Objective Initiation,growth,recurrence,and metastasis of head and neck squamous cell carcinoma (HNSCC) have been related to the cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH-1) activity.In this study,we try to prove that suspension culture can enrich ALDH-1 high expression cells within HNSCC cell lines and the enriched cells possess cancer stem cell properties.Methods Cells from five HNSCC cell lines were cultured in ultra-low attachment plates in serum-free Quantum 263 medium supplemented with 10 ng/ml EGF and 10 ng/ml bFGF,and ALDH-1 expression level was evaluated by ALDEFLUOR assay.ALDH-1 high expression cells were separated by FACS sorting,and their phenotypical and functional properties were characterized.Results Spheroids can be formed from all five HNSCC cell lines (UD-SCC1,UT-SCC22,UM-SCC11B,UT-SCC9 and UTSCC24A) under anchorage independent culture condition.The proportion of ALDH1 high expression cells were highly increased in speroids derived cells (SDCs) compared with their monolayers (P ＜ 0.05).The clones formed by ALDH1 high expression cells on average contained 197 (197 ±47) cells compared with 33 (33 ± 16) cells in clones generated from ALDH1 low expression cells (P ＜ 0.01).Single ALDH1 high expression cell could significantly better regenerate a spheroid (UT-SCC9:17.1％,UD-SCC1:19.3％),whereas under the same conditions single ALDH1 low expression cells regenerated only in one case a spheroid (P ＜ 0.01).SDCs from all five tested cell lines also showed a significantly increased invasion capacity (P ＜ 0.05).We also found that the mRNA levels of Oct-4,Sox2,and Nanog were all significantly increased in the SDC.The reactive oxygen species (ROS) levels in SDCs from UD-SCC1 and UT-SCC24A were significantly increased compared with their monolayer counterpart [(26.3 ± 4.9) ％ vs (8.6 ± 1.7) ％ and (72.1 ± 6.1) ％ vs (23.7 ± 7.5) ％,P ＜ 0.05)].Conclusion Cancer stem cells can be enriched by suspension culture,which may be of importance in investigation of their contribution to therapy resistance,tumor recurrence and metastasis.%目的 探讨通过悬浮培养法富集乙醛脱氢酶1(ALDH-1)高表达的头颈部鳞癌肿瘤细胞的可行性,鉴定富集的细胞是否具有肿瘤干细胞特性.方法 将头颈肿瘤细胞株接种于低黏附平板,在10 ng/ml重组表皮生长因子和10 ng/ml碱性成纤维细胞生长因子的无血清培养基中形成细胞微球,通过ALDEFLUOR方法对ALDH-1的表达水平进行半定量分析,采用流式细胞术分选出ALDH-1高表达细胞,分析其生物学特性.结果 UD-SCC1、UT-SCC22、UM-SCC11B、UT-SCC9和UT-SCC24A细胞在无血清培养液中悬浮培养5～1Od后均能形成细胞微球.在UD-SCC1、UT-SCC22、UM-SCC11B、UT-SCC9和UT-SCC24A细胞株细胞微球来源的细胞(SDC)中,ALDH-1的表达水平均高于其贴壁生长细胞(均P＜0.05).ALDH-1高表达和低表达细胞形成的克隆细胞数分别为(197±47)个和(33±16)个(P＜0.01).单个ALDH-1高表达和低表达UT-SCC9细胞的克隆形成率分别为17.1％和0.7％,差异有统计学意义(P＜0.05);单个ALDH-1高表达和低表达UD-SCC1细胞的克隆形成率分别为19.3％和0,差异有统计学意义(P＜0.01).UD-SCC1、UT-SCC22、UM-SCC11B、UT-SCC9和UT-SCC24A细胞的SDC侵袭力均高于其贴壁细胞(均P＜0.05).在UD-SCC1、UT-SCC22、UM-SCC11B、UT-SCC9和UT-SCC24A细胞的SDC中,Nanog、Sox2和Oct-4 mRNA的表达水平均高于其贴壁细胞(均P＜0.05).UD-SCC1和UT-SCC24A细胞的SDC中,低活性氧簇(ROS)水平细胞比例分别为(26.3±4.9)％和(72.1±6.1)％,均高于UD-SCC1和UT-SCC24A细胞的贴壁细胞中低ROS水平细胞比例[分别为(8.6±1.7)％和(23.7±7.5)％,均P＜0.05].结论 悬浮培养法可有效富集头颈部鳞癌细胞系内的ALDH-1高表达细胞,富集后的细胞具有肿瘤干细胞特性.
展开▼
