Over 80% of patients with advanced breast cancer will develop bone metastases for which there is no cure. Although thought to involve a complex cascade of cell-cell interactions, the factors controlling the development of bone metastases are still poorly understood. Osteoblasts may have an important role in mediating homing and proliferation of breast cancer cells to the bony environment. This study aimed to examine the potential role osteoblasts have in the migration of circulating tumour cells to bone and the factors involved in this attraction. Culture of osteoblasts and MDA-MB-231 breast cancer cells was performed. Breast cancer cell migration in response to osteoblasts was measured using Transwell Migration Inserts. Potential mediators of cell migration were detected using ChemiArray & ELISA assays. A luminometer based Vialight assay was used to measure breast cancer cell proliferation in response to factors secreted by osteoblasts. There was a 3-4 fold increase of MDA-MB-231 migration in response to osteoblasts. ChemiArray analysis of osteoblast-conditioned medium revealed a range of secreted chemokines including IL-6 & 8, TIMP 1 & 2 and MCP-1. Initially, MCP-1 was quantified at 282 pg/ml, but rose to over 9000 pg/ml when osteoprogenitor cells were differentiated into mature osteoblasts. Inclusion of a monoclonal antibody to MCP-1 in osteoblast-conditioned medium resulted in a significant decrease in breast cancer cell migration to osteoblasts. There was no significant change in proliferation of MDA-MB 231 cells when exposed to osteoblast-conditioned medium. Osteoblasts are capable of inducing breast cancer cell migration mediated at least in part by chemokine secretion. MCP-1 produced by the osteoblasts was shown to play a central role in mediating homing of the breast cancer cells. Increased understanding of the pathways involved in the development of bone metastases may provide new targets for therapeutic intervention

Purpose. Versican is a member of the large aggregating chondroitin sulfate proteoglycan family. Structurally, it is made up of an N-terminal G1 domain, a glycosamingoglycan attachment region, and a C-terminus containing a selectin-like (G3) domain. Versican is highly expressed in the interstitial tissues at the invasive margins of breast carcinoma and predictive of relapse and overall survival. The purpose of the study to investigate the role of of versican G3 domain in breast cancer bone metastasis. Method. Mouse mammary tumor cell lines 66c14, 4T07 and 4T1, and human breast cancer cell lines MT-1, MDA-MB-468 and MDA-MB-231 were stably transfected with versican G3. Effects of expression of versican G3 on cell proliferation, migration, invasion, cell cycle progression, and EGFR signaling were observed. The effects of G3 on cell viability in the conditional media of serum free, apoptotic agent C2-ceramide, and chemotherapeutic agents, including Docetaxel, Doxorubicin, Epirubicin were investigated. Colony formation assay and mammosphere formation assay were performed. A syngeneic orthotopic animal model was used to do the in vivo study. Results. In vitro, G3 enhanced breast cancer cell proliferation and migration by up-regulating EGFR signaling, and enhanced cell motility through chemotactic mechanisms to bone stromal cells, which was prevented by inhibitor AG 1478. Experiments in a syngeneic orthotopic animal model demonstrated that G3 promoted tumor growth and bone metastasis in vivo. Versican G3 domain enhanced tumor cell resistance to apoptosis in serum free medium, Doxorubicin, or Epirubicin by up-regulating pERK and GSK-3β (S9P), and promoted cell apoptosis induced by C2-ceramide or Docetaxel by enhanced expression of pSAPK/JNK and decreased GSK-3β (S9P). Versican expresses highly in the breast cancer mammosphere progenitor cells. Expression of versican G3 enhanced breast cancer self-renewal in vitro and in vivo. Conclusion. The activity of G3 on mouse mammary tumor cell growth, migration and its effect on spontaneous metastasis to bone in an orthotopic model was modulated by up-regulating the EGFR-mediated signaling. The dual roles of G3 in modulating breast cancer cell resistance to chemotherapeutic agents indicate a potential mechanism for breast cancer cell sensitivity or resistance to chemotherapy and EGFR therapy. GSK-3β (S9P) works as a key check point for the balance of apoptosis and anti-apoptosis. Over-expression of versican G3 domain enhanced breast cancer self-renewal, and resistant to chemo-drug treatments. Strategies designed to target versican mediated breast cancer self-renewal or GSK-3β (S9P) may lead to an effective therapy benefiting advanced breast cancer patients

