Marshall John
Barts Cancer Institute, Barts and The London, Queen Mary's School of Medicine and Dentistry, Queen Mary, University of London, London, UK.
Methods Mol Biol. 2011;769:97-110. doi: 10.1007/978-1-61779-207-6_8.
The need to identify inhibitors of cancer invasion has driven the development of quantitative in vitro invasion assays. The most common assays used are based on the original Boyden assay system. Today commercially available plastic inserts for multi-well plates, which possess a cell-permeable membrane, as typified by Transwell(®) Permeable Supports, permit accurate repeatable invasion assays. When placed in the well of a multi-well tissue culture plate these inserts create a two-chamber system separated by the cell-permeable membrane. To create an invasion assay the pores in the membrane are blocked with a gel composed of extracellular matrix that is meant to mimic the typical matrices that tumour cells encounter during the invasion process in vivo. By placing the cells on one side of the gel and a chemoattractant on the other side of the gel, invasion is determined by counting those cells that have traversed the cell-permeable membrane having invaded towards the higher concentration of chemoattractant. In this chapter, in addition to protocols for performing Transwell invasion assays, there is consideration of the limitations of current assay designs with regard to available matrices and the absence of tumour microenvironment cells.
鉴定癌症侵袭抑制剂的需求推动了定量体外侵袭试验的发展。最常用的试验基于最初的博伊登试验系统。如今，市售的用于多孔板的塑料插入物，如Transwell（®）可渗透支持物，具有可渗透细胞的膜，可进行准确、可重复的侵袭试验。将这些插入物放置在多孔组织培养板的孔中时，会形成一个由可渗透细胞的膜分隔的双室系统。为了进行侵袭试验，膜上的孔用一种由细胞外基质组成的凝胶封闭，该凝胶旨在模拟肿瘤细胞在体内侵袭过程中遇到的典型基质。通过将细胞置于凝胶的一侧，趋化剂置于凝胶的另一侧，通过计数那些穿过可渗透细胞膜并朝着趋化剂高浓度侵袭的细胞来确定侵袭情况。在本章中，除了进行Transwell侵袭试验的方案外，还考虑了当前试验设计在可用基质以及缺乏肿瘤微环境细胞方面的局限性。
