Breast cancer is the most frequent malignancy in women with an estimation of 2.1 million new diagnoses in 2018. Even though primary tumours are usually efficiently removed by surgery, 20–40% of patients will develop metastases in distant organs. Bone is one of the most frequent site of metastases from advanced breast cancer, accounting from 55 to 58% of all metastases. Currently, none of the therapeutic strategies used to manage breast cancer bone metastasis are really curative. Tailoring a suitable model to study and evaluate the disease pathophysiology and novel advanced therapies is one of the major challenges that will predict more effectively and efficiently the clinical response. Preclinical traditional models have been largely used as they can provide standardization and simplicity, moreover, further advancements have been made with 3D cultures, by spheroids and artificial matrices, patient derived xenografts and microfluidics. Despite these models recapitulate numerous aspects of tumour complexity, they do not completely mimic the clinical native microenvironment. Thus, to fulfil this need, in our study we developed a new, advanced and alternative model of human breast cancer bone metastasis as potential biologic assay for cancer research. The study involved breast cancer bone metastasis samples obtained from three female patients undergoing wide spinal decompression and stabilization through a posterior approach. Samples were cultured in a TubeSpin Bioreactor on a rolling apparatus under hypoxic conditions at time 0 and for up to 40 days and evaluated for viability by the Alamar Blue test, gene expression profile, histology and immunohistochemistry. Results showed the maintenance and preservation, at time 0 and after 40 days of culture, of the tissue viability, biological activity, as well as molecular markers, i.e. several key genes involved in the complex interactions between the tumour cells and bone able to drive cancer progression, cancer aggressiveness and metastasis to bone. A good tis sue morphological and microarchitectural preservation with the presence of lacunar osteolysis, fragmented trabeculae locally surrounded by osteoclast cells and malignant cells and an intense infiltration by tumour cells in bone marrow compartment in all examined samples. Histomorphometrical data on the levels of bone resorption and bone apposition parameters remained constant between T0 and T40 for all analysed patients. Additionally, immunohistochemistry showed homogeneous expression and location of CDH1, CDH2, KRT8, KRT18, Ki67, CASP3, ESR1, CD8 and CD68 between T0 and T40, thus further confirming the invasive behaviour of breast cancer cells and indicating the maintaining of the metastatic microenvironment. The novel tissue culture, set-up in this study, has significant advantages in comparison to the pre-existent 3D models: the tumour environment is the same of the clinical scenario, including all cell types as well as the native extracellular matrix; it can be quickly set-up employing only small samples of breast cancer bone metastasis tissue in a simple, ethically correct and cost-effective manner; it bypasses and/or decreases the necessity to use more complex preclinical model, thus reducing the ethical burden following the guiding principles aimed at replacing/reducing/refining (3R) animal use and their suffering for scientific purposes; it can allow the study of the interactions within the breast cancer bone metastasis tissue over a relatively long period of up to 40 days, preserving the tumour morphology and architecture and allowing also the evaluation of different biological factors, parameters and activities. Therefore, the study provides for the first time the feasibility and rationale for the use of a human-derived advanced alternative model for cancer research and testing of drugs and innovative strategies, taking into account patient individual characteristics and specific tumour subtypes so predicting patient specific responses