Background. 70% of breast cancer patients develop metastatic bone deposits, predominantly spinal metasases. Adult Mesenchymal Stem Cells (MSCs) are multiprogenitor stem cells found within the bone marow which have the ability to self-renew and differentiate into multiple cell types. MSCs home specifically to tumour sites, highlighting their potential as delivery vehicles for therapeutic agents. However studies show they may also increase tumour metastatic potential. Aim. To investigate interactions between MSCs and breast cancer cells to further elucidate their role in the tumour microenvironment and hence understand factors involved in stimulating the formation of bone metastases. Methods. MSCs harvested from the iliac crest of healthy volunteers were grown for collection of conditioned medium (CM), containing all factors secreted by the cells. Breast cancer cell lines (T47D, SK-BR-3, MDA-MB-231) were then cultured in MSC CM +/− antibodies to TGFβ, VEGF, MCP-1 and CCL5 for 72hrs. Cell proliferation was assessed using an Apoglow. (r). assay and RNA harvested for analysis of changes in Epithelial Mesenchymal Transition specific gene expression : N-Cadherin, E-Cadherin, Vimentin, Twist, Snail. Results. A significant down regulation of breast cancer cell proliferation in the presence of MSC secreted factors was observed (p< 0.05). There was a dramatic increase in expression of EMT specific genes in both cell lines following exposure to MSC-secreted factors. Inclusion of antibodies to TGF, VEGF, MCP-1 and CCL5 inhibited the effect seen, suggesting these paracrine factors played a role in the elevated expression levels. Conclusion. MSCs clearly have a distinct paracrine effect on breast cancer epithelial cells, mediated at least in part through secretion of growth factors and chemokines. These factors play an important role in the metastatic cascade and may represent potential therapeutic targets to inhibit MSC-breast cancer interactions, helping to prevent the formation of bone metastases in cancer

Background. 70% of Breast Cancer patients develop metastatic bone deposits, predominantly spinal metasases. Adult Mesenchymal Stem Cells (MSCs) are multiprogenitor stem cells found within the bone marow which have the ability to self renew and differentiate into multiple cell types. MSCs home specifically to tumour sites, highlighting their potential as delivery vehicles for therapeutic agents. However studies show they may also increase tumour metastatic potential. Aims. The aim of this study was to investigate interactions between MSCs and breast cancer cells to further elucidate their role in the tumour microenvironment and hence understand factors involved in stimulating the formation of bone metastases. Methods. MSCs harvested from the iliac crest of healthy volunteers were grown for collection of conditioned medium (CM), containing all factors secreted by the cells. Breast cancer cell lines (T47D, SK-BR-3, MDA-MB-231) were then cultured in MSC CM +/− antibodies to TGFβ, VEGF, MCP-1 and CCL5 for 72hrs. Cell proliferation was assessed using an Apoglow(r) assay and RNA harvested for analysis of changes in Epithelial Mesenchymal Transition specific gene expression : N-Cadherin, E-Cadherin, Vimentin, Twist, Snail. Results. A significant down regulation of breast cancer cell proliferation in the presence of MSC secreted factors was observed (p<0.05). There was a dramatic increase in expression of EMT specific genes in both cell lines following exposure to MSC-secreted factors. Inclusion of antibodies to TGF, VEGF, MCP-1 and CCL5 inhibited the effect seen, suggesting these paracrine factors played a role in the elevated expression levels. Conclusion. MSCs clearly have a distinct paracrine effect on breast cancer epithelial cells, mediated at least in part through secretion of growth factors and chemokines. These factors play an important role in the metastatic cascade and may represent potential therapeutic targets to inhibit MSC-breast cancer interactions, helping to prevent the formation of bone metastases in cancer

