Double-Layered Collagen Gel Hemisphere for Cell Invasion Assay: Successful Visualization and Quantification of Cell Invasion Activity

Abstract

Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b].

Curcumin inhibits LPS-induced EMT through downregulation of NF-κB-Snail signaling in breast cancer cells

“…Cell invasion assay. Cell invasion assay was performed and evaluated as recently described in detail by employing Boyden chambers equipped with 8 µm porosity polyvinylpyrrolidonefree polycarbonate filters that were coated with 50 µg/ml of Matrigel solution (20). The cells were first seeded in 12-well plates at a concentration of 2.5x10 5 /well and were cultured for 48 h following treatment with LPS (5 µg/ml).…”

Reconstitution of cell migration at a glance

“…In most cases, cells migrate parallel to the grooves, which biases the orientation of focal adhesions and actin fibers. Examples of cells that undertake such motility include rat myocytes (Kim et al, 2010;Pieuchot et al, 2018), SV40immortalized or primary human corneal epithelial cells (Clark and Vignjevic, 2015;Karuri et al, 2004;Teixeira et al, 2003), NIH 3T3 fibroblasts (Kim et al, 2009;Takata et al, 2007) and human pancreatic cancer cells (Blaha et al, 2017;Holle et al, 2017). Interestingly, contact guidance has also been observed in amoeboid migrating cells such as confined T cells (Gaggioli et al, 2007;Kwon et al, 2012) and the amoeba Dictyostelium discoideum (Driscoll et al, 2014).…”

“…Imaging of 3D migration and invasion can be achieved by viewing cells in collagen and gelatin gels. In such assays, cells are either platted on, embedded inside or sandwiched between gels (Takata et al, 2007). A novel microfluidic matrix invasion model (see poster) was recently employed to image cellcell and cell-matrix interactions driving metastatic cancer cell invasion (Blaha et al, 2017).…”

<em>Bex1</em> significantly contributes to the proliferation and invasiveness of malignant tumor cells

“…Previously, we established a simple 3-dimensional (3D) in vitro model of an invasion assay, which was referred to as the double-layered collagen gel hemisphere (DL-CGH) method ( 9 ). This method enables easy visualization of tumor cell invasion.…”

“…This method enables easy visualization of tumor cell invasion. Our previous study using the DL-CGH method demonstrated that the lung adenocarcinoma cell line A549 displayed no invasiveness on its own, but showed invasive migration in the presence of fibroblast cells ( 9 ). Previously, we also demonstrated the difference in cell invasiveness among MPM cell lines ( 10 ).…”