Isolation of Extracellular Vesicles for Cancer Diagnosis and Functional Studies

Methods Mol Biol. 2019;1882:229-237. doi: 10.1007/978-1-4939-8879-2_21.

Abstract

Extracellular vesicles (EVs) are a diverse category of cellular export products that are present in a variety of biofluids and cell culture media. EVs contain a wide variety of macromolecules that represent a sampling of the cytoplasmic or endosomal compartments and function in cell-to-cell paracrine and endocrine signaling; it has been demonstrated that pathological states such as oxidative stress, transformation, apoptosis, and various cell injuries induce cells to increase their EV release rate, simultaneously altering their composition to reflect the altered state of the cellular origin. Specifically, in patients with solid tumors, EVs are released from cancerous cells at a higher rate than from healthy cells and are enriched in tumor signature molecules. Because of their stability, increased concentration, and unique signatures in cancer patients, EVs have become the subject of investigation for diagnostic and prognostic purposes. Moreover, understanding EVs' biogenesis and biological role could lead to novel insights toward cellular cross talk and complex biological pathways in cancer research. To make use of EVs for diagnostic and mechanistic cancer research, standardized well-characterized methods are required. This chapter provides an overview of two EV isolation techniques and provides detailed instructions on the isolation of EVs by ultracentrifugation, the labor-intensive gold standard, and concentrated polymer precipitation, a faster, higher-yield technique that can be utilized in cancer research.

Keywords: Cancer; Exosomes; Extracellular vesicles; Microvesicles; Polymer precipitation; Tumors; Ultracentrifugation.

MeSH terms

  • Biomarkers, Tumor / analysis*
  • Biomarkers, Tumor / metabolism
  • Cell Culture Techniques / instrumentation
  • Cell Culture Techniques / methods
  • Cell Line, Tumor
  • Centrifugation, Density Gradient / instrumentation
  • Centrifugation, Density Gradient / methods*
  • Chemical Precipitation
  • Extracellular Vesicles / chemistry*
  • Extracellular Vesicles / metabolism
  • Extracellular Vesicles / ultrastructure
  • Flow Cytometry / methods
  • Humans
  • Microscopy, Electron, Transmission / methods
  • Neoplasms / blood
  • Neoplasms / diagnosis*
  • Neoplasms / pathology
  • Neoplasms / urine
  • Polymers / chemistry

Substances

  • Biomarkers, Tumor
  • Polymers