Horman Shane R, To Jeremy, Orth Anthony P
1Genomics Department, Genomics Institute of the Novartis Research Foundation (GNF), San Diego, CA, USA.
J Biomol Screen. 2013 Dec;18(10):1298-308. doi: 10.1177/1087057113499405. Epub 2013 Aug 5.
There has been increasing interest in the development of cellular behavior models that take advantage of three-dimensional (3D) cell culture. To enable assessment of differential perturbagen impacts on cell growth in 2D and 3D, we have miniaturized and adapted for high-throughput screening (HTS) the soft agar colony formation assay, employing a laser-scanning cytometer to image and quantify multiple cell types simultaneously. The assay is HTS compatible, providing high-quality, image-based, replicable data for multiple, co-cultured cell types. As proof of concept, we subjected colorectal carcinoma colonies in 3D soft agar to a mini screen of 1528 natural product compounds. Hit compounds from the primary screen were rescreened in an HTS 3D co-culture matrix containing colon stromal cells and cancer cells. By combining tumor cells and normal, nontransformed colon epithelial cells in one primary screening assay, we were able to obtain differential IC50 data, thereby distinguishing tumor-specific compounds from general cytotoxic compounds. Moreover, we were able to identify compounds that antagonized tumor colony formation in 3D only, highlighting the importance of this assay in identifying agents that interfere with 3D tumor structural growth. This screening platform provides a fast, simple, and robust method for identification of tumor-specific agents in a biologically relevant microenvironment.
利用三维（3D）细胞培养开发细胞行为模型的研究兴趣日益浓厚。为了能够评估差异诱变剂对二维和三维细胞生长的影响，我们对软琼脂集落形成试验进行了小型化并使其适用于高通量筛选（HTS），采用激光扫描细胞仪同时对多种细胞类型进行成像和定量分析。该试验与高通量筛选兼容，可为多种共培养细胞类型提供高质量的、基于图像的、可重复的数据。作为概念验证，我们对三维软琼脂中的结肠癌细胞集落进行了1528种天然产物化合物的小型筛选。在包含结肠基质细胞和癌细胞的高通量筛选三维共培养基质中对初筛得到的活性化合物进行了复筛。通过在一次初筛试验中结合肿瘤细胞与正常的、未转化的结肠上皮细胞，我们能够获得差异半数抑制浓度（IC50）数据，从而区分肿瘤特异性化合物与一般细胞毒性化合物。此外，我们能够鉴定出仅在三维环境中拮抗肿瘤集落形成的化合物，突出了该试验在鉴定干扰三维肿瘤结构生长的药物方面的重要性。这个筛选平台为在生物学相关微环境中鉴定肿瘤特异性药物提供了一种快速、简单且可靠的方法。
