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Introduction: Globally, 300 million adults have clinical obesity. Heightened adiposity and inadequate musculature secondary to obesity alter bipedal stance and gait, diminish musculoskeletal tissue quality, and compromise neuromuscular feedback; these physiological changes alter stability and increase injury risk from falls. Studies in the field focus on obese patients across a broad range of body mass indices (BMI >30 kg/m2) but without isolating the most morbidly obese subset (BMI ≥40 kg/m2). We investigated the impact of obesity in perturbing postural stability in morbidly obese subjects elected for bariatric intervention, harboring a higher-spectrum BMI. Subjects and Methods: Traditional force plate measurements and stabilograms are gold standards employed when measuring center of pressure (COP) and postural sway. To quantify the extent of postural instability in subjects with obesity before bariatric surgery, we assessed 17 obese subjects with an average BMI of 40 kg/m2 in contrast to 13 nonobese subjects with an average BMI of 30 kg/m2. COP and postural sway were measured from static and dynamic tasks. Involuntary movements were measured when patients performed static stances, with eyes either opened or closed. Two additional voluntary movements were measured when subjects performed dynamic, upper torso tasks with eyes opened. Results: Mean body weight was 85% (p < 0.001) greater in obese than nonobese subjects. Following static balance assessments, we observed greater sway displacement in the anteroposterior (AP) direction in obese subjects with eyes open (87%, p < 0.002) and eyes closed (76%, p = 0.04) versus nonobese subjects. Obese subjects also exhibited a higher COP velocity in static tests when subjects’ eyes were open (47%, p = 0.04). Dynamic tests demonstrated no differences between groups in sway displacement in either direction; however, COP velocity in the mediolateral (ML) direction was reduced (31%, p < 0.02) in obese subjects while voluntarily swaying in the AP direction, but increased in the same cohort when swaying in the ML direction (40%, p < 0.04). Discussion and Conclusion: Importantly, these data highlight obesity’s contribution towards increased postural instability. Obese subjects exhibited greater COP displacement at higher AP velocities versus nonobese subjects, suggesting that clinically obese individuals show greater instability than nonobese subjects. Identifying factors contributory to instability could encourage patient-specific physical therapies and presurgical measures to mitigate instability and monitor postsurgical balance improvements.

Osteoporosis, a condition of skeletal decline that undermines quality of life, is treated with pharmacological interventions that are associated with poor adherence and adverse effects. Complicating efforts to improve clinical outcomes, the incidence of obesity is increasing, predisposing the population to a range of musculoskeletal complications and metabolic disorders. Pharmacological management of obesity has yet to deliver notable reductions in weight and debilitating complications are rarely avoided. By contrast, exercise shows promise as a non-invasive and non-pharmacological method of regulating both osteoporosis and obesity. The principal components of exercise - mechanical signals - promote bone and muscle anabolism while limiting formation and expansion of fat mass. Mechanical regulation of bone and marrow fat might be achieved by regulating functions of differentiated cells in the skeletal tissue while biasing lineage selection of their common progenitors - mesenchymal stem cells. An inverse relationship between adipocyte versus osteoblast fate selection from stem cells is implicated in clinical conditions such as childhood obesity and increased marrow adiposity in type 2 diabetes mellitus, as well as contributing to skeletal frailty. Understanding how exercise-induced mechanical signals can be used to improve bone quality while decreasing fat mass and metabolic dysfunction should lead to new strategies to treat chronic diseases such as osteoporosis and obesity.

PURPOSE OF REVIEW: To highlight recent basic, translational, and clinical work demonstrating exercise and diet regulation of marrow adipose tissue (MAT) and bone, and how this informs current understanding of the relationship between marrow adiposity and musculoskeletal health. RECENT FINDINGS: Marrow adipocytes accumulate in bone in the setting of hypercaloric intake (calorie excess; e.g. diet-induced obesity), but also with hypocaloric intake (calorie restriction; e.g. anorexia), despite the fact that these states affect bone differently. With hypercaloric intake, bone quantity is largely unaffected whereas with hypocaloric intake, bone quantity and quality are greatly diminished. Voluntary running exercise in rodents was found to lower MAT and promote bone in eucaloric and hypercaloric states, while degrading bone in hypocaloric states, suggesting differential modulation of MAT and bone, dependent upon whole body energy status. Energy status alters bone metabolism and bioenergetics via substrate availability or excess, which plays a key role in the response of bone and MAT to mechanical stimuli. SUMMARY: Marrow adipose tissue (MAT) is a fat depot with a potential role in – as well as responsivity to – whole body energy metabolism. Understanding the localized function of this depot in bone cell bioenergetics and substrate storage, principally in the exercised state, will aid to uncover putative therapeutic targets for skeletal fragility.

