Establishment and characterization of multicellular spheroids from a human glioma cell line; Implications for tumor therapy.
Establishment and characterization of multicellular spheroids from a human glioma cell line; Implications for tumor therapy.
复制标题
来自人神经胶质瘤细胞系的多细胞球体的建立和表征；对肿瘤治疗的影响。
DOI：
10.1186/1479-5876-4-12
复制
发表时间：
2006-03-02
影响因子：
7.4
通讯作者：
Dwarakanath, BS
中科院分区：
文献类型：
--
作者：
Khaitan, D;Chandna, S;Arya, MB;Dwarakanath, BS
多细胞球体是一种用于模拟 3D 肿瘤微环境的合适体外系统，可用于评估和预测肿瘤对包括代谢抑制剂在内的治疗剂的反应。然而，为了以最佳精度使用这种独特的模型，需要详细了解细胞亚群对潜在治疗剂/策略的性质、分布和敏感性/反应。球体特征也可能因所用细胞系的来源和类型而有很大差异，并且活的和解离的神经胶质瘤细胞球体的彻底表征尚不完全清楚。为了评估神经胶质瘤对各种治疗策略的体内反应，特别是能够穿透血脑屏障的代谢抑制剂，我们使用显微镜、流式细胞术和酶测定对人神经胶质瘤细胞系（BMG-1）的持续生长球体的组织、生长、活力、细胞存活、细胞死亡、代谢和线粒体状态、氧化应激和辐射反应进行了表征。每天用新鲜培养基喂养球体，以维持外细胞层的营养供应，同时在扩大的球体的最内层细胞中出现缺氧/坏死。每天添加新鲜培养基的球体体积在 7-28 天的生长过程中通过三次群体倍增呈指数增长。 G1 期细胞的比例 (~60%) 高于指数生长的单层细胞 (~48%)。随着球体年龄的增加，很大一部分 S 期细胞变得代谢不活跃（脱离 DNA 合成），这与静态单层培养物不同，其中 S 期细胞的比例低于 5%。随着球体尺寸的增加，越来越多的细胞亚群变得无法生存，并进入细胞凋亡或坏死（Annexin-V-FITC/PI 染色显示）。 PI阳性（坏死）细胞并不局限于球体的中心，而是分布在某些离散的病灶处。与单层细胞相比，活球状细胞的平均葡萄糖消耗和乳酸产量高出 2-3 倍，这意味着糖酵解可能由于缺氧环境而出现补偿性增加。 HIF-1α 仅在球状体中表达，并以年龄依赖性方式增加，而 c-Myc（已知在葡萄糖剥夺的细胞中诱导细胞凋亡）水平比单层细胞高三倍。线粒体质量和活性在生长的前 14 天显着下降，但随着年龄的增长而增加，并且与 ROS 水平的增加无关。 Bcl-2 和 Bax 水平比单层细胞高（约 2 倍），而比率 (Bcl/Bax) 保持不变。与单层细胞相比，球状体中辐射诱导的氧化应激要少得多，并且与克隆形成试验证明的放射抗性的增加很好地对应，类似于在肿瘤中观察到的缺氧诱导的放射抗性。在球状体中观察到的 S 阴性细胞的发育、内源性和辐射诱导的 ROS 减少以及较高水平的抗 (Bcl2) 和亲 (Bax) 凋亡调节因子表明肿瘤中可能存在的内源性以及诱导的应激抵抗的复杂性/复杂性，这有助于治疗抵抗。
Multicellular spheroids, an appropriate in vitro system for simulating 3-D tumor micro-milieu can be used for evaluating and predicting tumor response to therapeutic agents including metabolic inhibitors. However, detailed understanding of the nature, distribution and sensitivity/responses of cellular sub-populations to potential therapeutic agents/strategies is required for using this unique model with optimal precision. Spheroid characteristics may also vary considerably with the origin and type of cell line used, and thorough characterization of viable and dissociated glioma cell spheroids is not yet completely known. In order to evaluate in vivo responses of gliomas to various therapeutic strategies, especially the metabolic inhibitors capable of penetrating the blood brain barrier, we have characterized continuously growing spheroids of a human glioma cell line (BMG-1) with respect to organization, growth, viability, cell survival, cell death, metabolic and mitochondrial status, oxidative stress and radiation response using microscopy, flow cytometry and enzymatic assays. Spheroids were fed daily with fresh medium in order to maintain nutrient supply to outer cellular layers while hypoxia/necrosis developed in the innermost cells of enlarging spheroids. Volume of spheroids, fed daily with fresh medium, increased exponentially during 7–28 days of growth through three population doublings. Proportion of G1-phase cells was higher (~60%) than exponentially growing monolayer cells (~48%). A significant fraction of S-phase cells turned metabolically inactive (disengaged in DNA synthesis) with increasing age of the spheroids, unlike in quiescent monolayer cultures, where the fraction of S-phase cells was less than 5%. With increasing spheroid size, increasing sub-populations of cells became non-viable and entered apoptosis or necrosis revealed by Annexin-V-FITC/PI staining. PI positive (necrotic) cells were not confined to the centre of the spheroid, but distributed at certain discrete foci. Average glucose consumption and lactate production were 2–3 folds higher in viable spheroid cells compared to monolayer cells, implying a compensatory increase in glycolysis possibly due to hypoxic environment. HIF-1α was expressed only in spheroids and increased in an age-dependent manner, whereas c-Myc (known to induce apoptosis in glucose-deprived cells) levels were three times higher than monolayer cells. Mitochondrial mass and activity decreased significantly during first 14 days of growth but increased with age, and were not associated with increase in ROS levels. Bcl-2 and Bax levels were higher (~2 folds) than monolayers, while the ratio (Bcl/Bax) remained unaltered. Radiation-induced oxidative stress was considerably less in spheroids as compared to monolayers, and corresponded well with increase in radioresistance demonstrated by the clonogenic assay, similar to hypoxia induced radioresistance observed in tumors. Development of S-negative cells and reduced endogenous and radiation-induced ROS coupled with higher levels of anti (Bcl2) as well as pro (Bax) apoptotic regulators observed in spheroids suggest the intricate/complex nature of endogenous as well as induced stress resistance that could exist in tumors, which contribute to the treatment resistance.
登录
查看更多内容
影响因子：
6.2
作者：
Kunkel, M;Reichert, TE;Whiteside, TL
通讯作者：
Whiteside, TL
DOI：
10.1073/pnas.78.4.2383
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
DARZYNKIEWICZ, Z;STAIANOCOICO, L;MELAMED, MR
通讯作者：
MELAMED, MR
DOI：
10.1016/s0360-3016(01)01534-6
发表时间：
2001-07-15
影响因子：
7
作者：
Dwarkanath, BS;Zolzer, F;Jain, V
通讯作者：
Jain, V
影响因子：
5.6
作者：
CASCIARI, JJ;SOTIRCHOS, SV;SUTHERLAND, RM
通讯作者：
SUTHERLAND, RM
影响因子：
8
作者：
Guenal, I;SidotideFraisse, C;Mignotte, B
通讯作者：
Mignotte, B