The interaction of PSGL-1 with P-selectin regulates extravasation and homing to the BM of MM cells in vivo. (A) Calcein-labeled MM1s cells were injected intravenously into SCID mice treated with either vehicle (Ctrl), anti–P-selectin Ab (250 μg/kg IV), or GMI-1070 (25 mg/kg IP) for 1 hour before injection of MM1s cells (n = 3 per group). The number of circulating cells was followed over time using in vivo flow cytometry. Cells were counted every 5 minutes for 40 minutes. Fluorescence signal was detected on an artery in the ear and digitized for analysis with MATLAB software. Inhibition of P-selectin using GMI-1070 or neutralizing Ab delayed the extravasation of MM cells. (B) MM1s cells were transfected with either PSGL-1 or scramble siRNA, labeled with Calcein, and injected intravenously into SCID mice (n = 3 per group), followed by IV injection of Evans blue. Homing to the BM of mice was imaged by in vivo confocal microscopy 30 minutes after injection. Inhibition of the homing of MM cells to the BM was observed with knockdown of PSGL-1, shown as an average of number of MM cells in 18 images taken from 3 different mice (P < .01; i) and in representative images of the BM (ii; green indicates MM cells; red, blood vessels). (C) Calcein-labeled MM1s cells were injected intravenously into SCID mice, which were treated with either vehicle (Ctrl) or GMI-1070 (25 mg/kg IP) for 1 hour before injection of MM1s cells (n = 3 per group), followed by IV injection of Evans blue. Homing to the BM of mice was imaged by in vivo confocal microscopy 30 minutes after injection. Inhibition of the homing of MM cells to the BM was observed in mice treated with GMI-1070, shown as an average of number of MM cells in 18 images taken from 3 different mice (P < .01; i) and in representative images of the BM (ii; green indicates MM cells; red, blood vessels).