Complications associated with advanced cancer are a major clinical challenge and, if associated with bone metastases, worsen the prognosis and compromise the survival of the patients. Breast and prostate cancer cells exhibit a high propensity to metastasize to bone. The bone microenvironment is unique, providing fertile soil for cancer cell propagation, while mineralized bone matrices store potent growth factors and cytokines. Biologically active transforming growth factor β (TGF-β), one of the most abundant growth factors, is released following tumor-induced osteoclastic bone resorption. TGF-β promotes tumor cell secretion of factors that accelerate bone loss and fuel tumor cells to colonize. Thus, TGF-β is critical for driving the feed-forward vicious cycle of tumor growth in bone. Further, TGF-β promotes epithelial-mesenchymal transition (EMT), increasing cell invasiveness, angiogenesis, and metastatic progression. Emerging evidence shows TGF-β suppresses immune responses, enabling opportunistic cancer cells to escape immune checkpoints and promote bone metastases. Blocking TGF-β signaling pathways could disrupt the vicious cycle, revert EMT, and enhance immune response. However, TGF-β’s dual role as both tumor suppressor and enhancer presents a significant challenge in developing therapeutics that target TGF-β signaling. This review presents TGF-β’s role in cancer progression and bone metastases, while highlighting current perspectives on the therapeutic potential of targeting TGF-β pathways.

