Search

When breast cancer metastasizes to bone, treatment options are limited. Failure to treat bone metastases is thought to be due to therapy-resistant features of the bone marrow microenvironment. Using a murine model of bone metastatic mammary carcinoma, we demonstrate that systemic delivery of polymer nanoparticles loaded with cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) inhibited tumor growth and bone destruction after 7 days of treatment. Each dose of STING-activating nanoparticles trafficked to the bone marrow compartment and was retained within the tumor microenvironment for over 24 hours, enhancing antitumor immunity through proinflammatory cytokine production and early T-cell activation. While acquired resistance mechanisms, including increased levels of immunosuppressive cytokines and the infiltration of regulatory T cells, ultimately limited antitumor efficacy after 2 weeks of treatment, bone protective effects remained. Overall, these studies demonstrate that STING pathway activation, here enabled using a nanomedicine approach to enhance CDN delivery to bone metastatic sites, can reprogram the immune contexture of the bone marrow to an antitumor phenotype that inhibits bone colonization of metastatic breast cancer cells and protects from tumor-mediated bone destruction., Significance: Bone metastases are difficult to treat due to the inaccessibility of the bone marrow compartment and the immunosuppressive microenvironment that protects resident stem cells. Packaging a STING agonist into a nanoparticle that enables systemic administration and drug accumulation at tumor sites overcomes both barriers to stymie metastatic breast cancer growth., Competing Interests: N.E. Bennett reports grants from NIH during the conduct of the study. A.R. Merkel reports grants from Verteran's Administration and non-financial support from Vanderbilt University Medical Center during the conduct of the study; grants from NIH Clinical and Translational Science Award and National Center for Advancing Translational Sciences outside the submitted work. M.A. Fischer reports grants from Incyte Corporation during the conduct of the study. M.R. Savona reports grants from ALX Oncology, Astex, Incyte, Takeda, TG Therapeutics; personal fees from BMS, CTI, Forma, Geron, Novartis, Sierra Oncology, Taiho; personal fees and other from Ryvu and Karyopharm outside the submitted work. J.A. Rhoades reports grants from Veterans Administration during the conduct of the study. S.A. Guelcher reports grants from Incyte Corporation during the conduct of the study. J.T. Wilson reports a patent to 10,696,985 issued and a patent to PCT/US2019/058945 pending. No disclosures were reported by the other authors., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2β1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2β1 in the context of bone metastasis. In this study, we aimed to understand the role of α2β1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2β1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2β1 expression and bone-metastatic potential. Inhibiting α2β1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.

EphA2 receptor tyrosine kinase (RTK) is highly expressed in breast tumor cells across multiple molecular subtypes and correlates with poor patient prognosis. In this study, the potential role of EphA2 in this clinically relevant phenomenon is investigated as metastasis of breast cancer to bone is a major cause of morbidity and mortality in patients. It was found that the EphA2 function in breast cancer cells promotes osteoclast activation and the development of osteolytic bone disease. Blocking EphA2 function molecularly and pharmacologically in breast tumors reduced the number and size of bone lesions and the degree of osteolytic disease in intratibial and intracardiac mouse models, which correlated with a significant decrease in the number of osteoclasts at the tumor-bone interface. EphA2 loss of function in tumor cells impaired osteoclast progenitor differentiation in coculture, which is mediated, at least in part, by reduced expression of IL-6. EPHA2 transcript levels are enriched in human breast cancer bone metastatic lesions relative to visceral metastatic sites; EphA2 protein expression was detected in breast tumor cells in bone metastases in patient samples, supporting the clinical relevance of the study's findings. These data provide a strong rationale for the development and application of molecularly targeted therapies against EphA2 for the treatment of breast cancer bone metastatic disease. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.)

Patients with advanced skeletal metastases arising from primary cancers including breast, lung, and prostate suffer from extreme pain, bone loss, and frequent fractures. While the importance of interactions between bone and tumors is well-established, our understanding of complex cell-cell and cell-microenvironment interactions remains limited in part due to a lack of appropriate 3D bone models. To improve our understanding of the influence of bone morphometric properties on the regulation of tumor-induced bone disease (TIBD), we utilized bone-like 3D scaffolds in vitro and in vivo. Scaffolds were seeded with tumor cells, and changes in cell motility, proliferation, and gene expression were measured. Genes associated with TIBD significantly increased with increasing scaffold rigidity. Drug response differed when tumors were cultured in 3D compared to 2D. Inhibitors for Integrin β3 and TGF-β Receptor II significantly reduced bone-metastatic gene expression in 2D but not 3D, while treatment with the Gli antagonist GANT58 significantly reduced gene expression in both 2D and 3D. When tumor-seeded 3D scaffolds were implanted into mice, infiltration of myeloid progenitors changed in response to pore size and rigidity. This study demonstrates a versatile 3D model of bone used to study the influence of mechanical and morphometric properties of bone on TIBD.

