Evaluating Melanoma Cell Migration/Invasion in Transwell Assays by Flow Cytometry

Cell migration and invasion are vital aspects of melanoma biology that are critical to the progression of this aggressive cancer. Numerous in vitro assays are available to study these hallmark properties of cancer, including two-dimensional wound healing ("scratch") and transwell (Boyden chamber) assays, as well as three-dimensional microfluidic-based video microscopy assays and matrix/skin organ construct-based assays. Though each of these assays has unique advantages depending on the experimental question at hand, transwell assays are highly adaptable and thus particularly well-suited for investigating the role of specific stimulators/inhibitors of cancer cell migration and invasion. Traditionally, quantification of migrating/invading cells that are capable of transiting the Boyden chamber utilized in these assays is achieved by manual counting of cells that have reached the bottom chamber or by colorimetric/fluorometric detection of transited cells that were preloaded with specific dyes prior to assay initiation. These approaches, while commonly used, have inherent limitations in their design, including the introduction of quantification artifacts that arise from human error during direct cell counting or uneven/inconsistent cell labeling within/across experiments. To overcome these limitations, we have optimized a non-fluorescent flow cytometry-based assay for quantifying melanoma transwell migration and invasion, and we present herein an application of this assay to assess the role of the FOXC2 transcription factor in these processes.