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

Metastatic bone disease (MBD) is a significant contributor to diminished quality of life in cancer patients, often leading to pathologic fractures, hypercalcaemia, intractable bone pain, and reduced functional independence. Standard of care management for MBD patients undergoing orthopaedic surgery is multi-disciplinary, includes regular surgical follow-up, case by case assessment for use of bone protective medications, and post-operative radiation therapy to the operative site. The number of patients in southern Alberta receiving standard of care post-operative management is currently unclear. Our aim is to develop a database of all patients in southern Alberta undergoing orthopaedic surgery for MBD and to assess for deficiencies and opportunities to ensure standard of care for this complex patient population. Patients were identified for database inclusion by a search query of the Alberta Cancer Registry of all patients with a diagnosis of metastatic cancer who underwent surgery for an impending or pathologic fracture in the Calgary, South and Central Alberta Zones. Demographic information, primary cancer history, previous local and systemic treatments, anatomical location of MBD event(s), surgical fixation techniques, and post-operative care details were collected. The rate of standard of care post-operative treatment was evaluated. A comparison of outcomes between tertiary urban centres and rural centres was also completed. Survival was calculated from time of first operation to date of death. Univariate and multivariate analyses were performed to identify the impact of post-operative care variables on survival amongst patients surviving longer than one month. We identified 402 patients who have undergone surgical treatment for MBD in southern Alberta from 2006-2018. Median age at time of surgery was 66.3 years and 52.7% of patients were female. Breast, lung, prostate, renal cell and multiple myeloma were the most common primary malignancies (n=328, 81.6%). Median post-operative survival was 6.8 months (95%CI: 5.7-8.3). 203 patients (52.5%) were treated with post-operative radiotherapy and 159 patients (50.8%) had post-operative surgical follow-up. Only 39 patients (11.3%) received bone protective agents in the peri-operative period. On multivariate survival analysis, post-operative surgical follow-up was associated with improved survival (p<0.001). Patients were treated at nine hospitals across southern Alberta with most patients treated in an urban center (65.9%). Post-operative survival was significantly longer amongst patients treated in an urban center (9.0 months, 95%CI: 6.9-12.3 versus 4.3 months, 95%CI: 3.4-5.6, p<0.001). The burden of MBD is significant and increasing. With treatment occurring at multiple provincial sites, there is a need for standardized, primary disease-specific peri- and post-operative protocols to ensure quality and efficacious patient care. To provide evidence informed treatment recommendations, we have developed a database of all patients in southern Alberta undergoing orthopaedic surgery for MBD. Our results demonstrate that many patients were not treated according to post-operative standard of care recommendations. Notably, half of the included patients did not have documented surgical follow-up, post-operative radiation treatment was low and only 11% were actively treated with bone protective agents. This data justifies the need for established surgical MBD care pathways and provides reference data to benchmark prospective QA and QI outcomes in this patient population