Osteoclasts (OCs) are multinucleated cells that play a pivotal role in skeletal development and bone remodeling. Abnormal activation of OCs contributes to the development of bone-related diseases, such as osteoporosis, bone metastasis and osteoarthritis. Restoring the normal function of OCs is crucial for bone homeostasis. Recently, RNA therapeutics emerged as a new field of research for osteoarticular diseases. The aim of this study is to use non-coding RNAs (ncRNAs) to molecularly engineer OCs and modulate their function. Specifically, we investigated the role of the microRNAs (namely miR-16) and long ncRNAs (namely DLEU1) in OCs differentiation and fusion. DLEU1/DLEU2 region, located at chromosome 13q14, also encodes miR-15 and miR-16. Our results show that levels of these ncRNA transcripts are differently expressed at distinct stages of the OCs differentiation. Specifically, silencing of DLEU1 by small interfering RNAs (siDLEU1) and overexpression of miR-16 by synthetic miRNA mimics (miR-16-mimics) led to a significant reduction in the number of OCs formed per field (OC/field), both at day 5 and 9 of the differentiation stage. Importantly, time-lapse analysis, used to track OCs behavior, revealed a significant decrease in fusion events after transfection with siDLEU1 or miR-16-mimics and an alteration in the fusion mode and partners. Next, we investigated the migration profile of these OCs, and the results show that only miR-16-mimics-OCs, but not siDLEU-OCs, have a lower percentage of immobile cells and an increase in cells with mobile regime, compared with controls. No differences in cell shape were found. Moreover, mass-spectrometry quantitative proteomic analysis revealed independent effects of siDLEU1 and miR-16-mimics at the protein levels. Importantly, DLEU1 and miR-16 act by distinct processes and pathways. Collectively, our findings support the ncRNAs DLEU1 and miR-16 as therapeutic targets to modulate early stages of OCs differentiation and, consequently, to impair OC fusion, advancing ncRNA-therapeutics for bone-related diseases. Acknowledgements: Authors would like to thank to AO CMF / AO Foundation (AOCMFS-21-23A). SRM and MIA are supported by FCT (SFRH/BD/147229/2019 and BiotechHealth Program; CEECINST/00091/2018/CP1500/CT0011, respectively)

Prophylactic treatment is advised for metastatic bone disease patients with a high risk of fracture. Clinicians face the task of identifying these patients with high fracture risk and determining the optimal surgical treatment method. Subject-specific finite element (FE) models can aid in this decision process by predicting the mechanical effect of surgical treatment. In this study, we specifically evaluated the potential of FE models to simulate femoroplasty, as uncertainty remains whether this prophylactic procedure provides sufficient mechanical strengthening to the weight-bearing femur. In eight pairs of human cadaveric femurs artificial metastatic lesions were created. In each pair, an identical defect was milled in the left and right femur. Four pairs received a spherical lesion in the neck and the other four an ellipsoidal lesion in the intertrochanteric region, each at the medial, superior/lateral, anterior and posterior side, respectively. One femur of each pair was augmented with polymethylmethacrylate (5–10 ml), while the contralateral femur was left untreated. CT scans were made at three different time points: from the unaffected intact femurs, the defect femurs with lesion and the augmented femurs. Bone strength was measured by mechanical testing until failure in eight defect and eight augmented femurs. Nonlinear CT-based FE models were developed and validated against the experimentally measured bone strength. Subsequently, the validated FE model was applied to the available CT scans for the three different cases: intact (16 scans), defect (16) and augmented (8). The FE predicted strength was compared for the three different cases. The FE models predicted the experimental bone strength with a strong correspondence, both for the defect (R. 2. = 0.97, RMSE= 0.75 kN) and the augmented femurs (R. 2. = 0.90, RMSE = 0.98 kN). Although all lesions had a “moderate” to “high” risk for fracture according to the Mirels’ scoring system (score 7 or 8), three defect femurs did not fracture through the lesion (intertrochanteric anterior, lateral and posterior), indicating that these lesions did not act as a critical weak spot. In accordance with the experimental findings, the FE models indicated almost no reduction in strength between the intact and defect state for these femurs (0.02 ± 0.1%). For the remaining “critical” lesions, bone strength was reduced with 15.7% (± 14.9%) on average. The largest reduction was observed for lesions on the medial side (up to 43.1%). For the femurs with critical lesions, augmentation increased bone strength with 29.5% (± 29.7%) as compared to the defect cases, reaching strength values that were 2.5% (± 3.7%) higher than the intact bone strength. Our findings demonstrate that FE models can accurately predict the experimental bone strength before and after augmentation, thereby enabling to quantify the mechanical benefit of femoroplasty. This way FE models could aid in identifying suitable patients for whom femoroplasty provides sufficient increase in strength. For all lesions evaluated in this study, femoroplasty effectively restored the initial bone strength. Yet, additional studies on larger datasets with a wide variation of lesion types are required to confirm these results

