| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 97 === Colorectal carcinoma (CRC) is a deadly malignancy due mainly to its metastasis. Recent discovery of colon cancer stem cells (Co-CSCs) that are responsible for tumor initiation, metastasis and relapse after therapy from the primary tumor tissues of CRC patients as well as several established tumor cell lines should be helpful in developing more effective therapies for this disease. Hence, distinguishing the differences between Co-CSC and their regular counterparts became an important task in cancer research. To enrich Co-CSCs from CT-26 mouse colon cancer cells, they were cultured in a serum-free RPMI medium containing N-2 supplement, EGF and bFGF. Floating spheres appeared one week after cells being cultured in this condition. We found that single spheroid cells could form secondary spheres which in turn could form tertiary spheres in self-renewal assays. After being incubated in 10 % FBS containing-RPMI, floating spheres became attached and differentiated into parental-like as well as multinucleated cells. Real-time RT-PCR was then performed to analyze the expression of several potential stem cell marker genes. We found that mRNA levels of CD44, CD29 and Msi-1 in CT-26 spheres were increased. We subcutaneously injected spheroid cells to the mice and tumors formed three weeks post-injection. However, the tumorigenecity of spheroid cells were lower than that of the parental cells. Interestingly, the tumorigenecity of spheroid cells increased 20% when injecting less cell number. To further elucidate the differences between CT-26 cells and spheres derived from them, microarray analysis was carried out. Among genes identified, the one encoding connective tissue growth factor (CTGF) appears to be most intriguing since its downregulation has been found in invasive colon cancer cells. Indeed, the mRNA levels of CTGF in CT-26 spheres were lower than those in the parental cells. We then examined several genes related to epithelial-mesenchymal-transition (EMT) since EMT usually accompany with metastasis. However, real-time RT-PCR analysis shown that except fibronectin and vimentin, the mRNA levels of N-cadherin and Slug in CT-26 spheres were lower than those in the parental cells. Indeed, both in vitro invasion and in vivo metastasis abilities of CT-26 spheroid cells were markedly lower than those of the parental cells, suggesting that downregulation of this growth factor in CT-26 spheres does not promote their invasion. Hence, the roles of CTGF in the formation and maintenance of CT-26 spheres are under investigation.
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