Gene therapy-mediated expression by tumor cells of the angiogenesis inhibitor flk-1 results in inhibition of neuroblastoma growth in vivo.

BACKGROUND/PURPOSE: Preventing tumors from forming new blood vessels appears to be an effective new anticancer approach. Antiangiogenic therapy usually is cytostatic, however, and, therefore, long-term angiogenesis inhibition is likely to be required. The objective of this study was to determine if sustained gene therapy-mediated expression of these agents from tumor cells could restrict tumor growth in vivo.

METHODS

Two replication-defective retroviral vectors were made, one encoding both the soluble, truncated vascular endothelial growth factor receptor (VEGF-R2), flk-1, together with green fluorescent protein (GFP), and the other encoding GFP alone. These vectors were then used to transduce murine neuroblastoma cells (NXS2). Stable, high expression of the flk-1 transgene was confirmed in the former population of cells by Western analysis. Flk-1 protein was isolated from cell culture supernatants and tested in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays to confirm that functional protein was being made. Finally, in vivo activity was assessed by injecting 10(6) tumor cells subcutaneously into SCID mice and monitoring subsequent tumor growth.

RESULTS

Purified flk-1 (0.1 micromol/L) was able to inhibit basic fibroblast growth factor (bFGF) stimulated HUVEC proliferation by 44% and VEGF-stimulated migration by 30%. In vitro growth rates for the transduced cell lines were similar to the unmodified cell line. In vivo, however, after 23 days, tumors from flk-1 expressing neuroblastoma cells were less than 33% the average volume of tumors from cells expressing only the GFP transgene (mean volume, 1.9 cm(3) v 5.8 cm(3), P<.001). GFP expression alone had no effect on tumor growth when compared with unmodified tumor cells.

CONCLUSIONS

Engineered expression of flk-1, a competitive inhibitor of VEGF, by tumor cells results in the production of an inhibitor of endothelial cell proliferation and migration that greatly restricts the growth of the tumor cells in vivo. Gene therapy-mediated delivery of angiogenesis inhibitors may provide an alternative approach to treating refractory tumors such as neuroblastoma.