The Role of Osteocytes in Pre-metastatic Niche Formation.

PURPOSE OF REVIEW: The formation of a pre-metastatic niche (PMN), in which primary cancer cells prime the distant site to be favorable to their engraftment and survival, may help explain the strong osteotropism observed in multiple cancers, such as breast and prostate. PMN formation, which includes extracellular matrix remodeling, increased angiogenesis and vascular permeability, enhanced bone marrow-derived cell recruitment and immune suppression, has mostly been described in soft tissues. In this review, we summarize current literature of PMN formation in bone. We also present evidence of a potential role for osteocytes to be the primary mediators of PMN development. RECENT FINDINGS: Osteocytes regulate the bone microenvironment in myriad ways beyond canonical bone tissue remodeling, including changes that contribute to PMN formation. Perilacunar tissue remodeling, which has been observed in both bone and non-bone metastatic cancers, is a potential mechanism by which osteocyte-cancer cell signaling stimulates changes to the bone microenvironment. Osteocytes also protect against endothelial permeability, including that induced by cancer cells, in a loading-mediated process. Finally, osteocytes are potent regulators of cells within the bone marrow, including progenitors and immune cells, and might be involved in this aspect of PMN formation. Osteocytes should be examined for their role in PMN formation.

Bone-homing metastatic breast cancer cells impair osteocytes' mechanoresponse in a 3D loading model.

Breast cancer predominantly metastasizes to the skeleton. Mechanical loading is reliably anabolic in bone, and also inhibits bone metastatic tumor formation and bone loss in vivo. To study the underlying mechanisms, we developed a 3D culture model for osteocytes, the primary bone mechanosensor. We verified that MLO-Y4s responded to perfusion by reducing their rankl and rankl:opg gene expression. We next cultured MLO-Y4s with tumor-conditioned media (TCM) collected from human breast cancer cells (MDA-MB-231s) and a corresponding bone-homing subclone to test the impacts on osteocytes' mechanosensation. We found that TCM from the bone-homing subclone was more detrimental to MLO-Y4 growth and viability, and it abrogated loading-induced changes to rankl:opg. Our studies demonstrate that MLO-Y4s, including their mechanoresponse to perfusion, were more negatively impacted by soluble factors from bone-homing breast cancer cells compared to those from parental cells.

Mechanobiology of Bone Metastatic Cancer.

PURPOSE OF REVIEW: In this review, we provide an overview of what is currently known about the impacts of mechanical stimuli on metastatic tumor-induced bone disease (TIBD). Further, we focus on the role of the osteocyte, the skeleton's primary mechanosensory cell, which is central to the skeleton's mechanoresponse, sensing and integrating local mechanical stimuli, and then controlling the downstream remodeling balance as appropriate. RECENT FINDINGS: Exercise and controlled mechanical loading have anabolic effects on bone tissue in models of bone metastasis. They also have anti-tumorigenic properties, in part due to offsetting the vicious cycle of osteolytic bone loss as well as regulating inflammatory signals. The impacts of metastatic cancer on the mechanosensory function of osteocytes remains unclear. Increased mechanical stimuli are a potential method for mitigating TIBD.

Application of machine learning classifiers for microcomputed tomography data assessment of mouse bone microarchitecture.

The current standard approach for analyzing cortical bone structure and trabecular bone microarchitecture from micro-computed tomography (microCT) is through classic parametric (e.g., ANOVA, Student's T-test) and nonparametric (e.g., Mann-Whitney U test) statistical tests and the reporting of p-values to indicate significance. However, on their own, these univariate assessments of significance fall prey to a number of weaknesses, including an increased chance of Type 1 error from multiple comparisons. Machine learning classification methods (e.g., unsupervised, k-means cluster analysis and supervised Support Vector Machine classification, SVM) simultaneously utilize an entire dataset comprised of many cortical structure or trabecular microarchitecture measures, thus minimizing bias and Type 1 error that are generated through multiple testing. Through simultaneous evaluation of an entire dataset, k-means and SVM thus provide a complementary approach to classic statistical analysis and enable a more robust assessment of microCT measures.

