972. Gene-Modified Human Glioma Cells Producing Soluble Human Plasminogen Kringle 5 Peptide Suppress Brain Tumor Progression

Abstract

Objective: Glioblastoma multiforme is one of the most highly vascularized tumors in humans. Therefore, the development of a potent anti-angiogenic gene therapy strategy for brain cancer represents an attractive alternative to existing therapeutic interventions and circumvents the pitfalls associated with direct recombinant anti-angiogenic protein delivery. Amongst the known human angiogenic inhibitors, the fifth kringle (K5) of plasminogen has been found to enhance the angiostatic potency of angiostatin, a cleavage product of human plasminogen. We propose that the K5 domain may serve as a potent angiostatic agent on its own and that it may act as a useful therapeutic transgene within a cancer gene therapy strategy. To test this hypothesis, we have developed a K5-expressing retroviral vector and have characterized the angiostatic activity of the de novo produced K5 peptide in vitro and tested its efficacy in vivo using an orthotopic brain cancer model. Methods: To assess the endothelial cell inhibitory properties of retrovector-generated K5 peptide, the 363bp human plasminogen K5 domain cDNA was His-tagged and cloned into a bicistronic retroviral vector comprising the enhanced green fluorescent protein reporter gene. Upon transfection of K5 retrovector plasmid into 293GPG retroviral packaging cells, single clones were drug selected and characterized. Kringle 5 retroparticles were then used to transduce U87 human glioma cells, whereby single clones were isolated and characterized. Results: Southern blot analysis on K5 transduced U87 glioma cells using a GFP cDNA-specific probe suggests unrearrangement upon integration into the genome of transduced glioma cells. Anti-His immunoblot analysis on conditioned culture media collected from K5 transduced U87 glioma cells revealed a major 18kDa protein consistent with the predicted MW of soluble K5, which was capable of inhibiting HUVEC migration up to 75±3% (p<0.0005) and disrupting capillary tube formation. Upon intracerebral implantation of U87-GFP and U87-K5 glioma cells in nude mice, hematoxylin and eosin-stained brain tissue sections reveal that U87-K5 implanted mice possess significantly reduced tumor volumes as compared to the mock implanted mice 32 days post-implantation (Figure 1).