FIGURE 5.

Exposure to breast cancer-derived factors enhances subsequent breast cancer cell attachment to immature osteoblasts. Bone marrow cells were grown for 9 days with AA (50 μg/ml) in the absence (AA, open bars) or presence of MDA-MB-231 CM (10%), combined with vehicle (AA+231, black bars) or the following inhibitors (gray bars): OPG (500 ng/ml), LiCl (10 mm), SB216763 (SB, 10 μm), DAPT (100 nm), or Compound E (CE, 100 nm). The MDA-MB-231 cells were labeled with Cell Tracker Green and added to bone marrow cultures for 40 min, and then the cultures were washed to remove nonattached cells, fixed, and analyzed. A, representative images demonstrate attachment of breast cancer cells (green) to mature osteoblasts (OB) in cultures treated with AA only (left); to immature osteoblast precursors (pOB) in cultures treated with AA and MDA-MB-231 CM (center); or to osteoclasts (OC, white outline) in cultures treated with AA and MDA-MB-231 CM (right). Scale bar is 20 μm. B, significantly more breast cancer cells attached to bone marrow cultures treated with AA and MDA-MB-231 CM compared with cultures treated with AA alone. Inhibitors of γ-secretase DAPT and CE prevented this effect of MDA-MB-231 CM, whereas glycogen synthase kinase inhibitors and OPG were ineffective. Data are means ± S.E. (error bars), n = 2–6 independent experiments; different letters indicate significant difference at p < 0.05. C, bone marrow cells were grown for 9 days with AA (50 μg/ml) in the absence (AA, open bars) or presence of 4T1, MCF7, or MCF10A CM (10%), either alone (CM, black bars) or with γ-secretase inhibitors DAPT (100 nm) or CE (100 nm). The same cells as were used for CM treatment were labeled with Cell Tracker Green and added to bone marrow cultures for 40 min. Treatment with 4T1 or MCF7 CM significantly increased attachment of these cells to bone marrow cultures, which was inhibited by γ-secretase inhibitors. Data are means ± S.E., n = 3 independent experiments, p < 0.05.