PSGL-1 regulated adhesion of MM cells to ECs. (A) Expression of E-, L-, and P-selectins evaluated on HUVECs and primary MM BM-derived ECs (n = 5) using flow cytometry and expressed as ratio between MFI of selectin to the MFI of the isotype control. ECs present with higher expression of E- and P-selectins. (B) HUVECs were transfected with siRNAs for E-, L-, or P-selectin. Scramble siRNA was used as a control (panel i) or cells were treated with anti–E-, anti–L-, or anti–P-neutralizing Abs. Isotype control Ab was used as a control (panel ii). Adhesion of MM cells on HUVECs was evaluated: significant inhibition of MM cells to HUVECs was observed in P-selectin knock-down cells (panel i) and in HUVECs treated with neutralizing Ab for P-selectin (panel ii), P < .01. All HUVECs shown in panel C were exposed to TNFα (30 U/mL for 3 hours) or to IL-4 (3 ng/mL for 24 hours) and histamine (2.25mM for 4 hours). Expression of E-, L-, and P-selectins was evaluated by flow cytometry. Induction of E- and P-selectin was observed after activation with TNFα or IL-4/histamine, respectively (panel i). Adhesion of nontreated MM cells to HUVECs was evaluated. MM cells showed increased adhesion to HUVECs with activation of P-selectin (panel ii), P < .01. (D) MM1s cells were transfected with either PSGL-1 siRNA or scramble siRNA. Adhesion of MM cells to HUVECs was evaluated: a significant inhibition of MM cells to HUVECs was observed in PSGL-1 knock-down cells (P < .05). (E) HUVECs were treated with increasing concentrations of GMI-1070 (0, 100, 250, and 500μM) for 1 hour, and adhesion of nontreated MM cells (H929, OPM1, and MM.1S) to HUVECs was evaluated: dose-dependent inhibition of MM cell adhesion to HUVECs was observed (P < .05). All data represent means ± SD of triplicate experiments. (F) MM1s cells were treated with either isotype control or PSGL-1–blocking Ab. Adhesion of MM cells to HUVECs was evaluated: significant inhibition of MM cells to HUVECs was observed in cells treated with PSGL-1–blocking Ab (P < .01).