Lung cancer is the most common cancer diagnosed, the leading cause of cancer-related deaths, and bone metastases occurs in 20–40% of lung cancer patients. They often present symptomatically with pain or skeletal related events (SREs), which are independently associated with decreased survival. Bone modifying agents (BMAs) such as Denosumab or bisphosphonates are routinely used, however no specific guidelines exist from the National Comprehensive Cancer Center or the European Society of Medical Oncologists. Perhaps preventing the formation of guidelines is the lack of a high-quality quantitative synthesis of randomized controlled trial (RCT) data to determine the optimal treatment for the patient important outcomes of 1) Overall survival (OS), 2) Time to SRE, 3) SRE incidence, and 4) Pain Resolution. The objective of this study was to perform the first systematic review and network meta-analysis (NMA) to assess the best BMA for treatment of metastatic lung cancer to bone. We conducted our study in accordance to the PRISMA protocol. We performed a librarian assisted search of MEDLINE, PubMed, EMBASE, and Cochrane Library and Chinese databases including CNKI and Wanfang Data. We included studies that are RCTs reporting outcomes specifically for lung cancer patients treated with a bisphosphonate or Denosumab. Screening, data extraction, risk of bias and GRADE were performed in duplicate. The NMA was performed using a Bayesian probability model with R. Results are reported as relative risks, odds ratios or mean differences, and the I2 value is reported for heterogeneity. We assessed all included articles for risk of bias and applied the novel GRADE framework for NMAs to rate the quality of evidence supporting each outcome. We included 132 RCTs comprising 11,161 patients with skeletal metastases from lung cancer. For OS, denosumab was ranked above zoledronic acid (ZA) and estimated to confer an average of 3.7 months (95%CI: −0.5 – 7.6) increased survival compared to untreated patients. For time to SRE, denosumab was ranked first with an average of 9.1 additional SRE-free months (95%CI: 4.0 – 14.0) compared to untreated patients, while ZA conferred an additional 4.8 SRE-free months (2.4 – 7.0). Patients treated with the combination of Ibandronate and systemic therapy were 2.3 times (95%CI: 1.7 – 3.2) more likely to obtain successful pain resolution, compared to untreated. Meta-regression showed no effect of heterogeneity length of follow-up or pain scales on the observed treatment effects. Heterogeneity in the network was considered moderate for overall survival and time to SRE, mild for SRE incidence, and low for pain resolution. While a generally high risk of bias was observed across studies, whether they were from Western or Chinese databases. The overall GRADE for the evidence underlying our results is High for Pain control and SRE incidence, and Moderate for OS and time to SRE. This study represents the most comprehensive synthesis of the best available evidence guiding pharmacological treatment of bone metastases from lung cancer. Denosumab is ranked above ZA for both overall survival and time to SRE, but both treatments are superior to no treatment. ZA was first among all bisphosphonates assessed for odds of reducing SRE incidence, while the combination of Ibandronate and radionuclide therapy was most effective at significantly reducing pain from metastases. Clinicians and policy makers may use this synthesis of all available RCT data as support for the use of a BMA in MBD for lung cancer

One out of nine Canadian males would suffer prostate cancer (PC) during his lifetime. Life expectancy of males with PC has increased with modern therapy and 90% live >10 years. However, 20% of PC-affected males would develop incurable metastatic diseases. Bone metastases (BM) are present in ~80% of metastatic PC patients, and are the most severe complication of PC, generating severe pain, fractures, spinal cord compression, and death. Interestingly, PC-BMs are mostly osteoblastic. However, the structure of this newly formed bone and how it relates to pain and fracture are unknown. Due to androgen antagonist treatment, different PC phenotypes develop with differential dependency on androgen receptor (AR) signaling: androgen-dependent (AR+), double negative (AR-) and neuroendocrine. How these phenotypes are related to changes in bone structure has not been studied. Here we show a state-of-the-art structural characterization of PCBM and how PC phenotypes are associated to abnormal bone formation in PCBM. Cadaveric samples (n=14) obtained from metastases of PC in thoracic or lumbar vertebrae (mean age 74yo) were used to analyze bone structure. We used micro-computed tomography (mCT) to analyze the three-dimensional structure of the bone samples. After imaging, the samples were sectioned and one 3mm thick section was embedded in epoxy-resin, ground and polished. Scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) and quantitative backscattering electron (qBSE) imaging were used to determine mineral morphology and composition. Another section was used for histological analysis of the PC-affected bone. Collagen structure, fibril orientation and extracellular matrix composition were characterized using histochemistry. Additionally, we obtained biopsies of 3 PCBM patients undergoing emergency decompression surgery following vertebral fracture and used them for immunohistological characterization. By using mCT, we observed three dysmorphic bone patterns: osteolytic pattern with thinned trabecula of otherwise well-organized structures, osteoblastic pattern defined as accumulation of disorganized matrix deposited on pre-existing trabecula, and osteoblastic pattern with minimum residual trabecula and bone space dominated by accumulation of disorganized mineralized matrix. Comparing mCT data with patho/clinical parameters revealed a trend for higher bone density in males with larger PSA increase. Through histological sections, we observed that PC-affected bone, lacks collagen alignment structure, have a higher number of lacunae and increased amount of proteoglycans as decorin. Immunohistochemistry of biopsies revealed that PC-cells inside bone organize into two manners: i) glandular-like structures where cells maintain their polarization in the expression of prostate markers, ii) diffuse infiltrate that spreads along bone surfaces, with loss of cell polarity. These cells take direct contact with osteoblasts in the surface of trabecula. We define that PCBM are mostly composed by AR+ with some double negative cells. We did not observe neuroendocrine phenotype cells. PCBMs generate predominantly osteoblastic lesions that are characterized by high lacunar density, lack of collagen organization and elevated proteoglycan content. These structural changes are associated with the infiltration of PC cells that are mostly androgen-dependent but have lost their polarization and contact directly with osteoblasts, perhaps altering their function. These changes could be associated with lower mechanical properties that led to fracture and weakness of the PCBM affected bone

