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Characterization and Analysis of Extracellular Matrix in Malignant Brain Tumors and Their Cellular Derivatives

  • Maksim Sinyuk
  • Justin D. Lathia
  • Mariano S. ViapianoEmail author
Protocol
Part of the Neuromethods book series (NM, volume 93)

Abstract

The neural extracellular matrix (ECM) is a key regulator of cellular phenotype in normal and diseased states. Analytical approaches have helped uncover the myriad of roles the ECM plays in a variety of diseases including malignant gliomas, the most prevalent and deadly primary brain tumors. Major components of the glioma ECM include secreted proteoglycans as well as basal lamina proteins such as laminins. Characterization of secreted proteoglycans by protein blotting is an invaluable tool to understand their molecular complexity. To provide complete characterization of these molecules, tissue specimens must be first processed to separate insoluble (structural) from soluble proteoglycans. Here, we describe the procedure to separate subcellular fractions by differential centrifugation and extract chondroitin sulfate proteoglycans. Further treatment of these fractions using glycosidases to remove specific carbohydrates, followed by protein electrophoresis and western blotting, provides rich information about the composition and variability of the glioma matrix. To determine ECM protein localization in vivo, immunofluorescence analysis techniques are required. Here we describe commonly used approaches for protein analysis using fluorescence antibody detection in primary human tumor tissue and patient-derived xenografts. Additionally, the characterization of a cancer stem cell fraction in these tumors has received much attention, and we provide the methodology for the visualization of ECM proteins and carbohydrates by immunofluorescence in three-dimensional tumorspheres. These techniques are particularly relevant to correlate matrix expression and changes with glioma growth and invasion or to follow up potential microenvironmental biomarkers during therapeutic interventions.

Key words

Chondroitin sulfate proteoglycan Chondroitinase Glycosidase Immunoblotting Glycovariants Immunofluorescence Integrins Laminins Tumorspheres Cancer stem cell 

Abbreviations

AP

Alkaline phosphatase

BCIP

5-Bromo-4-chloro-3-indolyl phosphate

CHAPS

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

CNS

Central nervous system

CSPG

Chondroitin sulfate proteoglycan

DTT

Dithiothreitol

ECM

Extracellular matrix

HA

Hyaluronic acid

HABP

Hyaluronic acid-binding protein

NBT

Nitroblue tetrazolium chloride

NEB

New England Biolabs

PBS

Phosphate-buffered saline

PFA

Paraformaldehyde

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Maksim Sinyuk
    • 1
  • Justin D. Lathia
    • 1
    • 2
  • Mariano S. Viapiano
    • 3
    Email author
  1. 1.Department of Cellular and Molecular Medicine, Lerner Research InstituteCleveland ClinicClevelandUSA
  2. 2.Case Comprehensive Cancer CenterClevelandUSA
  3. 3.Department of Neurosurgery, Brigham and Women’s HospitalHarvard Medical SchoolBostonUSA

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