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1.
Cancer Res Commun ; 3(2): 223-234, 2023 02.
Article in English | MEDLINE | ID: mdl-36968140

ABSTRACT

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.


Subject(s)
Nanoparticles , Neoplasms , Mice , Animals , Bone Marrow , Cytokines , Phenotype , Tumor Microenvironment
2.
J Clin Transl Sci ; 6(1): e142, 2022.
Article in English | MEDLINE | ID: mdl-36590348

ABSTRACT

Background: Coronavirus Disease 2019 (COVID-19) instigated a flurry of clinical research activity. The unprecedented pace with which trials were launched left an early void in data standardization, limiting the potential for subsequent data pooling. To facilitate data standardization across emerging studies, the National Heart, Lung, and Blood Institute (NHLBI) charged two groups with harmonizing data collection, and these groups collaborated to create a concise set of COVID-19 Common Data Elements (CDEs) for clinical research. Methods: Our iterative approach followed three guiding principles: 1) draw from existing multi-center COVID-19 clinical trials as precedents, 2) incorporate existing data elements and data standards whenever possible, and 3) alignment to data standards that facilitate data sharing and regulatory submission. We also supported rapid implementation of the CDEs in NHLBI-funded studies and iteratively refined the CDEs based on feedback from those study teams. Results: The NHLBI COVID-19 CDEs are publicly available and being used for current COVID-19 clinical trials. CDEs are organized into domains, and each data element is classified within a three-tiered prioritization system. The CDE manual is hosted publicly at https://nhlbi-connects.org/common_data_elements with an accompanying data dictionary and implementation guidance. Conclusions: The NHLBI COVID-19 CDEs are designed to aid data harmonization across studies to achieve the benefits of pooled analyses. We found that organizing CDE development around our three guiding principles focused our efforts and allowed us to adapt as COVID-19 knowledge advanced. As these CDEs continue to evolve, they could be generalized for use in other acute respiratory illnesses.

3.
Transl Oncol ; 14(11): 101213, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34461557

ABSTRACT

Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy (ADT) remains challenging. Many studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of CRPC, including resistance to the new generation of inhibitors of androgen receptor (AR) action. ARVs are constitutively active and lack the ligand-binding domain (LBD), thereby allowing prostate cancer (PC) to maintain AR activity despite therapies that target the AR (full-length AR; AR-FL). Previously, we have reported that long-term ADT increases the neuroendocrine (NE) hormone - Gastrin Releasing Peptide (GRP) and its receptor (GRP-R) expression in PC cells. Further, we demonstrated that activation of GRP/GRP-R signaling increases ARVs expression by activating NF-κB signaling, thereby promoting cancer progression to CRPC. Most importantly, as a cell surface protein, GRP-R is easily targeted by drugs to block GRP/GRP-R signaling. In this study, we tested if blocking GRP/GRP-R signaling by targeting GRP-R using GRP-R antagonist is sufficient to control CRPC progression. Our studies show that blocking GRP/GRP-R signaling by targeting GRP-R using RC-3095, a selective GRP-R antagonist, efficiently inhibits NF-κB activity and ARVs (AR-V7) expression in CRPC and therapy-induced NEPC (tNEPC) cells. In addition, blocking of GRP/GRP-R signaling by targeting GRP-R can sensitize CRPC cells to anti-androgen treatment (such as MDV3100). Further, preclinical animal studies indicate combination of GRP-R antagonist (targeting ARVs) with anti-androgen (targeting AR-FL) is sufficient to inhibit CRPC and tNEPC tumor growth.

4.
Int J Mol Sci ; 22(13)2021 Jun 27.
Article in English | MEDLINE | ID: mdl-34199096

ABSTRACT

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 α2ß1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2ß1 in the context of bone metastasis. In this study, we aimed to understand the role of α2ß1 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 α2ß1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2ß1 expression and bone-metastatic potential. Inhibiting α2ß1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.


