Figure 2

Nestin KD enhances melanoma invasion in vitro and decreases sphere formation. (a) Significantly diminished cell migration of nestin KD cells (NesKD, solid lines) as compared with the vector control cells (Vec, dash lines) of A2058 (left; P<0.001 at 6 and 8 h, respectively) and A375 (right; P<0.001 at 6 and 8 h, respectively). (b) Representative fields of migrating A2058 (left) and A375 (right) cells in Matrigel inserts (top panel) and control inserts (bottom panel). Scale bars: 50 μm. (c) Nestin KD cells (black bars) had significantly higher Matrigel invasion than vector control cells (white bars) of A2058 (left, 2.54-fold, P<0.001) and A375 (right, 4.02-fold, P<0.001). (d) Matrigel invasion of A2058 and A375 nestin KD cells was characterized in the presence of MMP3-specific inhibitor UK356618 or the broad-spectrum MMP inhibitor Marimastat. Compared with untreated A2058 nestin KD cells (black bar), UK356618 (20 nM, 33% decrease, P<0.05) and Marimastat (20 nM, 43% decrease, P<0.01) significantly suppressed Matrigel invasion of A2058 nestin KD cells. Combinatorial treatment of UK356618 (20 nM) and Marimastat (20 nM) additively suppressed Matrigel invasion of A2058 nestin KD cells (56% decrease, P<0.001). In addition, Matrigel invasion of A2058 nestin KD cells with combinatorial treatment of UK356618 and Marimastat was lower than monotherapy with UK356618 (65%, P=0.097). Likewise, in A375 nestin KD cells, Matrigel invasion of A375 nestin KD cells was not altered by UK356618 (20 nM), but was significantly suppressed by Marimastat (20 nM) (45% decrease, P<0.01) or by combinatorial treatment of Marimastat (20 nM) and UK356618 (20 nM) (55% decrease, P<0.001), as compared with untreated cells (black bar). In addition, Marimastat monotherapy and combinatorial treatment of Marimastat and UK356618 had comparable suppressive effects on A375 nestin KD Matrigel invasion. Results were compared by one-way ANOVA test with Tukey’s multiple comparison test. Representative cell migration and invasion results are shown from at least three independent assays. (e) Representative images of A2058 and A375 melanospheres in spherogenic suspension culture in DMEM supplemented with 10% FBS and 0.5% methyl cellulose for 21 days. Melanospheres were visualized with p-iodonitrotetrazolium violet staining. Marked decreased of sphere formation in both A2058 (top) and A375 (bottom) nestin KD cells as compared with vector control cells (both P<0.01, respectively). Representative results are shown from two independent experiments. (f) H&E and IHC images of spheres growing over Matrigel. A2058 vector control spheres grew mainly on the surface of Matrigel (top), whereas nestin KD spheres invaded into the lattice of Matrigel (bottom). Scale bars: 50 μm. Location of spheres on Matrigel. Majority (83.3%) of A2058 vector control spheres grew on the surface of Matrigel (white bar), whereas 16.7% partially invaded into Matrigel (gray bar). In contrast, 28.3% nestin KD spheres grew on the surface of Matrigel (white bar), 25% partially invaded into Matrigel (gray bar), and 46.7% completely invaded and embedded into Matrigel (black bar). Difference in Matrigel invasion between melanospheres derived from A2058 vector control (N=60) and nestin KD (N=60) cells was significant (P<0.0001, χ²-test). Data are represented from two independent Matrigel sphere experiments. Nestin was abundantly expressed in the cytoplasm of vector control A2058-derived melanospheres grown in association with Matrigel (top), whereas decreased nestin protein levels in nestin KD A2058-derived spheres correlated with a localized submembranous pattern (bottom, arrows). Left panels: low magnification; right panels: high magnification. Scale bars: 10 μm. Note: *P<0.05, **P<0.01, ***P<0.001.