Breast cancer conditioned media is capable of stimulating the development and/or survival of osteoclast progenitor cells. (A) Bone marrow flushes were cultured in α-MEM (Ctl) or MDA-MB-231 conditioned α-MEM (CM) and surviving cells were counted at days 3, 4, 5, and 6. Data are expressed as a mean +/− S.E. *, p < 0.02. (B) On day 6, cells from the culture in breast cancer conditioned α-MEM were then seeded into 48 well plates at 1 × 104, 2 × 104, or 4 × 104 cells per well and treated with 100 ng/ml RANKL and 10 ng/ml rM-CSF. Quantification of the osteoclastogenesis assays is shown in mean number of multinucleated TRAP-positive cells (>3 nuclei) per well. (C) Each condition had three replicates (wells) and was repeated 4 times. A representative area of the culture from each condition is shown (4× and 10× magnification). A separate set of cultures was continued 4 additional days to perform bone resorption assays. Resorption pits were then visualized by SEM. Magnification by SEM was 200x.