evaluation of tumor malignancy based on expression analysis of genome instability factors

我们利用胶质母细胞瘤源性U87MG细胞在神经干细胞培养基中进行球形培养，建立了肿瘤干细胞细胞系U87CS。U87CS细胞在裸鼠脑内移植后显示出高致瘤性，CD133免疫组化染色呈阳性，CD133是白血病和胶质母细胞瘤癌症干细胞亚群的标记物。U87CS细胞端粒酶明显高于原U87MG细胞。使用G2/S细胞分裂抑制剂ICRF193， U87CS细胞的十烷化检查点活性比U87MG细胞增加。与U87MG细胞相比，U87CS细胞中CD133和MDR (multidrug resistance， MDR) 1基因的表达量平均分别增加了47.18倍和8.51倍。U87CS细胞对多柔比星（Dox）、依托泊苷（VP-16）、BCNU等常规抗癌药物的耐药性较U87MG细胞强。单色流式细胞分析显示，U87CS细胞对Dox含量的摄取低于U87MG细胞，而ABC转运蛋白抑制剂维拉帕米增加了U87MG和U87CS细胞对Dox含量的摄取。双免疫荧光染色显示CD133和MDR1在裸鼠脑移植的U87CS细胞上共表达。此外，我们在手术切除和放化疗后复发的胶质母细胞瘤手术标本中发现了CD133和MDR1的交叉反应性。这些结果表明，CS细胞可能对包括抗癌药物在内的细胞损伤条件具有抗性，并代表了胶质母细胞瘤治疗的新靶点。

We established a cancer stem cell line, U87CS, by means of spheroid culture of U87MG cells derived from glioblastoma in neuronal stem cell medium. U87CS cells presented high tumorigenicity after intracranial transplantation into nude mice brains and positive immunohistochemical staining of CD133, a marker for a subset of leukemia and glioblastoma cancer stem cells. Telomerase of U87CS cells was higher than original U87MG cells. With ICRF193, an inhibitor of G2/S cell division, decatenation checkpoint activity increased in U87CS cells more than U87MG cells. The gene expression of CD133 and multidrug resistance (MDR) 1 on U87CS cells increased an average of 47.18 and 8.51 times, respectively, compared to the level on U87MG cells by Real time quantitative RT-PCR. U87CS cells possessed stronger drug-resistance to conventional anti-cancer drugs, such as Doxorubicin (Dox), etoposide (VP-16), and BCNU, than U87MG cells. Single-color flow cytometric analysis showed the uptake of Dox content into U87CS cells were lower than U87MGcells and verapamil, an inhibitor of ABC transporters, increased the uptake on both of U87MG and U87CS cells. Double immunofluoresence staining showed co-expression of CD133 and MDR1 on U87CS cells transplanted into nude mice brains. Furthermore, we identified the crossreactivity of CD133 and MDR1 in surgical specimens of recurrent glioblastomas after surgical removal and radiochemotherapy. These results suggest that CS cells may be resistant to cell damage conditions including anti-cancer drugs and represent a novel target for glioblastoma therapeutics.