Figure 1.

Scheme of the experimental flow for EV production, isolation, characterization, and data analysis. Conditioned medium (CM) isolated after 6 d of 3D cell culture and 2 d of 2D cell culture was used for EV isolation by differential centrifugation. Quality control techniques (NTA, TEM, and imaging flow cytometry) were performed on the isolated EVs prior to next‐generation sequencing of small RNAs and mass spectrometry of proteins. Resulting data were analyzed separately and further integrated for the identification of altered pathways and EV cargo in 3D cell culture conditions. Results were validated using independent samples and technical approaches (quantitative real‐time PCR and Western blotting).