C oncogene promotes mesenchymal-amoeboid transition A.Figure 1A. Top: graphs showing the change in cell diameter for MCF7 cells transfected with ZsGreen (left) or ZsRASSF1C (right) plasmids at 0 h and 12 h, connections indicate individual cell tracking. Bottom: example images diameter measurements (red line). Scale 20 μm. B. Single cell morphology of MCF7 and MDA-MB-231 cell lines, expressing or not a RASSF1C construct, cultured in 3D rat tail collagen I and analyzed for their rounded or elongated morphology 24 hours after seeding in 3D matrix. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. C. Confocal images of MDA-MB-231 cells transfected with ZsGreen or ZsRASSF1C grown in 3D-collagen matrix. Cells were imaged and stained with Phalloidin-568 (red), pMLCII/Alexa‑633 (false‑coloured green) or imaged for ZsGreen (false-coloured white). Scale 20 μm. D. Single cell morphology assay of MDA-MB-231 cells cultured in 3D-collagen and analysed for morphology 24 hours after transient knock-down using either a control sequence (NT, non-targeting) or a sequence targeting the RASSF1 locus. RASSF1 knock-down marks a reduction in rounded cells. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. E. Gelatine zymography assay shows downregulation of MMP-9 metalloprotease in MDA-MB-231 cells over‑expressing RASSF1C. F. 3D Matrigel Boyden chamber invasion with or without metalloproteases inhibitor GM6001 and 3D Hydrogel invasion of MDA-MB-231 control cells or cells over‑expressing RASSF1C (24 h). Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. G. Quantification of Boyden chamber invasion after 24 hours in 3D matrigel of MDA-MB-231 cells, transfected with ZsGreen (control) or ZsRASSF1C (RASSF1C) and treated with inhibitors against ROCK (Y27632, 10 μmol/L) or Rho (C3, 2 μg/ml). Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. H. Cartoon summarising intravital imaging experiments. I. Lung colonization of MDA-MB-231 CFP+ cells (MDA-MB-231CFP) stably expressing control or HA-RASSF1C in mammary gland tumors from 5 mice each, measured from intravital imaging time-lapse images. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. J. Average migration speed (expressed as μm/min) of n=30 randomly selected CFP positive cells in intravital images of Control and RASSF1C expressing MDA-MB-231 tumors. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. K. Quantification of CFP+ MDA-MB-231 cells displaying different types of morphology in vivo, expressed as average number of events, observed from a total of 30 positions in n=5 mice/group (Control or RASSF1C). Mesenchymal, amoeboid and hybrid morphology were scored manually based on diameter and circularity (distance between the two furthest points). The higher distance between cell edges was defined as mesenchymal morphology, while the lower distance was considered amoeboid morphology. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.