Department of Obstetrics and Gynecology, Innsbruck Medical University, Innsbruck, Austria.
Neoplasma. 2012;59(6):747-55. doi: 10.4149/neo_2012_094.
Because of its semi-solid character in dissemination and growth, advanced ovarian cancer with its hundreds of peritoneal tumor nodules and plaques appears to be an excellent in vivo model for studying the cancer stem cell hypothesis. The most important obstacle, however, is to adequately define and isolate these tumor-initiating cells endowed with the properties of anoikis-resistance and unlimited self-renewal. Until now, no universal single marker or marker constellation has been found to faithfully isolate (ovarian) cancer stem cells. As these multipotent cells are known to possess highly elaborated efflux systems for cytotoxic agents, these pump systems have been exploited to outline putative stem cells as a side-population (SP) via dye exclusion analysis. Furthermore, the cells in question have been isolated via flow cytometry on the basis of cell surface markers thought to be characteristic for stem cells.In the Vienna variant of the ovarian cancer cell line A2780 a proof-of-principle model with both a stable SP and a stable ALDH1A1+ cell population was established. Double staining clearly revealed that both cell fractions were not identical. Of note, A2780V cells were negative for expression of surface markers CD44 and CD117 (c-kit). When cultured on monolayers of healthy human mesothelial cells, green-fluorescence-protein (GFP)-transfected SP of A2780V exhibited spheroid-formation, whereas non-side-population (NSP) developed a spare monolayer growing over the healthy mesothelium. Furthermore, A2780V SP was found to be partially resistant to platinum. However, this resistance could not be explained by over-expression of the "excision repair cross-complementation group 1" (ERCC1) gene, which is essentially involved in the repair of platinated DNA damage. ERCC1 was, nonetheless, over-expressed in A2780V cells grown as spheres under stem cell-selective conditions as compared to adherent monolayers cultured under differentiating conditions. The same was true for the primary ovarian cancer cells B-57.In summary our investigations indicate that even in multi-passaged cancer cell lines hierarchic government of growth and differentiation is conserved and that the key cancer stem cell population may be composed of small overlapping cell fractions defined by various arbitrary markers.
由于其在传播和生长过程中的半固态特性，具有数百个腹膜肿瘤结节和斑块的晚期卵巢癌似乎是研究癌症干细胞假说的极佳体内模型。然而，最重要的障碍是充分定义和分离这些具有抗失巢凋亡和无限自我更新能力的肿瘤起始细胞。到目前为止，尚未发现通用的单一标志物或标志物组合能够忠实地分离（卵巢）癌症干细胞。由于这些多能细胞已知具有高度精细的细胞毒性药物外排系统，因此这些泵系统已被用于通过染料排除分析来描绘假定的干细胞作为侧群（SP）。此外，这些有问题的细胞已通过基于被认为是干细胞特征的细胞表面标志物的流式细胞术进行分离。在卵巢癌细胞系 A2780 的维也纳变体中，建立了一个具有稳定 SP 和稳定 ALDH1A1+细胞群体的原理验证模型。双重染色清楚地表明，这两个细胞群并不完全相同。值得注意的是，A2780V 细胞对表面标志物 CD44 和 CD117（c-kit）的表达呈阴性。当在健康人间皮细胞单层上培养时，GFP 转染的 A2780V SP 表现出球体形成，而非侧群（NSP）在健康间皮上形成单层生长。此外，发现 A2780V SP 对铂具有部分抗性。然而，这种抗性不能用“切除修复交叉互补组 1”（ERCC1）基因的过度表达来解释，该基因主要参与铂化 DNA 损伤的修复。然而，与在分化条件下培养的贴壁单层相比，在干细胞选择性条件下生长的 A2780V 细胞球体中 ERCC1 过度表达。原发性卵巢癌细胞 B-57 也是如此。总之，我们的研究表明，即使在多代传代的癌细胞系中，生长和分化的层次结构也得以保留，并且关键的癌症干细胞群体可能由通过各种任意标志物定义的小重叠细胞群组成。
