Methods, models and techniques

Isolation of endothelial cells from tissues

We have developed techniques for isolation of endothelial cells from tissues by immunomagnetic separation and fluorescence-activated cell sorting (FACS). EC isolated from tumors and corresponding normal tissues are a highly valuable resource for detailed molecular profiling of the properties of these phenotypically different cells (Van Beijnum et al. Nature Prot, 2008).

Molecular profiling of angiogenesis

This method has been developed to obtain the angiogenesis expression profile of >50 known angiogenesis factors and their receptors in tumor cells and tumor vasculature in vivo. This real-time PCR based technique, using species specific primers, can be used to determine the interactions between host- and tumor cells and allows the analysis of the effect of angiostatic treatment on tumor cells and on the tumor vasculature (Thijssen et al, Exp.Cell Res. 2004).

 

Endothelial cell migration

Migration and motility of endothelial cells is a fundamental necessity for the process of angiogenesis. We developed a high-throughput wound assay endothelial cell migration platform (Peira Scientific Instruments, Belgium) making use of a 96-well pin tool and a computer assisted automated digital analysis system. Images are segmented and scratches are detected and quantified using sophisticated software algorithms (Weiss et al. Angiogenesis, 2015).

Endothelial cell sprout formation

Endothelial cells of choice (freshly isolated or cell lines) are grown in spheroids of predefined size by hanging drop technology. Once spheroids are formed, which can be in as early as 4 hours, they are transferred into 3-dimensional collagen gels. Sprouting will be optimal after 16-20 hours. An ImageJ based method developed by drs Ballini and Nowak-Sliwinska (Lausanne, Switzerland) is available for quantification of sprouting.

In vivo angiogenesis. Chicken chorioallantoic membrane (CAM) assay. Angiogenesis is studied in vivo using this model. A little hatch is made in the shell on day 3 of development. The chorioallantoic membrane is visible and development of vasculature can be studied during embryo development, but also after transplantation of (human) tumor tissues (Nowak-Sliwinska et al. Angiogenesis, 2014).
Tumor transplantation on the CAM. Light microscopy (Weiss et al. Angiogenesis, 2015).
Tumor transplantation on the CAM. Fluorescence angiography (Weiss et al. Angiogenesis, 2015).