The interaction of PSGL-1 and P-selectin regulates proliferation of MM cells induced by BMSCs and ECs in vitro and tumor initiation in vivo. (A) Expression of E-, L-, and P-selectins evaluated in primary BMSCs (n = 5) using flow cytometry and expressed as ratio between MFI of selectin to the MFI of the isotype control. BMSCs presented with higher expression of E- and P-selectins. (B) MM1s cells were transfected with either PSGL-1 siRNA or scramble siRNA. Adhesion of MM cells to BMSCs was evaluated: significant inhibition of MM cell adhesion to BMSCs was observed in PSGL-1 knockdown cells (P = .006; i). BMSCs were treated with or without GMI-1070 (500μM for 1 hour) and adhesion of nontreated MM1s cells to BMSCs was evaluated: inhibition of MM cell adhesion to BMSCs was observed in HUVECs treated with GMI-1070 (P < .001; ii). Data represent means ± SD of triplicate experiments. (C) MM1s cells were transfected with either PSGL-1 siRNA or scramble siRNA and cultured with or without BMSCs (i) and HUVECs (ii). Cell proliferation was measured at 24 hours by bromodeoxyuridine incorporation and ELISA. Coculture of MM1s with HUVECs and BMSCs increased the proliferation of MM1s cells transfected with scramble siRNA, an effect that was reversed by PSGL-1 siRNA. Data represent means ± SD of triplicate experiments. (D) HUVECs and BMSCs were treated with or without GMI-1070 and nontreated MM cells were cultured with or without BMSCs (i) and HUVECs (ii). Cell proliferation was measured at 24 hours by bromodeoxyuridine incorporation and ELISA. Coculture of MM1s with nontreated HUVECs and BMSCs increased the proliferation of MM1s cells, an effect that was reversed by GMI-1070. Data represent means ± SD of triplicate experiments. (E) MM1s cells were transfected with either PSGL-1 or scramble siRNA and injected intravenously into SCID mice (n = 4 per group); after 1 week the BM was extracted from the femurs of the mice and tumor initiation was determined as the percentage of CD138⁺ cells in the BM. Inhibition of tumor initiation in the BM of the mice was observed with knockdown of PSGL-1 (P < .001). (F) MM1s cells were transfected with either PSGL-1 or scramble siRNA and injected into the BM of the tibia of SCID mice; after 1 week the BM was extracted from the tibias and tumor initiation was determined as the percentage of CD138⁺ cells in the BM. Inhibition of tumor initiation in the BM of the mice was observed with knockdown of PSGL-1, but not to the same extent as that observed after IV injection (P = .02).