Study design for the derivation of the human invasion signature (HIS).Schematic of experimental procedures for the selective profiling of migratory tumor cells in vivo. Migratory/invasive tumor cells were isolated from live primary tumors based on their chemotactic and highly motile properties toward known chemoattractants. The whole or "average" primary tumor cells were isolated with fluorescence-activated cell sorting (FACS) based on the tumor cells stably expressing green fluorescent protein (GFP). Both cell populations analyzed were >95% pure tumor cells (detailed discussion in Additional File 1). Comparative gene-expression analysis with microarrays was then used to derive a signature specific to in vivo migration and invasion of breast tumor cells. Methods and technical controls are discussed in more detail in Methods and in Additional File 1.