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

Cementoplasty, like vertebroplasty, is a technique whereby Polymetylmethacrylate is placed into a bone lesion either percutaneouly or by surgery under image intensifier guidance. Although there have been few studies with regard to cementoplasty percutaneously, there is no series in the literature to support the open surgical technique as a palliative procedure. In our series we describe four patients (1male and 3 females, age range 63-83) with metastatic bone cancer who have benefited from an open surgical procedure. The four patients presented to our hospital between January 2004 and December 2006. They all had gradually worsening hip pain at the time of presentation and pelvic radiographs revealed osteolytic lesions in the acetabulum. A 5 centimetre longitudinal incision proximal to the greater trochanter was made and the malignant lesion identified using the image intensifier. The malignant tissue was curetted and sent for microscopy, culture, sensitivity and histopathology and the remaining void filled with bone cement (via a gun or by hand) under x-ray control. Radiographs were taken in all patients post-operatively and were referred for adjuvant radiotherapy. All patients had immediate relief of pain and were able to mobilise within 48 hours. Two patients died within 6 weeks post-operatively due to complications from their primary malignancy (lung). One patient died at three months due to unknown primary. One patient remained pain free and fully ambulatory at one and a half years post surgery (breast primary). This procedure can be recommended for patients with metastatic bone disease as it provides adequate pain control and improves the quality of life in this group of patients. These patients need a multi-disciplinary approach to their care, but as orthopaedic surgeons, we can make a significant impact to such patients and their families

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

Surgical management for acute or impending pathologic fractures in metastatic bone disease (MBD) places patients at high-risk for post-operative venous thromboembolism (VTE). Due to the combination of malignancy, systemic cancer treatment, and surgical treatment, VTE-risk is increased 7-fold in patients with MBD compared to non-cancer patients undergoing the same procedure. The extent and duration of post-operative hypercoagulability in patients with MBD remains unknown and thromboprophylaxis guidelines were developed for non-cancer patients, limiting their applicability to address the elevated VTE-risk in cancer patients. Thrombelastography (TEG) analysis is a point-of-care test that measures clot formation, stabilization, and lysis in whole blood samples. The TEG parameter, maximal amplitude (MA), indicates clot strength and the threshold of ≥65 mm has been used to define hypercoagulability and predict VTE events in non-cancer patients requiring orthopaedic surgery. Therefore, this study aims to quantify the extent and duration of post-operative hypercoagulability in patients with MBD using serial TEG analysis. Consecutive adults (≥18 years) with MBD who required orthopaedic surgery for acute or impending pathologic fractures were enrolled into this single-centre, prospective cohort study. Serial TEG analysis was performed onsite using a TEG®6s haemostasis analyzer (Haemonetics Corporation, Boston, MA) on whole blood samples collected at seven timepoints: pre-operatively; on post-operative day (POD) 1, 3, and 5; and at 2-, 6-, and 12-weeks post-operatively. Hypercoagulability was defined as MA ≥65 mm. Participants received standardized thromboprophylaxis for four weeks and patient-reported compliance with thromboprophylaxis was recorded. VTE was defined as symptomatic DVT or PE, or asymptomatic proximal DVT, and all participants underwent a screening post-operative lower extremity Doppler ultrasound on POD3. Descriptive statistics were performed and difference between pre-operative MA values of participants with VTE versus no VTE was evaluated using Student's t-test (p≤0.05). Twenty-one participants (10 female; 47.6%) with a mean age of 70 ± 12 years were enrolled. Nine different primary cancers were identified amongst participants, with breast (23.8%), colorectal (19.0%), and lung cancer (14.3%) most frequently reported. Most participants (57.1%) were hypercoagulable pre-operatively, and nearly half remained hypercoagulable at 6- and 12-weeks post-operatively (47.1 and 46.7%, respectively). VTE occurred in 5 patients (23.8%) and mean MA was 68.1 ± 4.6 mm at the time of diagnosis. Mean pre-operative MA values were significantly higher (p=0.02) in patients who experienced VTE (68.9 ± 3.5 mm) compared to those who did not (62.7 ± 6.5 mm). VTE incidence was highest in the first week post-operatively, during which time four VTE events (80%) occurred. The proportion of patients in a hypercoagulable state increased at three consecutive timepoints, beginning on POD3 (85.0%), increasing on POD5 (87.5%), and peaking at 2-weeks post-operatively (88.9%). Current thromboprophylaxis guidelines do not consider cancer-associated risk factors that contribute to increased VTE incidence and prescription duration may be inadequate to address prolonged post-operative hypercoagulability in patients with MBD. The high rate of VTE events observed and sustained hypercoagulable state indicate that thromboprophylaxis may be prematurely terminated while patients remain at high risk for VTE. Therefore, extending thromboprophylaxis duration beyond 4-weeks post-operatively in patients with MBD warrants further investigation

