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Breast cancer bone metastasis develops as the result of a series of complex interactions between tumor cells, bone marrow cells, and resident bone cells. The net effect of these interactions are the disruption of normal bone homeostasis, often with significantly increased osteoclast and osteoblast activity, which has provided a rational target for controlling tumor progression, with little or no emphasis on tumor eradication. Indeed, the clinical course of metastatic breast cancer is relatively long, with patients likely to experience sequential skeletal-related events (SREs), often over lengthy periods of time, even up to decades. These SREs include bone pain, fractures, and spinal cord compression, all of which may profoundly impair a patient's quality-of-life. Our understanding of the contributions of the host bone and bone marrow cells to the control of tumor progression has grown over the years, yet the focus of virtually all available treatments remains on the control of resident bone cells, primarily osteoclasts. In this perspective, our focus is to move away from the current emphasis on the control of bone cells and focus our attention on the hallmarks of bone metastatic tumor cells and how these differ from primary tumor cells and normal host cells. In our opinion, there remains a largely unmet medical need to develop and utilize therapies that impede metastatic tumor cells while sparing normal host bone and bone marrow cells. This perspective examines the impact of metastatic tumor cells on the bone microenvironment and proposes potential new directions for uncovering the important mechanisms driving metastatic progression in bone based on the hallmarks of bone metastasis.
Differentiating osteoclast-rich lesions of bone (giant cell tumor of bone [GCTB], chondroblastoma [CBA], and aneurysmal bone cyst [ABC]) can be challenging, especially in small biopsies or fine-needle aspirations. Mutations affecting codons 34 and 36 of either H3 Histone Family Member 3A (H3F3A) and/or 3B (H3F3B) are characteristically seen in GCTB and CBAs. We devised a simple assay to identify these mutations and evaluated its applicability for routine clinical diagnosis. One hundred twenty-four tissue specimens from 108 patients (43 GCTBs, 38 CBAs and 27 ABCs) were collected from the archives of the Calgary Laboratory Services/University of Calgary and Vanderbilt University Medical Center. Histology was reviewed by an expert orthopedic pathologist. A single base extension assay (SNaPshot) is used to interrogate each nucleotide in codons 34 and 36 of H3F3A and codon 36 of H3F3B. Final diagnoses were generated after re-reviewing cases and incorporating molecular findings. Of 43 GCTBs, 38 (88%) had an H3F3A G34W mutation; 35 of 38 CBAs (92%) had a K36M mutation in either H3F3B (N = 31; 82%) or H3F3A (N = 4; 11%); none of 27 ABCs had a tested mutation. Molecular findings changed the histomorphologic diagnosis in 5 cases (3 GCTB changed to ABC, and 2 ABC changed to GCTB). These findings support the diagnostic utility of mutational analysis for this differential diagnosis in certain challenging cases when clinicoradiologic and histomorphologic features are not definitive, particularly for distinguishing cellular ABC versus GCTB with secondary ABC.
Copyright © 2017 Elsevier Inc. All rights reserved.
We used a murine model of acute, posttraumatic osteomyelitis to evaluate the virulence of two divergent Staphylococcus aureus clinical isolates (the USA300 strain LAC and the USA200 strain UAMS-1) and their isogenic sarA mutants. The results confirmed that both strains caused comparable degrees of osteolysis and reactive new bone formation in the acute phase of osteomyelitis. Conditioned medium (CM) from stationary-phase cultures of both strains was cytotoxic to cells of established cell lines (MC3TC-E1 and RAW 264.7 cells), primary murine calvarial osteoblasts, and bone marrow-derived osteoclasts. Both the cytotoxicity of CM and the reactive changes in bone were significantly reduced in the isogenic sarA mutants. These results confirm that sarA is required for the production and/or accumulation of extracellular virulence factors that limit osteoblast and osteoclast viability and that thereby promote bone destruction and reactive bone formation during the acute phase of S. aureus osteomyelitis. Proteomic analysis confirmed the reduced accumulation of multiple extracellular proteins in the LAC and UAMS-1 sarA mutants. Included among these were the alpha class of phenol-soluble modulins (PSMs), which were previously implicated as important determinants of osteoblast cytotoxicity and bone destruction and repair processes in osteomyelitis. Mutation of the corresponding operon reduced the cytotoxicity of CM from both UAMS-1 and LAC cultures for osteoblasts and osteoclasts. It also significantly reduced both reactive bone formation and cortical bone destruction by CM from LAC cultures. However, this was not true for CM from cultures of a UAMS-1 psmα mutant, thereby suggesting the involvement of additional virulence factors in such strains that remain to be identified.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Bone metastatic disease remains a significant and frequent problem for cancer patients that can lead to increased morbidity and mortality. Unfortunately, despite decades of research, bone metastases remain incurable. Current studies have demonstrated that many properties and cell types within the bone and bone marrow microenvironment contribute to tumor-induced bone disease. Furthermore, they have pointed to the importance of understanding how tumor cells interact with their microenvironment in order to help improve both the development of new therapeutics and the prediction of response to therapy.
