Department of Otolaryngology, Head and Neck Surgery, Charité-Universitätsmedizin Berlin, Berlin, Germany.
PLoS One. 2011 Jan 27;6(1):e16466. doi: 10.1371/journal.pone.0016466.
Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinomas (HNSCC) have been related to the behavior of cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH1) activity. We quantified and enriched ALDH1(+) cells within HNSCC cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). Spheroid culture enriched CSC from five HNSCC cell lines by up to 5-fold. In spheroid-derived cells (SDC) and the parental monolayer-derived cell line ALDH1, CD44, CD24, E-Cadherin, α-SMA, and Vimentin expression was compared by flow-cytometry and immunofluorescence together with proliferation and cell cycle analysis. Invasion activity was evaluated by Matrigel assay and expression of stemness-related transcription factors (TF) Nanog, Oct3/4, Sox2 and EMT-related genes Snail1 and 2, and Twist by real-time PCR. All cell lines formed spheroids that could self-renew and be serially re-passaged. ALDH1 expression was significantly higher in SDC. ALDH1(+) cells showed increased colony-formation. The proportion of cells with a putative CSC marker constellation of CD44(+)/CD24(-) was highly variable (0.5% to 96%) in monolayer and spheroid cultures and overlapped in 0%-33% with the CD44(+)/CD24(-)/ALDH1(+) cell subset. SDC had significantly higher invading activity. mRNA of the stemness-related genes Sox2, Nanog, and Oct3/4 was significantly increased in SDC of all cell lines. Twist was significantly increased in two while Snail2 showed a significant increase in one and a significant decrease in SDC of two cell lines. SDC had a higher G0 phase proportion, showed high-level expression of α-SMA and Vimentin, but significantly decreased E-Cadherin expression. HNSCC-lines harbor potential CSC, characterized by ALDH1 and stemness marker TF expression as well as properties like invasiveness, quiescence, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.
头颈部鳞状细胞癌（HNSCC）的起始、生长、复发和转移与癌症干细胞（CSC）的行为有关，CSC 可以通过其乙醛脱氢酶同工酶-1（ALDH1）活性来识别。我们定量并富集了 HNSCC 细胞系中的 ALDH1(+)细胞，随后对其侵袭能力和上皮-间充质转化（EMT）等表型和功能特性进行了表征。在 5 种 HNSCC 细胞系的球体培养中，CSC 的富集程度高达 5 倍。在球体衍生细胞（SDC）和母代单层衍生细胞系 ALDH1 中，通过流式细胞术和免疫荧光比较 CD44、CD24、E-钙粘蛋白、α-SMA 和波形蛋白的表达，并结合增殖和细胞周期分析。通过 Matrigel 测定评估侵袭活性，并通过实时 PCR 评估与干细胞相关的转录因子（TF）Nanog、Oct3/4、Sox2 和 EMT 相关基因 Snail1 和 2 以及 Twist 的表达。所有细胞系均形成可自我更新并可连续再传代的球体。SDC 中 ALDH1 的表达显著升高。ALDH1(+)细胞显示出更高的集落形成能力。在单层和球体培养中，具有假定 CSC 标志物组合 CD44(+)/CD24(-)的细胞比例高度可变（0.5%至 96%），并且在 0%-33%与 CD44(+)/CD24(-)/ALDH1(+)细胞亚群重叠。SDC 的侵袭活性显著升高。所有细胞系的 SDC 中与干细胞相关的基因 Sox2、Nanog 和 Oct3/4 的 mRNA 均显著增加。在两个细胞系中 Twist 显著增加，而在 SDC 中 Snail2 的表达在一个细胞系中显著增加，在两个细胞系中显著降低。SDC 具有更高的 G0 期比例，高水平表达 α-SMA 和波形蛋白，但 E-钙粘蛋白表达显著降低。HNSCC 细胞系中存在潜在的 CSC，其特征是 ALDH1 和干细胞标志物 TF 的表达以及侵袭性、静止和 EMT 等特性。可以通过非锚定依赖性培养技术来富集 CSC，这对于研究它们对治疗耐药性、肿瘤复发和转移的贡献可能很重要。
