Prostate cancer cells preferentially metastasize to bone, presumably through physical interaction with E-selectin present on human bone marrow endothelial cells. We examined the interaction between prostate cancer cell lines and circulating tumor cells (CTCs) isolated from prostate cancer patients with stimulated endothelial cells expressing E-selectin under physiological blood flow using a parallel flow chamber system. Prostate cancer cells, MDAPCa 2b were first labeled with anti-PSMA monoclonal antibody J591 conjugated with alexa fluor 488 (J591-488) that recognizes prostate specific membrane antigen (PSMA) and is internalized following binding to PSMA. We observed that the mean rolling velocity of MDAPCa 2b cells on HUVECs ranged from 4.2-6 m/s at 0.5-4 dyn/cm2 shear stress. Interestingly, MDAPCa2b cells did not show rolling behavior on both unstimulated-HUVECs and stimulated-HUVECs incubated with anti-E-selectin neutralizing antibody (p 0.005) at different shear stress. We then used J591-488 antibody to label 4 metastatic prostate cancer patient CTCs and found that in 3 patients, prostate CTCs tethered and stably interacted with activated HUVECs expressing E-selectin at 0.6 dyn/cm2 shear stress. Furthermore, in parallel CTC-Endothelial interactions in same patients were abrogated in the presence of anti-E-selectin neutralizing antibody. We also found the presence of sialyl-Lewis X (sLex), an epitope present on E-selectin ligands, on CTCs derived from metastatic prostate cancer patients. Fluorescence intensity analysis of sLex expression in prostate cancer patient CTCs showed a heterogeneous expression of sLex ranging from 44-75%. These findings confirmed that prostate CTCs exhibit E-selectin-dependent adhesive interactions with endothelial cells. These results highly suggest the presence of E-selectin ligands on the surface of prostate CTCs which might be eventually responsible for tumor metastasis.