Bone metastasis is a common, yet serious, complication of breast cancer. Breast cancer cells that extravasate from blood vessels to the bone devastate bone quality by interacting with bone cells and disrupting the bone remodeling balance. Although exercise is often suggested as a cancer intervention strategy and mechanical loading during exercise is known to regulate bone remodeling, its role in preventing bone metastasis remains unknown. We developed a novel in vitro microfluidic tissue model to investigate the role of osteocytes in the mechanical regulation of breast cancer bone metastasis. Metastatic MDA-MB-231 breast cancer cells were cultured inside a 3D microfluidic lumen lined with human umbilical vein endothelial cells (HUVECs), which is adjacent to a channel seeded with osteocyte-like MLO-Y4 cells. Physiologically relevant oscillatory fluid flow (OFF) (1 Pa, 1 Hz) was applied to mechanically stimulate the osteocytes. Hydrogel-filled side channels in-between the two channels allowed real-time, bi-directional cellular signaling and cancer cell extravasation over 3 days. The applied OFF was capable of inducing intracellular calcium responses in osteocytes (82.3% cells responding with a 3.71 fold increase average magnitude). Both extravasation distance and percentage of extravasated side-channels were significantly reduced with mechanically stimulated osteocytes (32.4% and 53.5% of control, respectively) compared to static osteocytes (102.1% and 107.3% of control, respectively). This is the first microfluidic device that has successfully integrated stimulatory bone fluid flow, and demonstrated that mechanically stimulated osteocytes reduced breast cancer extravasation. Future work with this platform will determine the specific mechanisms involved in osteocyte mechanoregulation of breast cancer bone metastasis, as well as other types of cancer metastasis and diseases.

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.

Osteocytes' mechano-regulation of bone formation and resorption is key to maintaining appropriate bone health. Although extensive in vitro studies have explored osteocyte mechanobiology using the well-established MLO-Y4 cell model, the low amount of sclerostin secreted by this cell line renders it inadequate for studying cross-talk between osteocytes and osteoblasts under mechanical loading. Here, we investigated the potential of the sclerostin-expressing OCY454 osteocyte cell model in fulfilling this role. Fully differentiated OCY454 cells were tested for mechano-sensitivity by measuring changes in protein secretion, total adenosine triphosphate (ATP) content, and intracellular calcium in response to oscillatory fluid flow. Increases in sclerostin expression and total ATP content were observed. However, very low levels of receptor activator of the nuclear factor κ-B ligand were detected, and there was a great inconsistency in calcium response. Conditioned medium (CM) from OCY454 cells was then used to culture osteoblast and osteoclast precursors. Osteoblast activity was quantified with alkaline phosphatase (ALP) and Alizarin Red S stain, while osteoclast differentiation was quantified with tartrate-resistant acid phosphatase (TRAP) staining. We demonstrated that mechanically stimulated OCY454 cells released soluble factors that increased osteoblasts' ALP activity (p < 0.05) and calcium deposition (p < 0.05). There was also a significant decrease of large-sized TRAP-positive osteoclasts when osteoclast precursors were treated with CM from flow-stimulated OCY454 cells (p < 0.05). Results from this study suggest that OCY454 cells respond to mechanical loading with the release of key factors such as sclerostin to regulate downstream bone cells, thus demonstrating its potential as a novel cell model for in vitro osteocyte mechanobiology studies.

Multiple myeloma (MM), the disorder of plasma cells, is the second most common type of hematological cancer and is responsible for approximately 20% of deaths from hematological malignancies. The current gold standard for MM diagnosis includes invasive bone marrow aspiration. However, it lacks the sensitivity to detect minimal residual disease, and the nonuniform distribution of clonal plasma cells (CPCs) within bone marrow also often results in inaccurate reporting. Serum and urine assessment of monoclonal proteins, such as Kappa light chains, is another commonly used approach for MM diagnosis. Although it is noninvasive, the level of paraprotein elevation is still too low for detecting minimal residual disease and nonsecretive MM. Circulating CPCs (cCPCs) have been reported to be present in the peripheral blood of MM patients, and high levels of cCPCs were shown to correlate with poor survival. This suggests a potential noninvasive approach for MM disease progress monitoring and prognosis. In this study, we developed a mechanical property-based microfluidic platform to capture cCPCs. Using human myeloma cancer cell lines spiked in healthy donor blood, the microfluidic platform demonstrates high enrichment ratio (&gt;500) and sufficient capture efficiency (40%–55%). Patient samples were also assessed to investigate the diagnostic potential of cCPCs for MM by correlating with the levels of Kappa light chains in patients.

Osteocytes are the major mechanosensing cells in bone remodeling. Current in vitro bone mechanotransduction research use macroscale devices such as flow chambers; however, in vitro microfluidic devices provide an optimal tool to better understand this biological process with its flexible design, physiologically relevant dimensions and high-throughput capabilities. This project aims to design and fabricate a multi-shear stress, co-culture platform to study the interaction between osteocytes and other bone cells under varying flow conditions. Standard microfluidic design utilizing changing geometric parameters is used to induce different flow rates that are directly proportional to the levels of shear stress, with devices fabricated from standard polydimethylsiloxane (PDMS)-based softlithography processes. Each osteocyte channel (OCY) is connected to an adjacent osteoclast channel (OC) by 20-μm perfusion channels for cellular signaling molecule transport. Significant differences in RANKL levels are observed between channels with different shear stress levels, and we observed that pre-osteoclast differentiation was directly affected by adjacent flow-stimulated osteocytes. Significant decrease in the number of differentiating osteoclasts is observed in the OC channel adjacent to the 2-Pa shear stress OCY channel, while differentiation adjacent to the 0.5-Pa shear stress OCY channel is unaffected compared with no-flow controls. Addition of zoledronic acid showed a significant decrease in osteoclast differentiation, compounding to effect instigated by increasing fluid shear stress. Using this platform, we are able to mimic the interaction between osteocytes and osteoclasts in vitro under physiologically relevant bone interstitial fluid flow shear stress. Our novel microfluidic co-culture platform provides an optimal tool for bone cell mechanistic studies and provides a platform for the discovery of potential drug targets for clinical treatments of bone-related diseases.