Extracellular vesicles (EVs) are released by nearly all cell types as part of normal cell physiology, transporting biological cargo, including nucleic acids and proteins, across the cell membrane. In pathological states such as cancer, EV-derived cargo may mirror the altered state of the cell of origin. Exosomes are the smaller, 50–150 nanometer-sized EVs released from fusion of multivesicular endosomes with the plasma membrane. Exosomes play important roles in cell-cell communication and participate in multiple cancer processes, including invasion and metastasis. Therefore, proteomic analysis of exosomes is a promising approach to discover potential cancer biomarkers, even though it is still at an early stage.
Researchers from the Pacific Northwest National Laboratory review the advances in exosome isolation methods and their compatibility with mass spectrometry (MS)-based proteomic analysis, as well as studies of exosomes in pathogenesis and progression of prostate and bladder cancer, two common urologic cancers whose incidence rates continue to rise annually. As urological tumors, both urine and blood samples are feasible for noninvasive or minimally invasive analysis. A better understanding of the biological cargo and functions of exosomes via high-throughput proteomics will help provide new insights into complex alterations in cancer and provide potential therapeutic targets and personalized treatment for patients.
The common methods for isolating exosomes
Blue particles: exosomes; orange particles: microvesicles and other debris; green particles: large debris. (A) Ultracentrifugation separation is based on size, and large-size debris and microvesicles are collected earlier at the bottom of the tube and at lower g forces than the smaller exosomes. (B) Density gradient centrifugation separation is based on density. Exosomes will travel to their equilibrium density in the centrifugation media. (C) Size exclusion chromatography uses a porous matrix (dotted circles) that separates based on size. Soluble components and particles smaller than pore size enter the porous matrix temporarily hence elute later than the particles larger than the pore size. (D) In ultrafiltration, soluble proteins and particles smaller than the molecular weight cutoff of the filter (e.g., 105 kDa) are centrifugated through the filter, and the exosomes are collected in the fluid retained by the filter. (E) In affinity isolation, exosomes are captured based on their immunophenotype or specific ligands on the surface. Exosomes are often captured using a monoclonal antibody or exosome-specific ligand conjugated to magnetic nanoparticles and captured by magnets. (F) In precipitation, the addition of a precipitating agent induces clumping of exosomes and the clumps can be collected by centrifugation. While performing method (A,C,D,F) large debris needs to be separated (e.g., by low-speed centrifugation) and discarded before exosome isolation.