Mechanical Regulation of Breast Cancer Bone Metastasis via Osteocytes

Ma, Yu-Heng 1 ;  Lam, Candy 1 ;  Dalmia, Shreyash 1 ;  Gao, Peter 1 ;  Young, Jacob 1 ;  Liu, Chao 1 ;  Middleton, Kevin 1 ;  You, Lidan 1, 2 ; 

1. Institute of Biomaterials & Biomedical Engineering, University of Toronto; 2. Department of Mechanical and Industrial Engineering, University of Toronto

Bone metastases, the migration of cancers to the bone, are severe cancer complications that occur in 65-80% of patients with advanced breast cancer. Metastasized cancer cells have devastating impacts on bone quality due to their ability to alter bone remodeling by interacting with other cells in the bone such as bone-resorbing osteoclasts. Exercise, often suggested as an intervention for cancer patients, regulates bone remodeling via osteocytes, the mechanosensors of the bone. Several factors secreted by osteocytes in response to mechanical loading have also been shown to affect endothelial permeability, and in turn cancer cell extravasation into the bone. Therefore, we hypothesize that mechanical loading may regulate bone metastases via osteocyte signaling. 

To investigate, osteocytes (MLO-Y4; gift of Dr. Lynda Bonewald, Indiana University) on glass slides were placed in parallel-plate flow chambers and subjected to oscillatory fluid flow (1Pa; 1Hz; 2 hours). Media were then extracted 24-hours after (conditioned media; CM). Osteoclast precursors (RAW264.7) were conditioned in osteocyte CM. Migration of breast cancer cells (MDA-MB-231) towards osteocyte/osteoclast CM was assayed using Transwell. Trans-endothelial migration was done similarly with a confluent layer of endothelial cells (HUVEC) on Transwells. Apoptosis of cancer cells in the CM were measured with APOPercentage. 

It was observed that CM from flow-stimulated osteocytes significantly increased the migration, while slightly reducing apoptosis, of breast cancer cells, compared to CM from non-stimulated osteocytes. In contrast, CM from osteoclasts conditioned in CM from flow-stimulated osteocytes reduced the migration and increased the apoptosis of breast cancer cells. TRAP-staining results confirmed that fewer osteoclasts differentiated when precursor cells were cultured in CM from osteocytes exposed to flow. Breast cancer cells migration across endothelial cells was also reduced towards CM from flow-stimulated osteocytes.

In conclusion, this study suggests that when only osteocytes and cancer cells are involved, osteocytes subjected to mechanical loading can promote metastases. However, with the incorporation of osteoclasts, mechanical stimulation on osteocytes seems to be anti-metastatic. This is likely because flow-stimulated osteocytes reduce osteoclastogenesis, and osteoclasts have been shown to support cancer cells. Mechanically stimulated osteocytes also reduce the extravasation of breast cancer cells across endothelial cells, thus reducing metastases. Micro-channel co-cultures may be used to more closely mimic real-time cell-cell interaction that exists in vivo. Animal studies will also be conducted to verify the observed pro- or anti-metastatic effect of bone mechanical loading. Examining the effect of mechanical loading on metastases and its mechanism will assist in designing cancer intervention programs that lowers the risk for bone metastases.