Background. Metastatic bone patients who require surgery needs to be evaluated in order to maximise quality of life and avoiding functional impairment, minimising the risks connected to the surgical procedures. The best surgical procedure needs to be tailored on survival estimation. There are no current available tool or method to evaluate survival estimation with accuracy in patients with bone metastasis. We recently developed a clinical decision support tool, capable of estimating the likelihood of survival at 3 and 12 months following surgery for patients with operable skeletal metastases. After making it publicly available on . www.PATHFx.org. , we attempted to externally validate it using independent, international data. Methods. We collected data from patients treated at 13 Italian orthopaedic oncology referral centers between 2008 and 2012, then applied to PATHFx, which generated a probability of survival at three and 12-months for each patient. We assessed accuracy using the area under the receiver-operating characteristic curve (AUC), clinical utility using Decision Curve Analysis DCA), and compared the Italian patient data to the training set (United States) and first external validation set (Scandinavia). Results. The Italian dataset contained 287 records with at least 12 months follow-up information. The AUCs for the three-month and 12-month estimates was 0.80 and 0.77, respectively. There were missing data, including the surgeon's estimate of survival that was missing in the majority of records. Physiologically, Italian patients were similar to patients in the training and first validation sets. However notable differences were observed in the proportion of those surviving three and 12-months, suggesting differences in referral patterns and perhaps indications for surgery. Conclusions. PATHFx was successfully validated in an Italian dataset containing missing data. This study demonstrates its broad applicability to European patients, even in centers with differing treatment philosophies from those previously studied

Background. Metastatic bone patients who require surgery needs to be evaluated in order to maximise quality of life and avoiding functional impairment, minimising the risks connected to the surgical procedures. The best surgical procedure needs to be tailored on survival estimation. There are no current available tool or method to evaluate survival estimation with accuracy in patients with bone metastasis. We recently developed a clinical decision support tool, capable of estimating the likelihood of survival at 3 and 12 months following surgery for patients with operable skeletal metastases. After making it publicly available on . www.PATHFx.org. , we attempted to externally validate it using independent, international data. Methods. We collected data from patients treated at 13 Italian orthopaedic oncology referral centers between 2008 and 2012, then applied to PATHFx, which generated a probability of survival at three and 12-months for each patient. We assessed accuracy using the area under the receiver-operating characteristic curve (AUC), clinical utility using Decision Curve Analysis DCA), and compared the Italian patient data to the training set (United States) and first external validation set (Scandinavia). Results. The Italian dataset contained 287 records with at least 12 months follow-up information. The AUCs for the three-month and 12-month estimates was 0.80 and 0.77, respectively. There were missing data, including the surgeon's estimate of survival that was missing in the majority of records. Physiologically, Italian patients were similar to patients in the training and first validation sets. However notable differences were observed in the proportion of those surviving three and 12-months, suggesting differences in referral patterns and perhaps indications for surgery. Conclusions. PATHFx was successfully validated in an Italian dataset containing missing data. This study demonstrates its broad applicability to European patients, even in centers with differing treatment philosophies from those previously studied. Level of Evidence. IV. None of the authors have financial disclosures or conflicts of interest to declare. The study presented did not need the approval by ethics committee