Breast cancer patients are at high risk for bone metastasis. Metastatic bone disease is a major clinical problem that leads to a reduction in mobility, increased risk of pathologic fracture, severe bone pain, and other skeletal-related events. The transcription factor Gli2 drives expression of parathyroid hormone-related protein (PTHrP), which activates osteoclast-mediated bone destruction, and previous studies showed that Gli2 genetic repression in bone-metastatic tumor cells significantly reduces tumor-induced bone destruction. Small molecule inhibitors of Gli2 have been identified; however, the lipophilicity and poor pharmacokinetic profile of these compounds have precluded their success in vivo . In this study, we designed a bone-targeted nanoparticle (BTNP) comprising an amphiphilic diblock copolymer of poly[(propylene sulfide)- block -(alendronate acrylamide- co - N , N -dimethylacrylamide)] [PPS- b -P(Aln- co -DMA)] to encapsulate and preferentially deliver a small molecule Gli2 inhibitor, GANT58, to bone-associated tumors. The mol % of the bisphosphonate Aln in the hydrophilic polymer block was varied in order to optimize BTNP targeting to tumor-associated bone by a combination of nonspecific tumor accumulation (presumably through the enhanced permeation and retention effect) and active bone binding. Although 100% functionalization with Aln created BTNPs with strong bone binding, these BTNPs had highly negative zeta-potential, resulting in shorter circulation time, greater liver uptake, and less distribution to metastatic tumors in bone. However, 10 mol % of Aln in the hydrophilic block generated a formulation with a favorable balance of systemic pharmacokinetics and bone binding, providing the highest bone/liver biodistribution ratio among formulations tested. In an intracardiac tumor cell injection model of breast cancer bone metastasis, treatment with the lead candidate GANT58-BTNP formulation decreased tumor-associated bone lesion area 3-fold and increased bone volume fraction in the tibiae of the mice 2.5-fold. Aln conferred bone targeting to the GANT58-BTNPs, which increased GANT58 concentration in the tumor-associated bone relative to untargeted NPs, and also provided benefit through the direct antiresorptive therapeutic function of Aln. The dual benefit of the Aln in the BTNPs was supported by the observations that drug-free Aln-containing BTNPs improved bone volume fraction in bone-tumor-bearing mice, while GANT58-BTNPs created better therapeutic outcomes than both unloaded BTNPs and GANT58-loaded untargeted NPs. These findings suggest GANT58-BTNPs have potential to potently inhibit tumor-driven osteoclast activation and resultant bone destruction in patients with bone-associated tumor metastases.

Solid tumors frequently metastasize to bone and induce bone destruction leading to severe pain, fractures, and other skeletal-related events (SREs). Osteoclast inhibitors such as bisphosphonates delay SREs but do not prevent skeletal complications or improve overall survival. Because bisphosphonates can cause adverse side effects and are contraindicated for some patients, we sought an alternative therapy to reduce tumor-associated bone destruction. Our previous studies identified the transcription factor Gli2 as a key regulator of parathyroid hormone-related protein (PTHrP), which is produced by bone metastatic tumor cells to promote osteoclast-mediated bone destruction. In this study, we tested the treatment effect of a Gli antagonist GANT58, which inhibits Gli2 nuclear translocation and PTHrP expression in tumor cells. In initial testing, GANT58 did not have efficacy in vivo due to its low water solubility and poor bioavailability. We therefore developed a micellar nanoparticle (NP) to encapsulate and colloidally stabilize GANT58, providing a fully aqueous, intravenously injectable formulation based on the polymer poly(propylene sulfide) 135 -b-poly[(oligoethylene glycol) 9 methyl ether acrylate] 17 (PPS 135 -b-POEGA 17 ). POEGA forms the hydrophilic NP surface while PPS forms the hydrophobic NP core that sequesters GANT58. In response to reactive oxygen species (ROS), PPS becomes hydrophilic and degrades to enable drug release. In an intratibial model of breast cancer bone metastasis, treatment with GANT58-NPs decreased bone lesion area by 49% (p<.01) and lesion number by 38% (p<.05) and resulted in a 2.5-fold increase in trabecular bone volume (p<.001). Similar results were observed in intracardiac and intratibial models of breast and lung cancer bone metastasis, respectively. Importantly, GANT58-NPs reduced tumor cell proliferation but did not alter mesenchymal stem cell proliferation or osteoblast mineralization in vitro, nor was there evidence of cytotoxicity after repeated in vivo treatment. Thus, inhibition of Gli2 using GANT58-NPs is a potential therapy to reduce bone destruction that should be considered for further testing and development toward clinical translation., (Published by Elsevier B.V.)

