Ghosh Sourabh, Spagnoli Giulio C, Martin Ivan, Ploegert Sabine, Demougin Philippe, Heberer Michael, Reschner Anca
Institut für Chirurgische Forschung und Spitalmanagement (ICFS) and Departement Forschung, University of Basel, Basel, Switzerland.
J Cell Physiol. 2005 Aug;204(2):522-31. doi: 10.1002/jcp.20320.
Growth in three-dimensional (3D) architectures has been suggested to play an important role in tumor expansion and in the resistance of cancers to treatment with drugs or cytokines or irradiation. To obtain an insight into underlying molecular mechanisms, we addressed gene expression profiles of NA8 melanoma cells cultured in bidimensional monolayers (2D) or in 3D multicellular tumor spheroids (MCTS). MCTS containing 10-30,000 cells were generated upon overnight culture in poly-Hydroxyethylmethacrylate (polyHEMA) coated plates. Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long-term culture (>10 days), however, MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with >/=threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, >/=threefold, upregulation of the expression of >100 transcripts while 73 were >/=threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO-alpha, -beta, and gamma), IL-8, CCL20 (MIP-3alpha), and Angiopoietin-like 4 were significantly upregulated, whereas basic FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real-time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process.
三维（3D）结构中的生长被认为在肿瘤扩展以及癌症对药物、细胞因子或辐射治疗的抗性中发挥重要作用。为深入了解潜在的分子机制，我们研究了在二维单层（2D）或三维多细胞肿瘤球体（MCTS）中培养的NA8黑色素瘤细胞的基因表达谱。在聚甲基丙烯酸羟乙酯（polyHEMA）包被的平板中过夜培养后，生成了含有10 - 30,000个细胞的MCTS。MCTS中的细胞增殖动力学明显慢于单层培养。然而，长期培养（>10天）后，MCTS在外层显示出高度紧凑且有序的细胞生长，并有坏死核心。对在标准2D条件下、存在胶原蛋白作为细胞外基质（ECM）模型的情况下或在MCTS中培养的细胞进行了超过20,000个基因表达的寡核苷酸微阵列分析。在有或没有ECM的情况下在2D中培养的细胞的基因表达谱高度相似，大于或等于三倍的差异仅限于五个基因。相比之下，在MCTS中培养导致超过100个转录本的表达显著上调（大于或等于三倍），而73个转录本被下调（大于或等于三倍）。特别是，编码CXCL1、2和3（GRO-α、-β和-γ）、IL-8、CCL20（MIP-3α）和血管生成素样4的基因显著上调，而编码碱性FGF和CD49d的基因显著下调。寡核苷酸芯片数据通过定量实时PCR、ELISA和细胞表面染色试验在基因和蛋白质水平上得到验证。综上所述，我们的数据表明肿瘤细胞培养结构的改变导致许多先前显示在黑色素瘤进展和转移过程中起作用的基因的表达显著上调。
