Dynamic analyses of interactions between leukemic stem cells and hemopoietic stromal cells visualized by molecular imaging technique, and their possible pharmaceutical applications.

通过分子成像技术可视化白血病干细胞和造血基质细胞之间相互作用的动态分析及其可能的药物应用。

• 批准号: 17209020
• 负责人: ASANO Shigetaka
• 金额: $ 31.37万
• 依托单位: Waseda University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17209020/
• 关键词: cobblestone area; leukemic stem cells; stromal cells; self renewal; niche; repopulating assay; drug resistance; bioimaging; 細胞間相互作用; 幹細胞; 悪性腫瘍; 分子イメージング; 蛍光標識

A cobblestone area (CA) formation is generally regarded as one and only way to maintain normal pluripotent hemopoietic stem cells for long term in vitro, and therefore considered to mimic normal constitutive hematopoiesis in vivo. We have applied this culture system as a model of leukemia development involving maintenance and persistence of leukemic stem cells after various therapies, and optimized it by using MS5 as a supportive stroma, and HEL and TF-1 as model leukemic cell lines. Although the both leukemic cell lines derived from the same classification of erythroleukemia, they differ considerably each other, i.e., essentially every HEL cells seemed to maintain ability of CA formation, whereas only 10% of TF-1 population revealed CA formation, suggesting the presence of hierarchy along the differentiation in the latter cell line. Expression of some differentiation antigens such as Glycophorin-A and CD42b was diminished in the cells forming CA compared with original suspension cultu … More re of both cell lines, conversely, expression of CD44 which has recently been reported to be required for the homing of leukemic stem cells to bone marrow niche, was elevated in CA forming cells relative to the original suspension culture. Furthermore, the frequency of self renewal increased upon CA formation, which was evident from the result of repopulating assay, in contrast to the elevation of resting cell proportion demonstrated by flow cytometry. These results indicate that the CA forming cells may retain the properties of leukemic stem cells. Those CA forming cells were also characterized by greatly reduced sensitivity to various chemotherapeutic agents, in comparison to suspension cultures of the cognate cells. Particularly, Daunorubicin, a frequently prescribed anti-leukemic agent whose fluorescent property can be utilized to visualize its intracellular localization, showed distinct accumulation into the compartment corresponding to lysosome of the CA forming cells, and this was entirely different from the subcellular distribution observed in the drug-sensitive culture of the same cells. In summary, leukemic cells acquire drug resistance through the formation of CA, and one of the mechanisms behind this acquisition may involve alterations in intracellular transport and/or metabolism of the drugs, rather than mere physical prevention of the drug penetration into hemopoietic niche where leukemic stem cells reside. The currently described system of heterologous CA formation using murine MS5 stromal cells was considered to be useful particularly as an evaluating system for desirable drugs in the forthcoming era of personalized medicine. Less

鹅卵石区（CA）的形成通常被认为是在体外长期维持正常多能造血干细胞的唯一途径，因此被认为是在体内模拟正常的组成性造血。我们将该培养系统作为白血病发展的模型，包括白血病干细胞在各种治疗后的维持和持久性，并通过使用MS5作为支持基质，HEL和TF-1作为模型白血病细胞系对其进行优化。虽然这两种白血病细胞系来源于相同的红白血病分类，但它们彼此之间存在很大差异，即基本上每个HEL细胞似乎都保持CA形成的能力，而只有10%的TF-1群体显示CA形成，这表明后一种细胞系在分化过程中存在等级。与原始悬浮培养相比，形成CA的细胞中某些分化抗原如糖蛋白a和CD42b的表达减少，相反，最近报道的白血病干细胞归巢到骨髓生态位所需的CD44的表达在形成CA的细胞中相对于原始悬浮培养提高。此外，自我更新的频率随着CA的形成而增加，从重新填充实验的结果可以看出，与流式细胞术显示的静止细胞比例升高相反。这些结果表明CA形成细胞可能保留了白血病干细胞的特性。与悬浮液培养的同源细胞相比，这些CA形成细胞对各种化疗药物的敏感性也大大降低。特别是，柔红霉素，一种常用的抗白血病药物，其荧光特性可以用来观察其细胞内定位，显示出明显的积聚到CA形成细胞的溶酶体对应的腔室中，这与在药物敏感培养中观察到的亚细胞分布完全不同。总之，白血病细胞通过CA的形成获得耐药性，这种获得背后的机制之一可能涉及药物在细胞内运输和/或代谢的改变，而不仅仅是物理上防止药物渗透到白血病干细胞所在的造血生态位。目前描述的使用小鼠MS5基质细胞形成异源CA的系统被认为是有用的，特别是作为即将到来的个性化医疗时代所需药物的评估系统。少