Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, PR China.
Oncol Rep. 2011 Jul;26(1):135-43. doi: 10.3892/or.2011.1291. Epub 2011 Apr 29.
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.
先前的研究已经证明，在视网膜母细胞瘤中存在一小部分癌症干细胞样细胞。为了提供研究该群体的模型，我们试图从具有癌症干细胞样特性的人视网膜母细胞瘤中建立长期培养物。将新鲜的肿瘤组织消化并在无血清培养基中培养。肿瘤球体形成并连续传代。通过免疫染色、实时定量 RT-PCR 和化学抗性测定来检查干细胞特性。通过异种移植实验证实了肿瘤球形成细胞的致瘤性。此外，我们还检查了细胞表面标志物 CD44 和 CD133 的表达。肿瘤细胞在悬浮球中扩增超过 30 代。球体形成细胞过度表达干细胞基因 Oct-4、Nestin 和 Pax6。球体的免疫染色显示 Nestin、Pax6 和 ABCG2 呈阳性。相比之下，源自这些球体的分化细胞表达高水平的成熟视网膜细胞标志物 MAP2、GFAP、恢复蛋白、Opsin B 和 Nrl，并对 NF200、GFAP、恢复蛋白和 PKCα 表现出免疫反应性。此外，与分化细胞相比，球体形成细胞中 CD44 和 CD133 的表达水平更高。与分化细胞相比，球体形成细胞对卡铂的化学抗性更高。此外，向 NOD/SCID 小鼠眼内注射仅 2x103 个球体形成细胞就会产生与原始患者肿瘤相似的新肿瘤。这些结果表明，球体形成细胞在长期培养后仍然保持其干细胞特性和致瘤性。这将是一种合适的体外模型，可用于研究视网膜母细胞瘤中的癌症干细胞样细胞，并开发化疗药物和策略。
