An In Vitro Inverted Vertical Invasion Assay to Avoid Manipulation of Rare or Sensitive Cell Types

Abstract

The ability to quantify cell migration and invasion is critical in the study of cancer metastasis. Current invasion assays, such as the Boyden Chamber, present difficulties in the measurement of the invasion of cells that are few in number and are intrinsically tied to the cell microenvironment. There exists a need for a three-dimensional invasion assay that is easily reproduced, accessible for most laboratories, and requires no displacement of cells from their original microenvironment. Here we present a simple design for an inverted vertical invasion assay able to assess the invasion capabilities of cells in a three dimensional, extracellular matrix-based environment without displacement from the original culture location. We used the assay to determine the migratory capacity of hybrids between mesenchymal/multipotent stem/stroma cells (MSCs) and breast cancer cells MCF7. These hybrids are formed reliably but rarely (1 in 1,000 cells) and for this reason require an invasion assay that does not involve extensive cell manipulation. Using this assay, we found that MSCs, breast cancer cells, and corresponding fusion products are able to migrate and invade through the extracellular matrix and that hybrids invade in a manner more similar to stromal cells than cancer cells. Thus, this assay can aid the study of the invasive capacity of both cancerous cells and associated fusion hybrids and could augment testing of therapeutic strategies to inhibit metastatic spread.

Cancer Cell Fusion: Mechanisms Slowly Unravel

“…This fusion-enhanced migration was associated with altered CCL21/CCR7 signaling, which was previously linked to the metastatic spreading of breast cancer to lymph nodes [ 28 ]. We found in our studies that breast tumor cells could spontaneously fuse with mesenchymal stem cells (MSCs) to form hybrids presenting increased invasion and migratory capacity [ 34 , 37 ].…”

Simple and Cost-Effective Assay for Isolating Invasive Living Cells

“…The iCT assay is similar to other methods in that it is also an inverted invasion assay [18,19]. The advantage of the iCT assay is that it allows the two collagen gel layers to be easily separated, with living invaded and noninvaded cells trapped in the upper and lower gels, respectively.…”

Moving Upwards: A Simple and Flexible <em>In Vitro</em> Three-dimensional Invasion Assay Protocol

“…Moreover, wound healing assays are difficult to reproduce and to yield quantitative results 7,8,9 . The Cell Exclusion Zone assay, which is a 3D modification of the wound healing assay, is a more physiologically relevant assay but it is not suitable for cells low in number such as hybrid cells resulting from cell fusion events 10,11 .…”

“…However, it requires cells to be removed from their original microenvironment and to be deposited into the upper chamber of the Boyden assay system to migrate and invade downwards toward the chemoattractant containing medium. This 3D approach is not appropriate in analyzing the migration and invasion capability of cells vulnerable to their microenvironment and/or limited in number 10,11,12 . A newer approach to analyze cell migration and invasion is the microfluidic gradient chamber assay 13 .…”