Liu Qiu-Xia, Tang Jing-Yan, Cai Jiao-Yang, Yin Min-Zhi, Li Ben-Shang
Department of Hematology/Oncology, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai 200127, P. R. China.
Chin J Cancer. 2010 Dec;29(12):1012-7. doi: 10.5732/cjc.010.10319.
Since the proposal of the tumor stem cell hypothesis, considerable interest has been devoted to the isolation and purification of tumor stem cells. Tumor stem cell enrichment from primary tumor derived cell spheres has been demonstrated in specific, serum-free media. This goal of this study is to establish a method of cultivating floating tumor spheres from neuroblastoma cells and to confirm that neuroblastoma spheres are rich in tumor stem cells.
Bone marrow aspirates were obtained from pediatric patients diagnosed with stage IV neuroblastoma. Primary tumor cells were isolated and cultivated in serum-free, stem cell-selective medium. Single sphere-forming cells were cultivated under serum-free conditions; their cloning efficiency and monoclonal tumor sphere formation rates were calculated. The expression of stem cell marker genes Oct-4 and Bmi-1 was detected by RT-PCR in sphere-forming cells and parental neurolastoma cells. Sphere-forming cells were injected into the armpit of nude mice with subsequent assessment for tumor growth. Sphere-forming cells were cultivated in differentiation medium containing 5 μmol/L 13-cis retinoic acid; changes in cell morphology were observed.
Neuroblastoma cells formed non-adherent neurospheres under serum-free, stem cell-selective conditions after a period of 4 to 6 days. A single cell dissociated from a neurosphere could reform a monoclonal sphere; cloning efficiency and monoclonal sphere formation rates were 55.3% and 26.3%, respectively. RT-PCR results revealed heightened tumor sphere expression of Oct-4 and Bmi-1 as compared with parental tumor cells. Fourteen days after injection of 10(4) sphere-forming cells into nude mice, a neuroblastoma xenograft formed. Treatment of sphere-forming cells with 13-cis retinoic acid induced a gradual differentiation to neuronal cell morphology.
Neuroblastoma derived tumor spheres enrich tumor stem cells and the cultivation of primary neuroblastoma cells in serum-free, stem cell-selective medium is an effective method to dissociate and purify tumor stem cells in vitro.
自肿瘤干细胞假说提出以来，人们对肿瘤干细胞的分离和纯化投入了大量关注。在特定的无血清培养基中已证实可从原发性肿瘤来源的细胞球中富集肿瘤干细胞。本研究的目的是建立一种从神经母细胞瘤细胞中培养悬浮肿瘤球的方法，并证实神经母细胞瘤球富含肿瘤干细胞。
从诊断为IV期神经母细胞瘤的儿科患者中获取骨髓抽吸物。分离原发性肿瘤细胞并在无血清、干细胞选择性培养基中培养。将单个成球细胞在无血清条件下培养；计算其克隆效率和单克隆肿瘤球形成率。通过RT-PCR检测成球细胞和亲代神经母细胞瘤细胞中干细胞标记基因Oct-4和Bmi-1的表达。将成球细胞注射到裸鼠腋窝，随后评估肿瘤生长情况。将成球细胞在含有5μmol/L 13-顺式维甲酸的分化培养基中培养；观察细胞形态变化。
神经母细胞瘤细胞在无血清、干细胞选择性条件下培养4至6天后形成非贴壁神经球。从神经球中分离出的单个细胞可重新形成单克隆球；克隆效率和单克隆球形成率分别为55.3%和26.3%。RT-PCR结果显示，与亲代肿瘤细胞相比，肿瘤球中Oct-4和Bmi-1的表达升高。将10⁴个成球细胞注射到裸鼠体内14天后，形成了神经母细胞瘤异种移植瘤。用13-顺式维甲酸处理成球细胞可诱导其逐渐分化为神经元细胞形态。
神经母细胞瘤来源的肿瘤球富集肿瘤干细胞，在无血清、干细胞选择性培养基中培养原发性神经母细胞瘤细胞是体外分离和纯化肿瘤干细胞的有效方法。
