br Materials and methods br
Materials and methods
Discussion To our best knowledge, a multi-modality approach to systematically characterize an OS in vivo orthotopic model, using an aggressive OS cell line has not been previously reported. The advantage of using multi-modality approach i.e. BLI, histopathology, immunohistochemistry, and electron microscopy allowed us to detect tumor cells and disease progression in real time, detect OS-specific biomarkers, and successfully detect the presence of myofibroblasts and extra-cellular membrane vesicles in the osteosarcoma tissue. The primary tumor generated by 143B cells in the BOOM model revealed histological features similar to those of clinical OS as detected by light microscopy, IHC and Goldner staining. Histological detection of newly formed osteoid in our studies indicates that the 143B cells in the BOOM model proliferate rapidly, secreting osteoid, and form osteoblastic lesions similar to neoplastic OS cells in the clinical disease [21,22]. Bone involvement of the experimental OS was also confirmed by μ-CT which revealed the presence of both osteoblastic and osteolytic lesions in tumor-bearing bones. Human OS cell line 143B is derived from TE85 (ras wild type) OS cell line. The metastatic ability of 143B cells is due to the activation of K-ras . The propensity of ras-activated tumor cells to lungs is known  but the underlying mechanisms that facilitate lung metastasis are not clear. While pulmonary metastasis is a common feature associated with OS, we neither know the frequency of ras mutations nor its association with pulmonary metastasis in clinical OS. Metastatic OS in the kidney was detected in the BOOM model. Renal metastasis is rare in clinical OS, but there are a few studies which report that renal metastasis develops subsequent to pulmonary metastasis [24–29]. Renal metastasis of OS is correlated with aggressive cancer and poor survival  and usually detected after death in about 10–12% OS autopsies [31,32].To our best knowledge, this is the first study that reports renal metastasis in OS orthotopic model using 143B cells. It is not clear whether the renal metastasis as observed in few animals in our study is cell-line specific or due to immune-incompetence, and/or the chosen animal. Ezrin is one of most consistent metastasis biomarker of OS and its role in metastasis is discussed in a number of investigative studies [33–35]. Ezrin is a protein that links cytoskeleton proteins like flunixin meglumine to the cell membrane. It plays an important role in cell–cell interaction and signal transduction . In pediatric OS patients, increased ezrin expression is correlated with poor prognosis [37,38] and down regulation of ezrin expression in murine OS model resulted in inhibition of pulmonary metastasis . The expression of ezrin in the tumor tissue of bones and lungs observed in our study supports the clinical relevance of the BOOM model. A key pathophysiological feature of OS is the cumulative defect in osteoblastic differentiation. Runx2 is a transcription factor which regulates osteogenic differentiation and bone formation. The role of Runx2 in OS is a subject of intense debate. While some studies report that OS cells express lower levels of Runx2 and suggest that Runx2 insufficiency contributes towards osteosarcomagenesis and progression,  others report high incidence of Runx2 genomic amplification,  and increased Runx2 expression (mRNA and protein) in OS biopsy samples [41,42]. Increased Runx2 expression also correlates with poor response to neoadjuvant chemotherapy in OS . Presence of increased expression of Runx2 in the tumor tissue obtained from the BOOM model confirmed our observation of increased expression of Runx2 in the osteoblastic type of OS in the bone cancer tissue microarrays. The presence of myofibroblasts in OS is not surprising as previous studies have reported the presence of myofibroblasts in bone tumors [43–46]. To our best knowledge, this is the first study that reports the presence of myofibroblasts in an in vivo OS experimental model, as identified by immunoexpression of α-SMA and characteristic ultrastructural features by TEM. Myofibroblasts of the reactive tumor stroma mediate ECM remodeling mainly by active synthesis of extracellular matrix (ECM) components like collagen type I, III, fibronectin, and enzymes like MMPs, urokinase plasminogen activator and fibronectin activation protein [47–50]. Expression of α-SMA in OS cells as seen in this study and others suggest that (a) highly aggressive OS cells may tend to become de-differentiated and pleomorphic , and (b) tumor cells may fuse with stromal cells such as myofibroblasts from tumor microenvironment . Previous studies suggest that expression of α-SMA in OS tissue may be of prognostic significance [51–53]. Previous studies report that the expression of desmin in OS is highly variable (absent to minimal). This could be attributed to heterogeneity of tumor stroma . Evidence of strongly positive staining for desmin expression in our OS tissue could be associated with K-ras mutational background of 143B cells as previous studies with soft tissue sarcomas have reported that expression of Kras in satellite cells induced expression of myogenic proteins such as Myo D and desmin .