion, we would like to prove the roles of mast cells during colitis with or without infliximab treatment using a mast cell-deficient mouse model (Sasaki, et al. BBRC 2021) in the next step. We modified the text (L338-343).
As pointed out by the reviewer, there may be a gap between the introduction and the conclusion of this study, we also modified the introduction to match the conclusions as follows: “we verified if anti TNF-α Ab inhibits carcinogenesis in mouse models of colitis-associated cancer in vivo. Furthermore, comprehensive gene expression analysis was carried out to identify the genes involved in colitis-associated cancer.” (Line 71-74).

Reviewer 3 Report

Very interesting laboratory study on the pros and cons of TNFab on colon cancer in cell lines and in mice.

I could not find the number of experiments leading to the numbers and figures, only in the methods section.To add this would help to review the data more clear.

Author Response

1. Comments and Suggestions for Authors from Reviewer 3

Very interesting laboratory study on the pros and cons of TNFab on colon cancer in cell lines and in mice. I could not find the number of experiments leading to the numbers and figures, only in the methods section. To add this would help to review the data more clear.

Thank you for critical comment. We put the number of experiments in Materials and Methods section (Line 94, 103, and 112).

Round 2

Reviewer 2 Report

Thank you for considering my comments and incorporating them into the manuscript. I appreciate the effort made to make these revisions, and I am confident the paper is ready for publication.