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1 DOI: /ncb2133 Figure S1 Actomyosin organisation in human squamous cell carcinoma. (a) Three examples of actomyosin organisation around the edges of squamous cell carcinoma biopsies are shown. Myosin IIa in green, E-cadherin in blue, F-actin in red and DAPI in white. Merge excludes DAPI channel. Scale bar is 20μm. (b) A431 squamous cell carcinoma cells were plated as single cells and fixed at various time-points. Upper panels show the F-actin (red) and ps19-mlc (green) distribution while lower panels show only areas of colocalisation of F-actin and ps19-mlc (thresholds set at pixel intensity >128 in both channels). Images show a single motile cell (top panels), three cells making initial cell contacts (upper mid panels), and two cells making more extensive contacts (lower mid panels) six hours after plating and four cells with established contacts (bottom panels) after 20 hours. Scale bar is 20μm. 1

2 Figure S2 Collective cell migration is disrupted by de-regulated ROCK1 activation. (a) Three images of collectively migrating control A431 cells (expressing Ezrin-GFP in green) taken at 5¼ hour intervals. Scale bar is 100μm. Coloured circles indicate the position of a specific cell at each time. In cases where mitosis occurred one daughter cell retains the same colour as the original cell while the other daughter has a hatched pattern in the same colour. Scale bar is 100μm. (b) Three images of collectively migrating ROCK1:ER A431 cells (CFSE (CarboxyFluorescein Succinimidyl Ester) labelled in green) taken at 5¼ hour intervals. Coloured circles indicate the position of a specific cell at each time. Cells were cultured in 1μM 4-OH tamoxifen to activate ROCK1:ER. Scale bar is 100μm. (c) Tracings of the cell paths from the series of images shown in (a) and (b) are shown. 2

3 Figure S3 sirna screening for regulators of A431 cell morphology. (a) Representative pictures showing the F-actin different phenotypes on a 2D substrate identified in an sirna screening against regulators of small GTPases, adhesion molecules and cytoskeletal regulators. (b) Genes identified for each phenotype are listed. (c) Quantification of F-actin organization at cell-cell contacts. A431 cells were transfected with the indicated sirna and 60 hours after transfection were fixed and stained for F-actin. The proportion of cells with tight F-actin at cell-cell contacts was scored (n=2, error bars are half the range, >200 cells for each data point). 3

4 Figure S4 Effects of DDR1 depletion. (a) Total ps19-mlc levels are shown by western blot in control and DDR1 sirna transfected A431 cells. (b) RhoA-GTP levels are shown in control and DDR1 sirna transfected A431 cells (average of two experiments, half range shown by error bars). (c) control or DDR1 depleted A431 cells were fixed 60 hours after transfection. The cell area around the edge of groups was quantified. (n=30, 36, 38 and 46 for non-transfected, control, DDR1#3 and DDR1#4 transfected data sets). Box and whisker plots show quartiles and extreme values. (d) Representative pictures showing F-actin and b-catenin in MCF10A cells transfected with control or DDR1 sirna. Scale bar is 20 mm. (e) Western blot analysis showing the levels of DDR1, β-tubulin, E-cadherin, β-catenin and p120 catenin in MCF10A cells transfected with control or DDR1 sirna. (f) Graph showing the percentage of cells in clusters in a cell-cell adhesion assay where cells were plated on poly Hema-coated plates and incubated under shaking for 30 minutes (an average of three independent experiments). (g) Graph showing quantification of control, DDR1 or integrin β1 sirna transfected cells attached to collagen-coated plates after incubation of 30 minutes followed by PBS wash (average of two experiments). (h) Western blot showing the efficiency of DDR1 depletion following transfection of SCC12 cells with either control or DDR1 sirna. 4

5 Figure S5 DDR1 expression and localisation in cancer cell lines. (a) Control or DDR1 sirna transfected A431 cells are stained for DDR1 (red) and F-actin (green). Scale bar is 10μm. (b) A range of normal, non-metastatic and metastatic breast cancer cell lines are stained for DDR1. Scale bar is 10μm. (c) Control and TGFβ(2ng/ml)+EGF(10nM) treated A431 cells are stained for DDR1 (white) and F-actin (green). Scale bar is 10μm. 5

6 Figure S6 DDR1 and Par3 expression in tumour tissue. (a) Upper panels show clinically normal oral mucosa adjacent to tumours stained in the same way. Lower panels show Par3 (red), DDR1 (green) and DAPI (blue) staining in head and neck squamous cell carcinomas. Scale bars are 20 mm. (b) Table summarises DDR1, Par3 and E-cadherin staining in 20 head and neck squamous cell carcinoma samples. ++ indicates strong staining or co-localisation, + indicates intermediate staining or co-localisation, - indicates no staining or co-localisation. (c-c) indicates cell-cell contact localisation of DDR1, nd indicates not determined. (c) Upper panels show Par3 (red) and DDR1 (green) staining in non-cancerous breast tissue. Lower panels show the same staining in invasive ductal breast carcinoma tissue. Scale bars are 20 mm. 6

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8 Figure S7 Full scans 8

9 Figure S7 continued 9

10 Supplementary Movie Legends Movie S1 This movie shows A431 (green) co-cultured with human carcinoma fibroblasts (red) moving collectively. The length of the movie is 11.5 h. Movie S2 This movie shows A431 ROCK:ER cells treated with tamoxifen in order to increase ROCK activity. The length of the movie is 11.5 h. Movie S3 This movie shows non transfected A431 moving on collagen gels for 12 h. Notice the cells moving as a group. Movie S4 This movie shows a clone of DDR1 sirna-transfected A431 cells (shddr1#3) moving on collagen gels for 12 h. Notice the loss of cohesion between cells. Movie S5 This movie shows a clone of DDR1 sirna-transfected A431 cells (shddr1#4) moving on collagen gels for 12 h. Notice the loss of cohesion between cells. Acknowledgements We thank Cancer Research UK and EMBO for funding and members of biological resources unit and histopathology laboratory for technical assistance. Finally, we thank Nic Tapon, Barry Thompson and lab members for discussion and comments. Supplementary References 1. Rheinwald, J.G. & Beckett, M.A. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultures from human squamous cell carcinomas. Cancer Res 41, (1981). 2. Gaggioli, C. et al. Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol 9, (2007). 3. Leitinger, B. Molecular analysis of collagen binding by the human discoidin domain receptors, DDR1 and DDR2. Identification of collagen binding sites in DDR2. J Biol Chem 278, (2003). 4. Croft, D.R. et al. Conditional ROCK activation in vivo induces tumor cell dissemination and angiogenesis. Cancer Res 64, (2004). 10