Tumor Cell Invasion Assay: A 3D In Vitro Native Matrix-based Method to Assess Epithelial-mesenchymal Cell Interactions

- To begin the assay, first, pipette a fibroblast cell suspension supplemented with ascorbic acid into a well plate and incubate. Ascorbic acid increases fibroblast proliferation. It also stimulates the secretion of native ECM components, thereby embedding the proliferating cells into the thick extracellular matrix.

Gently release the matrix from the plate surface and allow it to float in the media. The matrix contracts and becomes thicker with more matrix deposition and remodeling, giving rise to a tissue resembling the native dermis. Spread the matrix on a porous mesh. Next, seed the epithelial cancer cells over the matrix and incubate for the cells to settle down and attach.

Transfer the assembly onto a meshed platform pre-assembled in a dish. Add media to supplement just the bottom layer of the matrix to generate an air-liquid interface for the cells above. During culture, the interface promotes the proliferation and differentiation of tumor cells. The cells also secrete proteases that degrade the ECM, facilitating their migration and invasion into the matrix.

In the following protocol, we will demonstrate an invasion assay using cutaneous squamous cell carcinoma cells on a nat