High-grade osteosarcoma is characterized by extensive genetic instability, thereby hampering the identification of causative gene mutations and understanding of the underlying pathological processes. It lacks a benign precursor lesion and reports on associations with hereditary predisposition or germline mutations are uncommon, despite the early age of onset. Here we demonstrate a novel comprehensive approach for the study of premalignant stages of osteosarcoma development in a murine mesenchymal stem cell (MSC) system that formed osteosarcomas upon grafting. By parallel functional and phenotypic analysis of normal MSCs, transformed MSCs and derived osteosarcoma cells, we provide substantial evidence for a MSC origin of osteosarcoma. In a stepwise approach, using COBRA-FISH karyotyping and array CGH in different passages of MSCs, we identified aneuploidization, translocations and homozygous loss of the cdkn2 region as the key mediators of MSC malignant transformation. We then identified CDKN2A/p16 protein expression in 88 osteosarcoma patients as a sensitive prognostic marker, thereby bridging the murine MSCs model to human osteosarcoma. Moreover, occasional reports in patients mention osteosarcoma formation following bone marrow transplantation for an unrelated malignancy. Our findings suggest a possible hazard for the clinical use of MSCs; however, they also offer new opportunities to study early genetic events in osteosarcoma genesis and, more importantly, to modulate these events and record the effect on tumour progression. This could be instrumental for the identification of novel therapeutic strategies, since the success of the current therapies has reached a plateau phase.

Cancer cell lines represent in vitro models for studying malignancies, general cell biology, drug discovery and more. Whether they can be considered as exact representative models of the parental tumors remains uncertain given the acquisition of additional ex vivo changes of the cells and the lack of tissue architecture and stroma. Previously, within the EuroBoNeT consortium, we characterized a collection of bone sarcoma cell lines on genomic and proteomic level. Here, we address the phenotypical and functional characterization of the unique set of osteosarcoma cell lines (n ¼ 19) in vitro and in vivo. For functional analysis of differentiation capacity, cells were stimulated towards osteoblasts, adipocytes and chondrocytes. Furthermore, all cell lines were injected subcutaneously and intramuscularly into nude mice to assay their in vivo tumor formation capacity as well as for phenotypical analysis of the tumors. All formed tumors were further characterized histologically and immunohistochemically. Out of 19 cell lines, 17 (89%) showed adipogenic differentiation, 13/19 (68%) could differentiate towards osteoblasts and in 6/19 (32%) cell lines chondrogenic differentiation was evident. About half of the cell lines (8/19, 42%) produced tumors in vivo after subcutaneous and intramuscular injections. Several cell lines showed invasion into adjacent tissues and one tumor developed several lung metastases. The use of cell lines, especially in cancer research, is of paramount importance. Here, we identify comprehensively characterized osteosarcoma cell lines, which robustly represent clinical osteosarcoma providing researchers useful in vitro and in vivo models to study the genetics and functional characteristics of this highly malignant neoplasm.