Photodynamic therapy (PDT) uses the strong cytotoxicity of singlet oxygen and hyperthermia produced by irradiating excitation light on a photosensitizer. The phototoxic effects of indocyanine green (ICG) and near-infrared light (NIR) have been studied in different types of cancer cells. Plasma proteins bind strongly to ICG, followed by rapid clearance by the liver, resulting in no tumor-selective accumulation after systemic administration. Kimura et al. have proposed using a novel nanoparticle labeled with ICG (ICG-lactosome) that has tumor selective accumulation owing to enhanced permeability and retention (EPR) effect. In this study, we investigated the efficacy of PDT using ICG-lactosome and NIR for a bone metastatic mouse model of breast cancer. Cells from the human breast cancer cell line, MDA-MB-231 were injected into the right tibia of 26 anesthetized BALB/C nu/nu mice at a concentration. The mice were then randomly divided into three groups: the PDT group (n = 9), the laser (laser irradiation only) group (n = 9), and the control group (n = 8). PDT was performed thrice (7, 21, 35 days after cell inoculation) following ICG-lactosome administration via the tail vein 24 hours before irradiation. The mice were percutaneously irradiated with an 810-nm medical diode laser for 10 min. In the laser group, mice were irradiated following saline administration 24 hours before irradiation. Radiographic analysis was performed for 49 days after cell inoculation. The area of osteolytic lesion was quantified. The right hind legs of 3 mice were amputated 24 hours after the third treatment. Histological analysis was performed using hematoxylin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of sagittal sections. The data was analyzed using Tukey-Kramer post-hoc test. P-value of <0.05 was considered significant. X-ray on day 49 of the three groups are considered. The area of osteolytic lesion in the PDT group (7.9 ± 1.2 mm. 2. : mean ± SD) was significantly smaller than that of the control (11.4 ± 1.4 mm. 2. ) and laser (11.9 ± 1.2 mm. 2. ) groups. In histological findings, we observed many TUNEL-positive cells in the metastatic tissue 24 hours after PDT. In the control and laser groups, TUNEL-positive cells were occasionally observed. We have previously reported the effect of ICG-lactosome-enhanced PDT on the cytotoxicity of human breast cancer cells in vitroand on the delay of paralysis in a rat spinal metastasis model. In this study, we demonstrated the inhibitory effect of ICG-lactosome-enhanced PDT on bone destruction caused by human breast cancer cells in vivo. This PDT induced apoptosis and necrosis in the tumor cells. Intralesional resection is often performed for spinal metastases in an emergency. The residual tumor may regrow and cause neurological deficits. We believe that ICG-lactosome-enhanced PDT can decrease the rate of local recurrence through reduction of the residual tumor. PDT with ICG-lactosome and NIR had an inhibitory effect on the growth of bone metastasis of a human breast cancer

Demographics changes and the increasing incidence of metastatic bone disease are driving the significant issues of vertebral body (VB) fractures as an important consideration in the quality of life of the elderly. Whilst osteoporotic vertebral fractures have been widely studies both clinically and biomechanically, those fractures arising from metastatic infiltration in the spine are relatively poorly understood. Biomechanical in-vitro assessment of these structurally weaker specimens is an important methodology for gaining an understanding of the mechanics of such fractures in which a key aspect is the development of methodologies for predicting the failure load. Here we report on a method to predict the vertebral strength by combining computed tomography assessment with an engineering beam theory as an alternative to more complex finite element analyses and its verification within a laboratory scenario. Ninety-two human vertebral bodies with 3 different pathologies: osteoporosis, multiple myeloma (MM) and specimens containing cancer metastases were loaded using a define protocol and the failure loads recorded. Analysis of the resulting data demonstrated that the mean difference between predicted and experimental failure loads was 0.25kN, 0.41kN and 0.79 kN, with adjunct correlation coefficients of 0.93, 0.64 and 0.79 for osteoporotic, metastatic and MM VBs, respectively. Issues in predicting vertebral fracture arise from extra-vertebral bony formations which add to vertebral strength in osteoporotic VB but are structurally incompetent in metastatic disease. The methodology is currently used in providing better experimental design/benchmarking within in-vitro investigations together with further exploration of its utility in the clinical arena

