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 native matrix derived from primary stromal fibroblasts.

- Begin by seeding 200,000 fibroblasts per well in 6-well plates in fibroblast media supplemented with freshly prepared L-ascorbic acid 2-phosphate. Refeed the cells every 2 to 3 days with 2 to 5 milliliters of fresh media. A thick layer of cells embedded in extracellular matrix, visible to the naked eye, will form at the end of 6 weeks. Depending on the cells used, the visible matrix may form sooner or later and may begin to detach from the dish as seen here.

To release this layer from the tissue culture plate, gently scrape the circumference of the matrix with a 1 milliliter micropipette tip and then push the edges of the matrix towards the center of the well. The cells and native matrix should detach easily from the plate surface and now be floating in the media.

Spread the native matrix out within the media to avoid it folding up into itself. Let the native matrix float and remodel for 5 days, continuing to change the media every 2 to 3 days. Due to tensile strength and intrinsic remodeling, the matrix will contract drastically and become reduced to a smaller but thicker native matrix at which time it will be ready to be utilized for the invasion assay.

For the invasion assay, first, use blunt forceps to transfer the native matrix gently to a nylon net. Once on the net, use a 1 milliliter micropipette tip and blunt forceps to gently spread the matrix to lie as flat as possible. Next, smear a small amount of Vaseline onto one end of one sterile clonal cylinder per matrix. Then, place the cylinders onto the native matrices, Vaseline side down, to ensure a tight seal between the native matrix and the clonal rings.

Now, add 250,000 cutaneous squamous cell carcinoma, or SCC, cells in keratinocyte growth media to the cylinders. After the cutaneous SCC cells have settled onto the native matrix, remove the cylinders. Then, lift the nylon net, native matrix, and cutaneous SCC cells to the air-liquid interface and onto a bent stainless steel wire mesh support.

Finally, add keratinocyte growth media supplemented with ascorbic acid until the media level touches the bottom of the native matrix, changing the media every 2 to 3 days and harvesting at 7 and 14 days post-seeding of the cutaneous SCC cells.