Optimization of the chicken chorioallantoic membrane assay as reliable in vivo model for the analysis of osteosarcoma.

Survival rates of osteosarcoma patients could not be significantly improved by conventional chemotherapeutic treatment regimens since the introduction of high-dose chemotherapy 35 years ago. Therefore, there is a strong clinical need for new therapeutic targets and personalized treatment strategies, requiring reliable in vivo model systems for the identification and testing of potential new treatment approaches. Conventional in vivo rodent experiments face ethical issues, are time consuming and costly, being of particular relevance in orphan diseases like osteosarcoma. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay. The CAM is a highly vascularized, non-innervated extra-embryonic membrane that is perfectly suited for the engraftment of tumor cells. However, only few reports are available for osteosarcoma and reported data are inconsistent. Therefore, the aim of this study was the adaptation and optimization of the CAM assay for its application in osteosarcoma research. Tumor take rates and volumes of osteosarcoma that developed on the CAM were analyzed after modification of several experimental parameters, including egg windowing, CAM pretreatment, inoculation technique and many more. Eight osteosarcoma cell lines were investigated. Our optimized OS-CAM-assay was finally validated against a rat animal xenograft model. Using the cell line MNNG HOS as reference we could improve the tumor take rates from 51% to 94%, the viability of the embryos from initially 40% to >80% and achieved a threefold increase of the tumor volumes. We were able to generate solid tumors from all eight osteosarcoma cell lines used in this study and could reproduce results that were obtained using an osteosarcoma rat animal model. The CAM assay can bridge the gap between in vitro cell culture and in vivo animal experiments. As reliable in vivo model for osteosarcoma research the optimized CAM assay may speed up preclinical data collection and simplifies research on potential new agents towards personalized treatment strategies. Further, in accordance with Russell's and Burch's "Principles of Humane Experimental Technique" the reasonable use of this model provides a refinement by minimizing pain and suffering of animals and supports a considerable reduction and/or replacement of animal experiments.

MiR-127 and miR-376a act as tumor suppressors by in vivo targeting of COA1 and PDIA6 in giant cell tumor of bone.

Giant cell tumors of bone (GCTB) are generally benign bone tumors associated with expansive osteolytic defects, a high rate of recurrence and potential malignant transformation. We recently observed silencing of miR-127-3p and miR-376a-3p in GCTB and identified COA1 and PDIA6 as their target genes. Here, we investigate the impact of these microRNAs and their target genes on tumor engraftment and progression of giant cell tumor stromal cells (GCTSC) in vivo by xenotransplantation on the chorioallantoic membrane of chicken eggs. Prior to transplantation, the neoplastic GCTSCs were transfected with miRNA mimics or siRNAs directed against their target genes. Restoration of miR-127-3p and miR-376a-3p reduced the tumor take rate to 17% and 47% compared to 95% in the controls. The tumor volumes were significantly reduced to 29% by both miRNAs. Silencing of COA1 and PDIA6 significantly decreased the tumor volumes to 37.7% and 42.7%, while the tumor take rates remained stable. Our results indicate that re-expression of miR-127-3p and miR-376a-3p induces a strong tumor suppressor effect in GCTSC, which is partially mediated via COA1 and PDIA6.

Elevated ratio of MMP2/MMP9 activity is associated with poor response to chemotherapy in osteosarcoma.

BACKGROUND: Matrix metalloproteinases (MMPs) are crucially involved in the regulation of multiple stages of cancer progression. Elevated MMP levels have been associated with the development of metastases and poor prognosis in several types of cancer. However, the role of MMPs in osteosarcoma and their prognostic value is still unclear. Available data are conflicting, most likely due to different technical approaches. We hypothesized that in contrast to total mRNA or protein levels frequently analyzed in previous studies the enzymatic activities of MMPs and their inhibitors the tissue inhibitors of matrix metalloproteinases (TIMPs) are closer related to their biological functions. We therefore aimed to evaluate the reliability of different zymography techniques for the quantification of MMP and TIMP activities in osteosarcoma biopsies in order to investigate their distribution, possible regulation and prognostic value. METHODS: All analyses were done using cryo-conserved osteosarcoma pretreatment biopsies (n = 18). Gene and protein expression of MMPs and TIMPs were analyzed by RT-qPCR and western blot analysis, respectively. Overall MMP activity was analyzed by in situ zymography, individual MMP activities were analyzed by gelatin zymography. Reverse zymography was used to detect and quantify TIMP activities. RESULTS: Strong overall MMP activities could be detected in osteosarcoma pretreatment biopsies with MMP2 and MMP9 as predominant active MMPs. In contrast to total RNA or protein expression MMP2 and MMP9 activities showed significant quantitative differences between good and poor responders. While MMP9 activity was high in the good responder group and significantly decreased in the poor responder group, MMP2 activity showed a reverse distribution. Likewise, significant differences were detected concerning the activity of TIMPs resulting in a negative correlation of TIMP1 activity with MMP2 activity (p = 0.044) and negative correlations of TIMP2 and TIMP3 with MMP9 activity (p = 0.007 and p = 0.006). CONCLUSION: In contrast to mRNA or protein levels MMP and TIMP activities showed significant differences between the analyzed good and poor responder groups. A shift from MMP9 to predominant MMP2 activity is associated with poor response to chemotherapy suggesting that the ratio of MMP2/MMP9 activity might be a valuable and easily accessible marker to predict the response to chemotherapy in osteosarcoma.

