A mechanobiological model of bone metastasis reveals that mechanical stimulation inhibits the pro-osteolytic effects of breast cancer cells.

Bone is highly susceptible to cancer metastasis, and both tumor and bone cells enable tumor invasion through a "vicious cycle" of biochemical signaling. Tumor metastasis into bone also alters biophysical cues to both tumor and bone cells, which are highly sensitive to their mechanical environment. However, the mechanobiological feedback between these cells that perpetuate this cycle has not been studied. Here, we develop highly advanced in vitro and computational models to provide an advanced understanding of how tumor growth is regulated by the synergistic influence of tumor-bone cell signaling and mechanobiological cues. In particular, we develop a multicellular healthy and metastatic bone model that can account for physiological mechanical signals within a custom bioreactor. These models successfully recapitulated mineralization, mechanobiological responses, osteolysis, and metastatic activity. Ultimately, we demonstrate that mechanical stimulus provided protective effects against tumor-induced osteolysis, confirming the importance of mechanobiological factors in bone metastasis development.

Mechanoregulation may drive osteolysis during bone metastasis: A finite element analysis of the mechanical environment within bone tissue during bone metastasis and osteolytic resorption.

Metastatic bone disease occurs in 70-80% of advanced breast cancer patients and bone tissue is accepted to have attractive physical properties that facilitate cancer cell attraction, adhesion, and invasion. Bone cells also facilitate tumour invasion by biochemical signalling and through resorption of the bone matrix (osteolysis), which releases factors that further stimulate tumour cell activity. The evolving mechanical environment during tumour invasion might play an important role in these processes, as the activity of both bone and cancer cells is regulated by mechanical cues. In particular bone loss and altered mineralisation have been reported, yet how these alter the mechanical environment local to bone and tumour cells is unknown. The objective of this study is to quantify changes in the mechanical environment within bone tissue, during bone metastasis and osteolytic resorption, using finite element analysis (FEA) models reconstructed from high-resolution µCT images of metastatic mouse bone. In particular, we quantify time-dependent changes in mechanical stimuli, local to and distant from an invading tumour mass, to investigate putative mechanobiological cues for osteolysis during bone metastasis. We report here that in early metastasis (3 weeks after tumour inoculation), there was a decrease in strain distribution within the proximal femur trabecular and distal cortical bone tissue. These changes in the mechanical environment preceded extensive osteolytic destruction, but coincided with the onset of early osteolysis, cortical thickening and mineralisation of proximal and distal femur bone. We propose that early changes in the mechanical environment within bone tissue may activate resorption by osteoclast cells and thereby contribute to the extensive osteolytic bone loss at later stage (6 weeks) bone metastasis.

Temporal and spatial changes in bone mineral content and mechanical properties during breast-cancer bone metastases.

Cancer cells favour migration and metastasis to bone tissue for 70-80 % of advanced breast cancer patients and it has been proposed that bone tissue provides attractive physical properties that facilitate tumour invasion, resulting in osteolytic and or osteoblastic metastasis. However, it is not yet known how specific bone tissue composition is associated with tumour invasion. In particular, how compositional and nano-mechanical properties of bone tissue evolve during metastasis, and where in the bone they arise, may affect the overall aggressiveness of tumour invasion, but this is not well understood. The objective of this study is to develop an advanced understanding of temporal and spatial changes in nano-mechanical properties and composition of bone tissue during metastasis. Primary mammary tumours were induced by inoculation of immune-competent BALB/c mice with 4T1 breast cancer cells in the mammary fat pad local to the right femur. Microcomputed tomography and nanoindentation were conducted to quantify cortical and trabecular bone matrix mineralisation and nano-mechanical properties. Analysis was performed in proximal and distal femur regions (spatial analysis) of tumour-adjacent (ipsilateral) and contralateral femurs after 3 weeks and 6 weeks of tumour and metastasis development (temporal analysis). By 3 weeks post-inoculation there was no significant difference in bone volume fraction or nano-mechanical properties of bone tissue between the metastatic femora and healthy controls. However, early osteolysis was indicated by trabecular thinning in the distal and proximal trabecular compartment of tumour-bearing femora. Moreover, cortical thickness was significantly increased in the distal region, and the mean mineral density was significantly higher in cortical and trabecular bone tissue in both proximal and distal regions, of ipsilateral (tumour-bearing) femurs compared to healthy controls. By 6 weeks post-inoculation, overt osteolytic lesions were identified in all ipsilateral metastatic femora, but also in two of four contralateral femora of tumour-bearing mice. Bone volume fraction, cortical area, cortical and trabecular thickness were all significantly decreased in metastatic femora (both ipsilateral and contralateral). Trabecular bone tissue stiffness in the proximal femur decreased in the ipsilateral femurs compared to contralateral and control sites. Temporal and spatial analysis of bone nano-mechanical properties and mineralisation during breast cancer invasion reveals changes in bone tissue composition prior to and following overt metastatic osteolysis, local and distant from the primary tumour site. These changes may alter the mechanical environment of both the bone and tumour cells, and thereby play a role in perpetuating the cancer vicious cycle during breast cancer metastasis to bone tissue.

A Novel 3D Osteoblast and Osteocyte Model Revealing Changes in Mineralization and Pro-osteoclastogenic Paracrine Signaling During Estrogen Deficiency.

Recent in vitro studies have revealed that the mechanobiological responses of osteoblasts and osteocytes are fundamentally impaired during estrogen deficiency. However, these two-dimensional (2D) cell culture studies do not account for in vivo biophysical cues. Thus, the objectives of this study are to (1) develop a three-dimensional (3D) osteoblast and osteocyte model integrated into a bioreactor and (2) apply this model to investigate whether estrogen deficiency leads to changes in osteoblast to osteocyte transition, mechanosensation, mineralization, and paracrine signaling associated with bone resorption by osteoclasts. MC3T3-E1s were expanded in media supplemented with estrogen (17ß-estradiol). These cells were encapsulated in gelatin-mtgase before culture in (1) continued estrogen (E) or (2) no further estrogen supplementation. Constructs were placed in gas permeable and water impermeable cell culture bags and maintained at 5% CO2 and 37°C. These bags were either mechanically stimulated in a custom hydrostatic pressure (HP) bioreactor or maintained under static conditions (control). We report that osteocyte differentiation, characterized by the presence of dendrites and staining for osteocyte marker dentin matrix acidic phosphoprotein 1 (DMP1), was significantly greater under estrogen withdrawal (EW) compared to under continuous estrogen treatment (day 21). Mineralization [bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (ALP), calcium] and gene expression associated with paracrine signaling for osteoclastogenesis [receptor activator of nuclear factor kappa-ß ligand (RANKL)/osteoprotegerin OPG ratio] were significantly increased in estrogen deficient and mechanically stimulated cells. Interestingly, BSP and DMP-1 were also increased at day 1 and day 21, respectively, which play a role in regulation of biomineralization. Furthermore, the increase in pro-osteoclastogenic signaling may be explained by altered mechanoresponsiveness of osteoblasts or osteocytes during EW. These findings highlight the impact of estrogen deficiency on bone cell function and provide a novel in vitro model to investigate the mechanisms underpinning changes in bone cells after estrogen deficiency.