Bone metastases are the most common cause of cancer-related pain and often lead to other complications such as pathological fractures and spinal cord compression. Bisphosphonates (BP) are a class of potent anti-resorptive agents commonly prescribed to retard osteoporosis progression. Interestingly, BP may have indirect anti-tumour properties through negative effects on macrophages, osteoclasts, endothelial cells and their ability to suppress matrix metalloproteinase (MMP) activity. Currently, the use of bisphosphonates for cancer therapy is generally restricted to high dose systemic delivery. The purpose of this study was to investigate the effects of direct local delivery of Zoledronate at the metastatic site in a mouse model of breast cancer metastasis to bone. Seven days following intra-tibial inoculation with MDA-MB-231 (N = 1× 105) breast cancer cells in athymic mice, the experimental group (N = 11) was treated by direct infusion of 2µg of Zoledronate into the tibial lesion (three times/week for three weeks) and compared to vehicle-treated mice (N = 5). The formation of bone metastases and growth of the lesions were followed up by weekly bioluminescence imaging. In a subsequent experiment, a comparison of the effects of local versus systemic delivery of Zoledronate on the formation of osteolytic bone metastases was carried in athymic mice (N = 15). Seven days following intra-tibial inoculation with MDA-MB-231 breast cancer cells, the systemic group (N = 5) was treated with Zoledronate (0.025mg/kg) once per week for four weeks and compared to systemic delivery of vehicle (N = 4). Following treatment, the mice were sacrificed, and micro-CT images of the right tibia were obtained. Bone volume to tissue volume ratio (BV/TV%) was determined using µ-CT biomarkers. The first experiment showed a statistically significant increase in mean bone volume/tissue volume ratio% (BV/TV%) in the treated group (7.0±1.54%) as compared to the control group (3.8±0.48%) (P <0.001, 95%CI=1.9–4.3). This corresponded to a net increase of 84.21% in response to Zoledronate treatment. Comparison between the local and systemic effects of Zoledronate also revealed a significant increase in the BV/TV% in the locally treated group (6.69±0.62%) when compared to the cohort administered systemic bisphosphonate treatment (4.03±0.44%) (P<0.001, 95%CI=1.24–3.20), corresponding to a net increase of 66.0%. These preliminary results suggest that high dose sustained release of Zoledronate can lead to a significant inhibition of tumor-induced osteolysis. Moreover, comparison between local and systemic delivery revealed that the effect of local bisphosphonate administration exceeds the benefits of systemic delivery in terms of osteolysis inhibition. Lastly, the noted effects of Zoledronate local delivery triggers the need for further assessment of its anti-tumour activity

The presence of metastatic bone disease (MBD) often necessitates major orthopaedic surgery. Patients will enter surgical care either through emergent or electively scheduled care pathways. Patients in a pain crisis or with an acute fracture are generally admitted via emergent care pathways whereas patients with identified high-risk bone lesions are often booked for urgent yet scheduled elective procedures. The purpose of this study is to compare the post-operative outcomes of patients who present through emergent or electively scheduled care pathways in patients in a Canadian health care system. We have conducted a retrospective, multicenter cohort study of all patients presenting for surgery for MBD of the femur, humerus, tibia or pelvis in southern Alberta between 2006 and 2021. Patients were identified by a search query of all patients with a diagnosis of metastatic cancer who underwent surgery for an impending or actual pathologic fracture in the Calgary, South and Central Alberta Zones. Subsequent chart reviews were performed. Emergent surgeries were defined by patients admitted to hospital via urgent care mechanisms and managed via unscheduled surgical bookings (“on call list”). Elective surgeries were defined by patients seen by an orthopaedic surgeon at least once prior to surgery, and booked for a scheduled urgent, yet elective procedure. Outcomes include overall survival from the time of surgery, hospital length of stay, and 30-day hospital readmission rate. We have identified 402 patients to date for inclusion. 273 patients (67.9%) underwent surgery through emergent pathways and 129 patients (32.1%) were treated through urgent, electively scheduled pathways. Lung, prostate, renal cell, and breast cancer were the most common primary malignancies and there was no significant difference in these primaries amongst the groups (p=0.06). Not surprisingly, emergent patients were more likely to be treated for a pathologic fracture (p<0.001) whereas elective patients were more likely to be treated for an impending fracture (p<0.001). Overall survival was significantly shorter in the emergent group (5.0 months, 95%CI: 4.0-6.1) compared to the elective group (14.9 months 95%CI: 10.4-24.6) [p<0.001]. Hospital length of stay was significantly longer in the emergent group (13 days, 95%CI: 12-16 versus 5 days, 95%CI: 5-7 days). There was a significantly greater rate of 30-day hospital readmission in the emergent group (13.3% versus 7.8%) [p=0.01]. Electively managed MBD has multiple benefits including longer post-operative survival, shorter length of hospital stay, and a lower rate of 30-day hospital readmission. These findings from a Canadian healthcare system demonstrate clinical value in providing elective orthopaedic care when possible for patients with MBD. Furthermore, care delivery interventions capable of decreasing the footprint of emergent surgery through enhanced screening or follow-up of patients with MBD has the potential to significantly improve clinical outcomes in this population. This is an ongoing study that will justify refinements to the current surgical care pathways for MBD in order to identify patients prior to emergent presentation. Future directions will evaluate the costs associated with each care delivery method to provide opportunity for health economic efficiencies