Introduction Multiple myeloma (MM)-induced bone disease remains one of its most devastating complications, caused by increased bone resorption by overactivated osteoclasts coupled with impaired bone formation. MM cells produce osteoclast-activating factors that induce osteoclast activation and extensive bone resorption. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells (Fu J etc. JCI. 2016). We also identified that the checkpoint inhibitor, programmed death-1 homolog (PD-1H/VISTA), serves as the MMP-13 receptor in osteoclasts and mediates MMP-13-dependent osteoclastogenic function which is largely blocked in Pd-1h -/-osteoclasts (Fu J etc. ASH 2019, 2020). While the inhibitory role of PD-1H/VISTA in T-cells has recently been described (ElTanbouly MA etc. Science 2020), its cellular binding proteins remain unclear, and its role in osteoclast activation andMM bone disease have not been addressed. Methods and Results To identify its interacting proteins, PD-1H-His 6 recombinant protein was expressed in mouse bone marrow mononuclear cells, the associated proteins pulled down by Ni-NTA agarose beads from cell lysates and identified by mass spectrometry. Functional annotation charting of the 75 proteins enriched in PD-1H pull-down samples (with signal ratio of PD-1H-His 6 pull-down vs control >2) indicated that almost 30% of the interacting targets were either cytoskeletal or cytoskeleton-associated proteins. Given that the F-actin cytoskeleton undergoes dynamic reorganization during osteoclast differentiation and plays critical roles in bone resorption, we further addressed the role of PD-1H in F-actin cytoskeleton regulation. Initially, osteoclasts form F-actin-rich adhesive structures, termed podosomes. At later stages, podosomes collectively rearrange into clusters and rings and finally into sealing belts to mediate osteoclast spreading, migration and bone resorption (Teitelbaum SL. Ann N Y Acad Sci. 2011). By confocal immunofluorescence microscopy, we found that PD-1H co-localized with F-actin podosome clusters, rings and sealing belts during osteoclast differentiation (Figure 1A). The functional role of PD-1H in F-actin cytoskeleton reorganization was addressed using Pd-1h -/- osteoclast wherein Pd-1h knockout lead to the disruption of podosome clusters at early stages relative to WT controls, while at later stages, Pd-1h -/-osteoclasts exhibited significantly fewer F-actin rings and belts (Figure 1B). Further, binding of MMP-13 to PD-1H increased the number of osteoclasts forming F-actin rings and belts, as well as the size of F-actin belts, which was blocked in Pd-1h -/- osteoclasts. To determine the role of PD-1H in the development of myeloma-induced lytic bone lesions, 5TGM1 myeloma cells were bilaterally intratibially injected into Pd-1h wtRag2 -/- or Pd-1h -/-Rag2 -/- mice (n=10) to induce lytic bone lesions. Three weeks following intratibial 5TGM1 injection, tibiae were harvested for micro-computed tomography. Subsequent quantitative histomorphology analyses of the trabecular and cortical bones confirmed that the knockout of Pd-1h reduced MM-induced bone destruction with significantly less decrease in trabecular bone volume (Tb. [BV/TV]), trabecular bone number (Tb. N.), trabecular bone thickness (Tb. Th.), as well as less increase in trabecular bone spacing (Tb. Sp.) and bone specific surface (Tb. [BS/BV]) compared to Pd-1h wtRag2 -/- mice. Similar effects were observed in cortical bone with less decrease in cortical bone thickness (CT. Th.), cortical bone area fraction (CT. [BA/TA]), and cortical tissue mineral density (CT. TMD) in 5TGM1 bearing Pd-1h -/-Rag2 -/- mice vs Pd-1h wtRag2 -/- mice (Table 1). Conclusions Taken together, our study, for the first time, reveals the novel role of checkpoint inhibitor, PD-1H/VISTA, in osteoclasts and myeloma bone disease. PD-1H associates with cytoskeleton proteins and regulates the F-actin cytoskeleton reorganization which is critical for osteoclast bone resorption activity. Further, PD-1H mediates MMP-13-induced osteoclast fusion, F-actin belts formation, and osteoclast activation. Pd-1h -/-in recipient mice significantly impairs MM-induced bone loss, demonstrating that PD-1H/VISTA plays a critical role in MM bone disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues, and neurological deficits. MM cells secret pro-osteoclastogenic factors which lead to osteoclast (OCL) activation. Our previous work showed that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor highly secreted by MM cells and induces OCL fusion and bone resorption independently of its proteolytic activity (JCI 2016). We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed on OCLs and mediates MMP-13 induced OCL fusion and bone resorption activity (ASH 2019). Bone resorption activity is significantly impaired in Pd-1h-/- OCLs in vitro. However, the function of PD-1H in MM bone disease has not been defined. Methods The role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model. Pd-1h-/-Rag2-/- mice were generated by crossbreeding Pd-1h-/- with Rag2-/- mice. Firefly luciferase-expressing 5TGM1 cells were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=10). 3 weeks later, tibiae were harvested for quantitative micro-CT followed by histological analysis. Results Morphological analyses of trabecular and cortical bones confirmed that Pd-1h-/- recipient mice exhibited significantly less 5TGM1-induced bone loss (P Conclusions Taken together, our study, for the first time, reveals that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation, and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/- mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment.

Patients afflicted with or being treated for cancer constitute a distinct and alarming subpopulation who exhibit elevated fracture risk and heightened susceptibility to developing secondary osteoporosis. Cancer cells uncouple the regulatory processes central for the adequate regulation of musculoskeletal tissue. Systemically taxing treatments to target tumors or disrupt the molecular elements driving tumor growth place considerable strain on recovery efforts. Skeletal tissue is inherently sensitive to mechanical forces, therefore attention to exercise and mechanical loading as non-pharmacological means to preserve bone during treatment and in post-treatment rehabilitative efforts have been topics of recent focus. This review discusses the dysregulation that cancers and the ensuing metabolic dysfunction that confer adverse effects on musculoskeletal tissues. Additionally, we describe foundational mechanotransduction pathways and the mechanisms by which they influence both musculoskeletal and cancerous cells. Functional and biological implications of mechanical loading at the tissue and cellular levels will be discussed, highlighting the current understanding in the field. Herein, in vitro, translational, and clinical data are summarized to consider the positive impact of exercise and low magnitude mechanical loading on tumor-bearing skeletal tissue.

