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To begin to understand the role of IL33 in colon cancer liver metastasis, the expression levels of Il33 were validated in the colon cancer mouse model. The mRNA levels of Il33 and its receptor St2 were measured by real-time PCR. Il33 mRNA level in CT26-FL3 was found approximately 40-fold higher as compared to that in CT26 cells, which was consistent with the microarray data. On the other hand, the mRNA level for the St2 receptor in CT26-FL3 was only two-fold higher over that in CT26 cells (Figure 4.2a). Also the intracellular protein levels of IL33 from total cells extracts were determined by Western blotting. The results showed that intracellular levels of IL33 in CT26-FL3 cells were elevated only by approximately 2- to 3-fold, in spite of the 40-fold increase in mRNA levels (Figure 4.2b). This suggests that most of the IL33 is probably secreted from the cancer cells into the surrounding microenvironment where it could exert its effect on cells in the tumor stroma or target organ. Therefore, the protein levels of IL33 in blood serum from mice bearing tumors from CT26 or CT26-FL3 cells, or from sham control mice were determined by western blotting, using albumin as a control for equal loading in each lane. The results showed that sera from mice bearing CT26-FL3 derived tumors had a higher level of IL33 as compared to sera from mice bearing tumors from CT26 cells (Figure 4.2c) while sera from sham injected control mice had basal levels of IL33 (Figure 4.2c). Immunohistochemical staining for IL33 in primary tumor sections from the cecum further showed that IL33 levels were higher in tumors derived from CT26-FL3 as compared to those from CT26 (Figure 4.2d). Collectively, these results indicated that the highly metastatic cell line CT26-FL3 can secrete higher levels of

IL33 into circulation where it can potentially influence the host tumor microenvironment in a paracrine fashion.

To determine the stage in tumor development at which Il33 expression becomes elevated, its mRNA level was measured in early stage non-invasive, non-metastatic intestinal adenomas in the ApcMin/+ mouse, a genetic model of intestinal tumorigenesis.

Figure 4.2 Increased expression of IL33 in highly metastatic CT26-FL3 cells and in tumors derived from these cells. a. mRNA levels of Il33 and its receptor St2 in CT26 and CT26-FL3 were measured by qRT/PCR. The mRNA expression levels were normalized against β-actin mRNA. b. Total protein extracts from CT26 and CT26-FL3 cells were analyzed by Western blotting to detect IL33 protein levels. c. Sera taken from mice bearing tumors from CT26 or CT26-FL3 cells or sham injected mice at four weeks after cecal implantation were analyzed by Western blotting to detect serum levels of IL33. d. Immunohistochemical analysis of sections from primary cecal tumors derived from CT26 and CT26-FL3 using antibodies against IL33.

The ApcMin/+ mouse is derived from the C57BL/6J background and has a mutation in the tumor suppressor Apc (Adenomatous polyposis coli) gene. These mice spontaneously develop multiple adenomas in the small intestine with a few in the colon. Tumors and non-tumor regions of the small intestine were collected from ApcMin/+ mice and from normal intestinal tissues from wild type C57BL/6J mice.

mRNA was isolated from these tissues and Il33 mRNA levels were determined by real-time PCR (Figure 4.3a). IL33 protein levels were assessed by immunohistochemical staining of adenomas from ApcMin/+ mice and intestinal tissues from wild type mice

Figure 4.3 Increased expression of Il33 in tumor tissue from ApcMin/+ mice. a. mRNA levels of Il33 in tumor and intestine from ApcMin/+ mice, and intestine from C57BL/6J mice were measured by qRT/PCR. The mRNA expression levels were normalized against β-actin mRNA. b. Immunohistochemical analysis of sections from tumor tissue of ApcMin/+

(Figure 4.3b). The results showed that even in early stage adenomas, before the tumor becomes invasive, IL33 mRNA levels were already elevated by approximately 7-fold over non-tumor intestinal sections from ApcMin/+ mice or 17-fold over normal intestinal sections from non-tumor bearing C57BL/6 wild type mice. Immunohistochemical analysis further showed that IL33 was highly expressed in tumor tissue from ApcMin/+ mice as compared to normal intestinal sections from C57BL/6 mice.

To determine if the IL33 is similarly induced in human colorectal cancer, tissues representing different stages of cancer progression from colon cancer patients were acquired from tissue bank at the Center for Colon Cancer Research (CCCR) of the University of South Carolina. Analyses of these cells would validate the correlation of IL33 expression observed in the mouse model to that of colon cancer in the clinical setting. Tissue sections from non-tumor regions and from stage1, 2, 3, and 4 from colon cancer patients were analyzed by immunohistochemical staining to assess the levels of IL33 and ST2 proteins. Based on the staining intensity, the results indicated that the expression levels of both IL33 and ST2 are associated with colon cancer stages; they increase during the progression of colon cancer in patients (Figure 4.4a). To semi- quantify the expression levels of Il33 and St2, total RNA was isolated from tissues and measured by real-time PCR. Although variations in tissue samples from same stage existed, the trend of Il33 and St2 expression was consistent with results from immunohistochemical staining (Figure 4.4b). The low levels of Il33 and St2 observed in samples from stage 4 cancer may have resulted from the massive necrosis of cancer cells found inside the tumor lesion that typically occurs at the late stage of cancer. It is unclear why the mRNA expression of St2 is higher in non-tumor region of the patient samples. It

is possible that elevated levels of Il33 in the patient could induce the expression of St2; further studies and a larger number of tissue samples will need to be examined.

In summary the results from qRT/PCR and immunohistochemical staining indicate that both in mouse and human colon cancer tissues, Il33 mRNA and protein levels are elevated as early as the adenoma stage, suggesting an important and early role in the etiology of the disease.