CS1 mediates MM cell adhesion to BMSC, which is blocked by HuLuc63. (A) MM lines and freshly isolated patient MM cells were incubated with 10 μg/mL of CS1-AF488 (green), washed, fixed, mounted, and viewed on a Zeiss microscope with a 40× objective. Images were processed using Adobe Photoshop Software version 7.0 (Adobe, San Jose, CA). CS1 is concentrated in uropods of polarized MM cells that promote adhesion. (B) CS1 (green) is colocalized with CD138 (red), exhibiting yellow staining in uropods of the majority of polarized L636 MM cells. (C) Lentiviruses expressing CS1 siRNA or control (cnt) siRNA were generated and used to infect MM1S cells. Immunoblotting using ChLuc90 mAb confirmed CS1 knockdown in clone 1. 293tflagCS1 expressing CS1 and 293t without CS1 expression served as controls. DAPI staining indicates nuclei of cells. (D) Calcein-AM labeled MM1S and MM1R cells, as well as CS1-null counterparts (MM1S CS1 siRNA and MM1R CS1 siRNA), were added to BMSC-coated 96-well plates, in the presence of iso IgG₁ (□) or HuLuc63 (■; 0.1 μg/mL) for 4 hours. Unattached cells were washed and adherent cells were measured in a fluorescence plate reader. Shown is mean plus or minus SE of 3 independent experiments. A.U. indicates arbitrary unit. *P < .05. Error bars represent range of data. (E) Adhesion of MM1S and MM1R MM cells to BMSCs was assayed in the presence of HuLuc63 or iso IgG₁. Shown is mean plus or minus SE of triplicate wells from one representative of 3 independent experiments. (F) HuLuc63 specifically inhibits patient MM cells (■ indicates MM1; ◆;; MM2) binding to BMSCs. (G) CS1 + CD138 + MM cells from 2 patients (MM 1 and MM 2) were incubated with HuLuc63 (0-100 μg/mL). (H) CS1 + MM cells from 2 patients were cultured with (■) or without (□) BMSCs, in the presence of HuLuc63 (0-100 μg/mL). Cell viability was determined by MTT assay. Shown is mean plus or minus SE of 3 independent experiments.