Bone turnover and the accumulation of microdamage are impacted by the presence of skeletal metastases which can contribute to increased fracture risk. Treatments for metastatic disease may further impact bone quality. The present study aims to establish a preliminary understanding of microdamage accumulation and load to failure in osteolytic vertebrae following stereotactic body radiotherapy (SBRT), zoledronic acid (ZA), or docetaxel (DTX) treatment. Twenty-two six-week old athymic female rats (Hsd:RH-Foxn1rnu, Envigo, USA) were inoculated with HeLa cervical cancer cells through intracardiac injection (day 0). Institutional approval was obtained for this work and the ARRIVE guidelines were followed. Animals were randomly assigned to four groups: untreated (n=6), spine stereotactic body radiotherapy (SBRT) administered on day 14 (n=6), zoledronic acid (ZA) administered on day 7 (n=5), and docetaxel (DTX) administered on day 14 (n=5). Animals were euthanized on day 21. T13-L3 vertebral segments were collected immediately after sacrifice and stored in −20°C wrapped in saline soaked gauze until testing. µCT scans (µCT100, Scanco, Switzerland) of the T13-L3 segment confirmed tumour burden in all T13 and L2 vertebrae prior to testing. T13 was stained with BaSO. 4. to label microdamage. High resolution µCT scans were obtained (90kVp, 44uA, 4W, 4.9µm voxel size) to visualize stain location and volume. Segmentations of bone and BaSO. 4. were created using intensity thresholding at 3000HU (~736mgHA/cm. 3. ) and 10000HU (~2420mgHA/cm. 3. ), respectively. Non-specific BaSO. 4. was removed from the outer edge of the cortical shell by shrinking the segmentation by 105mm in 3D. Stain volume fraction was calculated as the ratio of BaSO. 4. volume to the sum of BaSO. 4. and bone volume. The L1-L3 motion segments were loaded under axial compression to failure using a µCT compatible loading device (Scanco) and force-displacement data was recorded. µCT scans were acquired unloaded, at 1500µm displacement and post-failure. Stereological analysis was performed on the L2 vertebrae in the unloaded µCT scans. Differences in mean stain volume fraction, mean load to failure, and mean bone volume/total volume (BV/TV) were compared between treatment groups using one-way ANOVAs. Pearson's correlation between stain volume fraction and load to failure by treatment was calculated using an adjusted load to failure divided by BV/TV. Stained damage fraction was significantly different between treatment groups (p=0.0029). Tukey post-hoc analysis showed untreated samples to have higher stain volume fraction (16.25±2.54%) than all treatment groups (p<0.05). The ZA group had the highest mean load to failure (195.60±84.49N), followed by untreated (142.33±53.08N), DTX (126.60±48.75N), and SBRT (95.50±44.96N), but differences did not reach significance (p=0.075). BV/TV was significantly higher in the ZA group (49.28±3.56%) compared to all others. The SBRT group had significantly lower BV/TV than the untreated group (p=0.018). Load divided by BV/TV was not significantly different between groups (p=0.24), but relative load to failure results were consistent (ZA>Untreated>DTX>SBRT). No correlations were found between stain volume fraction and load to failure. Focal and systemic cancer treatments effect microdamage accumulation and load to failure in osteolytic vertebrae. Current testing of healthy controls will help to further separate the effects of the tumour and cancer treatments on bone quality