Cancer-induced bone diseases are often associated with increased bone resorption and pathological fractures. In recent years, osteoprotective agents such as bisphosphonates have been studied extensively and have been shown to inhibit cancer-related bone resorption in experimental and clinical studies. The third-generation bisphosphonate, ibandronate (BM 21.0955), is a potent compound for controlling tumour osteolysis and hypercalcaemia in rats bearing Walker 256 carcinosarcoma. We have studied the effect of ibandronate given as an interventional treatment on bone strength and bone loss after the onset of tumour growth in bone. Our results suggest that it is capable of preserving bone quality in rats bearing Walker 256 carcinosarcoma cells. Since other bisphosphonates have produced comparable results in man after their success in the Walker 256 animal models our findings suggest that ibandronate may be a powerful treatment for maintaining skeletal integrity in patients with metastatic bone disease

Summary Statement. CXCR4 gene and protein expression is regulated in a dose and time-dependent manner by metallic wear debris but not polyethylene wear debris in vitro and in vivo. Introduction. Progressive osteolysis leading to aseptic loosening among metal-on-metal (MoM) total hip arthroplasties (THA's), and adverse reactions to metallic debris (ARMD) are increasing causes for concern among existing patients who have been implanted with MoM hip replacements. Close surveillance of these patients is necessary and difficulties lie in early detection as well as differentiating low-grade infection from ARMD in the early stages. Several inflammatory markers have been investigated in this context, but to date, none is specific with regards to the offending material. In earlier studies, it has been shown that osteoblastic phenotypes and differentiation are regulated by different types of wear particles. Methods. In vitro experiments were performed using MG63 and SaOs-2 osteoblast-like cells co-cultured with increasing concentrations of metallic (Co-35Ni-20Cr-10Mo and Co-28Cr-6Mo) and polyethylene (UHMWPE-GUR1020) particles simulating periprosthetic wear debris. Real-time Polymerase Chain Reaction (RT-PCR) and Western Blotting were used to quantify gene and protein expression of CXCR4. The expression of TNF-a and the effects of AMD3100 on both CXCR4 and TNF-a expression among these cells was also investigated. Immunohistochemical techniques were used to investigate the in-vivo expression of CXCR4 in retrieval tissues obtained from 2 cohorts of failed metal-on-metal and ceramic-on-polyethylene THA's. Results. In-vitro RT-PCR and experiments demonstrated a dose-dependent increase in CXCR4 mRNA (7.5 fold for MG63 and 4.0 fold for SaOs-2 cells) among cells co-cultured with metal alloy particles. Western blotting also showed a time-dependent increase in protein expression of CXCR4. No regulatory effects on CXCR4 gene expression were seen among cells co-cultured with UHMWPE particles. The attempted blockade of CXCR4 by it's known competitive receptor agonist AMD3100 (bicyclam) led to a significant inhibition of metal particle induced TNF-a mRNA expression. In-vivo immunohistochemical data from the 2 cohorts of patients with failed THA's showed CXCR4 positivity among 83% of patients with metal-on-metal hip replacements but none among ceramic-on-polyethylene hip replacements. Discussion/Conclusion. CXCR4, the chemokine receptor for the chemokine SDF-1 (stromal cell derived factor-1), has been shown to play a pivotal role in bone metastasis, inflammatory and autoimmune conditions but has not been investigated in the context of periprosthetic osteolysis in failed joint replacements. Our in-vivo and in-vitro findings collectively suggest that the CXCR4 chemokine is specifically upregulated in a dose and time-dependent manner in the presence of metallic (cobalt-chrome) wear debris but not by polyethylene wear debris. The CXCR4 chemokine receptor may be a selective and specific biomarker for progressive osteolysis seen in failed MoM hip replacements and this phenomenon could potentially have a translational effect on the practice of orthopaedic surgery. Further research is needed to evaluate the interactions of CXCR4 with osteoclast activation and signalling pathways