Osteosarcoma is the most common malignant bone tumour, with a peak incidence in children and young adolescents, suggesting a role of rapid bone growth in its pathogenesis. The Wnt/beta-catenin pathway plays a crucial role in skeletal development and is indispensable for osteoblasts' lineage determination. Previous studies suggesting an oncogenic role for the Wnt/beta-catenin pathway in osteosarcoma were based on cytoplasmic staining of beta-catenin or the detection of one component of this pathway. However, those approaches are inappropriate to address whether the Wnt/beta-catenin pathway is functionally active. Therefore, in this study, we examined nuclear beta-catenin expression in 52 human osteosarcoma biopsies, 15 osteoblastomas (benign bone tumours), and four human osteosarcoma cell lines by immunohistochemistry. Furthermore, we modulated Wnt/beta-catenin pathway activity using a GIN (GSK3beta inhibitor) and evaluated its effect on cell growth and osteogenic differentiation. Absence of nuclear beta-catenin staining was found in 90% of the biopsies and all osteosarcoma cell lines, whereas strong nuclear beta-catenin staining was observed in all osteoblastomas. Wnt-luciferase activity was comparable to the negative control in all osteosarcoma cell lines. GIN stimulated the Wnt/beta-catenin pathway, as shown by translocation of beta-catenin into the nucleus and increased Wnt-luciferase activity as well as mRNA expression of AXIN2, a specific downstream target gene. Stimulation of the Wnt/beta-catenin pathway by GIN significantly reduced cell proliferation in the cell lines MG-63 and U-2-OS and enhanced differentiation in the cell lines HOS and SJSA-1, as shown by an increase in alkaline phosphatase (ALP) activity and mineralization. In contrast with the oncogenic role of the Wnt/beta-catenin pathway in osteosarcoma as previous studies suggested, here we demonstrate that this pathway is inactivated in osteosarcoma. Moreover, activation of the Wnt/beta-catenin pathway inhibits cell proliferation or promotes osteogenic differentiation in osteosarcoma cell lines. Our data suggest that loss of Wnt/beta-catenin pathway activity, which is required for osteoblast differentiation, may contribute to osteosarcoma development.

Sarcomas are nonepithelial, nonhematopoietic malignant tumors that arise from the embryonic mesoderm. Despite their rarity, less than 10% of all cancers, sarcomas are accountable for relatively high morbidity and mortality especially in children and adolescents. Although there are some hereditary conditions predisposing sarcoma, such as the LiFraumeni and Retinoblastoma syndrome, the vast majority of these tumors are sporadic. Based on their histological morphology, sarcomas have been divided into a broad spectrum of subtypes recognized in the 2002 WHO classification of tumors. This wide lineage range suggests that sarcomas originate from either many committed different cell types or from a multipotent cell, subsequently driven into a certain lineage. Mesenchymal stem cells (MSCs) are able to differentiate into many cell types needed to create mature structures like vessels, muscle, and bone. These multipotent cells can be isolated from several adult human tissues and massively expanded in culture, making them both of use for research as well as potential beneficial therapeutical agents. For this reason MSCs are being extensively studied, however, concerns have raised about whether they are the putative originating cells of sarcoma and their questionable role in cancer progression. Recent accomplishments in the field have broadened our knowledge of MSCs in relation to sarcoma origin, sarcoma treatment and the safety of MSCs usage in therapeutic settings.

MSCs are hypothesized to potentially give rise to sarcomas after transformation and therefore serve as a good model to study sarcomagenesis. Both spontaneous and induced transformation of MSCs have been reported, however, spontaneous transformation has only been convincingly shown in mouse MSCs while induced transformation has been demonstrated in both mouse and human MSCs. Transformed MSCs of both species can give rise to pleomorphic sarcomas after transplantation into mice, indicating the potential MSC origin of so-called non-translocation induced sarcomas. Comparison of expression profiles and differentiation capacities between MSCs and sarcoma cells further supports this. Deregulation of P53- Retinoblastoma-, PI3K-AKT-and MAPK pathways has been implicated in transformation of MSCs. MSCs have also been indicated as cell of origin in several types of chromosomal translocation associated sarcomas. In mouse models the generated sarcoma type depends on amongst others the tissue origin of the MSCs, the targeted pathways and genes and the differentiation commitment status of MSCs. While some insights are glowing, it is clear that more studies are needed to thoroughly understand the molecular mechanism of sarcomagenesis from MSCs and mechanisms determining the sarcoma type, which will potentially give directions for targeted therapies.

