Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting

Exosomes are small, 40-130 nm secreted extracellular vesicles that recently have become the subject of intense focus as agents of intercellular communication, disease biomarkers and potential vehicles for drug delivery. It is currently unknown whether a cell produces different populations of exosomes with distinct cargo and separable functions. To address this question, high-resolution methods are needed.

Using a commercial flow cytometer and directly labelled fluorescent antibodies, researchers from the Vanderbilt University Medical Center show the feasibility of using fluorescence-activated vesicle sorting (FAVS) to analyse and sort individual exosomes isolated by sequential ultracentrifugation from the conditioned medium of DiFi cells, a human colorectal cancer cell line. EGFR and the exosomal marker, CD9, were detected on individual DiFi exosomes by FAVS; moreover, both markers were identified by high-resolution stochastic optical reconstruction microscopy on individual, approximately 100 nm vesicles from flow-sorted EGFR/CD9 double-positive exosomes. The researchers present evidence that the activation state of EGFR can be assessed in DiFi-derived exosomes using a monoclonal antibody (mAb) that recognizes “conformationally active” EGFR (mAb 806). Using human antigen-specific antibodies, FAVS was able to detect human EGFR and CD9 on exosomes isolated from the plasma of athymic nude mice bearing DiFi tumour xenografts. Multicolour FAVS was used to simultaneously identify CD9, EGFR and an EGFR ligand, amphiregulin (AREG), on human plasma-derived exosomes from 3 normal individuals. These studies demonstrate the feasibility of FAVS to both analyse and sort individual exosomes based on specific cell-surface markers. The researchers propose that FAVS may be a useful tool to monitor EGFR and AREG in circulating exosomes from individuals with colorectal cancer and possibly other solid tumours.

FAVS sorting of DiFi exosomes with subsequent immunoblot
and stochastic optical reconstruction microscopy (STORM) analysis.

exosome rna

(a) Dot plot of fluorescent intensities from FAVS analysis of DiFi exosomes stained with an Alexa-488-labelled CD9 antibody (y-axis) and Alexa-647-labelled CTX antibody for EGFR (x-axis). Percentages of gated populations from 10,000 total events are shown. Red box marks double-positive exosomes and blue box marks double-negative exosomes. (b) Post-sort analysis of double-positive exosomes. (c) Post-sort analysis of double-negative exosomes. (d) Immunoblot of flow-sorted exosomes. Blot was probed for EGFR and the exosomal markers, syntenin-1 and CD81. Equal quantities of protein were loaded in each lane. (e) Double-positive, flow-sorted exosomes were subjected to STORM (Materials and Methods). A representative image of an approximately 80 nm particle positive for EGFR (red) and CD9 (green) is shown.

Higginbotham JN, Zhang Q, Jeppesen DK, Scott AM, Manning HC, Ochieng J, Franklin JL, Coffey RJ. (2016) Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting. J Extracell Vesicles 5:29254. [article]

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