Interleukin-6 (IL-6) and interleukin-11 (IL-11) receptors (IL-6R and IL-11R, respectively) are both expressed in osteoclasts and transduce signal via the glycoprotein130 (gp130) co-receptor, but the physiological role of this pathway is unclear. To determine the critical roles of gp130 signalling in the osteoclast, we generated mice using cathepsin K Cre (CtskCre) to disrupt gp130 signalling in osteoclasts. Bone marrow macrophages from CtskCre.gp130(f/f) mice generated more osteoclasts in vitro than cells from CtskCre.gp130(w/w) mice; these osteoclasts were also larger and had more nuclei than controls. While no increase in osteoclast numbers was observed in vivo, osteoclasts on trabecular bone surfaces of CtskCre.gp130(f/f) mice were more spread out than in control mice, but had no functional defect detectable by serum CTX1 levels or trabecular bone cartilage remnants. However, trabecular osteoblast number and mineralising surfaces were significantly lower in male CtskCre.gp130(f/f) mice compared to controls, and this was associated with a significantly lower trabecular bone volume at 12 weeks of age. Furthermore, CtskCre.gp130(f/f) mice exhibited greatly suppressed periosteal bone formation at this age, indicated by significant reductions in both double-labelled surface and mineral apposition rate. By 26 weeks of age, CtskCre.gp130(f/f) mice exhibited narrower femora, with lower periosteal and endocortical perimeters than CtskCre.gp130(w/w) controls. Since IL-6 and IL-11R global knockout mice exhibited a similar reduction in femoral width, we also assessed periosteal bone formation in those strains, and found bone forming surfaces were also reduced in male IL-6 null mice. These data suggest that IL-6/gp130 signalling in the osteoclast is not essential for normal bone resorption in vivo, but maintains both trabecular and periosteal bone formation in male mice by promoting osteoblast activity through the stimulation of osteoclast-derived "coupling factors" and "osteotransmitters", respectively.
Copyright © 2015 Elsevier Inc. All rights reserved.
BACKGROUND - Infectious complications of musculoskeletal trauma are an important factor contributing to patient morbidity. Biofilm-dispersive bone grafts augmented with D-amino acids (D-AAs) prevent biofilm formation in vitro and in vivo, but the effects of D-AAs on osteocompatibility and new bone formation have not been investigated.
QUESTIONS/PURPOSES - We asked: (1) Do D-AAs hinder osteoblast and osteoclast differentiation in vitro? (2) Does local delivery of D-AAs from low-viscosity bone grafts inhibit new bone formation in a large-animal model?
METHODS - Methicillin-sensitive Staphylococcus aureus and methicillin-resistant S aureus clinical isolates, mouse bone marrow stromal cells, and osteoclast precursor cells were treated with an equal mass (1:1:1) mixture of D-Pro:D-Met:D-Phe. The effects of the D-AA dose on biofilm inhibition (n = 4), biofilm dispersion (n = 4), and bone marrow stromal cell proliferation (n = 3) were quantitatively measured by crystal violet staining. Osteoblast differentiation was quantitatively assessed by alkaline phosphatase staining, von Kossa staining, and quantitative reverse transcription for the osteogenic factors a1Col1 and Ocn (n = 3). Osteoclast differentiation was quantitatively measured by tartrate-resistant acid phosphatase staining (n = 3). Bone grafts augmented with 0 or 200 mmol/L D-AAs were injected in ovine femoral condyle defects in four sheep. New bone formation was evaluated by μCT and histology 4 months later. An a priori power analysis indicated that a sample size of four would detect a 7.5% difference of bone volume/total volume between groups assuming a mean and SD of 30% and 5%, respectively, with a power of 80% and an alpha level of 0.05 using a two-tailed t-test between the means of two independent samples.
RESULTS - Bone marrow stromal cell proliferation, osteoblast differentiation, and osteoclast differentiation were inhibited at D-AAs concentrations of 27 mmol/L or greater in a dose-responsive manner in vitro (p < 0.05). In methicillin-sensitive and methicillin-resistant S aureus clinical isolates, D-AAs inhibited biofilm formation at concentrations of 13.5 mmol/L or greater in vitro (p < 0.05). Local delivery of D-AAs from low-viscosity grafts did not inhibit new bone formation in a large-animal model pilot study (0 mmol/L D-AAs: bone volume/total volume = 26.9% ± 4.1%; 200 mmol/L D-AAs: bone volume/total volume = 28.3% ± 15.4%; mean difference with 95% CI = -1.4; p = 0.13).
