C cells produce pro-invasive Extracellular Vesicles A.Figure 3A. Left: Cartoon showing the principle behind the Cre-carrying EVs and how recipient cells switch from DsRed+ to eGFP+ expression upon Cre-mediated excision of the DsRed locus. Centre: representative images, and right: quantification of T47DDsRed cells that converted to eGFP+ in the bottom wells of a transwell system in the presence of MDA-MB-231CFP;Cre;Control or MDA‑MB‑231CFP;Cre;HA-RASSF1C seeded in the upper wells. Scale bars represent 100 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 ≤ 0.0001. B. Left: averaged Nanoparticle Tracking Analysis displaying the size distribution of EVs isolated from media of MDA-MB-231 cells expressing Control (pcDNA3) or FLAG-RASSF1C plasmid. Inserts: transmission electron microscopy images of single EVs. Scale bars represent 200 nm. Right: quantification of total secreted EVs (96 h, top) or at indicated times (below), as measured by NTA. 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 ≤ 0.0001. C. Western blot analysis of EVs isolated from MDA-MB-231 cells transfected with Control (pcDNA3) or FLAG-RASSF1C with indicated antibodies. Equal amounts of protein were loaded after normalization via microBCA assay. D. Quantification of EVs isolated from MDA-MB-231 cells transfected with Control (pcDNA3), RhoA‑CA, RhoA‑DN, FLAG‑RASSF1C, FLAG-RASSF1C‑R199F, or FLAG‑RASSF1C + RhoA‑DN using Nanoparticle Tracking Analysis. 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 ≤ 0.0001. E. Normalized values of real‑time migration rates in xCELLigence plates of MDA-MB-231 cells treated with 20 ng/μl EVs derived from MCF7 cells expressing a Control, FLAG-RASSF1C, or EGFP-RhoA plasmid, left to migrate for 24 h. Statistics were performed with 2-way ANOVA (Dunnett's multiple comparisons test). F. Left: Representative images of Boyden chamber invasion assay in 3D matrigel of MCF7 cells, treated with 20 ng/μl EVs derived from MDA-MB-231 cells, transfected the same constructs used in (e). Right: Quantification of Boyden chamber invasion 24 hours after EV treatment. Scale bars represent 200µm. 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 ≤ 0.0001. G. Top: graphical representation of the functional (invasion) assay performed using MDA‑MB‑231 derived EVs on MCF7 recipient cells. Bottom: Boyden chamber invasion assay in 3D matrigel of EVs isolated from different steps of ultracentrifugation. EVs were collected from conditional medium produced by MDA-MB-231 expressing Control (pcDNA3) or FLAG-RASSF1C. The 2K (large EVs), 10K (medium EVs or MVs) and 100K (small EVs, including exosomes) pellets were collected and equal amount of EVs (normalized according to protein content) were incubated with MCF7 cells. MCF7 were evaluated for invasion after 24 hours. 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 ≤ 0.0001.