MASSACHUSETTS GENERAL HOSPITAL HARVARD MEDICAL SCHOOL. January 4th, Dear Editors:

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1 MASSACHUSETTS GENERAL HOSPITAL HARVARD MEDICAL SCHOOL Department of Medicine Division of Gastroenterology Jackson 825D 55 Fruit Street, Boston, Massachusetts Telephone: ; Fax: Emiko Mizoguchi, M.D., Ph.D. Assistant Immunologist, Gastrointestinal Unit Investigator in Center for the Study of Inflammatory Bowel Disease (CSIBD) Massachusetts General Hospital Assistant Professor of Medicine Harvard Medical School January 4th, 2016 Dear Editors: Thank you very much for informing us that this review article will be acceptable after major revision by the Cancer Research Frontiers. I have revised the manuscript based on the reviewer s comments, and have highlighted the changes in yellow. The title and the format of this manuscript have also been updated in accordance with editorial suggestions. I hope that the revised manuscript will satisfy the issues raised by the editors and reviewers, and is now suitable for the publication in your journal. If you have any questions, please feel free to contact me. Sincerely yours, Emiko Mizoguchi, M.D., Ph.D. Massachusetts General Hospital and Harvard Medical School HealthCare System Member

2 Reviewer 1: This review summarizes the role of epithelial cells in immune cells in the mucosal layer of the intestinal tract in the pathogenesis of colitis-associated cancer. The focus appears to be on immune cells rather than on epithelial cells. Nonetheless, the review provides an up-to-date and highly relevant information in the field. 1.Major comments One of the obvious omissions in the review is the role of bacterial metabolites in shaping the mucosal signaling in reference to protection or pathogenesis of colonic inflammation and CAC. There has been growing-breaking research in this area in the past 5-6 years, identifying cell-surface G-protein-coupled receptors for bacterial fermentation products in epithelial cells and mucosal immune cells and describing their role in mucosal immune function. Similarly, there have been several recent studies highlighting the role of certain bacterial metabolites as HDAC inhibitors. This review does mention HDACs as targets for treatment of CAC, but does not say anything about the role of normal bacterial metabolites in the regulation of acetylation status of various signaling pathways that are related to inflammation and carcinogenesis in the colon. These aspects are directly relevant to the topic of the present review and should be included. Our response: Microbiota plays an important role in CAC and we have already mentioned about the bacteria induced CAC as a cause for IBD in section 1 and 4 of old version. We have added a brief paragraph on microbiota and bacterial metabolites in section 1 and 5 of the revised manuscript (on pages 7 and 26). 2.Fig. 2 needs to be modified to remove some misleading interpretations. The way Fig. 2 is drawn, one would think that caspase I is secreted into the circulation where it cleaves circulating pro-il1beta and pro-il18 into active molecules. This is obviously not true. Our response: We greatly appreciate the kind suggestion by Reviewer 1. Pro IL-1β and IL-18 conversion occurs inside the cell and mature IL-1β and IL-18 are secreted outside of the cells. We have changed this important point in the revised Figure 2. 3.It is not clear whether the different shapes of the mucosal cells and their brush border are supposed to mean something. Our response: We have edited the figure to have consistent shape of epithelial cells and brush border. 4.Is it known that RAGE is located on the lumen-facing apical membrane of the epithelial cells? If so, appropriate references should be given and also the authors need to describe or speculate why this receptor would be located on this membrane. Our response: RAGE is a multi-ligand pattern recognition receptor. So far, several RAGE ligands have been identified including AGE (advanced glycation end products), amyloid β-peptide, HMGB1 (DNA binding protein high mobility group box-1, S100/calgranulins and CHI3L1 (Chitinase 3-like 1) (Int J Inflam. 2013:403460; Oncotarget ; 6: ). Based on the function of RAGE and results from different research groups, RAGE is expressed on the apical surface of IECs. RAGE is expressed in IECs and primarily concentrated at lateral membranes of intestinal epithelial cells close to the apical cell

3 junction complexes (J Immunol. 2007:15;178: ). Findings from Raman et al revealed that RAGE signaling is involved in intestinal barrier dysfunction (Am J Physiol Gastrointest Liver Physiol. 2006: 291;G556-65). In addition, Heijimans et al showed that RAGE signaling is important for sporadic development of intestinal adenomas (Oncogene. 2013:32;1202-6). RAGE has different isoforms which include membrane bound full length form and its splice variants. Now we have included this information in the revised manuscript (on page 12). Minor comments 5.Page 4: The authors mention a UC susceptibility locus s It would be good if we know where this locus is located and whether anything is known on the protein product of the gene and its relevance to inflammation and/or carcinogenesis. Our response: Based on Khalili et al study, rs is located at chromosome 2 and it was correlated with the expression level of CXCR2. CXCR2 gene is located 10 kb downstream from rs CXCR2 is strongly associated with colitis-associated cancer (Carcinogenesis, 2015: 36; ). Information about the protein product of this gene and its direct role in inflammation/carcinogenesis is not available in the literature. We have added this information in the revised manuscript (on page 4). 6.Page 7: The authors mention that TLRs are hypo responsive in health individuals. It would be good to provide some explanation as why this is the case. Activations of TLRs is expected to happen in the colon owing to the presence of bacteria, but why this does not happen or happens in a muted manner not to induce inflammation. What are the underlying mechanisms? Is this similar to the tolerant phenotype of mucosal immune system, which is necessary for the symbiotic co-existence of the bacteria with the host? Our response: Delicate immunoregulatory balance is maintained in the intestine through unique expression pattern of TLRs and tightly controlled signaling pathways to prevent excessive inflammation in the gut. We have briefly elaborated the possible mechanisms in the revised manuscript (on page 8) 7.In several places, the results of a study is described without giving the appropriate reference (for example, page 9: A study by William et al...) but no reference is given. Our response: Now we have added references at the appropriate places. For Example, we described about the study by William and at the end we have given the reference (#41). We have moved the reference next to the sentence of A study by William as per reviewer s request. 8.For Fig. 2 and Fig. 3, there are no mentions of these figures in the text where relevant. The term "Fig. 2" does appear sometime much later in the manuscript, but the placement of Fig. 2 is suggested somewhere much earlier in the manuscript without any rationale. The authors have to place the figures in the manuscript and also refer to them when describing the information given in the figures. Our response: We have mentioned the Figures 2 and 3 in the text in the revised manuscript. 9.Page 24: How does primary sclerosing cholangitis lead to increased bile acids in the colonic lumen? One would expect the opposite because sclerosing cholangitis is expected to cause cholestasis. Our response: Sclerosing cholangitis cause cholestasis. Several studies have supported the fact that

