A method to assess target gene involvement in angiogenesis in vitro and in vivo using lentiviral vectors expressing shRNA.

Wayne Blosser,Julie Stewart,Louis Stancato,Courtney M Tate,Michelle L Swearingen,Lisa V Wyss,Eliza Vakana

doi:10.1371/journal.pone.0096036

Abstract

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.

Highlights

Tumor angiogenesis is a complex biological process that is both costly and difficult to study, often requiring extensive use of in vivo methodologies
VEGFR2 was chosen over other vascular endothelial growth factor (VEGF) receptors as it is exclusively expressed in endothelial cells and plays a major role in tumor vascularization, growth and metastasis [6]
Multiple studies have shown through the use of antisense oligomers and dominant-negative mutants that VEGF binding to VEGFR2 compared to VEGFR1 is a critical requirement to induce the full spectrum of VEGF driven biological responses [22,23,24]

Summary

Introduction

Tumor angiogenesis is a complex biological process that is both costly and difficult to study, often requiring extensive use of in vivo methodologies. Cord formation was assessed using High Content Imaging [19,20,21] following knockdown of VEGF and bFGF receptors (VEGFR2 and FGFR1, respectively) as well as the transcription factors FOXO1 in the ADSC/ECFC co-culture system and SP1 in a tumor driven coculture system using lentiviral delivered short hairpin RNA (shRNA).

Results

Conclusion