Myeloma-derived mtDAMPs induce a migratory signature in BM macrophages. (A) Schematic of experimental design. BMDMs were cultured with mtDAMPs (10 μg) for 24 hours. BMDM cell supernatant was cleared of cellular debris by centrifugation before cytokine array analysis (n = 3). (B) Quantification of cytokine array results segmented into inflammatory and chemoattractant-related factors. (C) Schematic of experimental design. Murine 5TGM1^{(GFP+ LUCI+)} cells (1 × 10⁶) were injected into C57BL/KaLwRij mice. On days 20, 22, 24, and 26 after engraftment, mice were injected intraperitoneally with either 200 μL H-151 (750 nM) or vehicle. Mice were euthanized at 27 days; BM was harvested, and myeloma-associated macrophages were isolated via FACS purification. (D) Relative gene expression of CXCL10, CXCL2, and CCL5 (n = 6 in each treatment group). Data indicate mean ± SD. Statistics presented as Kruskal-Wallis test with Dunn post hoc multiple comparisons test. (E) BMDMs were cultured with either mtDAMPs (10 μg) alone or mtDAMPs and H-151 (10 μM) for 24 hours. BMDM-CM was cleared of cellular debris by centrifugation and placed into the bottom chamber of the transwell. 5TGM1^(GFP+) cells were placed in the upper chamber and migrated 5TGM1^(GFP+) cells were counted at 4 and 24 hours. Data indicate mean ± SD. Statistics are presented as Mann-Whitney U test. (F) Murine 5TGM1^{(GFP+ LUCI+)} cells (1 × 10⁶) were injected into C57BL/KaLwRij mice. At 34 days after engraftment, PB samples were taken by tail vein bleed, then the mice were injected IP with H-151 (750 nM). On day 35, posttreatment blood samples were taken and mice were euthanized. The PB samples were analyzed for 5TGM1^(GFP+) cell presence via flow cytometry (n = 8 mice). Data indicate mean ± SD. Statistics are presented as Wilcoxon test. ∗∗P < .01; ∗P < .05.