An Asymmetric 3D In Vitro Assay for the Study of Tumor Cell Invasion

Abstract

Tumor cell invasion is an important step in tumor progression. There is a need for quantitative in vitro invasion assays that mimic as closely as possible in vivo tumor cell invasion. We describe a novel asymmetric 3D in vitro invasion assay. A monolayer of tumor cells is embedded between two layers of gel composed of extracellular matrix constituents that can differ in their composition and that may contain additional embedded cell types such as cell types recruited to the tumor microenvironment. This monolayer effectively represents a flattened 2D tumor embedded in a controlled 3D environment. The cells are then allowed to invade the upper and lower layers. The effects of various ECM constituents and of various stromal cell types on the invasive potential of different types of tumor cells can then be compared in a single experiment using one of the two gel layers as a control. In order to visualize the invading cells and to quantify the results the gels are sectioned perpendicular to the monolayer and frozen sections prepared. Because many tumor cells fail to invade the origin of invasion can be seen clearly as a thin line, which enables the precise evaluation of the rate and extent of invasion. DNA of highly invasive cells can then be recovered for further analysis by techniques such as laser microdissection.