β-2 Microglobulin (β2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but β2M may also have less understood chaperone-independent functions. Elevated plasma β2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. β2M mRNA is present in platelets at very high levels, and β2M is part of the activated platelet releasate. In addition to their more well-studied thrombotic functions, platelets are important immune regulatory cells that release inflammatory molecules and contribute to leukocyte trafficking, activation, and differentiation. We have now found that platelet-derived β2M is a mediator of monocyte proinflammatory differentiation through noncanonical TGFβ receptor signaling. Circulating monocytes from mice lacking β2M only in platelets (Plt-β2M-/-) had a more proreparative monocyte phenotype, in part dependent on increased platelet-derived TGFβ signaling in the absence of β2M. Using a mouse myocardial infarction (MI) model, Plt-β2M-/- mice had limited post-MI proinflammatory monocyte responses and, instead, demonstrated early proreparative monocyte differentiation, profibrotic myofibroblast responses, and a rapid decline in heart function compared with WT mice. These data demonstrate a potentially novel chaperone-independent, monocyte phenotype-regulatory function for platelet β2M and that platelet-derived 2M and TGFβ have opposing roles in monocyte differentiation that may be important in tissue injury responses.

Bone fractures at weight-bearing sites are challenging to treat due to the difficulty in maintaining articular congruency. An ideal biomaterial for fracture repair near articulating joints sets rapidly after implantation, stabilizes the fracture with minimal rigid implants, stimulates new bone formation, and remodels at a rate that maintains osseous integrity. Consequently, the design of biomaterials that mechanically stabilize fractures while remodeling to form new bone is an unmet challenge in bone tissue engineering. In this study, we investigated remodeling of resorbable bone cements in a stringent model of mechanically loaded tibial plateau defects in sheep. Nanocrystalline hydroxyapatite-poly(ester urethane) (nHA-PEUR) hybrid polymers were augmented with either ceramic granules (85% β-tricalcium phosphate/15% hydroxyapatite, CG) or a blend of CG and bioactive glass (BG) particles to form a settable bone cement. The initial compressive strength and fatigue properties of the cements were comparable to those of non-resorbable poly(methyl methacrylate) bone cement. In animals that tolerated the initial few weeks of early weight-bearing, CG/nHA-PEUR cements mechanically stabilized the tibial plateau defects and remodeled to form new bone at 16 weeks. In contrast, cements incorporating BG particles resorbed with fibrous tissue filling the defect. Furthermore, CG/nHA-PEUR cements remodeled significantly faster at the full weight-bearing tibial plateau site compared to the mechanically protected femoral condyle site in the same animal. These findings are the first to report a settable bone cement that remodels to form new bone while providing mechanical stability in a stringent large animal model of weight-bearing bone defects near an articulating joint., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The tumor-cell microenvironment is recognized as a dynamic place where critical cell interactions occur and play an important role in altering tumorigenesis. While many studies have investigated the effects of cellular cross-talk within distinct tumor microenvironments, these interactions have yet to be fully examined in bone. It is well-established that many common cancers metastasize to bone, resulting in the development of tumor-induced bone disease (TIBD), a multi-facetted illness that is driven by complex cell interactions within the bone marrow. Our group has previously published that myeloid progenitor cells expand in the presence of tumors in bone, aligning with the notion that myeloid cells can act as tumor promotors. Several groups, including ours, have established that transforming growth factor β (TGF-β), an abundant growth factor in bone, can regulate both TIBD and myeloid expansion. TGF-β inhibitors have been shown to increase bone volume, decrease bone destruction, and reduce but not eliminate tumor. Therefore, we hypothesize that inhibiting TGF-β will reduce myeloid expansion leading to a reduction of tumor burden in bone and osteoclast-mediated bone loss, causing to an overall reduction in TIBD. To address this hypothesis, two different mouse models of breast cancer bone colonization were pre-treated with the TGF-β neutralizing antibody, 1D11, prior to tumor inoculation (athymic: MDA-MB-231, BALB/c: 4T1) and continuously treated until sacrifice. Additionally, a genetically modified mouse model with a myeloid specific deletion of transforming growth factor beta receptor II (TGF-βRII) (TGF-βRII MyeKO ) was utilized in our studies. Systemic inhibition of TGF-β lead to fewer osteolytic lesions, and reduced tumor burden in bone as expected from previous studies. Additionally, early TGF-β inhibition affected expansion of distinct myeloid populations and shifted the cytokine profile of pro-tumorigenic factors in bone, 4T1 tumor cells, and bone-marrow derived macrophages. Similar observations were seen in tumor-bearing TGF-βRII MyeKO mice, where these mice contained fewer bone lesions and significantly less tumor burden in bone, suggesting that TGF-β inhibition regulates myeloid expansion leading to a significant reduction in TIBD., (Published by Elsevier Inc.)