Introduction Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues and neurological deficits. MM cells produce osteoclast-activating factors that induce osteoclast activation, thereby leading to excessive bone resorption and lytic bone lesions1. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption via a mechanism independent of its proteolytic activity2. We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed in osteoclasts at high levels3. Binding of MMP-13 to PD-1H/VISTA induces osteoclast fusion and bone resorption activity whereas knockdown or knockout of PD-1H/VISTA largely block MMP-13 mediated effects on osteoclasts3. However, the function of PD-1H inMM bone disease in vitro or in vivo has not been previously defined. Methods and Results To confirm the role of PD-1H in MMP-13 induced bone disease in MM, we first conducted MM-osteoclast trans-well co-culture assay using murine MM cell line, 5TGM1 cells, and bone marrow mononuclear cells from Pd-1h-/- or wild type (WT) mice. 5TGM1 control cells or MMP-13 knockdown 5TGM1 cells were seeded in the upper wells of the transwell plates; while WT or Pd-1h-/- bone marrow mononuclear cells were seeded in the lower wells and cultured for osteoclast differentiation assessed by TRAP staining. Results show that 5TGM1 induced differentiation of WT osteoclasts with significantly increased osteoclast size and nuclei number/osteoclast. Consistent with our previous results2, MMP-13 knockdown blocked the 5TGM1 MM cells-induced activation of WT osteoclasts. In contrast, neither 5TGM1 MM cells nor MMP-13 knockdown cells had significant effects on Pd-1h-/- osteoclasts. Hence, knockout of Pd-1h abrogated MMP-13 mediated MM induction of osteoclasts, indicating that MMP-13/PD-1H interactions are critically involved in MM-induced osteoclast activation. The in vivo role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model2. For this purpose, Pd-1h-/-Rag2-/- mice were generated by crossbreeding C57BL/6 Pd-1h-/- with C57BL/6 Rag2-/- mice. 3x105 firefly luciferase expressing 5TGM1 cells (5TGM1-luc) were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=5). Tumor progression was monitored by weekly bioluminescence imaging (BLI). 3 weeks after tumor inoculation, tibiae were harvested for quantitative micro-CT, followed by histological analysis. Histological staining showed that intratibial injection of 5TGM1-luc MM cells induced extensive lytic lesions and trabecular bone loss in Rag2-/- mice. In contrast, in Pd-1h-/-Rag2-/- mice,the bone structure was maintained with markedly less bone loss. Morphological analyses of trabecular bone across proximal tibiae further indicated that in Rag2-/- mice, 5TGM1 induced significant changes in bone microarchitecture, with decreased bone volume fraction (bone volume/tissue volume), connective density, trabecular bone numbers, and trabecular bone thickness, as well as increased trabecular bone spacing (Table 1). In contrast, in Pd-1h-/-Rag2-/- mice, 5TGM1 failed to induce significant loss of trabecular bone, confirming the critical role of PD-1H in MM induced bone disease in vivo. Conclusions Taken together, our study, for the first time, reveal that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/-mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment. References 1. Marino S, Petrusca DN, Roodman GD. Br J Pharmacol. 2019;10.1111/bph.14889. 2. Fu J, Li S, Feng R, et al. J Clin Invest. 2016;126(5):1759-1772. 3. Fu J, Li S, Yang C, et al. Blood. 2019; 134 (Supplement_1): 3072. Disclosures Lentzsch: Caelum Biosciences: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Celularity: Consultancy, Other; Magenta: Current equity holder in private company; Karyopharm: Research Funding; Mesoblast: Divested equity in a private or publicly-traded company in the past 24 months.

