Study On The Link Between Mucosal Type Mast Cells And The Development Of Colitis-associated Colon Cancer

Background:Recent data has established the concept that inflammation is a critical component of tumour progression. The strongest association of inflammation with malignant diseases is in colon carcinogenesis arising in individuals with inflammatory bowel diseases, for example, chronic ulcerative colitis and Crohn’s disease. The tumour microenvironment, which is largely infiltrated with inflammatory cells, is an essential participant in the neoplastic process. Among these immune cells, as a member of innate immune cell populations, mast cells play defensing roles in the frontline of intestinal barrier where there’s large quantities of commensal bacteria and pathogens. Recently most studies focused on the whole population of mast cells, however, intestinal mast cells are a heterogenous population, which at least has two main subsets:mucosal type mast cells (MMCs) and connective tissue mast cells (CTMCs), the biologic behavior of MMCs in CAC stromal environment has not been elucidated.Aim:To investigate whether mucosal mast cells play a role in the development of colon cancer from chronic inflammation, and what is the role of MMCs during the progression from chronic inflammation into colon cancer.Methods:Firstly 20 mice were delivered with AOM and 3 cycles of DSS with 4 mice as same aged control without AOM/DSS. At AD1, AD2, AD3, asending colon and descending colon tissues were collected respectively for further analysis.Then 70 mice were randomly divided into Sham group and DSCG-treated group. For pretreatment experiment, on week -1 and 0, treated group were delivered with 100 mg/kg DSCG dissolved in normal saline by gavage and Sham group with normal saline of the same amount. For MMC blockade experiment during dysplasia,30 mice were administered DSCG (100 mg/kg) at wk10 for a whole week and normal amout of Normal Saline(NS) as respective vehicle control, or nonselective mast cell stabilizer Doxantrazole for 3 days as a positive treatment control (5 mg/kg) and respective vehicle control 5%NaHCO3 group. For DSCG pretreatment, mice received a single AOM intraperitoneal injection on 1st day of week 0 and 5-day water with 2.5% DSS at week 1, and then changed into fresh water without intervention till sacrificed at 5 different timepoints(wk0, wk2, wk5, wk10, wk14). After sacrificed, blood, spleen, mesenteric lymphonodes (MLN) and colon were collected for analysis, such as evaluation of blockade of mast cells. Tumor numbers, size, load, and colon length were compared between two groups. For treatment experiment during dysplasia, mice were sacrificed at wkl4, and blockade of mast cell was assessed, and the macroscopical view of colon tumors was evaluated. CD11b+Gr-1+ MDSCs in IEL, LPL, SP and MLN were analyzed, and in vivo recruitment of CD11b+Gr-1+MDSCs were examined by flow cytometry 24 hr after intraperitoneal injection of mMCP-1.Results:In pretreatment experiment, activation of MMC was significantly blocked by DSCG pretreatment. Numbers of MMCs in DSCG-treated group has obviously reduced compared with Sham group. Serum level of mMCP-1 was markedly declined in DSCG-treated group. MMC specific transcripts of mMCP-1 and mMCP-2 were significantly downregulated in DSCG-treated group, but transcripts of mMCP-4 and mMCP-6 relating to CTMCs had a marginal difference. Blockade of mucosal type mast cells decreased colorectal tumorigenesis at week 14, showing decreased tumor numbers, tumor size and tumor load, but longer colon length. In treatment experiment during dysplasia, MMC was markedly inhibited by DSCG or Doxantrazole treatment, with the results demonstrating obviously less mast cells present in colon mucosa of DSCG group than NS group or Doxantrazole group than NaHC03 group; mMCP-1 level in serum and colonic interstitial fluid declined remarkably compared to respective vehicle group. FACS analysis showed that infiltration of CDllb+Gr-1+ MDSCs were reduced significantly in intraepithelium, lamina propria, spleen and mln in DSCG pretreated group; at wk14, there’s a significant difference, which was consistent with serum level of mMCP-1. A concentration of 15 ng/ml mMCP-1 significantly increased the recruitment CD11b+Gr-1+cells, which indicated that mMCP-1 can directly recruit CDllb+Gr-1+ MDSCs.Conclusion:These results suggest that MMCs may promote the development of CAC, partially via direct recruitment of CD11b+Gr-1+MDSCs by mMCP-1 released upon activation.