Identification of Cancer Stem Cells and Its Clinical Application for Treating Aggressive Neuroblastomas

1. 神经母细胞瘤干细胞样标记基因的鉴定在斯隆-凯特琳癌症医院从神经母细胞瘤细胞系中亚克隆了三种具有特征表型的细胞系，N型、i型和s型细胞。通过使用我们的内部cDNA微阵列携带5300个cDNA，我们已经确定了多个基因特异性表达在i型细胞中，这些基因被认为非常接近神经母细胞瘤干细胞。肿瘤干细胞标志物在原代培养神经母细胞瘤中的表达为了寻找神经母细胞瘤干细胞，我们使用了34个原代培养神经母细胞瘤和6个神经母细胞瘤细胞系。大多数原发性神经母细胞瘤细胞形成球形，其中部分细胞持续BrdU标记阳性，提示神经母细胞瘤存在癌干细胞。然后，我们在原代培养细胞中测试了神经母细胞瘤干细胞的潜在标记物的意义。巢蛋白和TUJ1在有利肿瘤细胞中优先呈阳性，而在不利肿瘤细胞中则优先呈阳性。特别是，在一岁以下患者的肿瘤细胞中，巢蛋白的表达明显高。p75NTR的表达也表现出类似的模式，但其表达水平相对较低。有趣的是，p63和p73，即p53肿瘤抑制因子的变体，在许多患者中都有高表达。原代培养的神经母细胞瘤细胞与肿瘤分期无关。因此，它们可能是引发神经母细胞瘤发生的新的干细胞标记物。需要进一步研究它们的变体TA型和delta-N型的功能作用。

1. Identification of neuroblastoma stem-like cells marker genesThree cell lines with characteristic phenotype, N-, I-and S-type cells, were subcloned from the neuroblastoma cell lines at Sloan-kettering Cancer Hospital. By using our in-house cDNA microarray carrying 5300 cDNAs, we have identified more than several genes specifically expressed in I-type cells which are believed to be very close to the neuroblastoma stem cell.2. Expression of cancer stem cells markers in neuroblastomas in primary cultureTo search for the neuroblastoma stem cells, we used 34 neuroblastomas in primary culture as well as 6 neuroblastoma cell lines. Most of the primary neuroblastoma cells formed spheres, in which some cells were positive for sustained BrdU labeling, suggesting the presence of cancer stem cells of neuroblastoma. We then tested the significance of the potential markers of neuroblastoma stem cells in primary culture cells. Both nestin and TUJ1 were preferentially positive in favorable tumor cells as compared to unfavorable cells. Especially, expression of nestin was significantly high in tumor cells obtained from patients under one year of age. Expression of p75NTR also showed the similar pattern, but its expression levels were relatively low. Interestingly, both p63 and p73, the variants of p53 tumor suppressor, were highly expressed in many. neuroblastoma cells in primary culture independently of the tumor stages. Therefore, they could be new stem cell markers which might initiate the genesis of neuroblastoma. The further study about the functional roles of their variants, the TA form and the delta-N form, should be needed.