Bone turnover and the accumulation of microdamage are impacted by the presence of skeletal metastases which can contribute to increased fracture risk. Treatments for metastatic disease may further impact bone quality. The present study aims to establish a preliminary understanding of microdamage accumulation and load to failure in osteolytic vertebrae following stereotactic body radiotherapy (SBRT), zoledronic acid (ZA), or docetaxel (DTX) treatment. Twenty-two six-week old athymic female rats (Hsd:RH-Foxn1rnu, Envigo, USA) were inoculated with HeLa cervical cancer cells through intracardiac injection (day 0). Institutional approval was obtained for this work and the ARRIVE guidelines were followed. Animals were randomly assigned to four groups: untreated (n=6), spine stereotactic body radiotherapy (SBRT) administered on day 14 (n=6), zoledronic acid (ZA) administered on day 7 (n=5), and docetaxel (DTX) administered on day 14 (n=5). Animals were euthanized on day 21. T13-L3 vertebral segments were collected immediately after sacrifice and stored in −20°C wrapped in saline soaked gauze until testing. µCT scans (µCT100, Scanco, Switzerland) of the T13-L3 segment confirmed tumour burden in all T13 and L2 vertebrae prior to testing. T13 was stained with BaSO. 4. to label microdamage. High resolution µCT scans were obtained (90kVp, 44uA, 4W, 4.9µm voxel size) to visualize stain location and volume. Segmentations of bone and BaSO. 4. were created using intensity thresholding at 3000HU (~736mgHA/cm. 3. ) and 10000HU (~2420mgHA/cm. 3. ), respectively. Non-specific BaSO. 4. was removed from the outer edge of the cortical shell by shrinking the segmentation by 105mm in 3D. Stain volume fraction was calculated as the ratio of BaSO. 4. volume to the sum of BaSO. 4. and bone volume. The L1-L3 motion segments were loaded under axial compression to failure using a µCT compatible loading device (Scanco) and force-displacement data was recorded. µCT scans were acquired unloaded, at 1500µm displacement and post-failure. Stereological analysis was performed on the L2 vertebrae in the unloaded µCT scans. Differences in mean stain volume fraction, mean load to failure, and mean bone volume/total volume (BV/TV) were compared between treatment groups using one-way ANOVAs. Pearson's correlation between stain volume fraction and load to failure by treatment was calculated using an adjusted load to failure divided by BV/TV. Stained damage fraction was significantly different between treatment groups (p=0.0029). Tukey post-hoc analysis showed untreated samples to have higher stain volume fraction (16.25±2.54%) than all treatment groups (p<0.05). The ZA group had the highest mean load to failure (195.60±84.49N), followed by untreated (142.33±53.08N), DTX (126.60±48.75N), and SBRT (95.50±44.96N), but differences did not reach significance (p=0.075). BV/TV was significantly higher in the ZA group (49.28±3.56%) compared to all others. The SBRT group had significantly lower BV/TV than the untreated group (p=0.018). Load divided by BV/TV was not significantly different between groups (p=0.24), but relative load to failure results were consistent (ZA>Untreated>DTX>SBRT). No correlations were found between stain volume fraction and load to failure. Focal and systemic cancer treatments effect microdamage accumulation and load to failure in osteolytic vertebrae. Current testing of healthy controls will help to further separate the effects of the tumour and cancer treatments on bone quality

