Abstract
Bone metastases are common and severe complications of cancers. It is estimated to occur in 65–75% of breast and prostate cancer patients and cause 80% of breast cancer-related deaths. Metastasised cancer cells have devastating impacts on bone due to their ability to alter bone remodeling by interacting with osteoblasts and osteoclasts. Exercise, often used as an intervention for cancer patients, regulates bone remodeling via osteocytes. Therefore, we hypothesise that bone mechanical loading may regulate bone metastases via osteocytes. This provides novel insights into the impact of exercises on bone metastases. It will assist in designing cancer intervention programs that lowers the risk for bone metastases. Investigating the mechanisms for the observed effects may also identify potential drug targets.
MLO-Y4 osteocyte-like cells (gift of Dr. Bonewald, University of Missouri-Kansas City) on glass slides were placed in flow chambers and subjected to oscillatory fluid flow (1Pa; 1Hz; 2 hours). Media were extracted (conditioned media; CM) post-flow. RAW264.7 osteoclast precursors were conditioned in MLO-Y4 CM for 7 days. Migration of MDA-MB-231 breast cancer cells and PC3 prostate cancer cells towards CM was assayed using Transwell. Viability, apoptosis, and proliferation of the cancer cells in the CM were measured with Fixable Viability Dye eFluor 450, APOPercentage, and BrDu, respectively. P-values were calculated using Student's t-test.
Significantly more MDA-MB-231 and PC3 cells migrated towards the CM from MLO-Y4 cells with exposure to flow in comparison to CM from MLO-Y4 cells not exposed to flow. The preferential migration is abolished with anti-VEGF antibodies. MDA-MB-231 cells apoptosis rate was slightly lower in CM from MLO-Y4 cells exposed to flow, while proliferation rate was slightly higher. The current data showed no difference in cancer cells viability and adhesion to collagen between any two groups. On the other hand, it was observed that less MDA-MB-231 cells migrated towards CM from RAW264.7 cells conditioned in CM from MLO-Y4 cells stimulated with flow in comparison to those conditioned in CM from MLO-Y4 cells not stimulated with flow. TRAP staining results confirmed that there were less differentiated osteoclasts when RAW264.7 cells were cultured in CM from MLO-Y4 cells exposed to flow.
Overall, this study suggests that when only osteocytes and cancer cells are involved, osteocytes subjected to mechanical loading can promote metastases due to the increased secretion of VEGF. However, with the incorporation of osteoclasts, mechanical loading on osteocytes seems to reduce MDA-MB-231 cell migration. This is likely because osteocytes reduce osteoclastogenesis in response to mechanical stimulation, and osteoclasts have been shown to support cancer cells. Animal studies will also be conducted to verify the pro- or anti-metastatic effect of mechanical loading that is observed in the in vitro part of this study.