Objective The aim of this study was to determine whether inclusion of a refractory period between bouts of low-magnitude mechanical stimulation (LMMS) can curb obesity-induced adipose tissue dysfunction and sequelae in adult mice. Methods A diet-induced obesity model that included a diet with 45% of kilocalories from fat was employed with intention to treat. C57BL/6J mice were weight matched into four groups: low-fat diet (LFD, n = 8), high-fat diet (HFD, n = 8), HFD with one bout of 30-minute LMMS (HFDv, n = 9), and HFD with two bouts of 15-minute LMMS with a 5-hour separation (refractory period, RHFDv, n = 9). Two weeks of diet was followed by 6 weeks of diet plus LMMS. Results HFD and HFDv mice continued gaining body weight and visceral adiposity throughout the experiment, which was mitigated in RHFDv mice. Compared with LFD mice, HFD and HFDv mice had increased rates of adipocyte hypertrophy, increased immune cell infiltration (B cells, T cells, and macrophages) into adipose tissue, increased adipose tissue inflammation (tumor necrosis factor alpha gene expression), and a decreased proportion of mesenchymal stem cells in adipose tissue, all of which were rescued in RHFDv mice. Glucose intolerance and insulin resistance were elevated in HFD and HFDv mice, but not in RHFDv mice, as compared with LFD mice. Conclusions Incorporating a 5-hour refractory period between bouts of LMMS attenuates obesity-induced adipose tissue dysfunction and improves glucose metabolism.

The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We used running exercise to ask whether obesity-associated MAT can be attenuated via exercise and whether this correlates with gains in bone quantity and quality. C57BL/6 mice were divided into diet-induced obesity (DIO, n = 14) versus low-fat diet (LFD, n = 14). After 3 months, 16-week-old mice were allocated to an exercise intervention (LFD-E, DIO-E) or a control group (LFD, DIO) for 6 weeks (4 groups, n = 7/group). Marrow adipocyte area was 44% higher with obesity (p

Autologus augmentation of wound remodeling with platelet concentrate is a burgeoning field with promising results. We hypothesized that the addition of filtered platelet concentrate (fPC) to an acellular biologic graft would improve crural healing and tissue integrity in hiatal hernia repair. Sixteen healthy Yorkshire female pigs were divided into three groups: hiatus repair (HR) (n = 7), HR with biologic graft (HRM; n = 8, and HR with biologic graft and fPC (fPC; n = 9). Surgeries were performed by a single surgeon. Animals were euthanized at 8 weeks, and the distal esophagus with hiatus was harvested en-block. Tissue was graded by a histopathologist on collagen deposition, vascularization, and inflammation at the graft–hiatal interface. Tensile strength testing was performed using the Teststar IIs (MTS), coupled with a strain extensometer (Epsilon). Samples of equal dimensions were preloaded to 1 N and deformed at a constant rate of 0.2 mm/s. Statistical analysis was performed via Kruskal–Wallis one-way analysis of variance. Aspirate analysis revealed a mean platelet count of 3 million platelets/1 mL of aspirate. Animals in the fPC group had significantly increased mean chronic inflammation (3.1 ± 1.1 vs. 1.8 ± 1.6, 1.2 ± 1.2, p = 0.04) compared to HR alone and HR + biologic graft. Vascular deposition did not differ between groups (p = 0.8). A trend toward increased collagen deposition was demonstrated for the fPC group (1.4 ± 1.1 vs. 2.0 ± 0.6 in HR group and 3.0 ± 1.2 in HRM group, p = 0.06). There was a statistically significant increase in tensile strength, yield force, and Young’s modulus in the fPC group compared with HR and HR + biologic mesh (p

Background and Hypothesis: Hyperglycemia is a major source of disease and morbidity among the adult population. Prior studies correlate long-term high fat diet (HFD) mediated hyperglycemia with bone fragility and muscle weakness. Furthermore, the mechanism driving hyperglycemia between sexes are unknown. Our group previously showed that HFDs induced insulin resistance in male mice and glucose intolerance in female mice. This establishes the need to study the impact of long-term HFDs on the bones and muscles using an older cohort of both male and female mice. For that, we hypothesized a long-term HFD mediated hyperglycemia will change bone and muscle structures and impair their functions in adult male and female mice. Experimental Design or Project Methods: 22-week C57Bl6 mice were fed either a HFD or low fat diet (LFD) for 25 weeks. After euthanasia, bones and muscles were harvested and evaluated using MicroCT, histology, and mechanical testing. Statistical analysis was performed using GraphPad Prism with p

