The Study Of Metastasis-related Characteristics Of Tumor Spheres Derived From Human Colorectal Cell Line HCT116

Background and purposeColorectal carcinoma (CRC) is a malignancy tumor of highprevalence worldwide.Recent data suggest that a rare subpopulation ofcancer cells, the cancer stem cells (CSCs), is capable of initiating,maintaining and expanding of tumor.The characteristics of stemcells,specifically pluripotency and self-renewal,are critical for sustainingthe lifelong functionality of organs.The colorectal cancer stem cells werealso found in Ricci-Vitiani’s research in2007.These cells grow in vitro asundifferentiated cancer spheres and are suggested to be responsible formetastasis and therapy resistance of CRC.Epithelial-mesenchymal transition was defined as a processcharacterized by loss of polarization and cell-cell adhesion and gain offibroblastoid phenotypes and cell motility.Recent studies have demonstrated that epithelial-mesenchymal transition was related withcancer stem cell,they both play an important role in invasion andmetastasis of all kind of epithelial maligancies.Therefore, this research isolates cancer spheres from five differentcolorectal cell lines. We observe the location of the cancer stem cellmarker CD133, and investigate the ratio of CD133+cells and therelationship between the Epithelial-Mesenchymal transition and metastasisability in spheres.Key mechanism identified in this study hold thepotential for the developing of novel therapeutic approaches.Methods1、 Human colorectal cells SW480, SW620, LOVO, HT29andHCT116were cultured in SFM with supplemented cell growth factors，these cell spheres were stable subcultured and induced to differentiate inserum supplemented medium.2、The location of the cancer stem cell marker CD133in spheres wasobserved by confocol. The expression of CD133in HCT116monolayercells and spheres were investigated by flow cytometry.3、Cell metastasis assay, immunofluorescence and RT-PCR wereapplied to examine cell metastasis ability in vitro and the expression ofEpithelial-Mesenchymal markers E-cadherin、 N-cadherin and Vimentinof both HCT116monolayer cells and spheres. Results1、Cancer spheres could be generated from five colorectal cancer celllines in SFM. These spheres were stably subcultured and induced todifferentiate by serum supplemented medium, and these differentiatedcells were in adherent growth and kept the same morphology with theparental cells.2、CD133was located in cell membrane and have higher expressionof CD133+cells in HCT116spheres.3、The expression of mesenchymal markers N-cadherin and Vimentinwere high, but epithelial marker N-cadherin was low expressed. Themetastasis ability of cell sphere was higher than monolayer cells.Conclusion1、Five different Colorectal cancer spheres can be generated in SFMculture, HCT116cancer spheres enriched CD133+cells, cancer stem cellmarker CD133was located in cell membrane in spheres, and these cancerspheres enriched cancer stem-like cells.2、HCT116cancer cell spheres might get higher metastasis abilitythrough Epithelial-Mesenchymal transition.