Subject(s)
Bone Neoplasms/secondary , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Gene Expression , Integrin alpha2beta1/genetics , Animals , Bone Neoplasms/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Disease Models, Animal , Female , Humans , Mice , Neoplasm Invasiveness , Osteolysis/genetics , Osteolysis/metabolism , Phenotype
5.
JBMR Plus ; 5(4): e10465, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33869989

ABSTRACT

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.

6.
Int J Mol Sci ; 21(18)2020 Sep 21.
Article in English | MEDLINE | ID: mdl-32967150

ABSTRACT

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 ß3 and TGF-ß 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.


Subject(s)
Bone Neoplasms , Cell Movement/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Models, Biological , Neoplasm Proteins/biosynthesis , Pyridines/pharmacology , Thiophenes/pharmacology , Tissue Scaffolds/chemistry , Animals , Bone Neoplasms/drug therapy , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cell Line, Tumor , Female , Humans , Mice , Mice, Nude , Neoplasm Metastasis
7.
ACS Nano ; 14(1): 311-327, 2020 01 28.
Article in English | MEDLINE | ID: mdl-31894963

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/pharmacology , Bone Neoplasms/drug therapy , Nanoparticles/chemistry , Polymers/pharmacology , Pyridines/pharmacology , Thiophenes/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Bone Neoplasms/diagnostic imaging , Bone Neoplasms/secondary , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Screening Assays, Antitumor , Female , Humans , Ligands , Mice , Mice, Inbred C57BL , Mice, Knockout , Optical Imaging , Particle Size , Polymers/chemical synthesis , Polymers/chemistry , Pyridines/chemistry , Surface Properties , Thiophenes/chemistry , X-Ray Microtomography
8.
J Control Release ; 311-312: 257-272, 2019 10.
Article in English | MEDLINE | ID: mdl-31494183

ABSTRACT

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 (PPS135-b-POEGA17). 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.


Subject(s)
Antineoplastic Agents/administration & dosage , Bone Neoplasms/drug therapy , Drug Carriers/administration & dosage , Mammary Neoplasms, Animal/drug therapy , Nanoparticles/administration & dosage , Pyridines/administration & dosage , Thiophenes/administration & dosage , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Apoptosis/drug effects , Bone Neoplasms/secondary , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Drug Liberation , Female , Gene Expression Regulation, Neoplastic , Humans , Mammary Neoplasms, Animal/pathology , Mesenchymal Stem Cells/drug effects , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Nanoparticles/chemistry , Osteogenesis/drug effects , Polymers/administration & dosage , Polymers/chemistry , Polymers/pharmacokinetics , Pyridines/chemistry , Pyridines/pharmacokinetics , Thiophenes/chemistry , Thiophenes/pharmacokinetics , Zinc Finger Protein Gli2/antagonists & inhibitors , Zinc Finger Protein Gli2/genetics
9.
JCI Insight ; 4(5)2019 03 07.
Article in English | MEDLINE | ID: mdl-30702442

ABSTRACT

ß-2 Microglobulin (ß2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but ß2M may also have less understood chaperone-independent functions. Elevated plasma ß2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. ß2M mRNA is present in platelets at very high levels, and ß2M 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 ß2M is a mediator of monocyte proinflammatory differentiation through noncanonical TGFß receptor signaling. Circulating monocytes from mice lacking ß2M only in platelets (Plt-ß2M-/-) had a more proreparative monocyte phenotype, in part dependent on increased platelet-derived TGFß signaling in the absence of ß2M. Using a mouse myocardial infarction (MI) model, Plt-ß2M-/- 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 ß2M and that platelet-derived 2M and TGFß have opposing roles in monocyte differentiation that may be important in tissue injury responses.


