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Abstract: This paper aims to screen and identify sphere clone cells with characteristics similar to cancer stem cells in human gallbladder cancer cell line GBC-SD. GBC-SD cells were cultured in a serum-free culture medium with different concentrations of the chemotherapeutic drug cisplatin for generating sphere clones. The mRNA expressions of stem cell-related genes CD133, OCT-4, Nanog, and drug resistance genes ABCG2 and MDR-1 in sphere clones were detected by quantitative real-time polymerase chain reaction (PCR). Stem cell markers were also analyzed by flow cytometry and immunofluorescent staining. Different amounts of sphere clones were injected into nude mice to test their abilities to form tumors. sphere clones were formed in serum-free culture medium containing cisplatin (30 μmol/L). Flow cytometry results demonstrated that the sphere clones expressed high levels of stem cell markers CD133⁺ (97.6%) and CD44⁺ (77.9%) and low levels of CD24⁺ (2.3%). These clones also overexpressed the drug resistance genes ABCG2 and MDR-1. Quantitative real-time PCR showed that sphere clones expressed stem cell genes Nanog and OCT-4 284 and 266 times, respectively, more than those in the original GBC-SD cells. Immunofluorescent staining showed that sphere clones overexpressed OCT-4, Nanog, and SOX-2, and low expressed MUC1 and vimentin. Tumor formation experiments showed that 1×10³ sphere clone cells could induce much larger tumors in nude mice than 1×10⁵ GBC-SD cells. In conclusion, sphere clones of gallbladder cancer with stem cell-like characteristics can be obtained using suspension cultures of GBC-SD cells in serum-free culture medium containing cisplatin.

[1]Al-Hajj, M., Wicha, M.S., Benito-Hernandez, A., Morrison, S.J., Clarke, M.F., 2003. Prospective identification of tumorigenic breast cancer cells. PNAS, 100(7):3983-3988.

[2]Chen, Y.L., Huang, Z.Q., Zhou, N.X., Zhang, W.Z., Huang, X.Q., Duan, W.D., Liu, R., Liu, Y., 2007. Clinical analysis of 110 patients with primary gallbladder carcinoma. Chin. J. Oncol., 29(9):704-706.

[3]Chumsri, S., Burger, A.M., 2008. Cancer stem cell targeted agents: therapeutic approaches and consequences. Curr. Opin. Mol. Ther., 10(4):323-333.

[4]Clarke, M.F., Dick, J.E., Dirks, P.B., Eaves, C.J., Jamieson, C.H., Jones, D.L., Visvader, J., Weissman, I.L., Wahl, G.M., 2006. Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res., 66(19):9339-9344.

[5]de Groen, P.C., Gores, G.J., LaRusso, N.F., Gunderson, L.L., Nagorney, D.M., 1999. Biliary tract cancers. N. Engl. J. Med., 341(18):1368-1378.

[6]Fillmore, C., Kuperwasser, C., 2007. Human breast cancer stem cell markers CD44 and CD24: enriching for cells with functional properties in mice or in man? Breast Cancer Res., 9(3):303.

[7]Gilbert, C.A., Ross, A.H., 2009. Cancer stem cells: cell culture, markers, and targets for new therapies. J. Cell. Biochem., 108(5):1031-1038.

[8]Hadnagy, A., Gaboury, L., Beaulieu, R., Balicki, D., 2006. SP analysis may be used to identify cancer stem cell populations. Exp. Cell Res., 312(19):3701-3710.

[9]Kobayashi, N., Navarro-Alvarez, N., Soto-Gutierrez, A., Kawamoto, H., Kondo, Y., Yamatsuji, T., Shirakawa, Y., Naomoto, Y., Tanaka, N., 2008. Cancer stem cell research: current situation and problems. Cell Transplant., 17(1-2):19-25.

[10]Li, H.Z., Yi, T.B., Wu, Z.Y., 2008. Suspension culture combined with chemotherapeutic agents for sorting of breast cancer stem cells. BMC Cancer, 8(1):135.

[11]Ma, S., Lee, T.K., Zheng, B.J., Chan, K.W., Guan, X.Y., 2008. CD133⁺ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene, 27(12):1749-1758.

[12]Moserle, L., Ghisi, M., Amadori, A., Indraccolo, S., 2010. Side population and cancer stem cells: therapeutic implications. Cancer Lett., 288(1):1-9.

[13]Petersen, O.W., Nielsen, H.L., Gudjonsson, T., Villadsen, R., Rank, F., Niebuhr, E., Bissell, M.J., Ronnov-Jessen, L., 2003. Epithelial to mesenchymal transition in human breast cancer can provide a nonmalignant stroma. Am. J. Pathol., 162(2):391-402.

[14]Ponti, D., Costa, A., Zaffaroni, N., Pratesi, G., Petrangolini, G., Coradini, D., Pilotti, S., Pierotti, M.A., Daidone, M.G., 2005. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res., 65(13):5506-5511.

[15]Puré, E., 2009. The road to integrative cancer therapies: emergence of a tumor-associated fibroblast protease as a potential therapeutic target in cancer. Expert Opin. Ther. Tar., 13(8):967-973.

[16]Singh, S.K., Hawkins, C., Clarke, I.D., Squire, J.A., Bayani, J., Hide, T., Henkelman, R.M., Cusimano, M.D., Dirks, P.B., 2004. Identification of human brain tumour initiating cells. Nature, 432(7015):396-401.

[17]Vander Griend, D.J., Karthaus, W.L., Dalrymple, S., Meeker, A., DeMarzo, A.M., Isaacs, J.T., 2008. The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells. Cancer Res., 68(23):9703-9711.

[18]Wang, M., Qin, R.Y., Shen, M., Jiang, J.X., Hu, J., Du, Z.Y., Shi, J.C., 2009. Cancer stem cell marker CD24, CD44, ESA and CD34 expression in biliary tract tumors. Chin. J. Exp. Surg., 26(12):1607-1609.

[19]Yu, S.C., Ping, Y.F., Yi, L., Zhou, Z.H., Chen, J.H., Yao, X.H., Gao, L., Wang, J.M., Bian, X.W., 2008. Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87. Cancer Lett., 265(1):124-134.