Surgery (St Vincent's) - Theses
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ItemThe role of RECK in sarcoma( 2011)Sarcomas are malignant tumours of connective tissue, and arise in bone, muscle, adipose, or fibrous tissue. Osteosarcoma and chondrosarcomas are the most common primary bone tumours, with treatment consisting of wide-margin surgery and, in osteosarcoma, neoadjuvant chemotherapy. Chemotherapy has improved survival, but not without systemic side effects, and it is not effective in chondrosarcoma. Liposarcoma is a common soft tissue sarcoma, which also has a variable response to chemotherapy. All sarcomas demonstrate angiogenesis, growth and invasion of surrounding tissues as part of their natural, destructive progression. Targeting the molecular pathways underlying these processes is likely to improve outcomes and also avoid systemic cytotoxicity. The RECK protein has shown promising therapeutic properties in this regard. In the present study, RECK was studied in human sarcoma to examine the expression pattern and relationship with prognosis. RECK was downregulated in osteosarcoma cells, and the average survival for RECK negative patients was below average. No relationship between RECK and prognosis was found for chondrosarcoma and liposarcoma, although larger studies are needed. A consistent finding across all sarcomas studied was the presence of RECK in sarcoma vessels, and this expression was up-regulated compared with non-tumour vessels. RECK was overexpressed in sarcoma cell lines by transfection. This inhibited the invasion of SaOS-2 (osteosarcoma), JJ012 (chondrosarcoma), and SW872 (liposarcoma). Furthermore, RECK inhibited JJ012 proliferation and supported adhesion to collagen I in SaOS-2, while colony formation was suppressed in both cell lines. Overexpressing RECK in HMEC-1 (microvascular endothelial cell line), supported mature tube formations and decreased HMEC-1 invasion. RECK is likely to exert these effects through inhibition of MMP-2. RECK-transfected SaOS-2 cells were co-cultured with RAW cells (pre-osteoclasts) and this inhibited osteoclast mineralised bone-resorbing capacity. RECK overexpression in an orthotopic animal model of osteosarcoma significantly inhibited tumour formation and lung metastasis. These finding were noted on serial assessment of tumour size, x-ray changes, micro CT, and macroscopic specimens. An orthotopic mouse model of chondrosarcoma, utilising periosteal and intratibial sites for JJ012, was established for testing RECK, although increased transfection toxicity in the JJ012 cell line precluded a definitive analysis. To assess therapeutic RECK delivery, recombinant partial and full-length rhRECK proteins were first tested in vitro, and this demonstrated reduced SaOS-2 invasion when treated with the partial-length protein, while SaOS-2 cell viability was significantly reduced when treated with the full-length protein. A vascularised model of osteosarcoma growing within a polycarbonate chamber inside a rat limb was developed to test delivery of rhRECK proteins via an osmotic pump and catheter. An initial study delivering partial-length RECK was hampered by high infection rates and mechanical disruption of the catheters. Revisions to the model allowed more adequate testing of full-length rhRECK delivery, which resulted in increased percentage tumour necrosis in comparison with control chambers. These collective findings indicate that RECK has important anti-tumour roles in sarcoma and may provide prognostic information for clinicians in the future. Further studies evaluating intravascular delivery of RECK to sarcoma tumours, within the newly described model may provide a new avenue of treatment for sarcoma patients.