A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Summary

A tube formation assay is used to evaluate vascular activity of tumor cells.

Abstract

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro^1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype^6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)⁹. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis^{6, 10-13}. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients^{8, 10, 11}. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

Protocol

1. A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells Preparation of tumor cells and microvascular endothelial cells: brain tumor cells such as U87 cells, melanoma cells B16F1, breast cancer MDA-MB-435, and colon cells HCT116 were grown in DMEM supplemented with 10% FBS and penicillin/streptomycin (all from Invitrogen). Endothelial cell line human microvascular endothelial cells (HMVECs) were cultured in EBM2 medium (Lonza) supplemented with 1 μg/ml hydrocortisone and…

Discussion

In order for this assay to succeed, the quality of Matrigel should be tested first. A small sample may be obtained from BD Bioscience to pre-run the assay using HMVECs. Different batch products may display dissimilar qualities in which some lots do not provide an optimal condition for tube formation. Second, any bubbles should be avoided when an aliquot of Matrigel is loaded in 96-well plates, because bubbles can disrupt tubule formation. If tiny bubbles are found in a well, the Matrigel can be immediately moved back to …

Materials

Name of the reagent	Company	Catalogue number
DMEM	Invitrogen	11995
FBS	Invitrogen	16000-044
Growth factor-reduced Matrigel	BD Bioscience	47743-720
EBM2 kit	Lonza	CC-3156
Nikon ECLIPSE TS100 microscope	Nikon