Screening assay for blood vessel maturation inhibitors

https://doi.org/10.1016/j.bbrc.2013.07.077Get rights and content

Highlights

  • We model the complex process of blood vessel maturation in vitro.

  • Process can be observed in real-time.

  • Format allows for rapid and easy imaging/data acquisition.

  • System validated by drugs targeting and endothelial cells and pericytes.

  • Semi-high-throughput allows for drug screening.

Abstract

In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.

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.

References (22)

  • L. Evensen et al.

    Mural cell associated VEGF is required for organotypic vessel formation

    PLoS One

    (2009)
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