Cell Culture Substrate Variation Alters Extracellular Vesicle Biogenesis Without Affecting Non‐Coding Repeat RNA Profile

ABSTRACT Tissue stiffening is central to disease progression and aggressiveness in pancreatic adenocarcinoma (PDAC). Here we report how the biomechanical characteristics of the cell culture conditions impact the amount, as well as the cargo, of extracellular vesicles (EVs) produced by pancreatic cancer cells. Cells grown on softer matrices (0.5 kPa) release upwards of 40 times more EVs than those grown on stiffer surfaces (8 kPa) and their cargo is more enriched in protein‐coding RNAs related with mitochondrial and cytoskeleton pathways. Moreover, cells grown in a 3D conformation release more EVs than those grown in 2D, with the repeat RNA content being maintained across all different cell culture conditions. Our study provides evidence to support that the mechanical microenvironment of cells influences EV generation, with distinct consequences for coding and non‐coding RNA packaged within the EVs.