3D printing enables the creation of scaffolds with precisely controlled morphometric properties for multiple tissue types, including musculoskeletal tissues such as cartilage and bone. Computed tomography (CT) imaging has been combined with 3D printing to fabricate anatomically scaled patient-specific scaffolds for bone regeneration. However, anatomically scaled scaffolds typically lack sufficient resolution to recapitulate the <100 micrometer-scale trabecular architecture essential for investigating the cellular response to the morphometric properties of bone. In this study, it is hypothesized that the architecture of trabecular bone regulates osteoblast differentiation and mineralization. To test this hypothesis, human bone-templated 3D constructs are fabricated via a new micro-CT/3D inkjet printing process. It is shown that this process reproducibly fabricates bone-templated constructs that recapitulate the anatomic site-specific morphometric properties of trabecular bone. A significant correlation is observed between the structure model index (a morphometric parameter related to surface curvature) and the degree of mineralization of human mesenchymal stem cells, with more concave surfaces promoting more extensive osteoblast differentiation and mineralization compared to predominately convex surfaces. These findings highlight the significant effects of trabecular architecture on osteoblast function., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The skeleton is a common site for breast cancer metastasis. Although significant progress has been made to manage osteolytic bone lesions, the mechanisms driving the early steps of the bone metastatic process are still not sufficiently understood to design efficacious strategies needed to inhibit this process and offer preventative therapeutic options. Progression and recurrence of breast cancer, as well as reduced survival of patients with breast cancer, are associated with chronic stress, a condition known to stimulate sympathetic nerve outflow. In this study, we show that stimulation of the beta 2-adrenergic receptor (β2AR) by isoproterenol, used as a pharmacological surrogate of sympathetic nerve activation, led to increased blood vessel density and Vegf-a expression in bone. It also raised levels of secreted Vegf-a in osteoblast cultures, and accordingly, the conditioned media from isoproterenol-treated osteoblast cultures promoted new vessel formation in two ex vivo models of angiogenesis. Blocking the interaction between Vegf-a and its receptor, Vegfr2, blunted the increase in vessel density induced by isoproterenol. Genetic loss of the β2AR globally, or specifically in type 1 collagen-expressing osteoblasts, diminished the increase in Vegf-positive osteoblast number and bone vessel density induced by isoproterenol, and reduced the higher incidence of bone metastatic lesions induced by isoproterenol after intracardiac injection of an osteotropic variant of MDA-MB-231 breast cancer cells. Inhibition of the interaction between Vegf-a and Vegfr2 with the blocking antibody mcr84 also prevented the increase in bone vascular density and bone metastasis triggered by isoproterenol. Together, these results indicate that stimulation of the β2AR in osteoblasts triggers a Vegf-dependent neo-angiogenic switch that promotes bone vascular density and the colonization of the bone microenvironment by metastatic breast cancer cells. © 2017 American Society for Bone and Mineral Research., (© 2017 American Society for Bone and Mineral Research.)

This corrects the article DOI: 10.1038/ncb3408.

