Changes in Global Gene Expression Associated with 3D Structure of Tumors: an ex vivo Matrix-Free Mesothelioma Spheroid Model

Tumor microenvironments present significant barriers to anti-tumor agents. Molecules involved in multicellular tumor microenvironments, however, are difficult to study ex vivo. Here, we generated a matrix-free tumor spheroid model using the NCI-H226 mesothelioma cell line and compared the gene expression profiles of spheroids and monolayers using microarray analysis. Microarray analysis revealed that 142 probe sets were differentially expressed between tumor spheroids and monolayers. Gene ontology analysis revealed that upregulated genes were primarily related to immune response, wound response, lymphocyte stimulation and response to cytokine stimulation, whereas downregulated genes were primarily associated with apoptosis. Among the 142 genes, 27 are located in the membrane and related to biologic processes of cellular movement, cell-to-cell signaling, cellular growth and proliferation and morphology. Western blot analysis validated elevation of MMP2, BAFF/BLyS/TNFSF13B, RANTES/CCL5 and TNFAIP6/TSG-6 protein expression in spheroids as compared to monolayers. Thus, we have reported the first large scale comparison of the transcriptional profiles using an ex vivo matrix-free spheroid model to identify genes specific to the three-dimensional biological structure of tumors. The method described here can be used for gene expression profiling of tumors other than mesothelioma. We generated a matrix-free tumor spheroid model using the NCI-H226 mesothelioma cell line and compared the gene expression profiles of spheroids and monolayers using microarray analysis. Microarray analysis experiments were performed in triplicates. A total of six samples (three for monolayers and three for spheroids) were analyzed.肿瘤微环境会对抗肿瘤药物构成显著障碍。然而，涉及多细胞肿瘤微环境的分子难以在离体（ex vivo）环境中开展研究。本研究利用NCI-H226间皮瘤细胞系构建了无基质肿瘤球体模型，并通过微阵列（microarray）分析对比了球体与单层培养细胞的基因表达谱。微阵列分析结果显示，肿瘤球体与单层培养细胞间共有142个探针组存在差异表达。基因本体（Gene Ontology, GO）分析显示，上调基因主要与免疫应答、创伤应答、淋巴细胞刺激以及细胞因子刺激应答相关，而下调基因则主要与细胞凋亡相关。在这142个差异基因中，有27个定位于细胞膜，且参与细胞迁移、细胞间信号传导、细胞生长增殖以及细胞形态发生等生物学过程。蛋白质印迹（Western blot）分析证实，与单层培养细胞相比，肿瘤球体中MMP2、BAFF/BLyS/TNFSF13B、RANTES/CCL5以及TNFAIP6/TSG-6的蛋白表达水平显著升高。综上，本研究首次利用离体无基质肿瘤球体模型对转录组谱进行大规模对比分析，筛选出肿瘤三维生物学结构特异性相关基因。本研究所用方法可应用于间皮瘤以外的其他肿瘤的基因表达谱分析。本研究利用NCI-H226间皮瘤细胞系构建无基质肿瘤球体模型，并通过微阵列分析对比球体与单层培养细胞的基因表达谱。微阵列分析实验设置三次生物学重复，共分析6份样本（单层培养细胞与肿瘤球体各3份）。