PMID- 27345057
OWN - NLM
STAT- PubMed-not-MEDLINE
DCOM- 20160627
LR  - 20220321
IS  - 2001-3078 (Print)
IS  - 2001-3078 (Electronic)
IS  - 2001-3078 (Linking)
VI  - 5
DP  - 2016
TI  - Identification and characterization of EGF receptor in individual exosomes by 
      fluorescence-activated vesicle sorting.
PG  - 29254
LID - 10.3402/jev.v5.29254 [doi]
AB  - 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.
FAU - Higginbotham, James N
AU  - Higginbotham JN
AD  - Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
FAU - Zhang, Qin
AU  - Zhang Q
AD  - Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
FAU - Jeppesen, Dennis K
AU  - Jeppesen DK
AD  - Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
FAU - Scott, Andrew M
AU  - Scott AM
AD  - Tumour Targeting Laboratory, Olivia Newton-John Cancer Research Institute, 
      Heidelberg, VIC, Australia.
AD  - Department of Molecular Imaging and Therapy, Austin Health, Heidelberg, VIC, 
      Australia.
AD  - School of Cancer Medicine, La Trobe University, Melbourne, VIC, Australia.
FAU - Manning, H Charles
AU  - Manning HC
AD  - Center for Molecular Probes, Vanderbilt University Institute of Imaging Science, 
      Nashville, TN, USA.
AD  - Department of Radiology and Radiological Sciences, Vanderbilt University Medical 
      Center, Nashville, TN, USA.
FAU - Ochieng, Josiah
AU  - Ochieng J
AD  - Departments of Biochemistry and Cancer Biology, Meharry Medical College, 
      Nashville, TN, USA.
FAU - Franklin, Jeffrey L
AU  - Franklin JL
AD  - Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
AD  - Department of Cell and Developmental Biology, Nashville, TN, USA.
AD  - Department of Veterans Affairs Medical Center, Nashville, TN, USA.
FAU - Coffey, Robert J
AU  - Coffey RJ
AD  - Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
AD  - Department of Cell and Developmental Biology, Nashville, TN, USA.
AD  - Department of Veterans Affairs Medical Center, Nashville, TN, USA; 
      robert.coffey@vanderbilt.edu.
LA  - eng
GR  - P50 CA095103/CA/NCI NIH HHS/United States
GR  - R01 CA046413/CA/NCI NIH HHS/United States
GR  - P30 DK058404/DK/NIDDK NIH HHS/United States
GR  - U19 CA179514/CA/NCI NIH HHS/United States
GR  - P30 CA068485/CA/NCI NIH HHS/United States
GR  - R01 CA163563/CA/NCI NIH HHS/United States
PT  - Journal Article
DEP - 20160624
PL  - United States
TA  - J Extracell Vesicles
JT  - Journal of extracellular vesicles
JID - 101610479
PMC - PMC4921784
OTO - NOTNLM
OT  - EGFR
OT  - amphiregulin
OT  - cetuximab
OT  - colorectal cancer
OT  - exosomes
OT  - extracellular vesicles
OT  - flow cytometry
OT  - mAb 806
OT  - xenograft models
EDAT- 2016/06/28 06:00
MHDA- 2016/06/28 06:01
PMCR- 2016/06/24
CRDT- 2016/06/28 06:00
PHST- 2015/07/23 00:00 [received]
PHST- 2016/04/22 00:00 [revised]
PHST- 2016/04/27 00:00 [accepted]
PHST- 2016/06/28 06:00 [entrez]
PHST- 2016/06/28 06:00 [pubmed]
PHST- 2016/06/28 06:01 [medline]
PHST- 2016/06/24 00:00 [pmc-release]
AID - 29254 [pii]
AID - 10.3402/jev.v5.29254 [doi]
PST - epublish
SO  - J Extracell Vesicles. 2016 Jun 24;5:29254. doi: 10.3402/jev.v5.29254. eCollection 
      2016.