Multiple myeloma (MM) patients frequently develop tumor-induced bone destruction, yet no therapy completely eliminates the tumor or fully reverses bone loss. Transforming growth factor-β (TGF-β) activity often contributes to tumor-induced bone disease, and pre-clinical studies have indicated that TGF-β inhibition improves bone volume and reduces tumor growth in bone metastatic breast cancer. We hypothesized that inhibition of TGF-β signaling also reduces tumor growth, increases bone volume, and improves vertebral body strength in MM-bearing mice. We treated myeloma tumor-bearing (immunocompetent KaLwRij and immunocompromised Rag2-/-) mice with a TGF-β inhibitory (1D11) or control (13C4) antibody, with or without the anti-myeloma drug bortezomib, for 4weeks after inoculation of murine 5TGM1 MM cells. TGF-β inhibition increased trabecular bone volume, improved trabecular architecture, increased tissue mineral density of the trabeculae as assessed by ex vivo micro-computed tomography, and was associated with significantly greater vertebral body strength in biomechanical compression tests. Serum monoclonal paraprotein titers and spleen weights showed that 1D11 monotherapy did not reduce overall MM tumor burden. Combination therapy with 1D11 and bortezomib increased vertebral body strength, reduced tumor burden, and reduced cortical lesions in the femoral metaphysis, although it did not significantly improve cortical bone strength in three-point bending tests of the mid-shaft femur. Overall, our data provides rationale for evaluating inhibition of TGF-β signaling in combination with existing anti-myeloma agents as a potential therapeutic strategy to improve outcomes in patients with myeloma bone disease., (Published by Elsevier Inc.)

Breast cancer cells frequently home to the bone marrow, where they may enter a dormant state before forming a bone metastasis. Several members of the interleukin-6 (IL-6) cytokine family are implicated in breast cancer bone colonization, but the role for the IL-6 cytokine leukaemia inhibitory factor (LIF) in this process is unknown. We tested the hypothesis that LIF provides a pro-dormancy signal to breast cancer cells in the bone. In breast cancer patients, LIF receptor (LIFR) levels are lower with bone metastases and are significantly and inversely correlated with patient outcome and hypoxia gene activity. Hypoxia also reduces the LIFR:STAT3:SOCS3 signalling pathway in breast cancer cells. Loss of the LIFR or STAT3 enables otherwise dormant breast cancer cells to downregulate dormancy-, quiescence- and cancer stem cell-associated genes, and to proliferate in and specifically colonize the bone, suggesting that LIFR:STAT3 signalling confers a dormancy phenotype in breast cancer cells disseminated to bone.

Individuals with type 2 diabetes (T2D) have a higher fracture risk compared to non-diabetics, even though their areal bone mineral density is normal to high. Identifying the mechanisms whereby diabetes lowers fracture resistance requires well-characterized rodent models of diabetic bone disease. Toward that end, we hypothesized that bone toughness, more so than bone strength, decreases with the duration of diabetes in ZDSD rats. Bones were harvested from male CD(SD) control rats and male ZDSD rats at 16 weeks (before the onset of hyperglycemia), at 22 weeks (5-6 weeks of hyperglycemia), and at 29 weeks (12-13 weeks of hyperglycemia). There were at least 12 rats per strain per age group. At 16 weeks, there was no difference in either body weight or glucose levels between the two rat groups. Within 2 weeks of switching all rats to a diet with 48 % of kcal from fat, only the ZDSD rats developed hyperglycemia (>250 mg/dL). They also began to lose body weight at 21 weeks. CD(SD) rats remained normoglycemic (<110 mg/dL) on the high-fat diet and became obese (>600 g). From micro-computed tomography (μCT) analysis of a lumbar vertebra and distal femur, trabecular bone volume did not vary with age among the non-diabetic rats but was lower at 29 weeks than at 16 weeks or at 22 weeks for the diabetic rats. Consistent with that finding, μCT-derived intra-cortical porosity (femur diaphysis) was higher for ZDSD following ~12 weeks of hyperglycemia than for age-matched CD(SD) rats. Despite an age-related increase in mineralization in both rat strains (μCT and Raman spectroscopy), material strength of cortical bone (from three-point bending tests) increased with age only in the non-diabetic CD(SD) rats. Moreover, two other material properties, toughness (radius) and fracture toughness (femur), significantly decreased with the duration of T2D in ZDSD rats. This was accompanied by the increase in the levels of the pentosidine (femur). However, pentosidine was not significantly higher in diabetic than in non-diabetic bone at any time point. The ZDSD rat, which has normal leptin signaling and becomes diabetic after skeletal maturity, provides a pre-clinical model of diabetic bone disease, but a decrease in body weight during prolonged diabetes and certain strain-related differences before the onset of hyperglycemia should be taken into consideration when interpreting diabetes-related differences.

