Fig. 1

HNSCC-derived cell lines show a high inter- and intratumoural invasive heterogeneity. A 3D matrigel invasion assays were performed using SAS, FaDu, Cal33 and XF354. Pictures were taken after 48 h. The invasive front is marked in turquoise (scale bar = 400 μm, n = 3). B Proteins of preconditioned media of the HNSC cell line panel were concentrated and loaded onto a gelantine containing zymogram gel. Reduced Coomassie G-250 staining intensity indicates the activity of MMP2 (~ 70 kDa; n = 2). C Flow chart of SAS subclone generation. D 3D matrigel invasion assay was performed with twenty SAS subclones as well as the parental SAS cell line and their relative invasiveness is displayed. Here, the invasion factor of each clone was normalized to the average of all clones. The median is shown with a red line, second and third quartiles are displayed with a blue box. One-way ANOVA was performed comparing each clone with all clones of the second and third quartiles (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). E Pictures (48 h) showing parental SAS and representative subclones with a low, median and high invasiveness. The invasion front is marked in turquoise (scale bar = 400 μm). F MMP2 activity was measured via zymogram in the subclones with the weakest (F & L) and highest (H & S) invasiveness as well as the parental cell line (n = 2). G Clones were ranked according to their invasion factor from weak (top, green) to strongly invasive (bottom, red). Expression of invasion associated genes and EMT markers in each clone were measured via RT-qPCR and normalized to RPLP0, PPIA as well as the average expression in all clones (n = 3). High relative expression of a gene is indicated by a red color, whereas blue indicates a low relative expression. Expression values were correlated with the invasion factor. Correlation coefficient R², the slope of the regression curve and the significance value p of the slope being non-zero of a correlation analysis to the invasion factor are shown. Since the invasion start correlates in a logarithmic manner to the invasion area, values of the slope are marked with “ln”. H Representative Western blot (n = 3) of the epithelial marker E-cadherin and the mesenchymal markers N-cadherin and Vimentin in relation to the invasion factor. Rpl7 served as loading control