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

The poor prognosis of patients with soft-tissue sarcoma as not changed in the past several decades, highlighting the necessity for new therapeutic approaches. T-cell based immunotherapies are a promising alternative to traditional cancer treatments due to their ability to target only malignant cells, leaving benign cells unharmed. The development of successful immunotherapy requires the identification and characterization of targetable immunogenic tumor antigens. Cancer-testis antigens (CTA) are a group of highly immunogenic tumor-associated proteins that have emerged as potential targets for CD8+ T-cell recognition. In addition to identifying a targetable antigen, it is crucial to understand the tumor immune microenvironment. The level of immune infiltration and mechanisms of immune suppression within the tumor play important roles in the outcome of immunotherapy. The goal of this study is to identify targetable immunogenic antigens for T-cell based immunotherapy and to characterize the tumor immune microenvironment in human dedifferentiated liposarcoma (DDLS) by Nanostring and IHC. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens we used the nCounter NanoString platform to generate a gene expression profile for hundreds of genes from RNA obtained from 29 DDLS and 10 control fat FFPE samples. To classify inflammatory status of DDLS tumors, we performed hierarchical clustering based on expression levels of selected tumor inflammatory signature genes (CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-E, IDO1, LAG3, PDCDILG2, PSMB10, STAT1, TIGIT). To confirm protein expression and distribution of identified antigens, we performed immunohistochemistry on human tissue micro-arrays encompassing DDLPS tumor tissues and matched normal control tissue from 63 patients. IHC for the cancer testis antigens PBK, SPA17, MAGE-A3, NY-ESO-1 and SSX2 was performed, and the staining results were scored by two authors based on maximal staining intensity on a scale of zero to three (absent=0, weak=1, moderate=2, or strong=3) and the percentage of tumor cells that stained. Hierarchical clustering of DDLS tumors based on expression of tumor inflammation signature genes revealed two distinct groups, consisting of 15 inflamed tumor and 14 non-inflamed tumors, demonstrating tumor heterogeneity within the DDLS sarcoma subtype. All antigens were found to be expressed in DDLS at an mRNA level. SPA17 was expressed at the highest levels in DDLS, however, this antigen was expressed at high levels in normal fat. Notably, antigens PBK and TTK had the largest fold change increase in expression in DDLS compared to normal fat controls. Immunohistochemical analysis of selected antigens revealed that PBK was found to be expressed in 96% (52/54) of DDLS samples at high levels. Other antigens were absent or expressed at low levels in DDLS; MAGEA3 in 15.87% (10/63) NY-ESO-1 in 6.35% (4/62) and SSX2 in 12.7% (8/63) and SPA17 in 5.5% (3/54). This data shows considerable inter-tumoral heterogeneity of inflammation, which should be taken into consideration when designing an immunotherapy for DDLS. To date, these results show promising expression of PBK antigen in DDLS, which may be used as a target in the future development of an immunotherapy for sarcoma

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

The progressive painful and disabling predicament of patients with severe osteoarthritis awaiting a total hip or knee arthroplasty (THA/TKA) results in a decline in muscle mass, strength and function also known as Sarcopenia. We conducted a cross-sectional, prospective study of patients on the waiting-list for a THA/TKA in the South Australian public healthcare system and compared the findings to healthy participants and patients newly referred from their general practitioners. Participants with a history of joint replacements, pacemakers and cancers were excluded from this study. Outcomes of this study included (i) sarcopenia screening (SARC-F ≥4); (ii) sarcopenia, defined as low muscle strength (hand grip strength M<27kg; F<16kg), low muscle quality (skeletal muscle index M<27%, F<22.1%) and low physical performance (short physical performance battery ≤8). Additional outcomes include descriptions of the recruitment feasibility, randomisation and suitability of the assessment tools. 29 healthy controls were recruited; following screening, 83% (24/29) met the inclusion criteria and 75% (18/24) were assessed. 42 newly referred patients were recruited; following screening, 67% (30/45) met the inclusion criteria and 63% (19/30) were assessed. 68 waiting list patients were recruited; following recruitment, 24% (16/68) met the inclusion criteria and 75% (12/16) were assessed. Preliminary data shows increasing waiting time is associated with higher SARC-F scores, lower hand grip strength and lower muscle quality. As a pilot study, preliminary data demonstrate that: (1) study subjects’ willingness to participate will enable a larger study to be conducted to establish the prevalence of sarcopenia and the diagnostic cut-off points for this patient group. (2) SARC-F is a suitable tool to screen for sarcopenia. (3) There is a positive correlation between waiting time for a THA/TKA and sarcopenia