Osteoblast differentiation of mesenchymal stem cells is regulated by both soluble factor (e.g., bone morphogenetic proteins (BMP)) and mechanically transduced signaling, but the mechanisms have only been partially elucidated. In this study, physical association of BMP Receptor I (BMPRI) with integrin β1 sub-unit (Iβ1) was hypothesized to mediate osteoblast differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) on bone-like substrates. The effects of substrate modulus on osteoblast differentiation of MSCs were investigated for 2D poly(ester urethane) films with moduli varying from 5 - 266 MPa, which spans the range from collagen fibrils to trabecular bone. SMAD1/5 and p44/42 MAPK signaling, expression of markers of osteoblast differentiation, and matrix mineralization increased with increasing substrate modulus. The effects of substrate modulus on osteoblast differentiation were mediated by Iβ1, which was also expressed at higher levels on increasingly rigid substrates. Förster resonance energy transfer (FRET) and immunoprecipitation (IP) experiments showed that physical association of Iβ1 and BMP Receptor I (BMRPRI) increased with substrate modulus, resulting in activation of the BMP signaling pathway. Thus, these studies showed that integrin and BMP signaling converge to regulate osteoblast differentiation of MSCs, which may potentially guide the design of scaffolds and rhBMP-2 delivery systems for bone regeneration.

The growing need for therapies to treat large cutaneous defects has driven recent interest in the design of scaffolds that stimulate regenerative wound healing. While many studies have investigated local delivery of biologics as a restorative approach, an increasing body of evidence highlights the contribution of the mechanical properties of implanted scaffolds to wound healing. In the present study, we designed poly(ester urethane) scaffolds using a templated-Fused Deposition Modeling (t-FDM) process to test the hypothesis that scaffolds with substrate modulus comparable to that of collagen fibers enhance a regenerative versus a fibrotic response. We fabricated t-FDM scaffolds with substrate moduli varying from 5 to 266 MPa to investigate the effects of substrate modulus on healing in a rat subcutaneous implant model. Angiogenesis, cellular infiltration, collagen deposition, and directional variance of collagen fibers were maximized for wounds treated with scaffolds having a substrate modulus (Ks = 24 MPa) comparable to that of collagen fibers. The enhanced regenerative response in these scaffolds was correlated with down-regulation of Wnt/β-catenin signaling in fibroblasts, as well as increased polarization of macrophages toward the restorative M2 phenotype. These observations highlight the substrate modulus of the scaffold as a key parameter regulating the regenerative versus scarring phenotype in wound healing. Our findings further point to the potential use of scaffolds with substrate moduli tuned to that of the native matrix as a therapeutic approach to improve cutaneous healing., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cancer patients frequently develop skeletal metastases that significantly impact quality of life. Since bone metastases remain incurable, a clearer understanding of molecular mechanisms regulating skeletal metastases is required to develop new therapeutics that block establishment of tumors in bone. While many studies have suggested that the microenvironment contributes to bone metastases, the factors mediating tumors to progress from a quiescent to a bone-destructive state remain unclear. In this study, we hypothesized that the "soil" of the bone microenvironment, specifically the rigid mineralized extracellular matrix, stimulates the transition of the tumor cells to a bone-destructive phenotype. To test this hypothesis, we synthesized 2D polyurethane (PUR) films with elastic moduli ranging from the basement membrane (70 MPa) to cortical bone (3800 MPa) and measured expression of genes associated with mechanotransduction and bone metastases. We found that expression of Integrin β3 (Iβ3), as well as tumor-produced factors associated with bone destruction (Gli2 and parathyroid hormone related protein (PTHrP)), significantly increased with matrix rigidity, and that blocking Iβ3 reduced Gli2 and PTHrP expression. To identify the mechanism by which Iβ3 regulates Gli2 and PTHrP (both are also known to be regulated by TGF-β), we performed Förster resonance energy transfer (FRET) and immunoprecipitation, which indicated that Iβ3 co-localized with TGF-β Receptor Type II (TGF-β RII) on rigid but not compliant films. Finally, transplantation of tumor cells expressing Iβ3 shRNA into the tibiae of athymic nude mice significantly reduced PTHrP and Gli2 expression, as well as bone destruction, suggesting a crucial role for tumor-produced Iβ3 in disease progression. This study demonstrates that the rigid mineralized bone matrix can alter gene expression and bone destruction in an Iβ3/TGF-β-dependent manner, and suggests that Iβ3 inhibitors are a potential therapeutic approach for blocking tumor transition to a bone destructive phenotype., (Published by Elsevier Ltd.)

