Metastatic colonization involves cancer cell lodgment or adherence in the microvasculature and subsequent migration of those cells across the endothelium into a secondary organ site. To study this process further, we analyzed both in vitro and in vivo migration of human PC-3 prostate cancer cells . We isolated 6 subpopulation of cells: TEM4-18 were isolated from in vitro transendothelial migration of PC-3 cells; GS672.Ug, GS683.LALN and JD1203.Lu are single passaged in vivo cell lines from TEM4-18; GS689.Li and GS694.LAd are twice passaged in vivo cell lines from PC-3 cells. All the subpopulations crossed an endothelial barrier more efficiently and more aggressive in a murine metastatic colonization model than parental PC-3 cells. Microarray and FACS analysis of these cells showed that the expression of many genes previously associated with leukocyte trafficking and cancer cell extravasation were either unchanged or down-regulated. These cells exhibited characteristic molecular markers of an epithelial-to-mesenchymal transition, including frank loss of E-cadherin expression and upregulation of the E-cadherin repressor ZEB1. We used microarray to detail the global programme of gene expression underlying cancer metastasis. Overall design: We wanted to compare the expression profiles of cells that had undergone transendothelial migration in vitro or metastasis in vivo. TEM4-18 was generated from the parental PC-3 that was plated onto a confluent human microvascular endothelial cell line from the lung and allowed to migrate across this monolayer. GS672.Ug were isolated from urogenital after intravenous injection of PC-3 cells. GS683.LALN and JD1203.Lu were isolated from lung after intravenous injection of TEM-418 cells. JD549.Ki were isolated from kidney after intravenous injection PC-3 cells. GS689.Li and GS694.LAd were isolated from liver and left adrenal after intravenous injection of JD549.Ki. All the cell lines are analyzed once except duplicate for TEM4-18 in this experiment for a total of 7 samples in this microarray. All other five sublines were then compared to TEM4-18 cells for changes in gene expression.