The αvβ6 integrin is transferred intercellularly via exosomes

Abstract

Exosomes, cell-derived vesicles of endosomal origin, are continuously released in the extracellular environment and play a key role in intercellular crosstalk. In this study, we have investigated whether transfer of integrins through exosomes between prostate cancer (PrCa) cells occurs and whether transferred integrins promote cell adhesion and migration. Among others, we have focused on the αvβ6 integrin, which is not detectable in normal human prostate but is highly expressed in human primary PrCa as well as murine PrCa in Pten(pc-/-) mice. After confirming the fidelity of the exosome preparations by electron microscopy, density gradient, and immunoblotting, we determined that the αvβ6 integrin is actively packaged into exosomes isolated from PC3 and RWPE PrCa cell lines. We also demonstrate that αvβ6 is efficiently transferred via exosomes from a donor cell to an αvβ6-negative recipient cell and localizes to the cell surface. De novo αvβ6 expression in an αvβ6-negative recipient cell is not a result of a change in mRNA levels but is a consequence of exosome-mediated transfer of this integrin between different PrCa cells. Recipient cells incubated with exosomes containing αvβ6 migrate on an αvβ6 specific substrate, latency-associated peptide-TGFβ, to a greater extent than cells treated with exosomes in which αvβ6 is stably or transiently down-regulated by shRNA or siRNA, respectively. Overall, this study shows that exosomes from PrCa cells may contribute to a horizontal propagation of integrin-associated phenotypes, which would promote cell migration, and consequently, metastasis in a paracrine fashion.

Keywords: Adhesion; Cell Adhesion; Exosome; Extracellular Matrix; Integrin; Prostate Cancer; Tetraspanin.

FIGURE 1.

The α_v, β₆, and β₁ integrin subunits are enriched in exosomes from PrCa cells. A, cell surface expression of α_v, β₆, and β₁ integrin subunits in PC3 and RWPE cell lines was evaluated by FACS using Abs to α_v (dashed lines), β₆ (gray continuous lines), β₁ (black continuous lines), or a non-immune mouse IgG (dotted lines) used as negative control. B, upper panel, electron microscopy of negatively stained PC3-derived exosomes. Scale bar = 100 nm. Bottom panel, IB analysis of PC3 TCL and exosome (Exo) lysates. CD63 and CD81 were used as markers enriched in exosomes, whereas CANX and GM130 were used as markers not found in exosomes and tested in non-reducing (bottom left) and reducing (bottom right) conditions. β₁ integrin subunit was used as loading control under reducing conditions. C and D, IB analysis of α_v, β₆, and β₁ integrin subunits in PC3 exosomes and of α_v and β₆ integrin subunits in RWPE exosomes. E, IB analysis of α_v, β₆, and β₁ integrin subunits in density gradient fractions. FLOT1 and CD81 were used as markers enriched in exosomes, whereas CANX was used as a marker not found in exosomes. PC3 lysate (TCL) and the Exo lysate used as starting material were also loaded. F, IB analysis of β₆ and β₁ in PC3, DU145, and C4-2B TCL and Exo.

FIGURE 2.

Exosomal transfer of α_vβ₆ between PrCa cells. A, DU145 cells were incubated with PKH26 Red dye alone (left) or with 5 μg/ml labeled PC3-derived exosomes (right) for 1 h, and a confocal analysis was carried out to evaluate exosomal internalization. DAPI was used to detect nuclei (blue), Alexa Fluor Phalloidin-488 was used to label actin (green), and PKH26 Red was used to label the exosomes (red). Scale bar = 20 μm. Z-stack analysis shows the presence of exosomes (representative exosomes shown by the arrow) inside the cells. B, FACS analysis of DU145 cells incubated for 24 h with PC3-derived exosomes and analyzed for α_vβ₆ integrin surface expression. Neg. Ctrl, negative control. C, IB analysis of lysates (75 μg per lane) of α_vβ₆-negative DU145 and C4-2B cells incubated with α_vβ₆-positive PC3 exosomes using Abs to β₆ or β₁ integrin subunit; CANX was used as loading control for the cell lysates. D, IB analysis of lysates (75 μg per lane) of DU145 cells incubated with or without PC3-derived exosomes, before and after acid wash. E, qRT-PCR analysis of β₆ mRNA levels in DU145 and C4-2B cells incubated with or without PC3-derived exosomes. Results were normalized to the β₆ mRNA levels in PC3 cells and are shown as average ± S.E.

FIGURE 3.

PC3 exosome uptake by DU145 cells enhances adhesion and migration on LAP-TGFβ in an α_vβ₆ integrin-dependent manner. A, adhesion (left) and migration assays (center) of DU145 cells either untreated or treated with 20 μg/ml PC3 exosomes in which β₆ integrin is either expressed (parental and Ctrl.shRNA (Ctrl)) or down-regulated (β₆ shRNA). Left, cells were seeded for 2 h on BSA (1%)-, FN (10 μg/ml)-, or LAP-TGFβ (10 μg/ml)-coated wells, and cell adhesion was quantified by crystal violet staining. Center, cells were seeded on Transwell chambers coated with BSA (1%), FN (10 μg/ml), or LAP-TGFβ (10 μg/ml), and cell migration was evaluated as described under “Experimental Procedures.” Right, IB analysis of β₆ integrin expression in exosomes secreted by PC3 cells [parental (Par.), Ctrl.shRNA (Ctrl), and β₆ shRNA (β₆)]. CD63 was used as marker enriched in exosomes, whereas CANX was used as a marker not found in exosomes. B, migration assay of DU145 cells either untreated or treated with increasing concentrations of PC3 exosomes (5–40 μg/ml). C, left, migration assay of DU145 cells treated with or without 5 μg/ml exosomes secreted by PC3 cells previously incubated either with a non-silencing siRNA (NS siRNA) or with an siRNA specific for β₆ mRNA(β₆ siRNA). Right, IB analysis of β₆ integrin expression in exosomes secreted by PC3 cells. FLOT1 was used as marker enriched in exosomes, whereas CANX was used as a marker not found in exosomes. In A, B, and C, bar graphs show the number of DU145 cells migrated on BSA (white bars), LAP-TGFβ (gray bars), and FN (black bars), expressed as the percentage of total number of cells attached on both filter layers. Values are reported as means ± S.E. *, p ≤ 0.001 as determined by a Student's t test and χ2 tests for adhesion and migration assays, respectively.