In contrast to filter-based methods that rely on counts of cells that traverse an ECM-coated membrane, this type of migration assay has high information content because it allows imaging of actively invading cells in the ECM compartment during the entire assay period. Since each cell is assigned an x,y coordinate, a variety of analysis methods can be employed including those based on distributions, such as median distance traveled, as well as total number of cells migrating (Figure 3).
Analyses based on distribution and cells migrated both yield satisfactory data quality for compound profiling and screening. Because a purely antiproliferative compound would not be expected to alter the distribution of cells within the channel, distribution-based analysis is less sensitive to effects on proliferation. Furthermore, relative effects on migration vs. proliferation can be assessed by measuring the density of cells in the loading area at the end of the assay. Migration then can be normalized for any proliferation and/or cell death that might occur during the course of the assay, allowing compounds to be binned based on their inhibition of migration relative to other activities such as proliferation and cytotoxicity.
In addition to number of cells migrating and distance traveled, imaging of actively invading cells can provide clues to the mode of migration and the underlying mechanisms. For instance, with a clearly visible gap between almost all cells, the migration pattern of PC3M cells (prostatic carcinoma) in type I collagen can be identified as individual, as opposed to collective.
More detailed information on migration mechanisms or pathways can be gleaned using post-assay molecular staining of cells in the channels. Both phalloidin staining and immunocytochemistry have been employed in this way. Though we have just begun to explore these methods, the ability to apply in situ high-content analysis approaches to cells migrating in 3-D ECM is a promising avenue for delineating and targeting specific cell invasion events and pathways.