Recombinant adeno‐associated virus (rAAV) expressing TFPI‐2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line

Recombinant adeno‐associated viruses (rAAV) have become the vector of choice for many gene therapy protocols. rAAVs have a number of attractive features including long‐term transgene expression and the ability to transduce both dividing and non‐dividing cells. We have shown previously the anti‐cancer role of tissue factor pathway inhibitor‐2 (TFPI‐2), a matrix‐associated serine protease inhibitor, in human glioblastomas. As a result of our present study, in which 0.8‐kb fragment of human TFPI‐2 was cloned into the adeno‐associated viral vectors (rAAA‐TFPI‐2), rAAV‐TFPI‐2 infection of SNB19 cells significantly increased TFPI‐2 as determined by Western blotting. As assessed by spheroid and Matrigel assays, infection of SNB19 cells with rAAV‐TFPI‐2 significantly reduced migration and invasion in a dose‐dependent manner. Tumor spheroids infected with rAAV‐TFPI‐2 and co‐cultured with fetal rat brain aggregates did not invade rat brain aggregates, whereas 90–95% of the mock and AAV‐CMV infected cells invaded rat brain aggregates. In vitro angiogenesis studies (tumor cells co‐cultured with endothelial cells or endothelial cells seeded on matrigel) showed reduction of capillary‐like structure formation in rAAV‐TFPI‐2‐treated cells as compared to parental and mock‐transfected cells. In in vivo angiogenesis results demonstrated the formation of microvessels in SNB19 parental cells and this formation was inhibited when the SNB19 cells were infected with rAAV‐TFPI‐2. Further, we observed a large reduction of tumor growth in SNB19 cells treated with rAAV‐TFPI‐2 virus injected intracerebrally when compared to controls. Our study demonstrates that rAAV‐TFPI‐2‐mediated gene therapy offers a novel tool for the treatment of brain tumors. © 2005 Wiley‐Liss, Inc.

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