Routine post-operative bloods following all elective arthroplasty may be unnecessary. This retrospective cohort study aims to define the proportion of post-operative tests altering clinical management. Clinical coding identified all elective hip or knee joint replacement under Hawkes Bay District Health Board contract between September 2019-December 2020 (N=373). Uni-compartmental and bilateral replacements, procedures performed for cancer, and those with insufficient data were excluded. Demographics, perioperative technique, and medical complication data was collected. Pre- and post-operative blood tests were assessed. Outcome measures included clinical intervention for abnormal post-operative sodium (Na), creatinine (Cr), haemoglobin (Hb), or potassium (K) levels. A cost-benefit analysis assessed unnecessary testing. 350 patients were Included. Median age was 71 (range 34-92), with 46.9% male. Only 26 abnormal post-operative results required intervention (7.1%). 11 interventions were for low Na, 4 for low K, and 4 for elevated Cr. Only 7 patients were transfused blood products. Older age (p=0.009) and higher ASA (p=0.02) were associated with intervention of any kind. Abnormal preoperative results significantly predicted intervention for Na (p<0.05) and Cr (p<0.05). All patients requiring treatment for K used diuretic medication. Preoperative Hb level was not associated with need for transfusion. Overall, there were 1027 unnecessary investigations resulting in $18,307 excess expenditure. Our study identified that the majority of elective arthroplasty patients do not require routine postoperative blood testing. We recommend investigations for patients with preoperative electrolyte abnormality, those taking diuretics, and patients with significant blood loss noted intra-operatively. In future, a larger, randomised controlled trial would be useful to confirm these factors

The optimal timing of when to perform manipulation under anesthesia (MUA) for stiffness following total knee arthroplasty (TKA) is unclear. This study aimed to identify the risk factors for MUA following primary TKA and whether performing an “early” MUA within 3 months results in a greater improvement in range of motion. Primary TKAs performed between January 2013 and December 2018 at three tertiary New Zealand hospitals were reviewed. International Classification of Diseases discharge coding was used to identify patients who underwent an MUA. Multivariate Cox regression was performed to identify patient and surgical risk factors for MUA. Pre- and post-MUA knee flexion angles were identified through manual review of operation notes. Multivariate linear regression was performed to compare the mean flexion angles pre- and post-MUA, as well as the mean gain in flexion, between patients undergoing “early” (<3 months) versus “late” MUA (>3 months). 7386 primary TKAs were analyzed in which 131 underwent subsequent MUA (1.8%). Patients aged <65 years were two times more likely to undergo MUA compared to patients aged ≥65 years (2.5% versus 1.3%, adjusted hazard ratio = 2.1, p<0.001). Gender, body mass index, patient comorbidities or a history of cancer were not associated with the risk of MUA. There was no difference in the final post-MUA flexion angle between patients who underwent early versus late MUA (104.7 versus 104.1 degrees, p = 0.819). However, patients who underwent early MUA had poorer pre-MUA flexion (72.3 versus 79.6 degrees, p = 0.012), and subsequently had a greater overall gain in flexion compared to patients who underwent late MUA (mean gain 33.1 versus 24.3 degrees, p<0.001). Younger age was the only patient risk factor for MUA. A greater overall gain in flexion was achieved in patients who underwent early MUA within 3 months