Great strides have been made in the early detection and treatment of cancer which is resulting in improved survivability and more Canadians living with cancer. Approximately 80% of primary breast, lung, and prostate cancers metastasize to the spine. Poly-methyl methacrylate (PMMA) bone cement is one of the most commonly used bone substitutes in spine surgery. In clinical practice it can be loaded with various drugs, such as antibiotics or chemotheraputic drugs, as a means of local drug delivery. However, studies have shown that drugs loaded into PMMA cement tend to release in small bursts in the first 48–72 hours, and the remaining drug is trapped without any significant release over time. The objective of this study is to develop a nanoparticle-functionalized PMMA cement for use as a sustained doxorubicin delivery device. We hypothesize that PMMA cement containing mesoporous silica nanoparticles will release more doxorubicin than regular PMMA. High viscosity SmartSet ™ PMMA cement by DePuy Synthes was used in this study. The experimental group consisted of 3 replicates each containing 0.24 g of mesoporous silica nanoparticles, 1.76 g of cement powder, 1ml of liquid cement monomer and 1 mg of doxorubicin. The control group consisted 3 replicates each containing 2.0 g of cement powder, 1ml of liquid cement monomer and 1 mg of doxorubicin. The experimental group contained an average of 8.18 ± 0.008 % (W/W) mesoporous silica nanoparticles. Each replicate was casted into a cylindrical block and incubated in a PBS solution which was changed at predetermined intervals for 45 days. The concentration of eluted doxorubicin in each solution was measured using a florescent plate reader. The mechanical properties of cement were assessed by unconfined compression testing. The effect of the doxorubicin released from cement on prostate and breast tumor cell metabolic activity was assessed using the Alamar Blue test. After 45 days the experimental group released 3.24 ± 0.25 % of the initially loaded doxorubicin which was more than the 2.12 ± 0.005% released by the control group (p 0.03). There was no statistically significant difference in Young's elasticity modulus between groups (p 0.53). Nanoparticle functionalized PMMA suppressed the metabolic activity of prostate cancer by more than 50 percent but did not reach statistical significance. Nanoparticle functionalized PMMA suppressed the metabolic activity of breast cancer cells by 69 % (p < 0.05). Nanoparticle-functionalized PMMA cement can release up to 1.53 times more doxorubicin than the standard PMMA. The use of mesoporous silica nanoparticles to improve drug release from PMMA cement shows promise. In the future, in vivo experiments are required to test the efficacy of released doxorubicin on tumor cell growth

Quantitative assessment of metastatic involvement of the bony spine is important for assessing disease progression and treatment response. Quantification of metastatic involvement is challenging as tumours may appear as osteolytic (bone resorbing), osteoblastic (bone forming) or mixed. This investigation aimed to develop an automated method to accurately segment osteoblastic lesions in a animal model of metastatically involved vertebrae, imaged with micro computed tomography (μCT). Radiomics seeks to apply standardized features extracted from medical images for the purpose of decision-support as well as diagnosis and treatment planning. Here we investigate the application of radiomic-based features for the delineation of osteoblastic vertebral metastases. Osteoblastic lesions affect bone deposition and bone quality, resulting in a change in the texture of bony material physically seen through μCT imaging. We hypothesize that radiomics based features will be sensitive to changes in osteoblastic lesion bone texture and that these changes will be useful for automating segmentation. Osteoblastic metastases were generated via intracardiac injection of human ZR-75-1 breast cancer cells into a preclinical athymic rat model (n=3). Four months post inoculation, ex-vivo μCT images (µCT100, Scanco) were acquired of each rodent spine focused on the metastatically involved third lumbar vertebra (L3) at 7µm/voxel and resampled to 34µm/voxel. The trabecular bone within each vertebra was isolated using an atlas and level-set based segmentation approach previously developed by our group. Pyradiomics, an open source Radiomics library written in python, was used to calculate 3D image features at each voxel location within the vertebral bone. Thresholding of each radiomic feature map was used to isolate the osteoblastic lesions. The utility of radiomic feature-based segmentation of osteoblastic bone tissue was evaluated on randomly selected 2D sagittal and axial slices of the μCT volume. Feature segmentations were compared to ground truth osteoblastic lesion segmentations by calculating the Dice Similarity Coefficient (DSC). Manually defined ground truth osteoblastic tumor segmentations on the μCT slices were informed by histological confirmation of the lesions. The radiomic based features that best segmented osteoblastic tissue while optimizing computational time were derived from the Neighbouring Gray Tone Difference Matrix (NGTDM). Measures of coarseness yielded the best agreement with the manual segmentations (DSC=707%) followed by contrast, strength and complexity (DSC=6513%, 5428%, and 4826%, respectively). This pilot study using a radiomic based approach demonstrates the utility of the NGTDM features for segmentation of vertebral osteoblastic lesions. This investigation looked at the utility of isolated features to segment osteoblastic lesions and found modest performance in isolation. In future work we will explore combining these features using machine learning based classifiers (i.e. decision forests, support vector machines, etc.) to improve segmentation performance

