Session 5: New Therapeutic Strategies

Abstract

Background: Osteosarcoma accounts for 60% of all malignant childhood bone tumors. Standard treatment traditionally involved limb amputation at the joint proximal to the affected bone. Limb salvage procedures now offer therapeutic alternatives. Moreover adjuvant and postoperative chemotherapy can reduce micrometastases and tumor size. Nevertheless, 40% of all osteosarcoma patients experience relapse within .s ye:irs. and 30% of relapsing patients develop lung metastasis withm the first year. The obstinate course of osteosarcoma and the aggressive therapy demands the investigation of innovative treatments. A promising approach uses autologous or allogenic cancer cells as :acci~es,. to i~prove anti-tumor immunity. Recent mvestigat10ns have shown that the vaccination with irradiated tumor cell engineered to secrete murine GM-CSF ~timulates po~ent systemic immune responses against poorly immunogemc murine tumors, like melanoma, renal carcinoma and prostate cancer. We used a lentiviral vector to transfer the murine GM-CSF and human GM-CSF to murine and canine osteosarcoma cells. We evaluated the ability of the viral construct to infect target cells, the biosafety of the vectors and the production of this cytokine. Material and methods: A third generation of Lentiviral vector~ were used. As a test transgene we used a sequence encodmg for the Green Fluorescent Protein (GFP) to evaluate efficiency of infection in osteosarcoma cells. The murine and human GM-CSF genes were used. We used three cell lines of murine osteosarcoma: Os43, Os48 and Os5 l. These cell lines differ by their metastatic capacity. The canine osteosarcoma cell line (D 17) was also used. All trasduced cell lines were tested for the absence of replicat.ion _competent-virus by monitoring p24 antigen expression m the culture medium. The production of GM-CSF was evaluated by an ELISA assay. Results: The murine osteosarcoma cell lines were characterized for their ability to induce osteosarcomas with a metastatic behavior in syngeneic mice. Prior to lentiviral infection all osteosarcoma cell lines were tested for production of endogenous GM-CSF. None of the cell lines showed any detectable amount of the cytokine. All the cell lines studied stably produced a good level GM-CSF in absence oflentiviral particles after a few passages in vitro culture. The-amount of GM-CSF ~ro~uced was adequate to obtain a good immunogenicity lnVIVO. Discussion: The lentiviral vector used demonstrated in our experiments its biosafety and efficiency of stable expression of the transgene used. These results will allow 39 ~s to. progress to the in vivo studies, namely protection by irradiated osteosarcoma cells producing GM-CSF of tumor implants and metastases in the experimental murine osteosarcoma system and in the sporadic canine osteosarcoma model.