br Approval statement br Role of the funding source br

Approval statement
Role of the funding source
Conflict of interest
Acknowledgments
Introduction Osteosarcoma (OS) is the most common malignancy of bone, mainly affecting children, adolescents and young adults. According to the American Cancer Society, 750–900 new cases of OS are diagnosed annually [1]. It usually occurs near rapidly growing areas of long bones, such as distal femur, proximal tibia and humerus near metaphyseal growth plates [2]. Osteosarcoma is a highly aggressive tumor and typically metastasizes to lungs. The majority of OS patients at presentation have pulmonary micrometastases that are mostly undetectable by current diagnostic tools [3,4]. Despite aggressive chemotherapy and surgical treatments, the current 5 year survival rate is 60–70%. To improve survival rates in patients with metastasis or relapsed OS, clinically relevant models are needed to understand OS pathobiology and develop more efficacious treatment strategies. In vitro studies using OS cell lines are greatly limited owing to biological complexity and tissue heterogeneity of the tumor. Clinical tissue samples from OS patients are suboptimal in providing important information on dynamic tumor–stroma interactions as intense chemotherapy often disrupts the cellular components of the extracellular matrix network and induces cytotoxic effects. Obtaining clinical samples from pediatric patients for research studies is a major limitation. Development and application of reliable animal models is essential to better understand the pathological and molecular basis of the disease and for preclinical screening of potential chemotherapeutic agents. Even though there are numerous studies reporting OS xenograft and allograft models using either mouse or human OS cells, [5–7] those models fail to provide information on the tumor microenvironment (TME). In this context, orthotopic models are extremely valuable as they faithfully recapitulate human disease in terms of tumor onset, progression, and metastasis, [8] and provide important information not only on tumor biology and responsiveness to chemotherapy, but also on TME. Genetically engineered models further advance our understanding of genetic basis of this aggressive disease [9,10]. The knowledge gained is potentially significant in discovering new targets to block metastasis in the pre-clinical setting and eventually design metastasis directed therapies for effective OS management. In recent years, BI 6015 in vivo imaging system (IVIS) has emerged as an important technology in cancer biology to track tumor progression in real time in a non-invasive manner, and in reducing the number of animals needed in in vivo studies. The bioluminescent IVIS system is a highly specific and sensitive imaging modality, and is based on the detection and quantitation of signal produced by an enzymatic reaction in which luciferin a substrate is oxidized by luciferase expressing tumor cells, in the presence of oxygen and ATP. To the best of our knowledge, there are very few reports which describe in vivo characterization of orthotopic bioluminescent OS models (143B-based) in a comprehensive manner [11–13]. Since multiple genetic risk factors are involved in the etiology of OS, it is important to develop and characterize especially those models that closely represent human disease. In this paper, we describe the development and characterization of a bioluminescent OS orthotopic mouse (BOOM) model using human OS cell line 143B. This cell line has a mutation in p53 and K-ras genes, and when injected orthotopically in immunocompromised mice, results in a highly aggressive form of OS. The main goal is to apply the BOOM model to advance our understanding of OS pathobiology, and to aid in the preclinical screening of potential therapeutic agents that effectively inhibit OS metastasis. In conjunction with bioluminescent imaging, we have employed micro-computed tomography (μCT) to quantify intra-osseous tumor growth and changes in the bone microarchitecture, and histopathology to assess histological changes at the primary and metastatic tumor sites. Micro-CT is a powerful and sophisticated tool to monitor structural and morphological changes in bone in 3D. The 3D nature of μCT allows a detailed assessment of changes occurring in the cortical and trabecular bone. Establishment of tumor and its subsequent metastasis to distant sites like lungs and kidneys was evaluated by histochemical staining for the detection of unmineralized osteoid and for the presence of tumor biomarkers such as Ki-67, Runx2, α-smooth muscle actin and ezrin by immunohistochemistry. We have used transmission electron microscopy (TEM) to detect the presence of extracellular membrane vesicles (EMVs) and myofibroblasts in the primary tumor tissue. Extracellular membrane vesicles are nano-sized membrane bound entities which mediate intercellular communication via horizontal transfer of proteins, mRNA and miRNA [14]. Tumor derived EMVs support tumor cell proliferation, survival, invasion and metastasis either directly or indirectly by modifying stromal microenvironment. It is our hypothesis that EMVs derived from cancer cells mediate transdifferentiation of normal stromal fibroblasts or mesenchymal stem (MSCs) cells to myofibroblasts.