Protocol for isolating extracellular vesicles from pancreatic cancer cells for liver metastatic niche research

Abstract

Extracellular vesicles (EVs) are secreted, cell-derived, membrane-bound compartments implicated in various diseases for their ability to influence distant targets and as carriers of biomarkers. Here, we present a protocol for separating EVs from mammalian pancreatic cancer cells and their characterization using western blot and electron microscopy. We then demonstrate how they are utilized to affect tumor development in a murine model of metastatic pancreatic cancer including a method to quantify hepatic tumor burden in histologic samples. For complete details on the use and execution of this protocol, please refer to Dudgeon et al.¹.

Keywords: Cancer; Cell Biology; Molecular Biology.

Graphical abstract

Figure 1

Netrin-1 on the surface of EVs conditions the metastatic niche in the liver to promote pancreatic cancer metastasis Top: schematic depicting EV pretreatment and subsequent splenic injection and resection for metastasis model. Bottom right: representative liver sections, with tumor areas highlighted (red). Bottom left: Students t-test analysis of total percentage tumor area. NTN-WT: met38 cell line wild type netrin-1, NTN-KO: met38 cell line with netrin-1 CRISPR knock-out.

Figure 2

Schematic depiction of EV expected outcomes visualized through nanoparticle tracking, western blot, and transmission electron microscopy