Flow inside a bone scaffold: Visualization using 3D phase contrast MRI and comparison with numerical simulations.

We report on results of experimental flow measurements inside a bone scaffold model, subjected to a uniform incoming flow (applied perfusion). Understanding the flow behavior inside a tissue engineered scaffold is essential for mechanistic studies of mechanobiology, particularly flow-sensitive bone cells. Nearly all existing studies that quantify interstitial flow inside engineered bone scaffolds have been based on numerical results, in part due to the difficulties associated with quantitative measurements and visualization of flow inside large, opaque bone or bone mimics. Thus, an experimental platform to complement and validate in silico studies is needed. Therefore, we developed a flow visualization method using Phase-Contrast Magnetic Resonance Imaging (PC-MRI) to measure flow velocities within a 3D-printed microCT-based rendering of a bone scaffold. We designed and built a non-magnetic recirculating water tunnel to apply uniform perfusion to the 3D-printed model and we measured flow distribution within the scaffold and compared these experimental results with CFD results. Both magnitude and distribution of flow velocities observed at different slices of the scaffold were in quantitative agreement numerically and experimentally. This experimental approach can be used to both validate numerical studies and provide insight into the flow behavior inside tissue-engineered scaffolds for a range of applications, including fundamental mechanobiology of healthy cells, and in the context of diseases, such as cancer.

Microgravity-induced alterations of mouse bones are compartment- and site-specific and vary with age.

The age at which astronauts experience microgravity is a critical consideration for skeletal health and similarly has clinical relevance for musculoskeletal disuse on Earth. While astronauts are extensively studied for bone and other physiological changes, rodent studies enable direct evaluation of skeletal changes with microgravity. Yet, mouse spaceflight studies have predominately evaluated tissues from young, growing mice. We evaluated bone microarchitecture in tibiae and femurs from Young (9-week-old) and Mature (32-weeks-old) female, C57BL/6N mice flown in microgravity for ~2 and ~3 weeks, respectively. Microgravity-induced changes were both compartment- and site-specific. Changes were greater in trabecular versus cortical bone in Mature mice exposed to microgravity (-40.0% Tb. BV/TV vs -4.4% Ct. BV/TV), and bone loss was greater in the proximal tibia as compared to the distal femur. Trabecular thickness in Young mice increased by +25.0% on Earth and no significant difference following microgravity. In Mature mice exposed to microgravity, trabecular thickness rapidly decreased (-24.5%) while no change was detected in age-matched mice that were maintained on Earth. Mature mice exposed to microgravity experienced greater bone loss than Young mice with net skeletal growth. Moreover, machine learning classification models confirmed that microgravity exposure-driven decrements in trabecular microarchitecture and cortical structure occurred disproportionately in Mature than in Young mice. Our results suggest that age of disuse onset may have clinical implications in osteoporotic or other at-risk populations on Earth and may contribute to understanding bone loss patterns in astronauts.

Multiphysics simulation of a compression-perfusion combined bioreactor to predict the mechanical microenvironment during bone metastatic breast cancer loading experiments.

Incurable breast cancer bone metastasis causes widespread bone loss, resulting in fragility, pain, increased fracture risk, and ultimately increased patient mortality. Increased mechanical signals in the skeleton are anabolic and protect against bone loss, and they may also do so during osteolytic bone metastasis. Skeletal mechanical signals include interdependent tissue deformations and interstitial fluid flow, but how metastatic tumor cells respond to each of these individual signals remains underinvestigated, a barrier to translation to the clinic. To delineate their respective roles, we report computed estimates of the internal mechanical field of a bone mimetic scaffold undergoing combinations of high and low compression and perfusion using multiphysics simulations. Simulations were conducted in advance of multimodal loading bioreactor experiments with bone metastatic breast cancer cells to ensure that mechanical stimuli occurring internally were physiological and anabolic. Our results show that mechanical stimuli throughout the scaffold were within the anabolic range of bone cells in all loading configurations, were homogenously distributed throughout, and that combined high magnitude compression and perfusion synergized to produce the largest wall shear stresses within the scaffold. These simulations, when combined with experiments, will shed light on how increased mechanical loading in the skeleton may confer anti-tumorigenic effects during metastasis.

