Mechanism of myeloma-mediated cell fusion of DCs to OCLs. (A) Myeloma cells (U266) and DCs were cocultured overnight at a tumor/DC ratio of 1:2. After 24 hours, CD11c⁺ DCs were sorted by flow cytometry to > 99% purity. The expression of TSP-1 mRNA was analyzed by real-time RT-PCR (TaqMan) and normalized to the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. (B) Immunofluorescence analysis of TSP-1 in DCs cultured overnight either alone or with tumor cells at a tumor/DC ratio of 1:2. Tumor cells alone were used as control. Acetone-fixed cells on poly-l-lysine–coated slides were stained with anti–TSP-1 mab followed by Alexa Flour 568 as a secondary antibody. DAPI was used as a nuclear stain. Note intense staining of TSP-1 in DCs that had been cocultured with tumor cells (arrows). Original magnification, ×10. (C) Myeloma cells (U266) and immature DCs were cultured in methylcellulose cultures with and without TSP-1 antibody (to block TSP-1 and CD47/integrin-associated protein interaction) for 2 weeks. MGCs were counted as described previously. *P < .05. (D) Tumor cells (U266) were electroporated with 20 μg of CD47 siRNA (CD47) or nontargeting siRNA (control). The tumor cells were harvested at 48 hours after electroporation and were phenotypically characterized for CD138 (FITC) and CD47 (phycoerythrin) by flow cytometry. Shown is percent reduction in mean fluorescence intensity of CD47 expression on tumor cells electroporated with CD47 siRNA compared with nontargeting siRNA 48 hours after electroporation. (E) Myeloma cells (U266) electroporated with either CD47 siRNA or nontargeting control siRNA were cocultured with monocyte-derived DCs at a tumor/DC ratio of 1:100 in clonogenic assays. After an incubation of 2 weeks, cells were harvested onto poly-l-lysine–coated slides and were stained for enumeration of MGCs as described previously. *P < .05.