TAMs exhibit proinflammatory phenotype in BM cluster of MM cells. (A-B) Intravital live imaging of GFP⁺ myeloma cells (green) in the injected tibia at 2 weeks after inoculation (n = 2 mice). (A) 3D analysis of distances between myeloma cells and anti-CD169-PE–labeled MPs (red) within a small cluster. (B) Examples of myeloma nanotube structures extending to BM autofluorescent (MP) cells (red). Yellow arrows show transfer of GFP signals from MM cells to surrounded microenvironment cells. (C) Gating strategy to identify GFP⁺ and GFP^– BM MPs and the CD206 subset and analysis of CD206^– (M1-like) subset of GFP⁺ and GFP^– BM MPs in tumor-bearing mice. (D) Kinetics of CD206^– MP subset as a function of tumor burden in the BM. (E) Samples of frequency of intracellular IL-6 and TNFα production in MPs, with fold increase in tumor-bearing mice normalized to that of naïve mice, analyzed by a Wilcoxon test. (F) Plot of TNFα-producing BM MPs vs tumor burden in the BM. All experiments were independently repeated at least 2 times; data were pooled and comparisons were analyzed by using a Mann-Whitney t test. Scale bars, 20 μm. *** is P < .001. Errors bars in panel C represent standard deviation. Horizontal lines in panels A and E are mean values.