title: Maintenance of retinal cancer stem cell-like properties through long-term serum-free culture from human retinoblastoma.
abstract: Previous studies have demonstrated that a small population of cancer stem cell-like cells exists in retinoblastoma. To provide a model for studying this population, we sought to establish a long-term culture from human retinoblastoma that have cancer stem cell-like properties. Fresh tumor tissue was digested and cultured in serum-free medium. Tumor spheres formed and were passaged continuously. Stem cell properties were examined through immunostaining, real-time quantitative RT-PCR and chemoresistance assay. Tumorigenicity of the tumor sphere-forming cells was confirmed by xenograft experiments. Furthermore, we examined the expression of cell surface markers CD44 and CD133. Tumor cells expanded as floating spheres for more than 30 passages. Sphere-forming cells overexpressed stem cell genes Oct‑4, Nestin and Pax6. Immunostaining of spheres showed positivity for Nestin, Pax6 and also ABCG2. In contrast, differentiated cells derived from these spheres expressed high levels of mature retinal cell markers MAP2, GFAP, recoverin, Opsin B and Nrl, and showed immunoreactivity for NF200, GFAP, recoverin and PKCα. Furthermore, both CD44 and CD133 were highly expressed in sphere-forming cells vs. differentiated cells. Sphere-forming cells displayed higher chemoresistance to carboplatin as opposed to differentiated cells. Moreover, intraocular injection of as few as 2x103 sphere-forming cells into NOD/SCID mice gave rise to new tumors similar to the original patient tumors. These results revealed that the sphere-forming cells preserved their stem cell properties and tumorigenicity, even after long-term culture. This would be a suitable in vitro model to study cancer stem-like cells in retinoblastoma and to develop chemotherapeutic drugs and strategies.
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Ma
authors.forename: Xia
authors.initials: X
authors.lastname: Lei
authors.forename: Yuan
authors.initials: Y
authors.lastname: Guan
authors.forename: Li-Sha
authors.initials: LS
authors.lastname: Mou
authors.forename: Yi-Fei
authors.initials: YF
authors.lastname: Yuan
authors.forename: Han
authors.initials: H
authors.lastname: Yue
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
authors.forename: Guo-Tong
authors.initials: GT
authors.lastname: Xu
authors.forename: Jiang
authors.initials: J
authors.lastname: Qian
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D016895:Culture Media, Serum-Free / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000166:cytology*; D010455:Peptides; D012175:Retinoblastoma / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; C497483:CD44 protein, human; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D006023:Glycoproteins; D018960:Hyaluronan Receptors; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse