Fig. 1.

Bone, marrow, and cancer cells survive in ex vivo co-cultures. a Experimental design of ex vivo bone cultures using mouse bone explants. b Metabolic activity of ex vivo bone cultures compared to the metabolic activity of a dead bone and media controls. CellTiter-Blue (cell viability) assay to test metabolic activity of an increasing number of mouse bone explants in ex vivo culture after 4 weeks in culture (p values obtained by Student’s t test, multiple comparisons). Lines show the mean and standard deviation. c H&E of a fresh, uncultured mouse bone sample (scale bar: 10 μm). d H&E of a cultured mouse bone cultured for 4 weeks (scale bar: 10 μm). e TRAP staining of a 4-week cultured mouse bone sample. Arrows show the localization of osteoclasts (scale bar: 10 μm). f Masson’s trichrome staining on a mouse bone sample after 4 weeks in culture showing retention of collagen depositions shown in blue (scale bar: 20 μm). g Experimental design of ex vivo mouse bone co-cultures grown with breast mammary epithelial cancer cells injected into the bone prior to culture. h Immunohistochemistry (IHC) for Pan-cytokeratin on a mouse bone co-cultured for 4 weeks with PyMT cancer cells (left) and an uninjected mouse bone cultured for 4 weeks (right). i Luciferase assay on a mouse bone sample colonized by a luciferase-expressing PyMT cell line. The intensity bars (rainbow) and scale information (Min/Max) for BLI signal are provided. Bioluminescent PyMT cancer cells co-localize with the bone in culture. j IHC of mouse bone marrow cells after co-culture with mammary epithelial cancer cells (left) compared to a fresh mouse bone sample (right) stained for CD4+ helper T cells, CD8+ cytotoxic T cells, CD20+ B cells, CD68+ macrophages, Ly6G/6C+ neutrophils, endomucin endothelial cells, CD61+ megakaryocytes, CD71+ erythroid precursors, and k Safranin-O staining of a mouse bone post 4 weeks in co-culture (scale bar: 10 μm). ***Significant at p < 0.001; ****significant at p < 0.0001; n.s. not significant