Figure 1: Nanoscale structures physically connect metastatic cells and the endothelium.

(a) Representative image of MDA-MB-231 cancer cells exhibiting an invasive phenotype in the presence of preformed endothelial tubes in co-culture. (b) Representative image of a mammosphere typically formed by MDA-MB-231 cells when cultured on 3D tumour matrix in the absence of endothelial cells. MDA-MB-231 cells were loaded with CFSE. Actin was labelled with rhodamine phalloidin and nuclei were counterstained with DAPI. (c) A representative SEM of epithelial (EPI) MDA-MB-231 cells aligning on HUVEC (ENDO) tubules in the co-culture. Lower panel shows higher magnification. (d) SEM image reveals nanoscale membrane bridges connecting (nCs) metastatic breast cancer (EPI) cells and endothelial vessels (arrows). (e) A representative transmission electron micrograph shows intercellular connectivity through the nanoscale membrane bridge between MDA-MB-231 and an endothelial cell. (f) A cartoon represents the types of homotypic and heterotypic intercellular nanoscale connections that an epithelial cell may form in the presence of endothelial tubules. Highly metastatic (MDA-MB-468, MDA-MB-231 or MDA-MB-435) or low metastatic (MCF7 and SkBr3) cancer cells were co-cultured with the endothelial tubes. Normal HMECs were used as control. Graphs show percentage of total population of epithelial cells that exhibit either homotyptic (Epi–Epi) or heterotypic (Epi–Endo) nanoscale connections and (g) average number of nanoscale connections formed per cell. Quantification analysis was done on >300 cells of each cell type. Data shown are mean±s.e.m. (n=6 replicates per study, with 2–3 independent experiments). **P<0.01, ***P<0.001 (analysis of variance followed by Bonferroni’s post-hoc test).