Tungsten has been increasing in demand for use in manufacturing and recently, medical devices, as it imparts flexibility, strength, and conductance of metal alloys. Given the surge in tungsten use, our population may be subjected to elevated exposures. For instance, embolism coils made of tungsten have been shown to degrade in some patients. In a cohort of breast cancer patients who received tungsten-based shielding for intraoperative radiotherapy, urinary tungsten levels remained over tenfold higher 20 months post-surgery. In vivo models have demonstrated that tungsten exposure increases tumor metastasis and enhances the adipogenesis of bone marrow-derived mesenchymal stem cells while inhibiting osteogenesis. We recently determined that when mice are exposed to tungsten [15 ppm] in their drinking water, it bioaccumulates in the intervertebral disc tissue and vertebrae. This study was performed to determine the toxicity of tungsten on intervertebral disc. Bovine nucleus pulposus (bNP) and annulus fibrosus (bAF) cells were isolated from bovine caudal tails. Cells were expanded in flasks then prepared for 3D culturing in alginate beads at a density of 1×10. ∧. 6 cells/mL. Beads were cultured in medium supplemented with increasing tungsten concentrations in the form of sodium tungstate [0, 0.5, 5, 15 ug/mL] for 12 days. A modified GAG assay was performed on the beads to determine proteoglycan content and Western blotting for type II collagen (Col II) synthesis. Cell viability was determined by counting live and dead cells in the beads following incubation with the Live/Dead Viability Assay kit (Thermo Fisher Scientific). Cell numbers in beads at the end of the incubation period was determined using Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific). Tungsten dose-dependently decreased the synthesis of proteoglycan in IVD cells, however, the effect was significant at the highest dose of 15 ug/mL. (n=3). Furthermore, although tungsten decreased the synthesis of Col II in IVD cells, it significantly increased the synthesis of Col I. Upregulation of catabolic enzymes ADAMTS4 and −5 were also observed in IVD cells treated with tungsten (n=3). Upon histological examination of spines from mice treated with tungsten [15 ug/mL] in their drinking water for 30 days, disc heights were diminished and Col I upregulation was observed (n=4). Cell viability was not markedly affected by tungsten in both bNP and bAF cells, but proliferation of bNP cells decreased at higher concentration. Surprisingly, histological examination of IVDs and gene expression analysis demonstrated upregulation of NGF expression in both NP and AF cells. In addition, endplate capillaries showed increases in CGRP and PGP9.5 expression as determined on histological sections of mouse IVDs, suggesting the development of sensory neuron invasion of the disc. We provide evidence that prolonged tungsten exposure can induce disc fibrosis and increase the expression of markers associated with pain. Tungsten toxicity may play a role in disc degeneration disease

