M1-MØ are cytotoxic to MM cells and inhibit MM cell proliferation and tumor development in vivo. (A) The indicated MM cell lines were cultured alone or in the presence of GM-MØ or M-MØ for 3 days. Cell death was measured and normalized by MM cell spontaneous death. Data represent mean ± standard error of the mean (SEM) of 6 independent experiments with different MØ donors. (B) MM cells were cultured for 72 hours in the absence or presence of M-MØ, and cell death was induced with bortezomib (10 nM) (n = 3 MØ donors). (C) Cell death analysis of patient CD138⁺ MM BM cells (dot plot) cultured alone or with GM-MØ or M-MØ (48 hours). (D) NCI-H929 cells were cocultured with GM-MØ or M-MØ (stained with CFSE; blue) and live-imaged for 4 hours. First and last frames are shown (bright field images). Rapid acquisition of AnV (green)/PI (red) staining represents necrotic cells (red circles). Blebbing-apoptotic cells are circled in yellow, and one magnified case is indicated (asterisks). Scale bars, 100 μm. (E) MM cell proliferation (CFSE dilution method) in the presence of GM-MØ or M-MØ. A representative experiment is shown on the left, and mean fluorescence intensity (MFI) values of 3 independent MØ donors normalized by NCI-H929 cultured alone are shown on the right. (F-G) NCI-H929 cells were injected (subcutaneously) alone or mixed with GM-MØ or M-MØ (1:1) in the flank of NSG mice. After 10 days, mice were sacrificed for tumor volume evaluation (F) and confocal microscopy analysis (G) by determining CD138/CD38, caspase 3, F4/80, CD163, and cd45, and Ki67 labeling. Scale bars, 50 μm. Percentage of proliferating (Ki67) and apoptotic cells (active caspase 3) along intratumoral areas is represented on the right. Data show media ± SEM of at least 4 mice per group. *P < .05; **P < .01; ***P < .001. (D,G) Scale bars as indicated.