title: Preliminary screening and identification of stem cell-like sphere clones in a gallbladder cancer cell line GBC-SD.
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 1×10(3) sphere clone cells could induce much larger tumors in nude mice than 1×10(5) 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.
authors.affiliation: Department of General Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China. yinbaobing@hotmail.com
authors.forename: Bao-bing
authors.initials: BB
authors.lastname: Yin
authors.forename: Shuang-jie
authors.initials: SJ
authors.lastname: Wu
authors.forename: Hua-jie
authors.initials: HJ
authors.lastname: Zong
authors.forename: Bao-jin
authors.initials: BJ
authors.lastname: Ma
authors.forename: Duan
authors.initials: D
authors.lastname: Cai
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D051927:CD24 Antigen / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005706:Gallbladder Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D020869:Gene Expression Profiling; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D018396:Mucin-1 / Q000235:genetics; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics; D018874:Spheroids, Cellular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D014183:Transplantation, Heterologous; D023041:Xenograft Model Antitumor Assays
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D051927:CD24 Antigen; C497889:CD24 protein, human; C497483:CD44 protein, human; D006023:Glycoproteins; D018960:Hyaluronan Receptors; C060192:MUC1 protein, human; D018396:Mucin-1; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D002945:Cisplatin