Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion

Salvatore Coniglio, Ian Miller, Marc Symons, Jeffrey E. Segall

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5-10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrixcoated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Original languageEnglish (US)
Article numbere53990
JournalJournal of Visualized Experiments
Volume2016
Issue number116
DOIs
StatePublished - Oct 20 2016

Fingerprint

Macrophages
Microglia
Glioblastoma
Coculture Techniques
Assays
polycarbonate
Tumors
Glioma
Brain
Neoplasms
Polycarbonates
Cell culture
Refractory materials
Drug Discovery
Brain Neoplasms
Microscopic examination
Microscopy
Proteins
Communication
Pharmacology

Keywords

  • Cancer research
  • Glioblastoma
  • Glioma
  • Invasion
  • Issue 116
  • Malignancy
  • Microenvironment
  • Microglia

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion. / Coniglio, Salvatore; Miller, Ian; Symons, Marc; Segall, Jeffrey E.

In: Journal of Visualized Experiments, Vol. 2016, No. 116, e53990, 20.10.2016.

Research output: Contribution to journalArticle

@article{77415cf533314e80b6223a9abce14213,
title = "Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion",
abstract = "Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5-10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrixcoated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.",
keywords = "Cancer research, Glioblastoma, Glioma, Invasion, Issue 116, Malignancy, Microenvironment, Microglia",
author = "Salvatore Coniglio and Ian Miller and Marc Symons and Segall, {Jeffrey E.}",
year = "2016",
month = "10",
day = "20",
doi = "10.3791/53990",
language = "English (US)",
volume = "2016",
journal = "Journal of Visualized Experiments",
issn = "1940-087X",
publisher = "MYJoVE Corporation",
number = "116",

}

TY - JOUR

T1 - Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion

AU - Coniglio, Salvatore

AU - Miller, Ian

AU - Symons, Marc

AU - Segall, Jeffrey E.

PY - 2016/10/20

Y1 - 2016/10/20

N2 - Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5-10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrixcoated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

AB - Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5-10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrixcoated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

KW - Cancer research

KW - Glioblastoma

KW - Glioma

KW - Invasion

KW - Issue 116

KW - Malignancy

KW - Microenvironment

KW - Microglia

UR - http://www.scopus.com/inward/record.url?scp=84992520895&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84992520895&partnerID=8YFLogxK

U2 - 10.3791/53990

DO - 10.3791/53990

M3 - Article

C2 - 27805587

AN - SCOPUS:84992520895

VL - 2016

JO - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

SN - 1940-087X

IS - 116

M1 - e53990

ER -