Fig. 4: Cells exhibit pathfinding behavior on exosome trails.

a Time-series images from live imaging of pHluo_M153R-CD63 (green)/mCherry-CaaX (magenta) dual-expressing HT1080 cells in 2D culture conditions (see Supplementary Movie 2). mCherry-CaaX is shown in magenta and pHluo_M153R-CD63 is shown in green in the merged image. n = 186 cells from 31 movies from four independent experiments. Arrowheads indicate exosome trails. Arrows indicate protrusions. pHluo_M-CD63, pHluo_M153R-CD63. Scale bar, 50 µm. b Pathfinding index from (a) shown as a scatter-plot with median and quartile range. c Time-series images from live imaging of pHluo_M153R-CD63 (green)/mCherry-CaaX (magenta) dual-expressing HT1080 cells in 3D collagen gels (see Supplementary Movie 3). n = 151 cells from 27 movies from four independent experiments. Arrowheads indicate exosome trails. Arrows indicate protrusions. Scale bar, 50 µm. d Pathfinding index from (c) shown as a scatter-plot with median and quartile range. e Representative intravital images of pHluo_M153R-CD63 (green in merge) and collagen fibers (white in merge) in mice from eight independent experiments. Multiphoton z-stacks shown as maximum intensity-projections. Arrows indicate adhesive protrusions. Arrowheads indicate exosome deposits. Examples of exosome deposits associated with collagen fibers are shown in dotted circles. Scale bar, 50 µm. f Representative intravital images of pHluo_M153R-CD63 in chicks from six independent experiments. Arrows indicate adhesive protrusions. Arrowheads indicate exosome deposits. Scale bar, 50 µm. g Half-life determination of pHluo_M153R-CD63 small EVs in vivo using the avian embryo model (n = 7 chick embryos from three independent experiments). Flow cytometry was performed on chick plasma collected at each time point upon intravenous injection of pHluo_M153R-CD63 small EVs. Gated pHluo_M153R-positive events (pHluo_M153R+) at each time were used for half-life calculations in GraphPad Prism. The gating strategy is shown in Supplementary Fig. 4. Source data are provided as a Source Data file.