Figure 3. ALCAM-silenced cells display reduced wound-gap closure speed, invasive capacity, and MMP-2 activation. Closure rate of cells in a wound-gap assay was determined for each cell line listed (minimum of 3 trials each; A). The speed of sh5 cells was significantly reduced compared to MUM-2B, sh6 control, and sh5rxd rescue cells (ANOVA; p,0.05). Invasive capacity of each cell line was assayed via transwell migration. The number of invasive cells was standardized relative to MUM-2B for each cell line shown (minimum of 3 trials each; B). The average number of invasive MUM-2B cells per three non-overlapping 106 fields was 806. The sh5 cell line displayed significantly reduced invasive capacity compared to MUM-2B and sh5rxd rescue (ANOVA; p,0.01). To ensure that differences in invasiveness were not due to differences in growth or survival after plating, an equivalent number of cells was plated for each line. Eight hours later, the number of cells was assayed, and found to be comparable between lines (C). Error bars are mean 6 S.E.M. doi:10.1371/journal.pone.0039330.g003 ALCAM in Melanoma Motility and Adhesion Because the invasion assays were performed over a period of 8 hours, it was formally possible that sh5 cells simply proliferated more slowly than MUM-2B, which might contribute to the difference in the number of cells counted on the underside of the transwell filter. To ascertain that this was not the case, we performed a cell survival assay by plating a known number of cells in standard tissue culture wells, incubating for 8 hours, and then counting the cells. No significant differences were found in the survival of MUM-2B, sh5, and sh6 cell lines after 8 hours (Fig. 3C) or in growth at 24 hours (data not shown). Thus, our experiments demonstrate that ALCAM expression is necessary for cell motility and invasiveness in MUM-2B uveal melanoma cells. ALCAM Overexpression is not Sufficient to Enhance Migration and Invasive Capacity in MUM-2C Cells If ALCAM expression is necessary for motility and invasiveness in the MUM-2B uveal melanoma cell line, is ALCAM expression sufficient to increase motility and confer invasiveness in the normally ALCAM-negative MUM-2C line? To test this, we created a stable cell line, termed 2C-ALC, by transducing MUM- 2C with a virus encoding full-length ALCAM. Expression of the full-length ALCAM construct was confirmed by both western blot (Fig. 4C) and immunohistochemistry (Fig. 4A, B). Expression level of ALCAM in 2C-ALC was roughly comparable to that of MUM- 2B. As expected, ALCAM localized to cell-cell contacts in 2C- ALC cells (Fig. 4B). Overexpression of ALCAM in the 2C-ALC cell line, however, failed to enhance the velocity of cells in the gap closure assay (Fig. 4D). 2C-ALC cells still often moved as individual cells (similar to native 2C cells; Fig. 1F), and not as a cohesive sheet like MUM-2B cells (data not shown). Overexpres- sion of ALCAM was also not sufficient to enhance the invasive capacity of 2C-ALC cells (Fig. 4E), nor did it affect the survival or proliferation of the cell line (Fig. 4F). ALCAM-silenced Cells Exhibit Reduced MMP-2 One likely way in which ALCAM could promote an invasive phenotype is through regulation of matrix metalloproteinases (MMPs). MMPs are zinc-dependent proteinases whose expression has been implicated in processes such as tissue remodeling and cancer metastasis. MMP-2, a 72 kDa protein also called gelatinase A, is the most abundant of the MMPs and is documented as a marker of poor prognosis in a variety of cancers [46–48]. Activation of MMP-2, and the additional gelatinase family protein MMP-9, allows degradation of type IV collagen basement membranes. MMPs are synthesized as pro-enzymes that must be processed to their active form by proteolytic cleavage. Pro-MMP-2 is recruited from the extracellular milieu and processed by a complex consisting of Type I MMP (MT1-MMP/MMP-14) and tissue inhibitor of metalloproteinase-2 (TIMP-2); this process is known to require full-length ALCAM [40]. Thus, we assayed MMP-2 levels in our panel of melanoma cell lines via gelatin zymography and western blot, where pro-MMP-2 appears as a 72 kDa band, and active MMP-2 appears as a ,64 kDa band. In gelatin zymography, active MMP-2 appears as a clear, Coomassie-negative band of ,64 kDa upon staining of the gel; the ‘‘pro-MMP2’’ band of 72 kDa is also active in this assay, in the presence of SDS [40]. Because the pro-MMP2 band was much more prominent in our conditioned media samples, we quantified this band as a measure of MMP2 levels secreted by cells; in many gels, we could see a faint ,64 kDa active band as well, which tracked levels of the clearer pro-MMP2 band (data not shown). Gelatin-clearing MMP-2 activity was strong in MUM-2B (Fig. 5A), C918, and M619 cells (data not shown), all of which highly express ALCAM (Fig. 1C), but not in the ALCAM-negative OCM-1A PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39330