Most patients who succumb to cancer have metastases to bone that contribute to their death. Cancer cells that metastasize to bone are regularly subjected to mechanical stimuli that may affect their proliferation, growth and protein expression. Understanding why some cancer cells thrive in this environment could provide insight into new approaches to prevent or treat metastasis to bone. We used 4T1 cells as a model of breast cancer cells, and implanted them in gelatin hydrogels with moduli of 1 or 2.7 kPa to mimic the properties of bone marrow. The constructs were subjected to either perfusion of media through the hydrogel or combined perfusion and cyclic mechanical compression for 1 h d −1 for 4 d. Controls were cultured in free-swelling conditions. The cells formed spheroids during the 4 d of culture, with larger spheroids in the statically cultured constructs than in perfusion or compressed constructs. In stiffer gelatin, smaller spheroids formed in compressed constructs than perfusion alone, while compression had no effect compared to perfusion in the softer gelatin. Immunostaining indicated that the spheroids expressed osteopontin, parathyroid hormone-related protein and fibronectin, which are all hallmarks of bone metastasis. The proliferative marker Ki67 was present in all spheroids on day 4. In the 1 kPa gelatin, Ki67 staining intensity was greater in the statically cultured, free-swelling constructs than in bioreactor culture, regardless of dynamic compression. By contrast, proliferation was higher in the compressed gelatins compared to perfusion alone in the 2.7 kPa constructs, although the spheroids were smaller, on average. This suggests the stiffer gelatin may restrict spheroid growth at the same time that it enhances mechanobiological signalling during compression. Taken together, 4T1 breast cancer cells are mechanically sensitive, and mechanical stimuli can alter their proliferation and protein expression within soft materials with mechanical properties similar to bone marrow. As such, both in vivo and in vitro models of cancer metastasis should consider the role of the mechanical environment in the bone.

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Tumor invasion into bone tissue during metastasis alters the biomechanical cues experienced by both tumor and native bone cells. These cues may drive bone lysis and tumor growth, yet how these govern tumor cell proliferation and the mechanobiological role bone cells (osteoblasts, osteoclasts and osteocytes) play in tumor invasion are not yet fully understood. Here, three-dimensional (3D) in vitro co-culture and computational models were first developed to investigate the coupled influence of tumor cell-bone cell signaling and growth induced matrix stress on tumor spheroid evolution. We next developed advanced in vitro engineered 3D models to mimic the complex in vivo multicellular and mechanical environments and confirmed the ability to recapitulate osteoblast, osteoclast and metastatic activity. We applied these models to study the development of osteolytic bone metastasis, in particular to investigate how interactions between tumor/bone cells and their surrounding environment dictate tumor invasion. Our results revealed that tumor spheroid growth is regulated by the synergistic influence of bone cell signaling and microenvironment stiffness. We also demonstrate for the first time in an in vitro 3D model of bone metastasis that the breast cancer cell driven pro-osteoclastogenic effects, which can facilitate cancer metastasis, is inhibited by mechanical compression.