Mechanism of myeloma-mediated MGC formation. (A) Cell-cell contact dependency. Myeloma cells (U266) were cultured in methylcellulose cultures with DCs as in Figure 1. DCs were either plated with U266 or separated from tumor cells by a Transwell insert. In addition, DCs also were cultured in the presence of supernatant from the myeloma cell line (U266). Cells were harvested onto slides after an incubation of 2 weeks to be stained (CD138, CD11c, and DAPI) and MGC enumeration. MGCs were counted in 10 random high-power fields (HPFs). Data are representative of 3 different experiments. *P < .05. (B) Myeloma cells (U266) were cultured with immature DCs or LPS-matured DCs in methylcellulose cultures. Immature DCs alone and mature DCs alone were used as the controls. Cells were harvested for cytospins after an incubation of 2 weeks. Slides were acetone-fixed and stained for CD138, CD11c, and DAPI. MGCs were counted in 10 random HPFs. Results are the mean ± SEM of 3 separate experiments. *P < .05. (C) Combined immunofluorescence and FISH analysis of MGCs. U266 cells (male origin) were cocultured with iDCs (female origin) in methylcellulose cultures as described previously. After 2 weeks, cells were harvested onto cytospins and stained for CD11c (green) and X (green):Y(Red) chromosome. Shown is a representative CD11c⁺ MGCs with 6 nuclei, all with XX chromosomes (arrow). Note U266 cell with Y (red) chromosome (dotted arrow). Original magnification, ×100.