The spine is one of the most common sites of bony metastasis, with 80% of prostate, lung, and breast cancers metastasizing to the vertebrae resulting in significant morbidity. Current treatment modalities are systemic chemotherapy, such as Doxorubicin (Dox), administered after resection to prevent cancer recurrence, and systemic antiresorptive medication, such as Zolendronate (Zol), to prevent tumor-induced bone destruction. The large systemic doses required to elicit an adequate effect in the spine often leads to significant side-effects by both drugs, limiting their prolonged use and effectiveness. Recently published work by our lab has shown that biocompatible 3D-printed porous polymer scaffolds are an effective way of delivering Dox locally over a sustained period while inhibiting tumor growth in vitro. Our lab has also generated promising results regarding antitumor properties of Zol in vitro. We aim to develop 3D-printed scaffolds to deliver a combination of Zol and Dox that can potentially allow for a synergistic antitumor activity while preventing concurrent bone loss locally at the site of a tumor, avoiding long systemic exposure to these drugs and decreasing side effects in the clinical setting. The PORO Lay polymer filaments are 3D-printed into 5mm diameter disks, washed with deionized water and loaded with Dox or Zol in aqueous buffer over 7 days. Dox or Zol-containing supernatant was collected daily and the drug release was analyzed over time in a fluorescence plate reader. The polymer-drug (Dox or Zol) release was tested in vitro on prostate and lung cancer cell lines and on prostate- or lung-induced bone metastases cells. Alternatively, direct drug treatment was also carried out on the same cells in vitro. Following treatment, all cells were subject to proliferation assay (MTT and alamar blue), viability assay (LIVE/DEAD), migration assay (Boyden chamber) and invasion assay (3D gel matrix). 3D-printed scaffolds loaded with both Dox and Zol will also be tested on cells. We have established an effective dose (EC50) for prostate and lung cancer cell lines and bone metastases cells with direct treatment with Zol or Dox. We have titrated the drug loading of scaffolds to allow for a release amount of Dox at the EC50 dose over 7 days. In ongoing experiments, we are testing the release of Zol. We have shown Dox releasing scaffolds inhibit cancer cell growth in a 2D culture over 7 days using the above cellular assays and testing the scaffolds with Zol is currently being analyzed. 3D-printed porous polymers like the PORO Lay series of products offer a novel and versatile opportunity for delivery of drugs in future clinical settings. They can decrease systemic exposure of drugs while at the same time concentrating the drugs effect at the site of tumors and consequently inhibit tumor proliferation. Their ability to be loaded with multiple drugs can allow for achieving multiple goals while taking advantage of synergistic effects of different drugs. The ability to 3D-print these polymers can allow for production of custom implants that offer better structural support for bone growth

Introduction:. 25% of patients with an unknown primary tumour present to the orthopaedic surgeon with skeletal metastases. The onus is on the orthopaedic surgeon to establish the diagnosis, not only to decrease the patient's anxiety but also because the median survival increases from 6–9 months to 23 months when the primary is identified and allows for specific cancer treatment. The diagnostic work up of an unknown primary includes a multitude of special investigations. Since PET/CT has high sensitivity and specificity for detecting the primary tumours, we asked the question: Can you diagnose the unknown primary in patients with skeletal metastases with a PET/CT?. Method:. We included all PET/CT scans done in our institution between 2010 and 2013 for patients with malignancies known to metastasize to bone (melanoma, breast, lung, head and neck, GIT, other) and all scans done in patients with unknown primaries. After reviewing 686 PET/CT scans, 492 showed metastatic disease, with 78 of these having either spinal or skeletal metastases. Results:. Of these 78 patients, 68 primaries could be detected on the PET/CT scan. Thus the PET/CT detected the primary in 87% of cases. This number could possibly be higher as some were melanoma and breast cancer patients who had already undergone surgical resection. The most common primary detected was lung, followed by a group of other and unknown primaries which included cervical, kidney and thyroid carcinoma. Conclusion:. PET/CT scan is a good modality to use when looking for a primary malignancy in patients who present to the orthopaedic surgeon with bone metastases. We postulate that this might be a possible first line investigation when looking for the primary

Bone metastases are common and severe complications of cancers. It is estimated to occur in 65–75% of breast and prostate cancer patients and cause 80% of breast cancer-related deaths. Metastasised cancer cells have devastating impacts on bone due to their ability to alter bone remodeling by interacting with osteoblasts and osteoclasts. Exercise, often used as an intervention for cancer patients, regulates bone remodeling via osteocytes. Therefore, we hypothesise that bone mechanical loading may regulate bone metastases via osteocytes. This provides novel insights into the impact of exercises on bone metastases. It will assist in designing cancer intervention programs that lowers the risk for bone metastases. Investigating the mechanisms for the observed effects may also identify potential drug targets. MLO-Y4 osteocyte-like cells (gift of Dr. Bonewald, University of Missouri-Kansas City) on glass slides were placed in flow chambers and subjected to oscillatory fluid flow (1Pa; 1Hz; 2 hours). Media were extracted (conditioned media; CM) post-flow. RAW264.7 osteoclast precursors were conditioned in MLO-Y4 CM for 7 days. Migration of MDA-MB-231 breast cancer cells and PC3 prostate cancer cells towards CM was assayed using Transwell. Viability, apoptosis, and proliferation of the cancer cells in the CM were measured with Fixable Viability Dye eFluor 450, APOPercentage, and BrDu, respectively. P-values were calculated using Student's t-test. Significantly more MDA-MB-231 and PC3 cells migrated towards the CM from MLO-Y4 cells with exposure to flow in comparison to CM from MLO-Y4 cells not exposed to flow. The preferential migration is abolished with anti-VEGF antibodies. MDA-MB-231 cells apoptosis rate was slightly lower in CM from MLO-Y4 cells exposed to flow, while proliferation rate was slightly higher. The current data showed no difference in cancer cells viability and adhesion to collagen between any two groups. On the other hand, it was observed that less MDA-MB-231 cells migrated towards CM from RAW264.7 cells conditioned in CM from MLO-Y4 cells stimulated with flow in comparison to those conditioned in CM from MLO-Y4 cells not stimulated with flow. TRAP staining results confirmed that there were less differentiated osteoclasts when RAW264.7 cells were cultured in CM from MLO-Y4 cells exposed to flow. Overall, this study suggests that when only osteocytes and cancer cells are involved, osteocytes subjected to mechanical loading can promote metastases due to the increased secretion of VEGF. However, with the incorporation of osteoclasts, mechanical loading on osteocytes seems to reduce MDA-MB-231 cell migration. This is likely because osteocytes reduce osteoclastogenesis in response to mechanical stimulation, and osteoclasts have been shown to support cancer cells. Animal studies will also be conducted to verify the pro- or anti-metastatic effect of mechanical loading that is observed in the in vitro part of this study