Mechanical loading prevents bone destruction and exerts anti-tumor effects in the MOPC315.BM.Luc model of myeloma bone disease.

Bone continually adapts to changing external loading conditions via (re)modeling (modeling and remodeling) processes. While physical activity is known to beneficially enhance bone mass in healthy individuals, little is known in how physical stimuli affect osteolytic bone destruction in patients suffering from multiple myeloma bone disease. Multiple myeloma (MM) is caused by malignant plasma cells in the bone marrow, shifting the balance in bone remodeling towards massive resorption. We hypothesized that in vivo tibial mechanical loading has anabolic effects in mice with locally injected MOPC315.BM.Luc cells. Conventional microCT analysis revealed enhanced cortical bone mass and microstructure in loaded compared to nonloaded mice. State-of-the-art time-lapse microCT based image analysis demonstrated bone (re)modeling processes at the endosteal and periosteal surfaces as the underlying causes of increased bone mass. Loading prevented the progression and development of osteolytic destruction. Physical stimuli also diminished local MM cell growth and dissemination evidenced by quantification of MM cell-specific immunoglobulin A levels in the serum of mice and by bioluminescence analysis. These data indicate that mechanical loading not only rescues the bone phenotype, but also exerts cell-extrinsic anti-myeloma effects in the MOPC315.BM.Luc model. In conclusion, the use of physical stimuli should be further investigated as an anabolic treatment for osteolytic bone destruction in patients with MM.

Mechanically-Loaded Breast Cancer Cells Modify Osteocyte Mechanosensitivity by Secreting Factors That Increase Osteocyte Dendrite Formation and Downstream Resorption.

Advanced breast cancer predominantly metastasizes to the skeleton, at which point patient prognosis significantly declines concomitant with bone loss, pain, and heightened fracture risk. Given the skeleton's sensitivity to mechanical signals, increased mechanical loading is well-documented to increase bone mass, and it also inhibited bone metastatic tumor formation and progression in vivo, though the underlying mechanisms remain under investigation. Here, we focus on the role of the osteocyte because it is the primary skeletal mechanosensor and in turn directs the remodeling balance between formation and resoprtion. In particular, osteocytic dendrites are important for mechanosensing, but how this function is altered during bone metastatic breast cancer is unknown. To examine how breast cancer cells modulate dendrite formation and function, we exposed osteocytes (MLO-Y4) to medium conditioned by breast cancer cells (MDA-MB231) and to applied fluid flow (2 h per day for 3 days, shear stress 1.1 Pa). When loading was applied to MLOs, dendrite formation increased despite the presence of tumor-derived factors while overall MLO cell number was reduced. We then exposed MLOs to fluid flow as well as media conditioned by MDAs that had been similarly loaded. When nonloaded MLOs were treated with conditioned media from loaded MDAs, their dendrite formation increased in a manner similar to that observed due to loading alone. When MLOs simultaneously underwent loading and treatment with loaded conditioned media, dendrite formation was greatest. To understand potential molecular mechanisms, we then investigated expression of genes related to osteocyte maturation and dendrite formation (E11) and remodeling (RANKL, OPG) as well as osteocyte apoptosis. E11 expression increased with loading, consistent with increased dendrite formation. Though loaded conditioned media decreased MLO cell number, apoptosis was not detected via TUNEL staining, suggesting an inhibition of growth instead. OPG expression was inhibited while RANKL expression was unaffected, leading to an overall increase in the RANKL/OPG ratio with conditioned media from loaded breast cancer cells. Taken together, our results suggest that skeletal mechanical loading stimulates breast cancer cells to alter osteocyte mechanosensing by increasing dendrite formation and downstream resorption.

Perfusion applied to a 3D model of bone metastasis results in uniformly dispersed mechanical stimuli.

