1Molecular and Cellular Biology Program, Morrill Science Center, University of Massachusetts, 2Pioneer Valley Life Sciences Institute, University of Massachusetts, 3Department of Veterinary and Animal Sciences, University of Massachusetts
This article is a part ofJoVE General. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Francescone III, R. A., Faibish, M., Shao, R. A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells. J. Vis. Exp. (55), e3040, doi:10.3791/3040 (2011).
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 vitro1-5. 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 phenotype6-8. 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 angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 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.
1. A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells
2. Representative Results:
HMVECs were used as a positive control, as these tubules were developed from clear elongated cell bodies that connect to form polygon network. B16F1, U87, and MDA-MB-435 cells developed vascular tubules similar to those formed by HMVECs, but HCT116 cells did not (Figure 1). A cell dose-dependent tube formation was tested in U87 cells. As demonstrated in Figure 2, 10,000 cells formed discontinued tubules. Once the cells were doubled, a solid vascular network was comparable to those seen in HMVECs. In contrast, cells less than 5,000 failed to form a vascular phenotype.
Figure 1. Tube formation induced by HMVECs, U87, MDA-MB-435, and B16F1 cells, but not HCT116 cells. All the cells (2 x 104) were loaded on Matrigel and incubated overnight. Tubules were imaged using phase contrast. HMVECs were used as a positive control. A representative of 3-5 fields was shown. Bar: 100 μm.
Figure 2. U87 cell-induced tubules in a cell number-dependent manner. Different numbers of U87 cells as indicated in the corners were used for tube formation. HMVECs were used for a positive control. Bar: 100 mm.
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 the original Matrigel tubes which should be kept on ice during the experiment. In addition, tested cells should be maintained in a growth rate at a log phase. When any cultured conditions indicating slow growth of the cells occur, they should be replaced with healthy cells. Finally, the analysis of tubes under a microscope should be no longer than one hour if the tubes are not fixed because decreased temperature (room temperature) could affect tube formation. The whole assay should be completed within 24 hours to avoid disruption of tubular structure induced by cell death. It is well known that the tube formation is characterized by multiple cellular-activated processes including cell migration, cell-cell adhesion, survival, and apoptosis. For example, cell migration and cell-cell adhesion are noticeable during the tube development. After 24 hr, however, significant cell apoptosis takes place as discontinued network is increasingly observed. This event occurs in both endothelial cell and tumor cell-derived tube formation.
It is emerging that this assay is critical for evaluating tumor cell vasculogenic activity in vitro, the event that is independent of endothelial cell-associated angiogenesis. Here, we have shown that melanoma cell line B16F1 and breast cancer line MDA-MB-435 developed vascular networks on Matrigel, consistent with vasculogenic behavior of these types of tumors in human as well as in animal models14-17. Although the failure to develop a vascular phenotype by HCT116 cells on Matrigel is mechanistically unknown, it is speculated that this vascular property may be tumor cell type dependent. It would be interesting to know if HCT116 cells lack the ability to generate VM in vivo as compared with B16F1 and MDA-MB-435 cells. Therefore, establishment of this assay is of paramount importance in the study of cancer biology, vascular biology as well as drug screening.
No conflicts of interest declared.
This work was supported by NCI R01 CA120659 (RS).
|Growth factor-reduced Matrigel||BD Biosciences||47743-720|
|EBM2 kit||Lonza Inc.||CC-3156|
|Nikon ECLIPSE TS100 microscope||Nikon Instruments|