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Comparative Study
. 2011 May 10;21(9):779-86.
doi: 10.1016/j.cub.2011.03.043. Epub 2011 Apr 21.

Amphiregulin exosomes increase cancer cell invasion

James N Higginbotham  1 , Michelle Demory Beckler, Jonathan D Gephart, Jeffrey L Franklin, Galina Bogatcheva, Gert-Jan Kremers, David W Piston, Gregory D Ayers, Russell E McConnell, Matthew J Tyska, Robert J Coffey
Affiliations
Comparative Study

Amphiregulin exosomes increase cancer cell invasion

James N Higginbotham et al. Curr Biol. .

Abstract

Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands including EGF, TGF-α (TGFα), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin, and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells 4-fold over TGFα or HB-EGF exosomes and 5-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGFα or HB-EGF. An average of 24 AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between nontransformed and transformed cells is unknown. Exosomes from DLD-1 colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, nontransformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes might contribute to diverse cancer phenomena such as field effect and priming of the metastatic niche.

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Figures

Figure 1
Figure 1
Biochemical analysis of exosomes isolated from MDA-MB-231 breast cancer cells, HCA-7 colon cancer cells and MDCK cells stably expressing EGFR ligands. (A) Extracellular vesicles were purified from MDA-MB-231 conditioned medium by sucrose gradient fractionation (Experimental Procedures). Fractions 1-11 and whole cell lysate (WCL), lane 12, were separated by SDS-PAGE, and equal volumes were immunoblotted with the indicated antibodies. (B) Representative histograms from FAVS analysis of exosomes isolated from HCA-7 cells using the indicated ectodomain antibodies. Horizontal bars represent the gate with the percent of positive exosomes indicated. (C) Relative size of AREG exosomes. Three independent exosome preparations from MDCK cells stably expressing AREG were negatively stained and viewed by TEM. A representative field image (upper) shows vesicles with a smooth, saucer-like morphology characteristic of exosomes. The mean diameter of 300 vesicles was calculated from TEM images. Scale bar represents 250 nm. (D) Topology of endogenous TGFα and heterologous AREG in exosomes. Exosomes from MDA-MB-231 cells (left panel) were subjected to FAVS analysis using antibodies against the ectodomain (top) and cytoplasmic tail (bottom) of TGFα. Exosomes from MDCK cells expressing full-length AREG with a C-terminal HA tag (middle panel) were subjected to FAVS analysis using an AREG ectodomain antibody (top) or an HA antibody (bottom). Horizontal bars represent the gate with the percent of positive exosomes indicated. Right panel, exosomes were isolated from conditioned medium of MDCK cells stably expressing HA-tagged AREG or HB-EGF and immunoblotted with an anti-HA antibody to detect full-length AREG (arrows).
Figure 2
Figure 2
Exosomes from AREG-expressing MDCK cells induce the highest invasive activity in recipient breast cancer cells. (A and B) LM2-4175 cells were incubated for 2 hrs with serum-free medium containing 100 μg/mL BSA (CTL), 100 μg/mL of MDCK-AREG WCL, 100 μg/mL of parental MDCK cell exosomes, TGFα exosomes, HB-EGF exosomes or AREG exosomes. Cells then were plated in a Matrigel-coated Boyden chamber, and the numbers of cells on the bottom of Transwell filters was imaged (A) or quantified (B) after 72 hrs (Experimental Procedures). (B) Data represent the mean +/− the SD. (C) LM2-4175 cells were incubated with exosomes containing 20 ng/mL of AREG as measured by ELISA or 20 ng/mL recombinant human ligands, a similar concentration of AREG found in MDCK exosomes as measured by ELISA. Data represent the mean +/− the SD. (D) LM2-4175 cells were treated with increasing concentrations of AREG exosomes with or without pretreatment with an AREG neutralizing monoclonal antibody, 6R1C2.4 (20 μg/mL). Data represent the mean +/− SD. p < 0.0001 (*).
Figure 3
Figure 3
Compact packaging and rapid internalization of AREG exosomes. (A) Exosomes were isolated from conditioned medium of MDCK cells stably expressing EGFP-tagged AREG. The number of GFP molecules per exosome was determined as described in Experimental Procedures. Each exosome contains approximately 24 AREG molecules. (B) DiD-stained exosomes were purified from wild-type AREG-expressing MDCK cells and incubated with LM2-4175 cells for the indicated times. Flow cytometric analysis was performed as described in Experimental Procedures. Data represent the mean +/− SD. p < 0.005 (**). (C) Exosomes from non-tagged AREG-expressing MDCK cells were incubated with the fluorescent membrane stain DiD. LM2-4175 recipient cells were incubated with DiD-labeled AREG exosomes for the indicated times and uptake visualized by confocal microscopy. Scale bars represent 5 μm. (D) Exosomes from C-terminally EGFP-tagged AREG-expressing MDCK cells were purified and incubated with LM2-4175 cells for 30 min and internalization monitored by GFP fluorescence using confocal microscopy. Two xz planes are shown: apical surface (upper) and mid-cell (lower). Scale bars represent 5 μm. (E) LM2-4175 cells were pretreated in the presence (EGFR mAb) or absence (CTL) of 20 μg/mL EGFR mAb 528 for one hr and then incubated with DiD-stained AREG exosomes for 10 min. Flow cytometric analysis was performed as described in Experimental Procedures. Data represent the mean +/− SD. p<0.0001 (*).
Figure 4
Figure 4
Mutant KRAS colon cancer cells have higher exosomal AREG protein levels, and mutant KRAS exosomes increase invasiveness of recipient cells. (A) Whole cell lysates (WCL) or exosomes were isolated from the indicated cell lines, and the concentration of AREG was determined by ELISA. Data represent the mean +/− SD. (B) AREG Western blots of exosomes or WCL. (C) LM2-4175 cells were pretreated with or without 20 μg/mL of AREG neutralizing antibody, and the previously described invasion assay was performed. Data represent the mean +/− SD. p < 0.001 (*)
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