Figure 5: The nanoscale membrane bridges act as conduits for intercellular transfer of miRNA between cancer and endothelial cells.

Representative confocal images show the transfer of Cy3-labelled miRNA from MDA-MB-231 cells (EPI) to endothelial cells (ENDO) at (a) 24 h and (b) 36 h of co-culture. Alexa Fluor 488-Ac-LDL (green)-labelled endothelial cells were co-cultured with Cy3-labelled miRNA-transfected MDA-MB-231. Co-cultures were counterstained with phalloidin (purple) and DAPI+WGA (blue). A 3D visualization shows the localization of miRNA within the nanoscale connections (white arrows), which act as conduits for horizontal transfer of miRNAs to endothelial cells. (c) Schema shows quantification of Cy3-labelled control miRNA and Cy3-labelled miR132 transfer between cancer cell and endothelium using flow cytometry. Endothelial cell populations were isolated from the co-cultures and percentage of miRNA+ve cells was determined. Dual cultures in Boyden chambers were included as controls. (d) Graph shows the effect of pharmacological disruption of nanoscale conduits on miRNA transfer. (e) Schema shows experimental design for reverse transcriptase–PCR-based detection of transferred miR-132 in endothelial cells under different experimental conditions. MDA-MB-231 cells transfected with miR-132 and α-miR-132 were co-cultured with endothelial tubes. FACS-isolated endothelial cell populations were analysed for the expression of miR-132. (f) Graph shows miR-132+ve cell populations (solid red) show 5 × increase compared with miR-132−ve populations (solid blue) (P<0.0001), whereas anti-miR-132+ve cells (striped red) show 26 × decrease in miR-132 expression (P<0.0001) compared with α-miR-132−ve cells (striped blue). Direct transfection of miR-132 (black) and α-miR-132 (light blue) in endothelial cells is used as positive and negative controls, respectively. Upregulation of miR-132 from baseline was observed in dual culture (solid green), which could be inhibited with anti-miR-132 (striped green). MiR-132 levels are increased compared with dual only in those cells that are positive for intercellular transfer. Fold change was determined compared with endothelial cell transfection with control miRNA (grey). (g) FACS analysis shows nanoscale bridges-mediated transfer of miRNAs leads to changes in p120RasGAP and pAkt (S473) expression downstream of the miR-132 pathway in endothelial cell populations isolated from co-cultures. (h) Graphs show p120RasGAP expression is decreased in the miR-132+ve cell populations and increased in the α-miR-132+ve cell populations, while further downstream miR-132 positively regulates pAkt expression. Data shown are mean±s.e.m. (N=2–5 independent studies, with 3 replicates per study, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, analysis of variance followed by Bonferroni’s post-hoc test).