C cells express cancer stemness features A.Figure 4A. Quantification of fluorescent intensity of CD133 and ALDH1 cancer stem cell markers in MDA‑MB‑231 control or RASSF1C transfected spheroids cultured on Matrigel 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. B. Confocal images of ZsGreen or ZsRASSF1C expressing MDA-MB-231 spheroids embedded in 3D rat tail collagen I. Cells were stained with NANOG (red). Zoom images show that RASSF1C expressing cells are also NANOG positive. Scale 100 μm. C. Western blot analysis of CD133 protein expression level in MDA-MB-231 cells treated with siNT or siRASSF1. D. Representative confocal images (upper row) and quantification of fluorescent intensity of ALDH1 expression in MDA-MB-231 cells expressing RASSF1C (black bar in the graph) or after siRNA treatment against RASSF1 (grey bar). Lower row shows merged images with DAPI (blue), ALDH1 (green) and RASSF1C (red). Scale bars represent 20 μm. 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. Quantification of the absolute number of NANOG positive cells (measured as average cell number/spheroid) per MDA-MB-231 ZsGreen (control) or ZsRASSF1C (RASSF1C) spheroids in 3D-collagen, treated with ROCK inhibitor (Y27632, 10 μmol/L) or Rho inhibitor (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. F. Quantification of the absolute number of NANOG positive cells (measured as average cell number/spheroid) per MDA-MB-231 ZsGreen (control, con) or ZsRASSF1C (RASSF1C) spheroids in 3D-collagen, co‑transfected with either RhoA WT, catalytically active (CA) or dominant negative (DN) constructs. 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. Fold change in qRT-PCR of NANOG, OCT4 and SOX2 mRNA from MCF7 cells expressing CRISPR‑dCas9 or dCas9‑VP64 plus single guide RNA for the RASSF1C promoter (sgRASSF1C). 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. Quantification of the percentage of double positive pMLCII and NANOG cells in MCF7 cells expressing CRISPR-dCas9, dCas9-VP64, dCas9‑SKD plus single guide RNA for the RASSF1C promoter (sgRASSF1C). At least 300 cells were counted per experiment. 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. I. Quantification of number of invaded MCF7 cells in 3D matrigel, after incubation with EVs from MDA-MB-231 cells sorted for Greenhigh expression. MDA-MB-231 cells stably expressing a pQXCIP‑zsGreen or pQXCIP‑zsGreen-RASSF1C construct were sorted and EVs were harvested 24 h later, prior to seeding with MCF7 cells in Boyden chambers. Invasive potential of MCF7 cells was evaluated 24 h after incubation with EVs. 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. qRT‑PCR expression of NANOG, OCT4, SOX2 in MCF7 cells 24 h after exposure to 50 ng/μl EVs derived from MDA‑MB‑231 cells (treated as indicated). 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.