Biochemical and Biophysical Research Communications
Screening assay for blood vessel maturation inhibitors
Introduction
Angiogenesis is critical for tumor progression and metastasis. Currently employed antiangiogenic therapies, however, are primarily focused on the vascular-endothelial growth factor (VEGF) pathway. While clinical benefit is being observed in several solid tumor types, unfortunately, the emergence of resistance is common. One of the potential mechanisms of resistance is the increased coverage of blood vessel with pericytes [1]. These mural cells are believed to make endothelial cells less dependent on growth factor support and protect the tumor vasculature from VEGF withdrawal [2]. Hence, targeting blood vessel maturation may sensitize tumors to VEGF pathway inhibition and prevent or delay the occurrence of resistance.
In order to screen chemical libraries for inhibitors of pericyte recruitment, an in vitro assay, which reliably produces lumenized structures of endothelial cells associated with mural cells, is required. Although three-dimensional endothelial cell and pericyte models have been developed and utilized for studying biological questions [3], the methods are too complex to be used in a semi-high throughput fashion [4]. We present here the development of a blood vessel maturation assay, which features the development of lumenized, vascular structures in one optical plane. This format allows for the study of endothelial cell/pericyte interactions and is suitable for the interrogation of chemical compound libraries in semi-high-throughput fashion at the same time.
Section snippets
Materials
Unless otherwise stated, Reagents were from Sigma–Aldrich (St. Louis, MO). Dulbecco’s Modified Medium (DMEM) was from Mediatech (Manassas, VA). RPMI Medium 1640 and trypsin were from Invitrogen (Grand Island, NY). Smooth muscle cell medium was from ScienCell (Carlsbad, CA). EGM2 Medium was from Lonza (Walkersville, MD). EX-CELL® 293 Serum-Free Medium was from SAFC Biosciences (Lenexa, KS). Fetal bovine serum (FBS) was from Gemini Bio-Products (West Sacramento, CA). VEGF, IL-3, IFN-γ and TGFβ
Results and discussion
With this work we introduce a novel in vitro assay for blood vessel maturation. To have a scalable model suitable for drug screening efforts we decided against previously described 3-dimensional assays and took our inspiration from an endothelial cell/stromal coculture format, which was originally introduced as an assay for the quantification of stimulatory and inhibitory agents on angiogenesis [7]. This stromal coculture model produces lumenized vascular structures on top of a stromal cell
Acknowledgments
This work was supported in part by funds from the Prostate Cancer Foundation (PCF), the Flight Attendant Medical Research Institute (FAMRI), the Cigarette Restitution Fund (CRF), the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, donations from the R Sullivan family and the Dena and Chris Battle family as well as their supporters, friends and allies against kidney cancer. (kidneycancerchronicles.com)
We want to thank Dr. William Brennen for sharing the mesenchymal stem cells.
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