Restoration of miR-127-3p and miR-376a-3p counteracts the neoplastic phenotype of giant cell tumor of bone derived stromal cells by targeting COA1, GLE1 and PDIA6.

Although generally benign, giant cell tumors of bone (GCTB) display an aggressive behavior associated with significant bone destruction and lung metastasis in rare cases. This and the very high recurrence rate observed after surgical resection ranging from 20 to 55% necessitates the development of more effective treatment strategies. To identify valuable therapeutic targets, we screened a previously identified microRNA signature consisting of 23 microRNAs predominantly down-regulated in GCTB. We preselected eight candidate microRNAs and analyzed the impact of their restored expression on the neoplastic phenotype of GCTB stromal cells (GCTSC). A consistent and significant inhibition of cell proliferation, migration, colony formation and spheroid formation could be induced by transfection of primary GCTSC cell lines with miR-127-3p and miR-376a-3p, respectively. Genome wide expression analysis of miR-127-3p and miR-376a-3p transfected cells revealed four novel target genes for each microRNA. Luciferase reporter assays demonstrated direct interactions of miR-127-3p with COA1 and direct interaction of miR-376a-3p with GLE1 and PDIA6, suggesting a pivotal role of these genes in the molecular etiology of GTCB. Interestingly, both microRNAs are located within a chromosomal region frequently silenced in GCTB and many other types of cancers, indicating that these microRNAs and their target genes are valuable therapeutic targets for the treatment of GCTB and possibly other tumor entities.

Rescue of silenced UCHL1 and IGFBP4 expression suppresses clonogenicity of giant cell tumor-derived stromal cells.

Giant cell tumor (GCT) of bone is a generally benign tumor with a locally aggressive behavior. Histologically, GCTs consist of multinucleated giant cells, mononuclear histiocytes and the neoplastic fibroblast-like stromal cells (GCTSC). Growing evidence exists that GCTSCs develop from mesenchymal stem cells (MSCs), but little is known about the underlying molecular mechanisms. In previous studies we observed inactivation of the ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene in primary GCTSC due to strong DNA hypermethylation, indicating that epigenetic silencing might be involved in neoplastic transformation of MSCs. Here we investigated further candidate genes and identified strong hypermethylation of the insulin-like growth factor binding protein 4 (IGFBP4) promoter, resulting in IGFBP4 downregulation in GCTs compared to MSCs. Overexpression of UCHL1 and IGFBP4 by stable transfection of GCTSC did not influence cell viability, proliferation, migration and chemosensitivity compared to parental cells. However, colony-formation was significantly decreased suggesting that rescue of UCHL1 and IFGBP4 suppresses clonogenicity of GCT stromal cells. The observation of reduced expression of the stem-cell-specific transcription factors OCT4 and SOX2 in these cell lines further supported our findings. Epigenetic silencing of UCHL1 and IGFBP4 in GCTs might thus be a crucial event during the malignant transformation of MSCs in the context of GCT development and represent promising targets for the development of new diagnostic and therapeutic strategies.

Overexpression of inosine 5'-monophosphate dehydrogenase type II mediates chemoresistance to human osteosarcoma cells.

BACKGROUND: Chemoresistance is the principal reason for poor survival and disease recurrence in osteosarcoma patients. Inosine 5'-monophosphate dehydrogenase type II (IMPDH2) encodes the rate-limiting enzyme in the de novo guanine nucleotide biosynthesis and has been linked to cell growth, differentiation, and malignant transformation. In a previous study we identified IMPDH2 as an independent prognostic factor and observed frequent IMPDH2 overexpression in osteosarcoma patients with poor response to chemotherapy. The aim of this study was to provide evidence for direct involvement of IMPDH2 in the development of chemoresistance. METHODOLOGY/PRINCIPAL FINDINGS: Stable cell lines overexpressing IMPDH2 and IMPDH2 knock-down cells were generated using the osteosarcoma cell line Saos-2 as parental cell line. Chemosensitivity, proliferation, and the expression of apoptosis-related proteins were analyzed by flow cytometry, WST-1-assay, and western blot analysis. Overexpression of IMPDH2 in Saos-2 cells induced strong chemoresistance against cisplatin and methotrexate. The observed chemoresistance was mediated at least in part by increased expression of the anti-apoptotic proteins Bcl-2, Mcl-1, and XIAP, reduced activation of caspase-9, and, consequently, reduced cleavage of the caspase substrate PARP. Pharmacological inhibition of IMPDH induced a moderate reduction of cell viability and a strong decrease of cell proliferation, but no increase in chemosensitivity. However, chemoresistant IMPDH2-overexpressing cells could be resensitized by RNA interference-mediated downregulation of IMPDH2. CONCLUSIONS: IMPDH2 is directly involved in the development of chemoresistance in osteosarcoma cells, suggesting that targeting of IMPDH2 by RNAi or more effective pharmacological inhibitors in combination with chemotherapy might be a promising means of overcoming chemoresistance in osteosarcomas with high IMPDH2 expression.