Breast cancer most frequently metastasizes to the skeleton. Bone metastatic cancer is incurable and induces wide-spread bone osteolysis, resulting in significant patient morbidity and mortality. Mechanical cues in the skeleton are an important microenvironmental parameter that modulate tumor formation, osteolysis, and tumor cell-bone cell signaling, but which mechanical signals are the most beneficial and the corresponding molecular mechanisms are unknown. We focused on interstitial fluid flow based on its well-known role in bone remodeling and in primary breast cancer. We created a full-scale, microCT-based computational model of a 3D model of bone metastasis undergoing applied perfusion to predict the internal mechanical environment during in vitro experimentation. Applied perfusion resulted in uniformly dispersed, heterogeneous fluid velocities, and wall shear stresses throughout the scaffold's interior. The distributions of fluid velocity and wall shear stress did not change within model sub-domains of varying diameter and location. Additionally, the magnitude of these stimuli is within the range of anabolic mechanical signals in the skeleton, verifying that our 3D model reflects previous in vivo studies using anabolic mechanical loading in the context of bone metastasis. Our results indicate that local populations of cells throughout the scaffold would experience similar mechanical microenvironments.

Three-Dimensional Mechanical Loading Modulates the Osteogenic Response of Mesenchymal Stem Cells to Tumor-Derived Soluble Signals.

Dynamic mechanical loading is a strong anabolic signal in the skeleton, increasing osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) and increasing the bone-forming activity of osteoblasts, but its role in bone metastatic cancer is relatively unknown. In this study, we integrated a hydroxyapatite-containing three-dimensional (3D) scaffold platform with controlled mechanical stimulation to investigate the effects of cyclic compression on the interplay between breast cancer cells and BM-MSCs as it pertains to bone metastasis. BM-MSCs cultured within mineral-containing 3D poly(lactide-co-glycolide) (PLG) scaffolds differentiated into mature osteoblasts, and exposure to tumor-derived soluble factors promoted this process. When BM-MSCs undergoing osteogenic differentiation were exposed to conditioned media collected from mechanically loaded breast cancer cells, their gene expression of osteopontin was increased. This was further enhanced when mechanical compression was simultaneously applied to BM-MSCs, leading to more uniformly deposited osteopontin within scaffold pores. These results suggest that mechanical loading of 3D scaffold-based culture models may be utilized to evaluate the role of physiologically relevant physical cues on bone metastatic breast cancer. Furthermore, our data imply that cyclic mechanical stimuli within the bone microenvironment modulate interactions between tumor cells and BM-MSCs that are relevant to bone metastasis.

Biomechanical forces in the skeleton and their relevance to bone metastasis: biology and engineering considerations.

Bone metastasis represents the leading cause of breast cancer related-deaths. However, the effect of skeleton-associated biomechanical signals on the initiation, progression, and therapy response of breast cancer bone metastasis is largely unknown. This review seeks to highlight possible functional connections between skeletal mechanical signals and breast cancer bone metastasis and their contribution to clinical outcome. It provides an introduction to the physical and biological signals underlying bone functional adaptation and discusses the modulatory roles of mechanical loading and breast cancer metastasis in this process. Following a definition of biophysical design criteria, in vitro and in vivo approaches from the fields of bone biomechanics and tissue engineering that may be suitable to investigate breast cancer bone metastasis as a function of varied mechano-signaling will be reviewed. Finally, an outlook of future opportunities and challenges associated with this newly emerging field will be provided.

Load-induced changes in bone stiffness and cancellous and cortical bone mass following tibial compression diminish with age in female mice.

The vertebrate skeleton is an adaptive structure that responds to mechanical stimuli by increasing bone mass under increased mechanical loads. Although experimental animal models have shown the anabolic cortical bone response to applied load decreases with age, no consensus exists regarding whether this adaptive mechanism is affected by age in cancellous bone, the tissue most impacted by age-related bone loss. We used an established murine in vivo tibial loading model to characterize the load-induced cancellous, cortical and whole-bone responses to mechanical stimuli in growing and mature female mice at 6, 10 and 16 weeks of age. The effects of applied load on tibial morphology and stiffness were determined using microcomputed tomography and in vivo bone strains measured at the medial tibial midshaft during applied loading. At all ages, 2 weeks of applied load produced larger midshaft cortical cross-sectional properties (+13-72%) and greater cancellous bone volume (+21-107%) and thicker trabeculae (+31-68%) in the proximal metaphyses of the loaded tibiae. The relative anabolic response decreased from 6 to 16 weeks of age in both the cancellous and cortical envelopes. Load-induced tibial stresses decreased more in 6-week-old mice following loading, which corresponded to increased in vivo tibial stiffness. Stiffness in the loaded tibiae of 16-week-old mice decreased despite moderately increased cortical cross-sectional geometry, suggesting load-induced changes in bone material properties. This study shows that the cancellous and cortical anabolic responses to mechanical stimuli decline with age into adulthood and that cortical cross-sectional geometry alone does not necessarily predict whole-bone functional stiffness.

