Figure 7.

Exosomes act as a cargo delivery system that transports Wnt10b from stroma to cancer cells. (a) Western blot analysis of Wnt10b in exosomes purified from various fibroblasts. (b) Flow cytometry of CD81 and Wnt10b expression in exosomes purified from various fibroblasts. (c) Immunofluorescence analysis of Wnt10b and CD81 co-location in p85α^−/− fibroblasts. Scale bar, 25μm. (d) ELISA analysis of Wnt10b in conditioned medium from p85α^−/− fibroblasts before (conditioned medium (CM)) and after (120 000 g) ultracentrifugation overnight. Data are the mean±s.e.m. *P-value⩽0.05. (e) The protein abundance of Wnt10b in conditioned medium and exosomes from p85α^−/− fibroblasts. Data are the mean±s.e.m. ***P-value⩽0.001. (f) Western blot analysis of Wnt activity in 4T1 cells cultured in original CM or after (120 000 g) ultracentrifugation overnight. (g) ELISA analysis of Wnt10b in CM from shNC or shCD81 p85α^−/− fibroblasts. Data are the mean±s.e.m. *P-value⩽0.05. (h) Western blot analysis of Wnt10b in cell lysates and concentrated conditioned medium from shNC or shCD81 p85α^−/− fibroblasts and p85α^−/− fibroblasts treated with DMSO or GW4869 (10 μM) for 2 days. (i) Flow cytometric analysis of Wnt10b intracellular accumulation in shNC or shCD81 p85α^−/− fibroblasts and p85α^−/− fibroblasts treated with DMSO or GW4869 as described in (I). (j) Immunofluorescence analysis of intracellular Wnt10b accumulation in shNC or shCD81 p85α^−/− fibroblasts. Scale bar, 10 μm. (k) Western blot analysis of Wnt pathway activation in MDA-MB-231 cells and 4T1 cells cultured in shNC or shCD81 p85α^−/− fibroblast-conditioned medium. (l) Representative images of β-catenin immunostaining in tumours from mice described in Figure 6m. Scale bar, 50 μm. (m) Western blot analysis of PI3K activity and Wnt10b in negative control vector (NC) and in p85α knockdown (shp85α) WT fibroblasts and exosomes. (n) Schematic representation of tumour-promoting effects provoked by fibroblasts instigated by loss expression of p85α.