Background. Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a role in the progression of several common solid cancers. Given that osteosarcoma features extensive local invasion and haematogenous metastases, we hypothesised that osteosarcoma cells utilise MT1-MMP to drive these processes. Moreover, since hypoxia regulates MT1-MMP expression in breast cancer we investigated the effects of hypoxia on MT1-MMP expression in osteosarcoma cells. Aims. Examination of MT1-MMP expression in osteosarcoma biopsy tissue in relation to clinical outcome. Assessment of MT1-MMP, together with hypoxia inducible factors HIF-1α and HIF-2α expression in a panel of osteosarcoma cell lines under normoxia and hypoxia. Methods. Immunohistochemistry: Formalin-fixed and paraffin embedded osteosarcoma biopsy samples from 71 patients were immunostained for MT1-MMP, HIF-1α and -2α and the data correlated with patient survival. Confocal microscopy: following 24 hours culture in 20% versus 1% oxygen, a panel of osteosarcoma cell lines were analysed for the subcellular distribution of MT1-MMP, HIF-1α and -2α. Subcellular fractionation: following 48 hours culture in 20% versus 1% oxygen, the U2OS cell line was fractionated and the compartmental lysates immunoblotted for MT1-MMP, HIF-1α and -2α. Results. Immunohistochemistry showed MT1-MMP immunopositive cytoplasmic and nuclear staining. Biopsy samples with the highest MT1-MMP and HIF-2α intranuclear staining correlated with reduced patient survival: HR 16.10; (95% CI: 5.1–40.3); p< 0.0001. In vitro studies confirmed the intranuclear MT1-MMP presence with an increased nuclear fraction in hypoxia and evidence of nuclear co-localisation with HIF-2α. Conclusions. MT1-MMP expression in osteosarcoma tissue correlates with patient survival. The functional significance of the increased intranuclear presence in hypoxia warrants further investigation

Aim. To identify patient, tumour or treatment factors that influence outcome in patients with radiation induced sarcoma of bone. Method. A retrospective review of an oncology database supplemented by referral back to original records. Results. We identified 42 patients who presented to our Unit over a 25 year period with a new sarcoma of bone following previous radiotherapy. The age of the patients at presentation ranged from 10 to 84 years of age (mean: 17 years) and the time interval from previous radiotherapy ranged from 4 to 50 years (median: 14 yrs; mean: 17 years). The median dose of radiotherapy given had been 50 Gy but there was no correlation of radiation dose with time to development of sarcoma. The pelvis was the most common site for development of sarcoma (14 cases) but breast cancer was the most common primary tumour (8 cases). 9 of the patients had metastases at the time of diagnosis of the sarcoma. Osteosarcoma was the most common diagnosis (30). Treatment was by surgery and chemotherapy when indicated and 30 of the patients had treatment with curative intent. The survival rate was 41% at 5 years for those treated with curative intent but in those treated palliatively median survival was only 6 months and all had died by one year. The only factor found to be significant for survival was the ability to completely resect the tumour; thus, limb sarcomas had a better prognosis (66% survival at 5 years) than central ones (12%)(p=0.009). Conclusion. Radiation induced sarcoma is a rare complication of radiotherapy. Both surgical and oncological treatment is likely to be compromised by previous treatment the patient has received. Despite this 40% of patients will survive more than 5 years with aggressive modern treatment