4 IBD patients with primary sclerosing cholangitis are highly susceptible to develop colon cancer. However, the underlying mechanism is still unclear. Results from different animal studies showed that excess bile acids (particularly secondary bile acids) in the colon promote tumor development. We have clarified this point in the revised manuscript (on page 25). 10.Page 24: Is Ursodeoxycholic acid abbreviated as UCDA or UDCA? Our response: Ursodeoxycholic acid should be abbreviated as UDCA. Thanks to the reviewer to pointing it out. 11.Page 37: The authors seem to use CEC (colonic epithelial cells) and IEC (intestinal epithelial cells) synonymously. Our response: Sorry for the confusion. We are have used IECs consistently instead of CECs in the revised manuscript. Reviewer 2: 1.I thought the title was slightly misleading. Our response: Title has been modified as Mechanistic roles of epithelial and immune cell signaling during the development of colitis-associated cancer. 2.The article is long, and includes 130 references. The trouble is as follows: It came across in the format of an introduction chapter to a thesis (may be it was?). The problem with this is although thorough; people who read theses really have to (i.e. examiners) or are really driven to do so. It is very difficult to pick out global themes easily. I found after I read it, I still could not easily organize such a bulk of information. Our response: Thank you for your advice. This manuscript is not prepared for a thesis but we have tried to briefly summarize the factors which significantly contribute to colitis-associated cancer. We believe our review article complies with the published review article in the Cancer Research Frontiers. 3. There needs to be clear distinctions between mouse / animal work and human work. Of course these are complimentary but not necessarily the same. Our response: Animal and human work has been added wherever it s relevant in the revised manuscript. 4. Some of the statements are implausible or not true, and this then adds a huge weight of doubt. For instance, page 4 CAC development occurs between 15 and 30 years old. You can straw pole any number of paediatric gastroenterologists: CRC in teenagers is vanishingly rare: most will have never seen it. Our response: Based on the retrospective cohort study from by Bailey et al, incidence

5 rates of colon cancer and rectal cancer among the patients who are years of age increased by 90% and 124.2%, respectively (JAMA Surg. 2015;150(1):17-22 ). Based on the literature, it seems that young people are more likely to develop colon cancer particularly colitis associated cancer (CAC) than adults. We have toned down the statement with precise information in the revised manuscript (on Page 4). 5.On page 16: Presence of CD3+ CD8+ CD45RO+ and granzyme B in human colonic tumours is positively associated with disease severity as well as poor prognosis. This is in defiance of high profile reports about lymphocyte infiltrations and good outcome from CRC over the last 70 years. Our response: This sentence has been modified in the revised version of our manuscript (on page 17). We have already mentioned the anti-tumor role of CD8 T cells. 6. I found myself writing endless question marks because I did not believe statements or simply could not follow the text. This is a difficult area to review, and there was not a clear marker in the biology separating spontaneous sporadic CRC and IBD-related inflammation driven CRC. Our response: Although there is no clear distinction at the later stage of CRC and IBD- related inflammation driven CRC, the initiation and pathogenesis, in particular genetic alterations, of CRC and IBD-related CRC are distinct at early stage. Several studies have supported this conclusion. Reviewer 3: The review entitled "Mechanisms of mucosal signaling in colitis-associated cancer" by Subramaniam et al. described the important molecules, cells and microenvironment in the development of colitisassociated cancer, as well as the signaling molecules as potential targets for the treatment of such disease. This review provides valuable information for those working on colorectal cancer and drug discovery. The following comments should be addressed prior to acceptance for publication. 1. The authors introduced several inducible molecules during the development of CAC in section 2 with lots of words, such as TLRs, NLRs, CHI3L1 and MLCK. The authors also need to describe the potential of these molecules in the treatment of CAC as potential therapeutic targets in section 5. Our response: We have described the possibility of using CHI3L1, MLCK, TLRs and NLRs in the revised version (on page 26). 2. For the treatment of CAC, the therapies described in section 5 need to be combined with other conventional therapies including surgery, chemotherapy and radiation therapy. The authors may discuss such combination therapies in more details, if any. Our response: We have briefly mentioned about other possible therapies (on page 24). The scope of the treatment section is to describe the potential signaling molecules as therapeutic targets. 3. Adding a conclusion section at the end of the manuscript would improve it. Our response: We have added the conclusion section in our revised manuscript.

RE: Title: Practical fecal calprotectin cut-off value for Japanese patients with ulcerative colitis

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