Subject(s)
Blood Platelets/metabolism , Monocytes/metabolism , beta 2-Microglobulin/metabolism , Animals , Cell Differentiation , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Chaperones , Platelet Activation , Receptor, Transforming Growth Factor-beta Type II/genetics , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction , THP-1 Cells , beta 2-Microglobulin/genetics
10.
Biomaterials ; 179: 29-45, 2018 10.
Article in English | MEDLINE | ID: mdl-29960822

ABSTRACT

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% ß-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.


Subject(s)
Bone Cements/chemistry , Polymers/chemistry , Tibia/injuries , Animals , Bone Transplantation , Ceramics/chemistry , Compressive Strength , Female , Glass/chemistry , Immunohistochemistry , Polymethyl Methacrylate/chemistry , Sheep , Tibia/surgery , Weight-Bearing/physiology , X-Ray Microtomography
11.
Bone ; 113: 77-88, 2018 08.
Article in English | MEDLINE | ID: mdl-29753718

ABSTRACT

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 ß (TGF-ß), an abundant growth factor in bone, can regulate both TIBD and myeloid expansion. TGF-ß inhibitors have been shown to increase bone volume, decrease bone destruction, and reduce but not eliminate tumor. Therefore, we hypothesize that inhibiting TGF-ß 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-ß 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-ßRII) (TGF-ßRIIMyeKO) was utilized in our studies. Systemic inhibition of TGF-ß lead to fewer osteolytic lesions, and reduced tumor burden in bone as expected from previous studies. Additionally, early TGF-ß 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-ßRIIMyeKO mice, where these mice contained fewer bone lesions and significantly less tumor burden in bone, suggesting that TGF-ß inhibition regulates myeloid expansion leading to a significant reduction in TIBD.


Subject(s)
Bone Neoplasms/secondary , Mammary Neoplasms, Experimental/secondary , Myeloid Progenitor Cells/pathology , Transforming Growth Factor beta/antagonists & inhibitors , Tumor Microenvironment/physiology , Animals , Female , Humans , Mice , Mice, Knockout
12.
Adv Healthc Mater ; 6(22)2017 Nov.
Article in English | MEDLINE | ID: mdl-28892261

ABSTRACT

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.


Subject(s)
Cancellous Bone/cytology , Biocompatible Materials/chemistry , Bone Regeneration/physiology , Cartilage/cytology , Cell Differentiation/physiology , Cells, Cultured , Humans , Materials Testing/methods , Mesenchymal Stem Cells/cytology , Osteogenesis/physiology , Printing, Three-Dimensional , Tissue Engineering/methods , Tissue Scaffolds/chemistry
13.
J Bone Miner Res ; 32(7): 1442-1454, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28300321

ABSTRACT

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 (ß2AR) 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 ß2AR 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 ß2AR 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.


Subject(s)
Bone and Bones/metabolism , Breast Neoplasms/metabolism , Neoplasm Proteins/metabolism , Neovascularization, Pathologic/metabolism , Osteoblasts/metabolism , Receptors, Adrenergic, beta-2/metabolism , Animals , Bone and Bones/pathology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Coculture Techniques , Female , Humans , Mice , Mice, Knockout , Neoplasm Metastasis , Neoplasm Proteins/genetics , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Osteoblasts/pathology , Receptors, Adrenergic, beta-2/genetics , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism
15.
Nat Cell Biol ; 18(10): 1078-1089, 2016 10.
Article in English | MEDLINE | ID: mdl-27642788

ABSTRACT

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.

16.
Bone ; 91: 81-91, 2016 10.
Article in English | MEDLINE | ID: mdl-27423464

ABSTRACT

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-ß (TGF-ß) activity often contributes to tumor-induced bone disease, and pre-clinical studies have indicated that TGF-ß inhibition improves bone volume and reduces tumor growth in bone metastatic breast cancer. We hypothesized that inhibition of TGF-ß 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-ß inhibitory (1D11) or control (13C4) antibody, with or without the anti-myeloma drug bortezomib, for 4weeks after inoculation of murine 5TGM1 MM cells. TGF-ß 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-ß signaling in combination with existing anti-myeloma agents as a potential therapeutic strategy to improve outcomes in patients with myeloma bone disease.


