Metastatic melanoma has a poor prognosis with a 5-year survival rate ranging between 5–10% due to its resistance to available cancer therapies [23]. As such, understanding the molecular mechanisms that underlie melanoma metastasis is essential for the development of new prognostic indicators as well as strategies for disease treatment. Consistent with the central role played by MT1-MMP in mediating the pericellular ECM degradation necessary for cell invasion, the expression of the protease has been linked to the development of metastatic lesions in melanoma [4]. Interestingly, MT3-MMP is specifically upregulated in nodular melanoma, the most aggressive melanoma type, comprising about 15% of all melanoma cases [24,25]. Unlike the more common, superficially spreading form of melanoma that is characterized by a pattern of slow radial growth eventually followed by vertical growth phase and metastasis, nodular melanomas grow rapidly in thickness, and are often metastasized at the time of diagnosis [26]. As the functional contribution of MT3-MMP in melanoma progression has remained undefined, we have here examined its function using gene-silencing and overexpression in different types of melanoma cells. The invasive and growth properties of these cells were defined within the MT3-MMP in Matrix-Dependent Melanoma Invasion confines of the three-dimensional (3D) type I collagen or fibrin matrices that typify the surrounding host ECM environment. Results MT3-MMP is overexpressed in human melanoma metastases and metastatic melanoma cell lines MT1-MMP is frequently overexpressed in different types of melanoma [4,27], whereas mean MT3-MMP expression is not increased in primary melanoma compared to normal skin (GeneSapiens, www.genesapiens.org; [28]). However, increased levels of MT3-MMP are associated with the most aggressive nodular melanoma [24,25]. To assess if MT3-MMP expression is more generally linked to melanoma progression, we analyzed MT1-MMP and MT3-MMP mRNA expression in human tissue biopsies of normal skin (n = 8), benign nevi (n = 11), and melanoma metastases (n = 77). Significantly, MT3-MMP was .8-fold upregulated in the lymph node metastases as compared to normal skin (p = 0.028; Fig. 1A) whereas low MT3-MMP expression observed in benign nevi was comparable to normal skin (Fig. 1A). MT3-MMP was also upregulated in melanoma Figure 1. MT3-MMP is expressed in melanoma lymph node metastases and metastatic cell lines. MT3-MMP (A) and MT1-MMP (B) mRNA expression levels in human tissue biopsies from normal skin (n = 8), nevus (n = 11), and melanoma metastases (n = 77) were assessed by qPCR. cDNAs were normalized against beta-actin. Soft tissue comprise metastases obtained from subcutaneous tissue of groin (n = 2), pelvis (n = 1) and thigh (n = 1). Other organs comprise single or double biopsies from melanoma metastases to skin, stomach, liver, mediastinum, ovary, kidney and brain. *p = 0.028 (p = 0.031 if the highest outlying lymph node metastasis value of 84.4 is excluded). (C) Relative values of average MT1-, MT2- and MT3-MMP mRNA expression were analyzed by qPCR in 7 melanoma cell lines as indicated. (D) The melanoma cells were embedded inside 3D collagen type I gels. After 4-d culture, the 3D cell/matrix constructs were fixed and stained with TRITC-phalloidin (red) and DAPI (blue). Fluorescence micrographs of the collagen gels show representative cell colonies. doi:10.1371/journal.pone.0028325.g001 PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28325