Figure 7. Transient MT3-MMP expression in the MT3-MMP knockdown cells rescues nodular-type growth in collagen in conjunction with increased fibrin invasion. (A) MT3-MMP was detected by immunoblotting in COS-1 cells transiently expressing mock vector (Mock), MT3-MMP (MT3), or rescue constructs rescMT3-1 and rescMT3-2. Ponceau staining served as a loading control. Asterix marks non-specific bands. (B) WM852 cells stably expressing MT3-MMP targeting shRNA (shMT3-2) transfected to express MT3-MMP rescue vectors (rescMT3-1 and rescMT3-2) or mock vector were embedded in 3D collagen type I and fibrin gels. Chart shows relative percentage of invasive colonies formed after 5 d, n = 3. (C) Fluorescent micrographs show representative colonies stained for F-actin (red) to visualize the cells. (D) WM852 cell pools after lentiviral expression of scrambled shRNA (Scr) and shMT3-2 as well as knockdown cells transfected with MT3-MMP rescue vectors (rescMT3-1 and rescMT3-2) were allowed to invade into collagen type I gels for 7 d followed by imaging and quantification, n = 3. doi:10.1371/journal.pone.0028325.g007 adhesion receptors and/or their ligands [43,44]. In both COS-1 and Bowes cells, MT3-MMP overexpression decreased cell surface MT1-MMP concomitantly with increased MT1-MMP processing to the inactive fragments. In contrast, such processing of endogenous MT1-MMP or MT3-MMP was less evident in WM852 cells, which could be at least partially due to the higher expression levels of TIMP2 and TIMP3 that could inhibit and thus stabilize the MT-MMPs in these cells [29]. However, MT1-MMP levels were increased also on the WM852 melanoma cell surface after knockdown of endogenous MT3-MMP. Thus, the reduced MT1-MMP surface localization was a consistent property of the MT3-MMP in Matrix-Dependent Melanoma Invasion growing, but modestly invasive cell phenotype induced by MT3- MMP in 3D collagen. Stabilized intratumoral fibrin networks have been implicated in the progression of several human neoplasms, including brain tumors that are almost devoid of type I collagen [45,46]. Since MT3-MMP expression is upregulated frequently in tumors that arise in collagen-poor tissues ([28,47]; www.oncomine.org; www. genesapiens.org), and the effects of MT1-MMP and MT3-MMP were additive promoting invasion in fibrin, MT3-MMP expression in primary tumors associated with fibrin accumulation may promote local tumor spread. This hypothesis and the importance of MT3-MMP for fibrin invasion is strongly supported by our finding that silencing endogenous MT3-MMP in two different melanoma cell lines reduced dramatically their ability to invade and form sprouts in 3D fibrin. Although melanoma arises in collagen-rich skin, abundant fibrin accumulation is frequently found in these tumors within perivascular spaces as well as the vascular and lymphatic vessel-rich tumor-host tissue interface [32]. Within these fibrin-rich sites, MT3-MMP-dependent proteolysis could enhance the passage of melanoma cells into lymphatic vessels, where discontinued basement membranes do not form significant barriers for cell invasion [48,49]. In addition to primary tumor sites, the accumulation of fibrin- rich provisional matrices around the circulating melanoma cell embolus promotes their metastatic capacity in lungs and lymph nodes [50,51]. As such, MT3-MMP could further accelerate the metastatic process in such environments via its ability to affect pericellular fibrin remodeling. Consistent with these proposed pro- metastatic functions, MT3-MMP expression was increased in biopsies of human melanoma metastasis. While the nodular vs. superficially spreading origin of the biopsied tissues of the metastatic lesions had not been defined, MT3-MMP expression was not restricted to nodular melanoma-derived cell lines. Rather, WM165 and WM239 cells derived from the metastases of superficially spreading melanoma as well as in WM793 cells isolated from vertical growth phase primary melanoma also expressed the protease. In this regard, it is interesting to note that the formation of metastatic foci may also depend on the ability of tumor cells to form adhesive expansively growing colonies [52]. Since MT3-MMP had similar effects in the invasive activities of WM852 cells from nodular melanoma metastases and WM165 cells originated from superficially spreading melanoma metastases, we postulate that the protease may play previously unappreciated role in the metastatic process. To date, minimal information is available about any functions of endogenous MT3-MMP in tumor cells, and the relevant ECM and non-ECM substrates for MT3-MMP remain poorly defined. As supported by a recent report of Nogo receptor 1 shedding by endogenous MT3-MMP in neurons [53], MT3-MMP may modulate also melanoma cell invasion by cleaving transmembrane or pericellular proteins. Like MT1-MMP, MT3-MMP is also efficient cell surface activator of proMMP2 [21]. However, MMP2 is inefficient in driving cell invasion in collagen or fibrin alone [15,18]. In addition, current results revealed opposing effects of MT3-MMP on fibrin, that is its own substrate, and on collagen type I, that is a poor substrate for MT3-MMP but is instead efficiently cleaved by MT1-MMP. Since MT3-MMP concomi- tantly reduced MT1-MMP from the surface and MT1-MMP- dependent collagen invasion, current results suggest that MT3- MMP restricts the proinvasive MT1-MMP activity and cell invasion in type I collagen-rich tissues, thus promoting the expansion of nodular melanoma cell colonies. However, as endogenous MT3-MMP activity remains unaffected by MT1- MMP, its fibrinolytic potential could provide melanoma cells with PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28325