Flow Cytometry To maintain a homogeneous population of ALCAM-expressing or ALCAM-silenced cells, the sh5, 2C-ALC, and sh5rxd cell lines were flow-sorted using a FACSDiva (Becton Dickinson, Franklin Lakes, NJ, USA). Both sh5 and 2C-ALC were sorted using the anti-ALCAM 3A6 antibody (Millipore, Billerica, MA, USA) and phycoerythrin anti-mouse secondary antibody. For sh5, low- ALCAM cells were kept and high ALCAM cells were discarded. For 2C-ALC, ALCAM-positive cells were kept, and ALCAM- negative cells were discarded. The sh5rxd cell line was sorted using the cytoplasmic GFP tag on the full-length ALCAM construct they were transduced with; GFP-positive cells were kept and GFP- negative cells were discarded. Results ALCAM Expression Correlates with Cell Motility in Uveal Melanoma Cell Lines To begin to address the role of ALCAM in tumor cell behavior, we assembled a panel of five uveal melanoma cell line stocks: OCM-1A, MUM-2B, MUM-2C, C918, and M619. All cell lines had been previously characterized by cell phenotype, invasive potential, and vasculogenic mimicry [38–42]. Two of the lines, OCM-1A and MUM-2C, are poorly invasive and resemble normal uveal melanocytes, while the remaining three cell lines were characterized as highly invasive, based on the ability to invade a collagenous matrix-coated polycarbonate membrane. MUM-2B and MUM-2C were initially reported to be isolated from the same metastasis from a primary uveal melanoma, and found to be phenotypically divergent: MUM-2B is epithelioid, while MUM-2C is spindle-shaped [38]. Microarray analysis of these two lines revealed that ALCAM was one of the most up- regulated genes (8.3-fold) in highly invasive MUM-2B cells versus poorly invasive MUM-2C cells [38]. Subsequent analysis of short tandem repeats in the genomic DNA of these uveal melanoma lines indicates that MUM-2B and MUM-2C are, in fact, unlikely to have derived from the same metastasis [43]. Folberg and colleagues (2008) additionally present evidence that OCM-1A and MUM-2C share the same origin, as do MUM-2B, M619, and C918; our data below are consistent with this. Therefore, while we initially examined all five cell lines, most of our work has focused on MUM-2B and MUM-2C as exemplars. We first utilized a gap-closure assay as one measure of the motility of each cell line. Freshly confluent monolayers were inscribed with a gap using a micropipet tip, and movement of cells back into the gap was monitored by time-lapse imaging. MUM-2B cells moved more quickly to close the gap than did MUM-2C cells, and had completely closed the gap by 8 hours (Fig. 1A). Time- lapse analysis showed that MUM-2B cells appeared to move as a cohesive sheet across the empty space of the gap (Fig. 1A). Contrastingly, MUM-2C cells failed to completely close the gap by 8 hours, and, unlike MUM-2B, individual cells could be seen breaking away from the cell front and moving across the gap space individually (Fig. 1A). Gap closure analysis of the remaining cell lines revealed that M619 and C918 were fast-moving like MUM- 2B, while OCM-1A was slow-moving like MUM-2C. The average speed of each cell line was calculated from three independent gap closure trials (Fig. 1B). MUM-2B, C918, and M619 all moved at speeds 2–3 fold greater than OCM-1A and MUM-2C (Fig. 1B). Next, we assayed ALCAM expression in the five cell lines by both western blot (Fig. 1C) and RT-PCR (Fig. 1D). ALCAM protein expression was undetectable in OCM-1A and MUM-2C; in contrast, it was similarly high in MUM-2B, C918, and M619 (Fig. 1C). ALCAM can be shed from the membrane via the action ALCAM in Melanoma Motility and Adhesion of ADAM-10 and ADAM-17 metalloproteinases [44,45]; therefore we asked whether the lack of ALCAM protein in MUM-2C and OCM-1A indicated a true lack of gene expression or simply accelerated shedding and/or degradation. RT-PCR analysis of cDNA demonstrated that few, if any, ALCAM transcripts are present in these two cell lines, supporting the former possibility (Fig. 1D). Finally, we analyzed the mode of migration of each cell line, by plotting the cumulative percent of the initial gap closed versus time: Three separate trials of the gap-closure assay in MUM-2B (Fig. 1E) and MUM-2C (Fig. 1F) are shown. As noted above, MUM-2B appeared to move as a cohesive sheet to close the gap, and this linear mode of movement is reflected in the plots (Fig. 1E). In contrast, MUM-2C did not seem to move as a cohesive sheet, but instead, individual cells extended filopodia and the cell front moved discontinuously to close the gap. This is apparent in Figure 1F, where discrete ‘‘jumps’’ on the Y-axis demonstrate this stop-and-start movement. Together, these data demonstrate that ALCAM expression positively correlates with cell motility in uveal melanoma cell lines. Establishment of ALCAM-silenced MUM-2B Cell Lines by shRNA Knockdown To determine whether ALCAM regulates uveal melanoma cell behavior, we began by knocking down ALCAM levels in MUM- 2B cells, which normally express high levels of ALCAM. This was accomplished via transduction with retroviral constructs encoding shRNAs targeted against the Alcam transcript. We tested a total of 6 different shRNA sequences, and focus here on two such constructs, termed sh5 and sh6. Initial immunostaining of MUM- 2B cells infected with virus particles showed that many, though not all, sh5-expressing cells completely lost detectable ALCAM, while sh6 expression failed to silence ALCAM expression detectably (data not shown). To isolate a purified population of silenced MUM-2B cells, we performed FACS sorting using an antibody against the ALCAM ectodomain, keeping only the population of sh5-expressing cells that lacked detectable ALCAM. These cells, termed sh5 cells, were utilized for experiments, using sh6- expressing cells as well as parental MUM-2B cells as control groups (Fig 2A, C–H). To confirm that any phenotypes observed in sh5 cells were due to ALCAM-silencing and not to off-target effects of the shRNA, we established the sh5rxd ‘‘rescue’’ line. The sh5rxd cells were transduced with retroviral constructs encoding both sh5 and a full- length, C-terminally GFP-tagged human ALCAM construct containing three silent point mutations, rendering it resistant to sh5 knockdown. Expression of the GFP-tagged ALCAM was validated by western blot using antibodies against either ALCAM or GFP, which confirmed that sh5rxd expressed the higher molecular weight GFP-tagged ALCAM but little, if any, endog- enous ALCAM (Fig. 2B). As expected sh5rxd cells exhibited GFP- and ALCAM-positive cell-cell junctions (Fig. 2I–K and data not shown). ALCAM-silenced Cells Display Reduced Motility and Invasive Capacity We first tested sh5 ALCAM-silenced cells in the gap closure assay previously described, comparing them to both native MUM-2B cells and sh6 control cells. Silencing ALCAM results in a significant reduction in motility: sh5 cells exhibit a closure rate nearly 50% lower than that of parental MUM-2B or non- silenced sh6 cells (Fig. 3A). Although their velocity was markedly reduced, the sh5 cells still appeared to move as a cohesive sheet, and individual cells did not detach from the invasion front as in PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39330