Cellular myxoma and grade I myxofibrosarcoma are mesenchymal tumours that are characterized by their abundant myxoid extracellular matrix (ECM). Despite their histological overlap, they differ clinically. Diagnosis is therefore difficult though important. We investigated their (cyto) genetics and ECM. GNAS1-activating mutations have been described in intramuscular myxoma, and lead to downstream activation of cFos. KRAS and TP53 mutations are commonly involved in sarcomagenesis whereby KRAS subsequently activates c-Fos. A well-documented series of intramuscular myxoma (three typical cases and seven cases of the more challenging cellular variant) and grade I myxofibrosarcoma (n= 10) cases were karyotyped, analyzed for GNAS1, KRAS and TP53 mutations and downstream activation of c-Fos mRNA and protein expression. ECM was studied by liquid chromatography mass spectrometry and expression of proteins identified was validated by immunohistochemistry and qPCR. Grade I myxofibrosarcoma showed variable, non-specific cyto-genetic aberrations in 83,5% of cases (n= 6) whereas karyotypes of intramuscular myxoma were all normal (n= 7). GNAS1-activating mutations were exclusively found in 50% of intramuscular myxoma. Both tumour types showed over-expression of c-Fos mRNA and protein. No mutations in KRAS codon 12/13 or in TP53 were detected. Liquid chromatography mass spectrometry revealed structural proteins (collagen types I, VI, XII, XIV and decorin) in grade I myxofibrosarcoma lacking in intramuscular myxoma. This was confirmed by immunohistochemistry and qPCR. Intramuscular/cellular myxoma and grade I myxofibrosarcoma show different molecular genetic aberrations and different composition of their ECM that probably contribute to their diverse clinical behaviour. GNAS1 mutation analysis can be helpful to distinguish intramuscular myxoma from grade I myxofibrosarcoma in selected cases.

Conventional osteosarcoma is characterized by rapid growth, high local aggressiveness, and metastasizing potential. Patients developing lung metastases experience poor prognosis despite extensive chemotherapy regimens and surgical interventions. Previously we identified a subgroup of osteosarcoma patients with loss of CDKN2A/p16 protein expression in the primary tumor biopsies which was significantly predictive of a very poor prognosis. Here we aimed to identify the underlying mechanism(s) of this protein loss in relation to osteosarcoma behavior. The CDKN2A locus was analyzed in osteosarcoma cases with total loss of CDKN2A/p16 expression and in cases with high protein expression using melting curve analysis-methylation assay (MCA-Meth), fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and mutation analysis. All cases with complete CDKN2A/p16 protein loss showed homozygous deletions at the CDKN2A locus. In none of the cases hyper methylation of the promoter region was seen which was confirmed by sequencing this region. Taken together we show that large or smaller deletions of the CDKN2A locus are evident in patient samples and underlie the CDKN2A/p16 protein expression loss while promoter methylation does not appear to be a mechanism of this expression loss. Genomic loss of CDKN2A instead of promoter methylation might be a plausible explanation for the rapid proliferation and high aggressiveness of osteosarcoma by simultaneous impairment CDKN2A/p14(ARF) function.

High-grade conventional osteosarcoma is a malignant tumour predominantly affecting adolescents and, despite multimodal intensive therapy, lethal for one third of the patients. Although there is currently detailed knowledge of normal skeletal development, this has not been integrated into research on the genesis of osteosarcoma. Recently we showed that the canonical Wnt pathway is not active in osteosarcoma and that its reactivation is disadvantageous to osteosarcoma cells. Since Wnt is regulating normal skeletogenesis together with other pathways, here we report on the activities of the bone morphogenic protein (BMP), the transforming growth factor beta (TGFβ) and the hedgehog (Hh) pathways in osteosarcoma. Human osteosarcoma samples (n=210), benign bone tumours of osteoblastic lineage called osteoblastoma (n=25) and osteosarcoma cell lines (n=19) were examined. For pathway activity luciferase transcriptional reporter assays and gene and protein expression analyses were performed. Immunohistochemical analysis of phosphorylated Smad1 and Smad2, the intracellular effectors of BMP and TGFβ, respectively, showed nuclear expression of both proteins in 70% of the osteosarcoma samples at levels comparable to osteoblastoma. Interestingly cases with lower expression showed significantly worse disease free survival. This may imply that drugs restoring impaired signalling pathways in osteosarcoma might change the tumour's aggressive clinical course, however targeted pathway modulation in vitro did not affect cell proliferation.