Li Hai-zhi, Yi Tong-bo, Wu Zheng-yan
Department of General Surgery, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
BMC Cancer. 2008 May 14;8:135. doi: 10.1186/1471-2407-8-135.
Cancer stem cell (CSC) hypothesis has not been well demonstrated by the lack of the most convincing evidence concerning a single cell capable of giving rise to a tumor. The scarcity in quantity and improper approaches for isolation and purification of CSCs have become the major obstacles for great development in CSCs. Here we adopted suspension culture combined with anticancer regimens as a strategy for screening breast cancer stem cells (BrCSCs). BrCSCs could survive and be highly enriched in non-adherent suspension culture while chemotherapeutic agents could destroy most rapidly dividing cancer cells and spare relatively quiescent BrCSCs.
TM40D murine breast cancer cells were cultured in serum-free medium. The expression of CD44+CD24- was measured by flow cytometry. Cells of passage 10 were treated in combination with anticancer agents pacilitaxel and epirubicin at different peak plasma concentrations for 24 hours, and then maintained under suspension culture. The rate of apoptosis was examined by flow cytometry with Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining method. Selected cells in different amounts were injected subcutaneously into BALB/C mice to observe tumor formation.
Cells of passage 10 in suspension culture had the highest percentage of CD44+CD24- (about 77 percent). A single tumor cell in 0.35 PPC could generate tumors in 3 of 20 BALB/C mice.
Suspension culture combined with anticancer regimens provides an effective means of isolating, culturing and purifying BrCSCs.
癌症干细胞（CSC）假说尚未得到充分证实，因为缺乏关于单个细胞能够引发肿瘤的最有说服力的证据。CSC数量稀少以及分离和纯化方法不当已成为CSC研究取得重大进展的主要障碍。在此，我们采用悬浮培养结合抗癌方案作为筛选乳腺癌干细胞（BrCSC）的策略。BrCSC能够在非贴壁悬浮培养中存活并高度富集，而化疗药物可以破坏大多数快速分裂的癌细胞，使相对静止的BrCSC得以保留。
将TM40D小鼠乳腺癌细胞在无血清培养基中培养。通过流式细胞术检测CD44+CD24-的表达。将第10代细胞与不同血浆峰浓度的抗癌药物紫杉醇和表柔比星联合处理24小时，然后在悬浮培养条件下维持。采用膜联蛋白-V异硫氰酸荧光素（FITC）/碘化丙啶（PI）双染法通过流式细胞术检测细胞凋亡率。将不同数量的筛选细胞皮下注射到BALB/C小鼠体内观察肿瘤形成情况。
悬浮培养的第10代细胞中CD44+CD24-的百分比最高（约77%）。0.35血浆峰浓度下的单个肿瘤细胞可使20只BALB/C小鼠中的3只形成肿瘤。
悬浮培养结合抗癌方案为分离、培养和纯化BrCSC提供了一种有效的方法。
