Cargando…

Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs

microRNAs (miRNA) in extracellular vesicles (EVs) have been investigated as potential biomarkers for pancreatic ductal adenocarcinoma (PDAC). However, a mixed population of EVs is often obtained using conventional exosome isolation methods for biomarker development. EVs are derived from different ce...

Descripción completa

Detalles Bibliográficos
Autores principales: Xu, Yi-Fan, Xu, Xiaohui, Bhandari, Kritisha, Gin, Amy, Rao, Chinthalapally V., Morris, Katherine T., Hannafon, Bethany N., Ding, Wei-Qun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8594802/
https://www.ncbi.nlm.nih.gov/pubmed/34784377
http://dx.doi.org/10.1371/journal.pone.0259563
_version_ 1784600059079294976
author Xu, Yi-Fan
Xu, Xiaohui
Bhandari, Kritisha
Gin, Amy
Rao, Chinthalapally V.
Morris, Katherine T.
Hannafon, Bethany N.
Ding, Wei-Qun
author_facet Xu, Yi-Fan
Xu, Xiaohui
Bhandari, Kritisha
Gin, Amy
Rao, Chinthalapally V.
Morris, Katherine T.
Hannafon, Bethany N.
Ding, Wei-Qun
author_sort Xu, Yi-Fan
collection PubMed
description microRNAs (miRNA) in extracellular vesicles (EVs) have been investigated as potential biomarkers for pancreatic ductal adenocarcinoma (PDAC). However, a mixed population of EVs is often obtained using conventional exosome isolation methods for biomarker development. EVs are derived from different cellular processes and present in various sizes, therefore miRNA expression among them is undoubtedly different. We developed a simple protocol utilizing sequential filtration and ultracentrifugation to separate PDAC EVs into three groups, one with an average diameter of more than 220 nm, named operational 3 (OP3); one with average diameters between 100–220 nm, named operational 2 (OP2); and another with average diameters around 100 nm, named operational 1 (OP1)). EVs were isolated from conditioned cell culture media and plasma of human PDAC xenograft mice and early stage PDAC patients, and verified by nanoparticle tracking, western blot, and electronic microscopy. We demonstrate that exosome specific markers are only enriched in the OP1 group. qRT-PCR analysis of miRNA expression in EVs from PDAC cells revealed that expression of miR-196a and miR-1246, two previously identified miRNAs highly enriched in PDAC cell-derived exosomes, is significantly elevated in the OP1 group relative to the other EV groups. This was confirmed using plasma EVs from PDAC xenograft mice and patients with localized PDAC. Our results indicate that OP1 can be utilized for the identification of circulating EV miRNA signatures as potential biomarkers for PDAC.
format Online
Article
Text
id pubmed-8594802
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-85948022021-11-17 Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs Xu, Yi-Fan Xu, Xiaohui Bhandari, Kritisha Gin, Amy Rao, Chinthalapally V. Morris, Katherine T. Hannafon, Bethany N. Ding, Wei-Qun PLoS One Research Article microRNAs (miRNA) in extracellular vesicles (EVs) have been investigated as potential biomarkers for pancreatic ductal adenocarcinoma (PDAC). However, a mixed population of EVs is often obtained using conventional exosome isolation methods for biomarker development. EVs are derived from different cellular processes and present in various sizes, therefore miRNA expression among them is undoubtedly different. We developed a simple protocol utilizing sequential filtration and ultracentrifugation to separate PDAC EVs into three groups, one with an average diameter of more than 220 nm, named operational 3 (OP3); one with average diameters between 100–220 nm, named operational 2 (OP2); and another with average diameters around 100 nm, named operational 1 (OP1)). EVs were isolated from conditioned cell culture media and plasma of human PDAC xenograft mice and early stage PDAC patients, and verified by nanoparticle tracking, western blot, and electronic microscopy. We demonstrate that exosome specific markers are only enriched in the OP1 group. qRT-PCR analysis of miRNA expression in EVs from PDAC cells revealed that expression of miR-196a and miR-1246, two previously identified miRNAs highly enriched in PDAC cell-derived exosomes, is significantly elevated in the OP1 group relative to the other EV groups. This was confirmed using plasma EVs from PDAC xenograft mice and patients with localized PDAC. Our results indicate that OP1 can be utilized for the identification of circulating EV miRNA signatures as potential biomarkers for PDAC. Public Library of Science 2021-11-16 /pmc/articles/PMC8594802/ /pubmed/34784377 http://dx.doi.org/10.1371/journal.pone.0259563 Text en © 2021 Xu et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Xu, Yi-Fan
Xu, Xiaohui
Bhandari, Kritisha
Gin, Amy
Rao, Chinthalapally V.
Morris, Katherine T.
Hannafon, Bethany N.
Ding, Wei-Qun
Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs
title Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs
title_full Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs
title_fullStr Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs
title_full_unstemmed Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs
title_short Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs
title_sort isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: addition of one stringent filtration step improves recovery of specific micrornas
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8594802/
https://www.ncbi.nlm.nih.gov/pubmed/34784377
http://dx.doi.org/10.1371/journal.pone.0259563
work_keys_str_mv AT xuyifan isolationofextracellularvesiclesinthecontextofpancreaticadenocarcinomasadditionofonestringentfiltrationstepimprovesrecoveryofspecificmicrornas
AT xuxiaohui isolationofextracellularvesiclesinthecontextofpancreaticadenocarcinomasadditionofonestringentfiltrationstepimprovesrecoveryofspecificmicrornas
AT bhandarikritisha isolationofextracellularvesiclesinthecontextofpancreaticadenocarcinomasadditionofonestringentfiltrationstepimprovesrecoveryofspecificmicrornas
AT ginamy isolationofextracellularvesiclesinthecontextofpancreaticadenocarcinomasadditionofonestringentfiltrationstepimprovesrecoveryofspecificmicrornas
AT raochinthalapallyv isolationofextracellularvesiclesinthecontextofpancreaticadenocarcinomasadditionofonestringentfiltrationstepimprovesrecoveryofspecificmicrornas
AT morriskatherinet isolationofextracellularvesiclesinthecontextofpancreaticadenocarcinomasadditionofonestringentfiltrationstepimprovesrecoveryofspecificmicrornas
AT hannafonbethanyn isolationofextracellularvesiclesinthecontextofpancreaticadenocarcinomasadditionofonestringentfiltrationstepimprovesrecoveryofspecificmicrornas
AT dingweiqun isolationofextracellularvesiclesinthecontextofpancreaticadenocarcinomasadditionofonestringentfiltrationstepimprovesrecoveryofspecificmicrornas