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Current methods to study angiogenesis in cancer growth and development can be difficult and costly, requiring extensive use of  in vivo  methodologies. Here, we utilized an  in vitro  adipocyte derived stem cell and endothelial colony forming cell (ADSC/ECFC) co-culture system to investigate the effect of lentiviral-driven shRNA knockdown of target genes compared to a non-targeting shRNA control on cord formation using High Content Imaging. Cord formation was significantly reduced following knockdown of the VEGF receptor VEGFR2 in VEGF-driven cord formation and the FGF receptor FGFR1 in basic FGF (bFGF)-driven cord formation. In addition, cord formation was signifcantly reduced following knockdown of the transcription factor forkhead box protein O1 (FOXO1), a protein with known positive effects on angiogenesis and blood vessel stabilization in VEGF- and bFGF-driven cord formation. Lentiviral shRNA also demonstrated utility for stable knockdown of VEGFR2 and FOXO1 in ECFCs, allowing for interrogation of protein knockdown effects on  in vivo  neoangiogenesis in a Matrigel plug assay. In addition to interrogating the effect of gene knockdown in endothelial cells, we utilized lentiviral shRNA to knockdown specificity protein 1 (SP1), a transcription factor involved in the expression of VEGF, in U-87 MG tumor cells to demonstrate the ability to analyze angiogenesis  in vitro  in a tumor-driven transwell cord formation system and in tumor angiogenesis  in vivo . A significant reduction in tumor-driven cord formation, VEGF secretion, and  in vivo  tumor angiogenesis was observed upon SP1 knockdown. Therefore, evaluation of target gene knockdown effects in the  in vitro  co-culture cord formation assay in the ADSC/ECFC co-culture, ECFCs alone, and in tumor cells translated directly to  in vivo  results, indicating the  in vitro  method as a robust, cost-effective and efficient  in vitro  surrogate assay to investigate target gene involvement in endothelial or tumor cell function in angiogenesis.

A Method to Assess Target Gene Involvement in Angiogenesis In Vitro and In Vivo Using Lentiviral Vectors Expressing shRNA