Figure1: Effects of saracatinib on HNSCC proliferation, cell cycle progression, Erk1/2 activation and in vitro invasion. A: IC₅₀ values for cell growth determined by 5 day MTT assays for the indicated HNSCC lines treated with 0-10 μM saracatinib. Mean values are shown for each line from three independent assays. B: Impact of saracatinib on HNSCC cell cycle progression. HNSCC cell lines were treated with the indicated amounts of saracatinib for 24 hours, fixed labeled with propidium iodide to assess DNA content, and analyzed for cell cycle status by fluorescence-activated cell sorting. Results show the average percentage of cells in each cell cycle phase as indicated on the left. Bars, SD of two independent experiments. C: Effects of saracatinib on Erk1/2 activity. HNSCC cells were treated with saracatinib for 24 hours at the indicated doses, lysed and analyzed by Western blotting with phosphorylation-specific (pErk1/2) and total Erk1/2 antibodies. Blots shown are representative of three different experiments, with indicated band intensities shown relative to no treatment (0 μM) for each cell line. D: Saracatinib inhibits in vitro HNSCC invasion. HNSCC cells (1x10⁵) were plated in Matrigel-coated transwells alone or with increasing concentrations of saracatinib. After 2 h, invasion was stimulated with 5% FBS and cells were allowed to invade for 12 h (UMSCC1) or 24 h (HN31 and 1483). Invaded cells were quantified by brightfield microscopy. Bars, SEM of three independent experiments.

Figure2: Saracatinib inhibits Src activity and downstream Src substrate phosphorylation in HNSCC cell lines. HN31, UMSCC1 and 1483 cells were treated with DMSO vehicle or saracatinib (0.01-1 μM) for 24 h. Cells were lysed and total protein amounts were analyzed by Western blotting with total or phosphorylation site-specific antibodies for Src and the indicated substrates. Blots shown are representative of at least four independent experiments, with band intensities for each substrate quantified relative to the untreated (0 μM) condition for each cell line.

Figure3: Saracatinib inhibits Src activity, perineural invasion and cervical lymph node metastasis in orthotopic UMSCC1 tongue tumors. A: UMSCC1 tongue tumors from representative control-treated or saracatinib-treated mice were sectioned and stained with hematoxylin and eosin (H&E) or by IHC with the indicated antibodies (left). A case of human HNSCC was evaluated in parallel as a positive control. The pY416 SFK and pY421 cortactin ratios from saracatinib treated to control levels are indicated. Bars, 100 μm. B: Locoregional invasion and lymph node metastasis is inhibited by saracatinib. Submental and associated tracheoesophageal tissue from control treated and saracatinib treated mice was immunostained for cytokeratin 14 to detect cells of epithelial origin. Magnified regions containing a single sublingual nerve and superficial cervical lymph node are shown for clarity. Inset shows a magnified cortical region of superficial cervical lymph nodes from control and saracatinib treated mice. Arrowheads denote metastasized UMSCC1 cells. N; sublingual nerve, ED; excretory duct. Bars 100 μm; inset, 50 μm.

Figure4: Saracatinib inhibits invadopodia formation and ECM degradation. A: Representative images of UMSCC1 cells treated with different saracatinib concentrations. UMSCC1 cells plated on FITC-gelatin coated coverslips (pseudocolored white) for 2 h were treated with saracatinib as indicated (left) for 6 h. Cells were labeled to visualize F-actin (red), cortactin (green) and phosphotyrosine (blue). Arrows denote invadopodia and corresponding colocalized areas of focal matrix degradation with invadopodia components. Treatment with 1.0 μM saracatinib resulted in F-actin aggregates lacking cortactin but accumulated at cytoplasmic sites where invadopodia typically occur (arrowheads). Bar, 10 μm. B: UMSCC1 cells treated with increasing concentrations of saracatinib were stained as in A and quantified to determine the percentage of cells that produced functional invadopodia, presented as the mean ± SEM. All treatment groups were significantly different from each another based on a one-way ANOVA (p<0.05) except 0 and 0.01 μM, and 0.5 and 1.0 μM pairs. C: Saracatinib decreases the ability of UMSCC1 cells to degrade ECM. The percentage of gelatin degradation per cell area for the cell population analyzed in B is shown with the mean ± SEM.

Figure5: MMP9 secretion and ECM degradation activity in HNSCC cells is blocked by saracatinib. A: Localization of MMP9-containing vesicles in UMSCC1 invadopodia. Top: UMSCC1 cells plated on FITC-coated gelatin coverslips for 2 h were fixed and labeled with antibodies against cortactin and MMP9. The merged image indicates areas of cortactin and MMP9 co-localization (yellow; white arrows) that correspond with sites of focal gelatin degradation (black arrows). Bar; 10 μM, Asterisk; regions of global matrix degradation due to secreted protease activity. Bottom: Magnified view of indicated Top region. B: Inhibition of MMP9 secretion by saracatinib. Total cell lysates (cell) and aliquots of normalized conditioned media containing secreted MMPs (sec) from 1483 and UMSCC1 cells treated with increasing doses of saracatinib (bottom) were assayed for the presence of MMP2 and MMP9 by immunoblotting. Band intensities relative to control (0 μM) are shown for each treatment condition; secreted MMP2 was not detected and therefore not quantified. C: Gelatin zymography of MMP9 activity. Representative zymograms from conditioned media of 1483 or UMSCC1 cells cultured with the indicated saracatinib concentrations (bottom). DMEM was used as a negative control (M), DMEM containing 10% FBS (FBS) was used as a positive control for zymography. Graphs, densitometric analysis of MMP9 zymography. Percentage of MMP9 gelatin clearing is represented and the mean ± SEM for each cell line from three independent experiments.