Engineered Autologous Stromal Cells for the Delivery of Kringle 5, A Potent Endothelial Cell Specific Inhibitor, for Anti-Angiogenic Breast Cancer Therapy
Annual summary rept.
MCGILL UNIV MONTREAL (QUEBEC) LADY DAVIS INST FOR MEDICAL RESEARCH
Pagination or Media Count:
Neoangiogenesis has been strongly correlated with aggressive breast tumor growth and metastasis. The fifth kringle K5 domain of human plasminogen is distinct from angiostatin K1-4, and has been shown - on its own - to act as a potent suppressor of angiogenesis. We propose mat the K5 domain may serve as a potent angiostatic agent and that it may act as a useful therapeutic tranagene within a breast cancer gene therapy strategy. To test this hypothesis, we have developed a K5-expressing retroviral vector, gene-modified murine DA3 mammary cells to produce soluble human K5 protein and characterized the anti-tumor potency of the de novo produced K5 peptide in vivo. The 381bp K5 domain cDNA was His-tagged at the C terminus and cloned into a bicistronic retroviral vector comprising the enhanced green fluorescent protein reporter GFP gene. Upon transfection of the K5 retrovector plasmid into 293GPG retroviral packaging cells, single clones were drug selected and characterized. Stable K5-expressing 293GPG cells were utilized to transduce murine DA3 mammary cancer cells. Gene-modified polyclonal DA3-K5-GFP cells were GFP positive as assessed by flow cytometry analysis and capable of secreting soluble K5 protein as detected by anti-His immunoblot analysis. Upon subcutaneous implantation of one million DA3-K5-GFP cells in immunocompetent BALBc mice, tumor growth was strongly suppressed as early as 7 days post-implantation P 0.01 by f test as compared to DA3 control implanted mice and the anti-tumor effect remained sustained for over 6 months. As a result, the DA3-K5-GFP implanted mice possessed a clear survival advantage with 100 of mice surviving over 6 months as compared to 20 in the control cohort P 0.0002 by log-rank.
- Medicine and Medical Research