Research Institute on Signaling, Developmental Biology and Cancer, Centre A. Lacassagne, Nice, France Genetic, Immunology and Human Pathology Laboratory, Tunis Science Faculty, University El Manar, Tunis, Tunisia.
Bosn J Basic Med Sci. 2018 Nov 7;18(4):336-346. doi: 10.17305/bjbms.2018.3243.
Tumor microenvironment provides a specialized niche in which a population of stem-like cells is enriched and contributes to cancer progression. Moreover, cancer stem cell (CSC) phenotype has been associated with epithelial-mesenchymal transition (EMT). Here we investigated the effect of tumor microenvironment on the phenotypic characteristics of head and neck cancer cells and expression of CSC markers using a three-dimensional (3D), spheroid, culture system of CAL33 cell line from human tongue squamous cell carcinoma. CAL33 cells derived from 2D monolayer cultures were grown in spheroid cultures containing serum-free medium (epidermal growth factor [EGF], fibroblast growth factor [FGF], and insulin). Adherent CAL33 cells from spheroids or standard control cultures were grown in the presence/absence of serum in combination with hypoxia/normoxia. Markers of EMT, CSC, and hypoxia were analyzed either by Western blotting, immunofluorescence, or reverse transcription quantitative PCR. Spheroid cultures showed hypoxic microenvironment (high carbonic anhydrase IX [CAIX] expression), mesenchymal-like characteristics (reduced E-cadherin and increased vimentin and N-cadherin expression, presence of larger colonies comprised of larger, spread cells with lower density), and increased expression of the CSC marker glioma-associated oncogene homolog 1 (Gli1). These effects were recapitulated in serum-free adherent CAL33 cells maintained for prolonged periods in hypoxia (1% O2) but, in contrast, were completely abolished by the presence of serum. Overall, we found that a combination of hypoxia, EGF and FGF was essential to induce the EMT in adherent CAL33 cell cultures. The addition of serum rapidly reverts the EMT of cells, affects CSC phenotype and, thus, prevents the detection of such cells in tumor cell lines.
肿瘤微环境提供了一个特化的小生境，其中富集了一群具有干细胞样特性的细胞，并促进癌症的进展。此外，癌症干细胞（CSC）表型与上皮-间充质转化（EMT）有关。在这里，我们使用人舌鳞状细胞癌 CAL33 细胞系的三维（3D）球体培养系统，研究了肿瘤微环境对口腔癌细胞表型特征和 CSC 标志物表达的影响。CAL33 细胞源自 2D 单层培养，在含有无血清培养基（表皮生长因子[EGF]、成纤维细胞生长因子[FGF]和胰岛素）的球体培养中生长。来自球体或标准对照培养物的贴壁 CAL33 细胞在存在/不存在血清的情况下，在缺氧/常氧条件下生长。通过 Western blot、免疫荧光或逆转录定量 PCR 分析 EMT、CSC 和缺氧标志物。球体培养显示出低氧微环境（碳酸酐酶 IX [CAIX]高表达）、间充质样特征（E-钙粘蛋白减少，波形蛋白和 N-钙粘蛋白增加，存在由较大、展开、密度较低的细胞组成的较大菌落），并且 CSC 标志物神经胶质瘤相关癌基因同源物 1（Gli1）的表达增加。这些效应在无血清贴壁 CAL33 细胞中被重现，这些细胞在低氧（1%O2）下被维持较长时间，但与之相反，血清的存在完全消除了这些效应。总的来说，我们发现低氧、EGF 和 FGF 的组合对于诱导贴壁 CAL33 细胞培养中的 EMT 是必不可少的。血清的添加迅速逆转了细胞的 EMT，影响了 CSC 表型，从而阻止了在肿瘤细胞系中检测到此类细胞。