Scaffolds with tunable mechanical and topological properties fabricated by templated-fused deposition modeling promote increased osteogenic differentiation of bone marrow stem cells with increasing substrate modulus and decreasing pore size. These findings guide the rational design of cell-responsive scaffolds that recapitulate the bone microenvironment for repair of bone damaged by trauma or disease., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The contents of this data in brief are related to the article titled "Matrix Rigidity Regulates the Transition of Tumor Cells to a Bone-Destructive Phenotype through Integrin β3 and TGF-β Receptor Type II". In this DIB we will present our supplemental data investigating Integrin expression, attachment of cells to various adhesion molecules, and changes in gene expression in multiple cancer cell lines. Since the interactions of Integrins with adsorbed matrix proteins are thought to affect the ability of cancer cells to interact with their underlying substrates, we examined the expression of Integrin β1, β3, and β5 in response to matrix rigidity. We found that only Iβ3 increased with increasing substrate modulus. While it was shown that fibronectin greatly affects the expression of tumor-produced factors associated with bone destruction (parathyroid hormone-related protein, PTHrP, and Gli2), poly-l-lysine, vitronectin and type I collagen were also analyzed as potential matrix proteins. Each of the proteins was independently adsorbed on both rigid and compliant polyurethane films which were subsequently used to culture cancer cells. Poly-l-lysine, vitronectin and type I collagen all had negligible effects on PTHrP or Gli2 expression, but fibronectin was shown to have a dose dependent effect. Finally, altering the expression of Iβ3 demonstrated that it is required for tumor cells to respond to the rigidity of the matrix, but does not affect other cell growth or viability. Together these data support the data presented in our manuscript to show that the rigidity of bone drives Integrinβ3/TGF-β crosstalk, leading to increased expression of Gli2 and PTHrP.

As in clinical studies, finite element analysis (FEA) developed from computed tomography (CT) images of bones are useful in pre-clinical rodent studies assessing treatment effects on vertebral body (VB) strength. Since strength predictions from microCT-derived FEAs (μFEA) have not been validated against experimental measurements of mouse VB strength, a parametric analysis exploring material and failure definitions was performed to determine whether elastic μFEAs with linear failure criteria could reasonably assess VB strength in two studies, treatment and genetic, with differences in bone volume fraction between the control and the experimental groups. VBs were scanned with a 12-μm voxel size, and voxels were directly converted to 8-node, hexahedral elements. The coefficient of determination or R (2) between predicted VB strength and experimental VB strength, as determined from compression tests, was 62.3% for the treatment study and 85.3% for the genetic study when using a homogenous tissue modulus (E t) of 18 GPa for all elements, a failure volume of 2%, and an equivalent failure strain of 0.007. The difference between prediction and measurement (that is, error) increased when lowering the failure volume to 0.1% or increasing it to 4%. Using inhomogeneous tissue density-specific moduli improved the R (2) between predicted and experimental strength when compared with uniform E t=18 GPa. Also, the optimum failure volume is higher for the inhomogeneous than for the homogeneous material definition. Regardless of model assumptions, μFEA can assess differences in murine VB strength between experimental groups when the expected difference in strength is at least 20%.

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-β) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF-β in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by β-catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF-β and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched β-catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a β-catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF-β signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF-β signaling.

Prostate cancer is the most common primary tumor and the second leading cause of cancer-related deaths in men in the United States. Prostate cancer bone metastases are characterized by abnormal bone remodeling processes and result in a variety of skeletal morbidities. Prevention of skeletal complications is a crucial element in prostate cancer management. This study investigated prostate cancer-induced alterations in the molecular composition and morphological structure of metastasis-bearing bones in a mouse model of prostate cancer using Raman spectroscopy and micro-computed tomography (microCT). LNCaP C4-2B prostate cancer cells were injected into the right tibiae of 5-week old male SCID mice. Upon sacrifice at 8weeks post tumor inoculation, two out of the ten tumor-bearing tibiae showed only osteoblastic lesions in the radiographs, 4 osteolytic lesions only and 4 mixed with osteoblastic and osteolytic lesions. Carbonate substitution was significantly increased while there was a marked reduction in the level of collagen mineralization, mineral crystallinity, and carbonate:matrix ratio in the cortex of the intact tumor-bearing tibiae compared to contralateral controls. MicroCT analysis revealed a significant reduction in bone volume/total volume, trabecular number and trabecular thickness, as well as significant increase in bone surface/volume ratio in tibiae with osteolytic lesions, suggesting active bone remodeling and bone loss. None of the changes in bone compositional properties were correlated with lesion area from radiographs or the changes in bone architecture from microCT. This study indicates that LNCaP C4-2B prostate cancer metastases alter bone tissue composition independent of changes in architecture, and altered bone quality may be an important contributor to fracture risk in these patients. Raman spectroscopy may provide a new avenue of investigation into interactions between tumor and bone microenvironment., (© 2013.)