Subject(s)
Bone Diseases/drug therapy , Bone Diseases/etiology , Bone and Bones/pathology , Bortezomib/therapeutic use , Multiple Myeloma/complications , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Bone Diseases/pathology , Bone and Bones/drug effects , Bortezomib/pharmacology , Cancellous Bone/pathology , Cancellous Bone/physiopathology , Cell Count , Cell Line, Tumor , Disease Models, Animal , Drug Therapy, Combination , Mice, Inbred C57BL , Multiple Myeloma/pathology , Osteoblasts/pathology , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction/drug effects , Transforming Growth Factor beta/metabolism , Tumor Burden/drug effects
17.
Calcif Tissue Int ; 99(3): 289-301, 2016 09.
Article in English | MEDLINE | ID: mdl-27209312

ABSTRACT

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.


Subject(s)
Bone Density/physiology , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 2/complications , Fractures, Bone/physiopathology , Animals , Blood Glucose/biosynthesis , Body Weight/physiology , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 2/physiopathology , Disease Models, Animal , Fractures, Bone/prevention & control , Male , Rats , X-Ray Microtomography/methods
18.
Data Brief ; 4: 440-6, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26306316

ABSTRACT

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 ß3 and TGF-ß 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 ß1, ß3, and ß5 in response to matrix rigidity. We found that only Iß3 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 Iß3 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 Integrinß3/TGF-ß crosstalk, leading to increased expression of Gli2 and PTHrP.

19.
Biomaterials ; 64: 33-44, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26115412

ABSTRACT

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 ß3 (Iß3), 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 Iß3 reduced Gli2 and PTHrP expression. To identify the mechanism by which Iß3 regulates Gli2 and PTHrP (both are also known to be regulated by TGF-ß), we performed Förster resonance energy transfer (FRET) and immunoprecipitation, which indicated that Iß3 co-localized with TGF-ß Receptor Type II (TGF-ß RII) on rigid but not compliant films. Finally, transplantation of tumor cells expressing Iß3 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 Iß3 in disease progression. This study demonstrates that the rigid mineralized bone matrix can alter gene expression and bone destruction in an Iß3/TGF-ß-dependent manner, and suggests that Iß3 inhibitors are a potential therapeutic approach for blocking tumor transition to a bone destructive phenotype.


Subject(s)
Integrin beta3/physiology , Neoplasm Proteins/physiology , Osteolysis/etiology , Pliability , Protein Serine-Threonine Kinases/physiology , Receptors, Transforming Growth Factor beta/physiology , Transforming Growth Factor beta/physiology , Tumor Microenvironment/physiology , Adenocarcinoma/pathology , Adenocarcinoma/secondary , Animals , Bone Neoplasms/complications , Bone Neoplasms/secondary , Breast Neoplasms/pathology , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/secondary , Cell Line, Tumor , Elastic Modulus , Extracellular Matrix/physiology , Female , Gene Expression Regulation, Neoplastic , Humans , Integrin beta3/drug effects , Integrin beta3/genetics , Kruppel-Like Transcription Factors/biosynthesis , Kruppel-Like Transcription Factors/genetics , Lung Neoplasms/pathology , Mice , Mice, Nude , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Receptor, Transforming Growth Factor-beta Type II , Transfection , Xenograft Model Antitumor Assays , Zinc Finger Protein Gli2
20.
Adv Healthc Mater ; 4(12): 1826-32, 2015 Aug 26.
Article in English | MEDLINE | ID: mdl-26121662

ABSTRACT

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.


Subject(s)
Cell Differentiation , Osteogenesis , Tissue Scaffolds/chemistry , Animals , Cell Movement , Cell Proliferation , Cells, Cultured , Mesenchymal Stem Cells , Rats , Rats, Sprague-Dawley
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