Mechanically stimulated osteocytes reduce the bone-metastatic potential of breast cancer cells in vitro by signaling through endothelial cells

Bone metastases occur in 65% to 75% of patients with advanced breast cancer and significantly worsen their survival and quality of life. We previously showed that conditioned medium (CM) from osteocytes stimulated with oscillatory fluid flow, mimicking bone mechanical loading during routine physical activities, reduced the transendothelial migration of breast cancer cells. Endothelial cells are situated at an ideal location to mediate signals between osteocytes in the bone matrix and metastasizing cancer cells in the blood vessels. In this study, we investigated the specific effects of flow-stimulated osteocytes on the interaction between endothelial cells and breast cancer cells in vitro. We observed that CM from flow-stimulated osteocytes reduced endothelial permeability by 15% and breast cancer cell adhesion onto endothelial monolayers by 18%. The difference in adhesion was abolished with anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing antibodies. Furthermore, CM from endothelial cells conditioned in CM from flow-stimulated osteocytes significantly altered the gene expression in bone-metastatic breast cancer cells, as shown by RNA sequencing. Specifically, breast cancer cell expression of matrix metallopeptidase 9 (MMP-9) was downregulated by 62%, and frizzled-4 (FZD4) by 61%, when the osteocytes were stimulated with flow. The invasion of these breast cancer cells across Matrigel was also reduced by 47%, and this difference was abolished by MMP-9 inhibitors. In conclusion, we demonstrated that flow-stimulated osteocytes downregulate the bone-metastatic potential of breast cancer cells by signaling through endothelial cells. This provides insights into the capability of bone mechanical regulation in preventing bone metastases; and may assist in prescribing exercise or bone-loading regimens to patients with breast cancers.