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 exosome...

Full description

Bibliographic Details
Main Authors:	Higginbotham, James N., Zhang, Qin, Jeppesen, Dennis K., Scott, Andrew M., Manning, H. Charles, Ochieng, Josiah, Franklin, Jeffrey L., Coffey, Robert J.
Format:	Online
Language:	English
Published:	Co-Action Publishing 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921784/

id	pubmed-4921784
recordtype	oai_dc
spelling	pubmed-49217842016-07-15 Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting Higginbotham, James N. Zhang, Qin Jeppesen, Dennis K. Scott, Andrew M. Manning, H. Charles Ochieng, Josiah Franklin, Jeffrey L. Coffey, Robert J. Original Research Article 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, we 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. We 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. We 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. Co-Action Publishing 2016-06-24 /pmc/articles/PMC4921784/ /pubmed/27345057 http://dx.doi.org/10.3402/jev.v5.29254 Text en © 2016 James N. Higginbotham et al. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Higginbotham, James N. Zhang, Qin Jeppesen, Dennis K. Scott, Andrew M. Manning, H. Charles Ochieng, Josiah Franklin, Jeffrey L. Coffey, Robert J.
spellingShingle	Higginbotham, James N. Zhang, Qin Jeppesen, Dennis K. Scott, Andrew M. Manning, H. Charles Ochieng, Josiah Franklin, Jeffrey L. Coffey, Robert J. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting
author_facet	Higginbotham, James N. Zhang, Qin Jeppesen, Dennis K. Scott, Andrew M. Manning, H. Charles Ochieng, Josiah Franklin, Jeffrey L. Coffey, Robert J.
author_sort	Higginbotham, James N.
title	Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting
title_short	Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting
title_full	Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting
title_fullStr	Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting
title_full_unstemmed	Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting
title_sort	identification and characterization of egf receptor in individual exosomes by fluorescence-activated vesicle sorting
description	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, we 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. We 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. We 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.
publisher	Co-Action Publishing
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921784/
_version_	1613599878964838400

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

Similar Items