Introduction. Transfemoral osseointegration (TFOI) for amputees has substantial literature proving superior quality of life and mobility versus a socketed prosthesis. Some amputees have hip arthritis that would be relieved by a total hip replacement (THR). No other group has reported performing a THR in association with TFOI (THR+TFOI). We report the outcomes of eight patients who had THR+TFOI, followed for an average 5.2 years. Materials & Methods. Our osseointegration registry was retrospectively reviewed to identify all patients who had TFOI and also had THR, performed at least two years prior. Six patients had TFOI then THR, one simultaneous, one THR then TFOI. All constructs were in continuity from hip to prosthetic limb. Outcomes were: complications prompting surgical intervention, and changes in subjective hip pain, K-level, daily prosthesis wear hours, Questionnaire for Persons with a Transfemoral Amputation (QTFA), and Short Form 36 (SF36). All patients had clinical follow-up, but one patient did not have complete mobility and quality of life survey data at both time periods. Results. Four (50%) were male, average age 52.7±14.8 years. Three patients (38%) had amputation for trauma, three for osteosarcoma, one each (13%) infected total knee and persistent infection after deformity surgery. One patient died one year after THR+TOFA from subsequently diagnosed pancreatic cancer. One patient had superficial debridement for infection with implant retention after five years. No implants were removed, no fractures occurred. All patients reported severe hip pain preoperatively versus full relief of hip pain afterwards. K-level improved from 0/8=0% K>2 (six were wheelchair-bound) to 5/8=63% (p=.026). At least 8 hours of prosthesis wear was reported by 2/7=29% before TOFA vs 5/7=71% after (p=.286). The QTFA improved in all categories, but not significantly: Global (40.0±21.6 vs 60.0±10.9, p=.136), Problem (50.2±33.2 vs 15.4±8.4, p=.079), and Mobility (35.9±26.8 vs 58.3±30.7, p=.150). The SF36 also improved minimally and not significantly: Mental (53.6±12.0 vs 54.7±4.6, p=.849) and Physical (32.5±10.9 vs 36.3±11.2, p=.634). Conclusions. THR+TFOI is a successful reconstruction option for amputees who desire relief from severe pain related to hip joint degeneration, and also the opportunity for improved mobility and quality of life that TFOI typically confers. In our cohort, the procedure proved safe: no associated deaths, no removals, one soft tissue debridement. Mobility improved markedly. Quality of life improved, but not to significant thresholds as measured by the surveys. THR+TFOI appears safe and reasonable to offer to transfemoral amputees with painful hip joint degeneration

Giant cell tumors of bone (GCTs) are locally aggressive tumors with recurrence potential that represent up to 10% of primary tumors of the bone. GCTs pathogenesis is driven by neoplastic mononuclear stromal cells that overexpress receptor activator of nuclear factor kappa-B/ligand (RANKL). Treatment with specific anti-RANKL antibody (denosumab) was recently introduced, used either as a neo-adjuvant in resectable tumors or as a stand-alone treatment in unresectable tumors. While denosumab has been increasingly used, a percentage of patients do not improve after treatment. Here, we aim to determine molecular and histological patterns that would help predicting GCTs response to denosumab to improve personalized treatment. Nine pre-treatment biopsies of patients with spinal GCT were collected at 2 centres. In 4 patients denosumab was used as a neo-adjuvant, 3 as a stand-alone and 2 received denosumab as adjuvant treatment. Clinical data was extracted retrospectively. Total mRNA was extracted by using a formalin-fixed paraffin-embedded extraction kit and we determined the transcript profile of 730 immune-oncology related genes by using the Pan Cancer Immune Profiling panel (Nanostring). The gene expression was compared between patients with good and poor response to Denosumab treatment by using the nSolver Analysis Software (Nanostring). Immunohistochemistry was performed in the tissue slides to characterize cell populations and immune response in CGTs. Two out of 9 patients showed poor clinical response with tumor progression and metastasis. Our analysis using unsupervised hierarchical clustering determined differences in gene expression between poor responders and good responders before denosumab treatment. Poor responding lesions are characterized by increased expression of inflammatory cytokines as IL8, IL1, interferon a and g, among a myriad of cytokines and chemokines (CCL25, IL5, IL26, IL25, IL13, CCL20, IL24, IL22, etc.), while good responders are characterized by elevated expression of platelets (CD31 and PECAM), coagulation (CD74, F13A1), and complement classic pathway (C1QB, C1R, C1QBP, C1S, C2) markers, together with extracellular matrix proteins (COL3A1, FN1,. Interestingly the T-cell response is also different between groups. Poor responding lesions have increased Th1 and Th2 component, but good responders have an increased Th17 component. Interestingly, the checkpoint inhibitor of the immune response PD1 (PDCD1) is increased ~10 fold in poor responders. This preliminary study using a novel experimental approach revealed differences in the immune response in GCTs associated with clinical response to denosumab. The increased activity of checkpoint inhibitor PD1 in poor responders to denosumab treatment may have implications for therapy, raising the potential to investigate immunotherapy as is currently used in other neoplasms. Further validation using a larger independent cohort will be required but these results could potentially identify the patients who would most benefit from denosumab therapy