The management of pathological fractures due to Metastatic Bone Disease (MBD) and Primary Bone Tumours (PBTs) has implications for the Trauma service due to the extra pressures on staff, service delivery and budgets. We undertook an analysis of a cohort of patients presenting with MBD and PBTs. A retrospective chart review of all cases with MBD and PBTs admitted to a 40-bed Trauma Unit between 2005 and 2009 was conducted. The study looked at frequency, primary pathology, and site of pathology/fracture, time from primary diagnosis to referral, subsequent interventions and others. The results identified 34 patients, 21 females (62%) and 13 males (38%) (mean age: 64.6 years) with MBD or PBTs. Metastases secondary to breast cancer (n=13, 38%) and Myeloma (n=5, 15%) were the most common with the majority being found in the femur (n=22, 65%) and the Humerus (n=6, 18%). The mean time from primary tumour diagnosis to fracture referral was 29.6 months with 27 (79%) patients undergoing definitive surgical management within the unit. The conclusions of the study demonstrate that a wide variety of pathology presented to the unit over a 5 year period. Considerable variation was noted in the time from primary tumour diagnosis to presentation with a fracture. This could be due to improvements in treatments of specific cancers or a lack of understanding of what an Orthopaedic surgeon can offer the cancer patient. No definitive increase in pathological fractures was seen. The consensus opinion is that prompt and appropriate management of pathological fractures in cancer patients is cost effective. Management of these injuries, in a Trauma Unit, represents a small, but significant part of the annual work-load. While no significant trend has been seen, with respect to an increased incidence, it is noted that a proportion of these patients were a number of years from their initial diagnosis. With improvements in the survivorship of cancer patients, close scrutiny will be required to determine whether this ultimately translates into an increased fracture burden

The management of skeletal metastases can be challenging for the orthopaedic surgeon. They represent a significant source of pain and disability for cancer patients, adding to the morbidity of their condition. Treatment is directed at the alleviation of symptoms and the restoration of function. Metastatic involvement of the proximal humerus can be especially debilitating, having the potential to cause severe pain which leads to loss of function, and may also be complicated by pathological fracture and hence attenuate upper limb function. We present a report of four cases where the use of reverse geometry proximal shoulder prostheses has provided excellent symptomatic relief and a pain free functional range of movement in metastatic proximal humerus disease. To demonstrate a novel, effective surgical strategy for the management of proximal humeral metastatic disease in elderly patients with concomitant poor rotator cuff function, a review of the medical records and radiographic imaging who underwent reverse geometry shoulder replacement for metastatic disease of the proximal humerus was performed. Two cases were secondary to breast cancer, the other two of unknown primary. All four patients were referred with severe shoulder pain significantly limiting range of movement, in one case pathological fracture was demonstrated. In all cases significant symptomatic relief was achieved in the post operative phase, signified by a marked reduction in analgesic requirements. Two patients were completely pain-free at follow up, whilst the remaining two used only minimal oral analgesia. Upper limb function was preserved in all cases, with demonstration of a satisfactory range of motion adequate for activities of daily living in all patients. No surgical complications were noted. The use of reverse geometry shoulder prostheses in proximal humeral metastases (either with or without an associated proximal humeral fracture) demonstrates a reliable and effective method of pain relief with excellent restoration of upper limb function. The unique implant geometry allows the patient to achieve a functional range of motion without reliance on the rotator cuff musculature, which is often defunct in elderly patient groups

Aims

The exact risk to patients undergoing surgery who develop COVID-19 is not yet fully known. This study aims to provide the current data to allow adequate consent regarding the risks of post-surgery COVID-19 infection and subsequent COVID-19-related mortality.

Plots are an elegant and effective way to represent data. At their best they encourage the reader and promote comprehension. A graphical representation can give a far more intuitive feel to the pattern of results in the study than a list of numerical data, or the result of a statistical calculation.

The temptation to exaggerate differences or relationships between variables by using broken axes, overlaid axes, or inconsistent scaling between plots should be avoided.

A plot should be self-explanatory and not complicated. It should make good use of the available space. The axes should be scaled appropriately and labelled with an appropriate dimension.

Plots are recognised statistical methods of presenting data and usually require specialised statistical software to create them. The statistical analysis and methods to generate the plots are as important as the methodology of the study itself. The software, including dates and version numbers, as well as statistical tests should be appropriately referenced.

Following some of the guidance provided in this article will enhance a manuscript.

Cite this article: Bone Joint J 2015;97-B:1593–1603.