CONCLUSIONS - D-AAs inhibit biofilm formation, bone marrow stromal cell proliferation, osteoblast differentiation, and osteoclast differentiation in vitro in a dose-responsive manner. Local delivery of D-AAs from bone grafts did not inhibit new bone formation in vivo at clinically relevant doses.
CLINICAL RELEVANCE - Local delivery of D-AAs is an effective antibiofilm strategy that does not appear to inhibit bone repair. Longitudinal studies investigating bacterial burden, bone formation, and bone remodeling in contaminated defects as a function of D-AA dose are required to further support the use of D-AAs in the clinical management of infected open fractures.
Aberrant fibroblast growth factor receptor 3 (FGFR3) signaling disrupts chondrocyte proliferation and growth plate size and architecture, leading to various chondrodysplasias or bone overgrowth. These observations suggest that the duration, intensity and cellular context of FGFR signaling during growth plate chondrocyte maturation require tight, regulated control for proper bone elongation. However, the machinery fine-tuning FGFR signaling in chondrocytes is incompletely defined. We report here that neurofibromin, a Ras-GAP encoded by Nf1, has an overlapping expression pattern with FGFR1 and FGFR3 in prehypertrophic chondrocytes, and with FGFR1 in hypertrophic chondrocytes during endochondral ossification. Based on previous evidence that neurofibromin inhibits Ras-ERK signaling in chondrocytes and phenotypic analogies between mice with constitutive FGFR1 activation and Nf1 deficiency in Col2a1-positive chondrocytes, we asked whether neurofibromin is required to control FGFR1-Ras-ERK signaling in maturing chondrocytes in vivo. Genetic Nf1 ablation in Fgfr1-deficient chondrocytes reactivated Ras-ERK1/2 signaling in hypertrophic chondrocytes and reversed the expansion of the hypertrophic zone observed in mice lacking Fgfr1 in Col2a1-positive chondrocytes. Histomorphometric and gene expression analyses suggested that neurofibromin, by inhibiting Rankl expression, attenuates pro-osteoclastogenic FGFR1 signaling in hypertrophic chondrocytes. We also provide evidence suggesting that neurofibromin in prehypertrophic chondrocytes, downstream of FGFRs and via an indirect mechanism, is required for normal extension and organization of proliferative columns. Collectively, this study indicates that FGFR signaling provides an important input into the Ras-Raf-MEK-ERK1/2 signaling axis in chondrocytes, and that this input is differentially regulated during chondrocyte maturation by a complex intracellular machinery, of which neurofibromin is a critical component.
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: email@example.com.
Rheumatoid arthritis is a chronic inflammatory disease characterized by synovial hyperplasia, inflammatory cell infiltration, irreversible cartilage and bone destruction, and exuberant coagulation system activity within joint tissue. Here, we demonstrate that the coagulation transglutaminase, factor XIII (fXIII), drives arthritis pathogenesis by promoting local inflammatory and tissue degradative and remodeling events. All pathological features of collagen-induced arthritis (CIA) were significantly reduced in fXIII-deficient mice. However, the most striking difference in outcome was the preservation of cartilage and bone in fXIIIA(-/-) mice concurrent with reduced osteoclast numbers and activity. The local expression of osteoclast effectors receptor activator of nuclear factor-κB ligand (RANKL) and tartrate resistant acid phosphatase were significantly diminished in CIA-challenged and even unchallenged fXIIIA(-/-) mice relative to wild-type animals, but were similar in wild-type and fibrinogen-deficient mice. Impaired osteoclast formation in fXIIIA(-/-) mice was not due to an inherent deficiency of monocyte precursors, but it was linked to reduced RANKL-driven osteoclast formation. Furthermore, treatment of mice with the pan-transglutaminase inhibitor cystamine resulted in significantly diminished CIA pathology and local markers of osteoclastogenesis. Thus, eliminating fXIIIA limits inflammatory arthritis and protects from cartilage and bone destruction in part through mechanisms linked to reduced RANKL-mediated osteoclastogenesis. In summary, therapeutic strategies targeting fXIII activity may prove beneficial in limiting arthropathies and other degenerative bone diseases.
© 2015 by The American Society of Hematology.
Tumor-induced bone disease is a dynamic process that involves interactions with many cell types. Once metastatic cancer cells reach the bone, they are in contact with many different cell types that are present in the cell-rich bone marrow. These cells include the immune cells, myeloid cells, fibroblasts, osteoblasts, osteoclasts, and mesenchymal stem cells. Each of these cell populations can influence the behavior or gene expression of both the tumor cells and the bone microenvironment. Additionally, the tumor itself can alter the behavior of these bone marrow cells which further alters both the microenvironment and the tumor cells. While many groups focus on studying these interactions, much remains unknown. A better understanding of the interactions between the tumor cells and the bone microenvironment will improve our knowledge on how tumors establish in bone and may lead to improvements in diagnosing and treating bone metastases. This review details our current knowledge on the interactions between tumor cells that reside in bone and their microenvironment.