Selective capture of cancer-derived extracellular vesicles
Hugo Markus (TNW-BioEE), Edwin de Jong (TNW-BioEE), Armagan Kocer (TNW-BioEE)
Abstract
Not only the tumor itself, but also its micro-environment has a great influence on cancer progression and therapy resistance. Tumor cells communicate with their surroundings via the release of biological material including extracellular vesicles (EVs), which are small (30 – 1000 nm) lipid-bilayer enclosed vesicles that carry nucleic acids, metabolites, lipids, and proteins. In response to these EVs, stromal cells can become pro-tumorigenic and release factors that stimulate metastasis and development of therapy resistance. In this project, we will unravel the roles of different EV contents with the aim to identify novel biomarkers and therapeutic targets. To analyze EVs from specific cell types in culture, they need to be distinguished from those pre-existing in serum culture supplements or conditioned media. To this end, newly produced macromolecules and phospholipids are metabolically labeled with click-chemistry reactive groups to enable selective visualization, affinity purification, and protein identification. We demonstrate that a cell line of the childhood cancer neuroblastoma accepts several “clickable” substrate analogs, while the cell viability and EV proteome remain largely unaffected. The clickable groups on the surface of EVs facilitate their selective isolation for downstream analyses such as cryogenic electron microscopy and mass spectrometry. This methodology allowed us to map the surface proteome of EVs released by bone marrow metastatic neuroblastoma cells, which can guide future studies that require selective capture of cancer-derived EVs from patient samples.