Engineered culture models for studies of tumor-microenvironment interactions.

Heterogeneous microenvironmental conditions play critical roles in cancer pathogenesis and therapy resistance and arise from changes in tissue dimensionality, cell-extracellular matrix (ECM) interactions, soluble factor signaling, oxygen as well as metabolic gradients, and exogeneous biomechanical cues. Traditional cell culture approaches are restricted in their ability to mimic this complexity with physiological relevance, offering only partial explanation as to why novel therapeutic compounds are frequently efficacious in vitro but disappoint in preclinical and clinical studies. In an effort to overcome these limitations, physical sciences-based strategies have been employed to model specific aspects of the cancer microenvironment. Although these strategies offer promise to reveal the contributions of microenvironmental parameters on tumor initiation, progression, and therapy resistance, they, too, frequently suffer from limitations. This review highlights physicochemical and biological key features of the tumor microenvironment, critically discusses advantages and limitations of current engineering strategies, and provides a perspective on future opportunities for engineered tumor models.

In vivo tibial compression decreases osteolysis and tumor formation in a human metastatic breast cancer model.

Bone metastasis, the leading cause of breast cancer-related deaths, is characterized by bone degradation due to increased osteoclastic activity. In contrast, mechanical stimulation in healthy individuals upregulates osteoblastic activity, leading to new bone formation. However, the effect of mechanical loading on the development and progression of metastatic breast cancer in bone remains unclear. Here, we developed a new in vivo model to investigate the role of skeletal mechanical stimuli on the development and osteolytic capability of secondary breast tumors. Specifically, we applied compressive loading to the tibia following intratibial injection of metastatic breast cancer cells (MDA-MB231) into the proximal compartment of female immunocompromised (SCID) mice. In the absence of loading, tibiae developed histologically-detectable tumors with associated osteolysis and excessive degradation of the proximal bone tissue. In contrast, mechanical loading dramatically reduced osteolysis and tumor formation and increased tibial cancellous mass due to trabecular thickening. These loading effects were similar to the baseline response we observed in non-injected SCID mice. In vitro mechanical loading of MDA-MB231 in a pathologically relevant 3D culture model suggested that the observed effects were not due to loading-induced tumor cell death, but rather mediated via decreased expression of genes interfering with bone homeostasis. Collectively, our results suggest that mechanical loading inhibits the growth and osteolytic capability of secondary breast tumors after their homing to the bone, which may inform future treatment of breast cancer patients with advanced disease.

Tibial compression is anabolic in the adult mouse skeleton despite reduced responsiveness with aging.

The ability of the skeleton to adapt to mechanical stimuli diminishes with age in diaphyseal cortical bone, making bone formation difficult for adults. However, the effect of aging on adaptation in cancellous bone, tissue which is preferentially lost with age, is not well characterized. To develop a model for early post-menopausal women and determine the effect of aging on cancellous bone adaptation in the adult mouse skeleton, in vivo tibial compression was applied to adult (26 week old) osteopenic female mice using loading parameters, peak applied load and peak diaphyseal strain magnitude, that were previously found to be osteogenic in young, growing (10 week old) mice. A Load-Matched group received the same peak applied loads (corresponding to +2100 µÎµ at the medial diaphysis of the tibia) and a Strain-Matched group received the same peak diaphyseal strains (+1200 µÎµ, requiring half the load) as the young mice. The effects of mechanical loading on bone mass and architecture in adult mice were assessed using micro-computed tomography and in vivo structural stiffness measures. Adaptation occurred only in the Load-Matched group in both the metaphyseal and diaphyseal compartments. Cancellous bone mass increased 54% through trabecular thickening, and cortical area increased 41% through medullary contraction and periosteal expansion. Adult mice were able to respond to an anabolic stimulus and recover bone mass to levels seen in growing mice; however, the adaptive response was reduced relative to that in 10 week old female mice for the same applied load. Using this osteogenic loading protocol, other factors affecting pathological bone loss can be addressed using an adult osteopenic mouse model.