Background and Hypothesis: Camurati-Engelmann Disease (CED) is characterized by extreme bone turnover and excess TGF-β release. We previously showed that bone-derived TGF-β causes glucose intolerance, increases skeletal muscle weakness, and exacerbates diet-induced obesity in CED mice. However, it is unknown whether glucose intolerance and obesity alter bone and muscle phenotypes. Thus, we hypothesized that impaired glucose metabolism and diet-induced obesity exacerbate bone and muscle loss in a mouse model of CED. Experimental Design: 45-week WT and CED mice were fed either high-fat diet (HFD) or low-fat diet (LFD) for 15 weeks. Ex vivo bone micro-CT and histomorphometry were used to evaluate bone and muscle. Statistical analysis was performed using GraphPad Prism with p

Background and Hypothesis: Post-menopausal, estrogen-receptor positive breast cancer patients are treated with aromatase inhibitors (AIs) to limit tumor progression; however, this causes adverse musculoskeletal effects. Zoledronic acid (ZA) is prescribed to inhibit bone resorption. Mechanical signals, delivered via low intensity vibration (LIV), stimulate bone formation. We hypothesize that combining LIV with ZA will mitigate bone loss in a murine model of complete estrogen-deprivation more effectively than either treatment alone. Project Methods: 21-week-old C57BL/6 mice (n=20/group) were ovariectomized, receiving daily letrozole injections (OVX/AI) with LIV (OVX/AI+LIV), ZA (OVX/AI+ZA), LIV and ZA (OVX/AI+LIV/ZA) or underwent sham surgery with daily PBS (vehicle) injections (SH-OVX) for 22 weeks. Longitudinal dual energy X-ray absorptiometry (DEXA) and micro-computed tomography scans were analyzed for changes in body composition and bone microarchitecture, respectively. Results: OVX/AI reduced whole body (p< 0.0001) and lumbar spine (p

Bone metastases cause significant morbidity and mortality in late-stage breast cancer patients and are currently considered incurable. Investigators rely on translational models to better understand the pathogenesis of skeletal complications of malignancy in order to identify therapeutic targets that may ultimately prevent and treat solid tumor metastasis to bone. Many experimental models of breast cancer bone metastases are in use today, each with its own caveats. In this methods review, we characterize the bone phenotype of commonly utilized human- and murine-derived breast cell lines that elicit osteoblastic and/or osteolytic destruction of bone in mice and report methods for optimizing tumor-take in murine models of bone metastasis. We then provide protocols for four of the most common xenograft and syngeneic inoculation routes for modeling breast cancer metastasis to the skeleton in mice, including the intra-cardiac, intra-arterial, orthotopic and intra-tibial methods of tumor cell injection. Recommendations for in vivo and ex vivo assessment of tumor progression and bone destruction are provided, followed by discussion of the strengths and limitations of the available tools and translational models that aid investigators in the study of breast cancer metastasis to bone.

Despite association with low bone density and skeletal fractures, marrow adipose tissue (MAT) remains poorly understood. The marrow adipocyte originates from the mesenchymal stem cell (MSC) pool that also gives rise to osteoblasts, chondrocytes, and myocytes, among other cell types. To date, the presence of MAT has been attributed to preferential biasing of MSC into the adipocyte rather than osteoblast lineage, thus negatively impacting bone formation. Here, we focus on understanding the physiology of MAT in the setting of exercise, dietary interventions, and pharmacologic agents that alter fat metabolism. The beneficial effect of exercise on musculoskeletal strength is known: exercise induces bone formation, encourages growth of skeletally supportive tissues, inhibits bone resorption, and alters skeletal architecture through direct and indirect effects on a multiplicity of cells involved in skeletal adaptation. MAT is less well studied due to the lack of reproducible quantification techniques. In recent work, osmium-based 3D quantification shows a robust response of MAT to both dietary and exercise intervention in that MAT is elevated in response to high-fat diet and can be suppressed following daily exercise. Exercise-induced bone formation correlates with suppression of MAT, such that exercise effects might be due to either calorie expenditure from this depot or from mechanical biasing of MSC lineage away from fat and toward bone, or a combination thereof. Following treatment with the anti-diabetes drug rosiglitazone – a PPARγ-agonist known to increase MAT and fracture risk – mice demonstrate a fivefold higher femur MAT volume compared to the controls. In addition to preventing MAT accumulation in control mice, exercise intervention significantly lowers MAT accumulation in rosiglitazone-treated mice. Importantly, exercise induction of trabecular bone volume is unhindered by rosiglitazone. Thus, despite rosiglitazone augmentation of MAT, exercise significantly suppresses MAT volume and induces bone formation. That exercise can both suppress MAT volume and increase bone quantity, notwithstanding the skeletal harm induced by rosiglitazone, underscores exercise as a powerful regulator of bone remodeling, encouraging marrow stem cells toward the osteogenic lineage to fulfill an adaptive need for bone formation. Thus, exercise represents an effective strategy to mitigate the deleterious effects of overeating and iatrogenic etiologies on bone and fat.

