Angiogenesis is the formation of new blood vessels from the existing vasculature. It is a fundamental process in developmental biology but also a pathological event that initiates or aggravates many diseases. In this complex multistep process, endothelial cells are activated by angiogenic stimuli; undergo specialization in response to VEGF/Notch signaling; degrade the basement membrane of the parent vessel; sprout, migrate, and proliferate to form capillary tubes that branch; and ultimately anastomose with adjacent vessels. Here we describe an assay that mimics the invasion step in vitro. Human microvascular endothelial cells are confronted by a VEGF-enriched basement membrane material in a three-dimensional environment that promotes endothelial cell sprouting, tube formation, and anastomosis. After a few hours, endothelial cells have become tip cells, and vascular sprouts can be observed by phase contrast, fluorescence, or time-lapse microscopy. Sprouting endothelial cells express tip cell markers, display podosomes and filopodia, and exhibit cell dynamics similar to those of angiogenic endothelial cells in vivo. This model provides a system that can be manipulated genetically to study physiological or pathological angiogenesis and that can be used to screen compounds for pro-/anti-angiogenic properties. In this chapter, we describe the key steps in setting up this assay.

In order to protrude within a dense tissue, tumor cells have to develop the ability to digest the extracellular matrix (ECM). Melanoma cells, similarly to other types of tumor cells, form invadopodia, membranous invaginations rich in filamentous actin and several other proteins including matrix metalloproteinases (MMPs). MMPs degrade ECM structural proteins such as collagens, fibronectin, or laminin. Here we describe an assay that allows the detection of gelatinase activity exhibited by tumor cells under 2D conditions and methods to present obtained data in both a quantitative and a qualitative manner.

Calcium and calcium-binding proteins play crucial roles in the regulation of actin dynamics, which contributes to cancer cell migration and invasion. In this chapter, we have focused on a three-dimensional imaging method to explore the pathophysiological function of EF-hand domain-containing protein D2 (EFHD2), a novel actin-binding protein. To overcome the limitations of two-dimensional imaging on substrate-coated cover glass for examination of invasive protrusions of cancer cells, we suggest three-dimensional reconstruction from optical z-sections of cells cultured on substrate-impregnated membrane filters of Transwell.

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

The study of cell behavior in constricted environment is particularly relevant to our understanding of the mechanisms of cell invasion. In this regard, microfluidic systems offer promising platforms as microfabricated fluidic chips provide well-controlled physical, chemical and confined environments to study cell phenotype and behavior. Here, we report a fast and effective manufacturing process of user-friendly microfluidic chips ideally suited for quantitative live cell analysis in combination with immunofluorescence microscopy. The chip body, made of polydimethylsiloxane, is composed of two incubation chambers connected by one rectangular intermediate entry channel which provides access to a series of transversal slits where the observation can be made. The height of the slit is designed to be slightly smaller than that of the cells under study. To validate the chip performance, we analyzed the reorganization of the cytoskeleton of endothelial cells under various degree of spatial confinement. We illustrate how the constricted environment affects endothelial cell behavior in inducing the formation of podosomes. Moreover, the process was stimulated further when the surface of the slit was coated with a thin layer of fibronectin. The study demonstrates the suitability of this technological process for cost-effective fabrication of custom-made single-use chips for biological applications.