Myeloid-derived suppressor cells (MDSCs), identified as Gr1(+)CD11b(+) cells in mice, expand during cancer and promote tumor growth, recurrence and burden. However, little is known about their role in bone metastases. We hypothesized that MDSCs may contribute to tumor-induced bone disease, and inoculated breast cancer cells into the left cardiac ventricle of nude mice. Disease progression was monitored weekly by X-ray and fluorescence imaging and MDSCs expansion by fluorescence-activated cell sorting. To explore the contribution of MDSCs to bone metastasis, we co-injected mice with tumor cells or PBS into the left cardiac ventricle and Gr1(+)CD11b(+) cells isolated from healthy or tumor-bearing mice into the left tibia. MDSCs didn't induce bone resorption in normal mice, but increased resorption and tumor burden significantly in tumor-bearing mice. In vitro experiments showed that Gr1(+)CD11b(+) cells isolated from normal and tumor-bearing mice differentiate into osteoclasts when cultured with RANK ligand and macrophage colony-stimulating factor, and that MDSCs from tumor-bearing mice upregulate parathyroid hormone-related protein (PTHrP) mRNA levels in cancer cells. PTHrP upregulation is likely due to the 2-fold increase in transforming growth factor β expression that we observed in MDSCs isolated from tumor-bearing mice. Importantly, using MDSCs isolated from GFP-expressing animals, we found that MDSCs differentiate into osteoclast-like cells in tumor-bearing mice as evidenced by the presence of GFP(+)TRAP(+) cells. These results demonstrate that MDSCs expand in breast cancer bone metastases and induce bone destruction. Furthermore, our data strongly suggest that MDSCs are able to differentiate into osteoclasts in vivo and that this is stimulated in the presence of tumors.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio, that has since been transfected for GFP expression and re-cloned by our group. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis, in certain instances intratibial or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception. This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Background: Breast cancer cells frequently metastasize to bone, where they up-regulate their expression of the transcription factor GLI2 and the downstream osteolytic factor parathyroid hormone-related protein (PTHrP). The guanosine nucleotide 6-thioguanine (6-TG) inhibits PTHrP expression and blocks osteolytic bone destruction in mice inoculated with bone metastatic cells; however, the mechanism by which 6-TG inhibits PTHrP remains unclear. We hypothesized that 6-TG inhibition of PTHrP is mediated through GLI2 signaling., Materials and Methods: Human MDA-MB-231 breast cancer cells and RWGT2 squamous-cell lung carcinoma cells were treated with 100 μM 6-TG and examined for GLI2 mRNA expression and stability by Q-PCR, promoter activity by luciferase assay, and protein expression by Western blot., Results: 6-TG significantly blocked GLI2 mRNA and protein expression, but did not affect stability. Additionally, 6-TG directly inhibited GLI2 promoter activity, and when cells were transfected with constitutively expressed GLI2, the inhibitory effect of 6-TG on PTHrP expression was abolished., Conclusion: Taken together, these data indicate that 6-TG regulates PTHrP in part through GLI2 transcription, and therefore the clinical use of 6-TG or other guanosine nucleotides may be a viable therapeutic option in tumor types expressing elevated levels of GLI proteins.

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone-related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-β-stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep-expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.

Nearly 70% of breast cancer patients with advanced disease will develop bone metastases. Once established in bone, tumor cells produce factors that cause changes in normal bone remodeling, such as parathyroid hormone-related protein (PTHrP). While enhanced expression of PTHrP is known to stimulate osteoclasts to resorb bone, the environmental factors driving tumor cells to express PTHrP in the early stages of development of metastatic bone disease are unknown. In this study, we have shown that tumor cells known to metastasize to bone respond to 2D substrates with rigidities comparable to that of the bone microenvironment by increasing expression and production of PTHrP. The cellular response is regulated by Rho-dependent actomyosin contractility mediated by TGF-ß signaling. Inhibition of Rho-associated kinase (ROCK) using both pharmacological and genetic approaches decreased PTHrP expression. Furthermore, cells expressing a dominant negative form of the TGF-ß receptor did not respond to substrate rigidity, and inhibition of ROCK decreased PTHrP expression induced by exogenous TGF-ß. These observations suggest a role for the differential rigidity of the mineralized bone microenvironment in early stages of tumor-induced osteolysis, which is especially important in metastatic cancer since many cancers (such as those of the breast and lung) preferentially metastasize to bone.