In vivo tibial stiffness is maintained by whole bone morphology and cross-sectional geometry in growing female mice.

Whole bone morphology, cortical geometry, and tissue material properties modulate skeletal stresses and strains that in turn influence skeletal physiology and remodeling. Understanding how bone stiffness, the relationship between applied load and tissue strain, is regulated by developmental changes in bone structure and tissue material properties is important in implementing biophysical strategies for promoting healthy bone growth and preventing bone loss. The goal of this study was to relate developmental patterns of in vivo whole bone stiffness to whole bone morphology, cross-sectional geometry, and tissue properties using a mouse axial loading model. We measured in vivo tibial stiffness in three age groups (6, 10, 16 wk old) of female C57Bl/6 mice during cyclic tibial compression. Tibial stiffness was then related to cortical geometry, longitudinal bone curvature, and tissue mineral density using microcomputed tomography (microCT). Tibial stiffness and the stresses induced by axial compression were generally maintained from 6 to 16 wks of age. Growth-related increases in cortical cross-sectional geometry and longitudinal bone curvature had counteracting effects on induced bone stresses and, therefore, maintained tibial stiffness similarly with growth. Tissue mineral density increased slightly from 6 to 16 wks of age, and although the effects of this increase on tibial stiffness were not directly measured, its role in the modulation of whole bone stiffness was likely minor over the age range examined. Thus, whole bone morphology, as characterized by longitudinal curvature, along with cortical geometry, plays an important role in modulating bone stiffness during development and should be considered when evaluating and designing in vivo loading studies and biophysical skeletal therapies.

Cancellous bone adaptation to tibial compression is not sex dependent in growing mice.

Mechanical loading can be used to increase bone mass and thus attenuate pathological bone loss. Because the skeleton's adaptive response to loading is most robust before adulthood, elucidating sex-specific responses during growth may help maximize peak bone mass. This study investigated the effect of sex on the response to controlled, in vivo mechanical loading in growing mice. Ten-week-old male and female C57Bl/6 mice underwent noninvasive compression of the left tibia. Peak loads of -11.5 N were applied, corresponding to +1,200 microepsilon at the tibial midshaft in both sexes. Cancellous bone mass, architecture, and dynamic formation in the proximal metaphysis were compared between loaded and control limbs via micro-computed tomography and histomorphometry. The strain environment of the proximal metaphysis during loading was characterized using finite element analysis. Both sexes responded to tibial compression through increased bone mass and altered architecture. Cancellous bone mass and tissue density were enhanced in loaded limbs relative to control limbs in both sexes through trabecular thickening and reduced separation. Changes in mass were due to increased cellular activity in loaded limbs compared with control limbs. Adaptation to loading increased the proportion of load transferred by the cancellous bone in the proximal metaphysis. For all cancellous measures, the response to tibial compression did not differ between male and female mice. When similar strains are engendered in males and females, the adaptive response in cancellous bone to mechanical loading does not depend on sex.

Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons.

Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin-like growth factor-I (IGF-I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs, as well as IGF-I gene-enhanced MSCs (AdIGF-MSCs) on tendon healing in vivo. Collagenase-induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Tendons were treated with 10 x 10(6) MSCs or 10 x 10(6) AdIGF-MSCs. Control limbs were injected with 1 mL of phosphate-buffered saline (PBS). Ultrasound examinations were performed at t = 0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure and evaluated for biochemical composition and histologic characteristics. Expression of collagen types I and III, IGF-I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), and aggrecanase-1 (ADAMTS-4) were similar in MSC and control tendons. Both MSC and AdIGF-MSC injection resulted in significantly improved tendon histological scores. These findings indicate a benefit to the use of MSCs and AdIGF-MSCs for the treatment of tendinitis.