A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication

Venugopal Thayanithy, Patrick O'Hare, Phillip Wong, Xianda Zhao, Clifford J. Steer, Subbaya Subramanian, Emil Lou

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Background: Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication. Methods: We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps. Results: The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles. Conclusions: This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.

Original languageEnglish (US)
Article number46
JournalCell Communication and Signaling
Volume15
Issue number1
DOIs
StatePublished - Nov 13 2017

Fingerprint

Exosomes
Nanotubes
Assays
Communication
Oncolytic Viruses
Gap Junctions
Viruses
Cell Communication
Nanoparticles
Heparin
Actins
Cells
Pharmaceutical Preparations
Extracellular Vesicles

Keywords

  • Exosomes
  • Extracellular vesicles
  • Intercellular communication
  • Intercellular transfer
  • Membrane nanotubes
  • Microvesicles
  • Transwell assay
  • Tunneling nanotubes

Cite this

A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication. / Thayanithy, Venugopal; O'Hare, Patrick; Wong, Phillip; Zhao, Xianda; Steer, Clifford J.; Subramanian, Subbaya; Lou, Emil.

In: Cell Communication and Signaling, Vol. 15, No. 1, 46, 13.11.2017.

Research output: Contribution to journalArticle

@article{fe4670b3e9494362b1cc340ed478ba14,
title = "A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication",
abstract = "Background: Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication. Methods: We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps. Results: The experimental approach outlined here effectively reduced exosome trafficking by >95{\%}; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles. Conclusions: This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.",
keywords = "Exosomes, Extracellular vesicles, Intercellular communication, Intercellular transfer, Membrane nanotubes, Microvesicles, Transwell assay, Tunneling nanotubes",
author = "Venugopal Thayanithy and Patrick O'Hare and Phillip Wong and Xianda Zhao and Steer, {Clifford J.} and Subbaya Subramanian and Emil Lou",
year = "2017",
month = "11",
day = "13",
doi = "10.1186/s12964-017-0201-2",
language = "English (US)",
volume = "15",
journal = "Cell Communication and Signaling",
issn = "1478-811X",
publisher = "Signal Transduction Society",
number = "1",

}

TY - JOUR

T1 - A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication

AU - Thayanithy, Venugopal

AU - O'Hare, Patrick

AU - Wong, Phillip

AU - Zhao, Xianda

AU - Steer, Clifford J.

AU - Subramanian, Subbaya

AU - Lou, Emil

PY - 2017/11/13

Y1 - 2017/11/13

N2 - Background: Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication. Methods: We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps. Results: The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles. Conclusions: This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.

AB - Background: Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication. Methods: We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps. Results: The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles. Conclusions: This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.

KW - Exosomes

KW - Extracellular vesicles

KW - Intercellular communication

KW - Intercellular transfer

KW - Membrane nanotubes

KW - Microvesicles

KW - Transwell assay

KW - Tunneling nanotubes

UR - http://www.scopus.com/inward/record.url?scp=85034072277&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85034072277&partnerID=8YFLogxK

U2 - 10.1186/s12964-017-0201-2

DO - 10.1186/s12964-017-0201-2

M3 - Article

C2 - 29132390

AN - SCOPUS:85034072277

VL - 15

JO - Cell Communication and Signaling

JF - Cell Communication and Signaling

SN - 1478-811X

IS - 1

M1 - 46

ER -