The contribution of marrow adipose tissue (MAT) to skeletal fragility is poorly understood. Peroxisome proliferator-activated receptor (PPAR)γ agonists, associated with increased fractures in diabetic patients, increase MAT. Here, we asked whether exercise could limit the MAT accrual and increase bone formation in the setting of PPARγ agonist treatment. Eight-week-old female C57BL/6 mice were treated with 20-mg/kg·d rosiglitazone (Rosi) and compared with control (CTL) animals. Exercise groups ran 12 km/d when provided access to running wheels (CTL exercise [CTL-E], Rosi-E). After 6 weeks, femoral MAT (volume of lipid binder osmium) and tibial bone morphology were assessed by microcomputer tomography. Rosi was associated with 40% higher femur MAT volume compared with CTL (P < .0001). Exercise suppressed MAT volume by half in CTL-E mice compared with CTL (P < .01) and 19% in Rosi-E compared with Rosi (P < .0001). Rosi treatment increased fat markers perilipin and fatty acid synthase mRNA by 4-fold (P < .01). Exercise was associated with increased uncoupling protein 1 mRNA expression in both CTL-E and Rosi-E groups (P < .05), suggestive of increased brown fat. Rosi increased cortical porosity (P < .0001) but did not significantly impact trabecular or cortical bone quantity. Importantly, exercise induction of trabecular bone volume was not prevented by Rosi (CTL-E 21% > CTL, P < .05; Rosi-E 26% > Rosi, P < .01). In summary, despite the Rosi induction of MAT extending well into the femoral diaphysis, exercise was able to significantly suppress MAT volume and induce bone formation. Our results suggest that the impact of PPARγ agonists on bone and marrow health can be partially mitigated by exercise.

Background As the association between sleeve gastrectomy (SG) and gastroesophageal reflux disease remains unclear, the aim of this study was to evaluate whether performance of SG impacts the development and severity of esophagitis in a rodent model. Setting: University Hospital. Methods Wistar rats (Charles River Institute, Wilmington, MA) were fed a high fat diet (HFD) for 4 months and then were divided into 3 cohorts of nearly equal mean weight: HFD only (n = 25), sham operation+HFD (n = 29), and SG+HFD (n = 19). Animals were euthanized at 12 weeks. The esophagus was harvested en-bloc and processed for histologic assessment by a board certified pathologist, blinded to the animal treatment group. Reflux was graded by severity and defined as the presence of inflammation in the esophageal squamous mucosa. Results Rats who underwent SG had significantly increased reflux severity, compared with sham and HFD alone (21.1% versus 0% versus 4.5%, P = .02), respectively. No difference was demonstrated in negative, mild, or moderate esophagitis between the control, sham, and sleeve groups. Using nonparametric ANOVA, the mean severity score for severe esophagitis was significantly increased in the SG group versus sham or HFD group (1.5 versus .81 versus 1.36, P = .0202) respectively. Following multinomial logistic regression to assess for confounding variables to the severity scores, final weight, and change in weight, had no effect on severity of esophagitis between the 3 groups (P> .373). Conclusions SG is independently associated with histopathologic changes consistent with severe esophagitis in an animal model, likely secondary to gastroesophageal reflux.