Angiogenesis plays an essential role in tumor growth and metastasis and is a promising therapeutic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogenesis as well as in angiogenesis.TC71human Ewing's sarcoma cells overexpressVEGF, with a shift in isoform production from membrane-bound VEGF 189 to the more soluble VEGF 165 . Transfection of TC71cells with a vector-based VEGF targeted small interfering RNA expression system (VEGFsi) inhibited VEGF 165 expression by 80% and VEGF 165 protein production by 98%, with no alteration in VEGF 189 expression. Human microvascular endothelial cell proliferation and migration induced by conditioned medium from VEGFsi-transfected TC71 cells was significantly less than that induced by conditioned medium fromTC71cells and control vector-transfected TC71cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressingTC71 cells was significantly less than that of parental or control vector-transfected cells.Vessel density as assessed by CD31 immunohistochemical analysis and VEGF 165 expression as assessed by Northern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsi also resulted in tumor growth suppression.When inoculated into the tibias of nude mice,VEGFsiexpressingTC71cells induced osteolytic bone lesions that were less severe than those induced by control groups. These data suggest that targeting VEGF 165 may provide a therapeutic option for Ewing's sarcoma.

Osterix is a novel zinc finger-containing transcription factor that is essential for osteoblast differentiation and bone formation. We hypothesized that osterix might have a role in osteosarcoma tumor growth and metastasis. Northern blot analysis showed that the mRNA level of osterix was decreased in two mouse osteosarcoma cell lines compared with its level in normal mouse osteoblasts. Osterix expression was also decreased in three human osteosarcoma cell lines. Transfection of the osx gene into the mouse osteosarcoma cells inhibited tumor cell growth in vitro and in vivo and significantly reduced tumor incidence, tumor volume, and lung metastasis following intratibial injection. Osterix expression was also associated with decreased osteolysis. Using an in vitro migration assay, osterix suppressed the migration of tumor cells to lung extracts. These results suggest that osterix expression may play a role in osteosarcoma tumor growth and metastasis.

It is concluded that L-MTP-PE has specific biologic effects in patients with osteosarcoma that may be important to the drug's immunostimulatory capacity and its effectiveness as an antitumor agent.

Purpose: Pulmonary metastases continue to be a significant problem in osteosarcoma. Apoptosis dysfunction is known to influence tumor development. Fas (CD95, APO-1)/FasL is one of the most extensively studied apoptotic pathways. Because FasL is constitutively expressed in the lung, cells that express Fas should be eliminated by lung endothelium. Cells with low or no cell surface Fas expression may be able to evade this innate defense mechanism. The purpose of these studies was to evaluate Fas expression in osteosarcoma lung metastases and the effect of gemcitabine on Fas expression and tumor growth. Experimental Design and Results: Using the K7M2 murine osteosarcoma model, Fas expression was quantified using immunohistochemistry. High levels of Fas were present in primary tumors, but no Fas expression was present in actively growing lung metastases. Blocking the Fas pathway using Fas-associated death domain dominant-negative delayed tumor cell clearance from the lung and increased metastatic potential. Treatment of mice with aerosol gemcitabine resulted in increased Fas expression and subsequent tumor regression. Conclusions: We conclude that corruption of the Fas pathway is critical to the ability of osteosarcoma cells to grow in the lung. Agents such as gemcitabine that up-regulate cell surface Fas expression may therefore be effective in treating osteosarcoma lung metastases. These data also suggest that an additional mechanism by which gemcitabine induces regression of osteosarcoma lung metastases is mediated by enhancing the sensitivity of the tumor cells to the constitutive FasL in the lung.

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