Assaying Wnt5A-mediated invasion in melanoma cells

Abstract

Wnt5A has been implicated in melanoma metastasis, and the progression of other cancers including pancreatic, gastric, prostate, and lung cancers. Assays to test motility and invasion include both in vivo assays and in vitro assays. The in vivo assays include the use of tail vein or footpad injections of metastatic cells, and are often laborious and expensive. In vitro invasion assays provide quick readouts that can help to establish conditions that either activate or inhibit melanoma cell motility, and to assess whether the conditions in question are worth translating into an in vivo model. Here we describe two standard methods for assaying motility and invasion in vitro including wound healing assays and Matrigel invasion assays (Boyden chamber assays). In addition, we and several other laboratories have previously shown that melanoma cells require matrix metalloproteinase (MMP)-2 for their invasion, and have recently shown that Wnt5A treatment can increase the levels of this enzyme in melanoma cells, as demonstrated by gelatin zymography. The use of these techniques can help to assess the migratory capacity of melanoma cells in response to Wnt treatment.

Figure 1. Inhibiting PKC and Wnt5A in melanoma cells results in an inhibition of melanoma cell motility in a wound-healing assay

A. UACC647 melanoma cells (highly invasive, high Wnt5A) were treated with either a vehicle control or a PKC inhibitor, and subjected to a wound-healing assay. 24 hours post-treatment, vehicle controls had healed the wound, while PKC-inhibited cells could not. B. Treating highly invasive Wnt5A-high UACC903 melanoma cells with an siRNA against Wnt5A results in a decrease in the ability of these cells to close a wound.

Figure 2. Treatment of melanoma cells with recombinant Wnt5A increases MMP-2 secretion as determined by gelatin zymography

Wnt5A-low UACC1273EV and G361 cells were treated with recombinant Wnt5A at different concentrations, and subjected to gelatin zymography. Wnt5A treatment increases the secretion of MMP-2.