Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening
Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening
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DOI：
10.3791/57476
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发表时间：
2018-09-01
影响因子：
1.2
通讯作者：
Silver, Serena J.
中科院分区：
文献类型：
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作者：
Griner, Lesley Mathews;Gampa, Kalyani;Silver, Serena J.
癌细胞通常在塑料表面的二维（2D）中培养。然而，这种技术缺乏肿瘤块在体内暴露的真实环境。实体瘤不是附着在塑料上的薄片，而是在三维（3D）空间中与相邻细胞相互作用的克隆细胞的集合，并且具有独特的空间特性，例如正常细胞极性的破坏。这些相互作用导致3d培养的细胞获得与体内肿瘤更相关的形态学和细胞特征。此外，肿瘤块与其他细胞类型直接接触，如基质细胞和免疫细胞，以及所有其他细胞类型的细胞外基质。沉积的基质由胶原蛋白和纤维连接蛋白等大分子组成。为了增加肿瘤学研究成果从实验室到临床的转化，许多研究小组已经开始研究在药物开发策略中使用3D模型系统。这些系统被认为与生理更相关，因为它们试图概括肿瘤的复杂和异质性环境。然而，这些系统可能相当复杂，而且，尽管可以在96孔格式下生长，有些甚至可以在384孔格式下生长，但它们很少有大规模生长和筛选的选择。这种观察到的差距导致了这里详细描述的方法的发展，以1536孔板的高通量能力培养肿瘤球体。这些方法代表了对难以筛选的高度复杂的基于矩阵的系统和传统二维分析的妥协。成功筛选了多种具有不同基因突变的癌细胞系，通过使用针对丝裂原活化蛋白激酶或MAPK途径的精心策划的化合物库来检测化合物的功效。然后将球体培养反应与在2D中生长的细胞的反应进行比较，并报告不同的活性。这些方法为在高通量3D环境下测试化合物活性提供了独特的方案。
Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.