Nest expansion assay: a cancer systems biology approach to in vitro invasion measurements

The NEA was applied to two human-derived breast cell lines, MCF10A and MCF10A-CA1d, which exhibit opposite degrees of tumorigenicity and invasion in vivo. Assays were performed to incorporate various microenvironmental conditions, in order to test their influence on cell behavior and measures. Two types of computer-driven image analysis were performed using Java's freely available ImageJ software and its FracLac plugin to capture nest expansion and fractal dimension, respectively – which are both taken as indicators of invasiveness. Both analyses confirmed that the NEA is highly reproducible, and that the ECM component is key in defining invasive cell behavior. Interestingly, both analyses also detected significant differences between non-invasive and invasive cell lines, across various microenvironments, and over time.The spatial nature of the NEA makes its outcome susceptible to the global influence of many cellular parameters at once (e.g., motility, protease secretion, cell-cell adhesion). We propose the NEA as a mid-throughput technique for screening and simultaneous examination of factors contributing to cancer cell invasion, particularly suitable for parameterizing and validating Cancer Systems Biology approaches such as mathematical modeling.Classical wound-healing, cell migration, and cancer invasion assays have been carried out in tissue culture for decades, primarily to generate information about the relationship between cell motility and invasion [1-3]. However, a number of these techniques are encumbered with problems of quantification, reproducibility, and flexibility. For example, traditional wound-healing, or "scratch" assays include creation of an artificial wound (i.e., a scratch) within a monolayer of cells using a blunt object (e.g., pipet tip), and subsequent quantification of cells repopulating the scratch over time [1]. Not surprisingly, such assays often produce crude quantitative data, since they are typically difficult to standardize and re