Substrate stiffness modulates extracellular vesicles’ release in a triple-negative breast cancer model

Aim: Small extracellular vesicles (sEVs, 30 to 200 nm) are increasingly recognized as potent messengers in intercellular communication and have been shown to be involved in several cancer related processes as development, progression, niche preparation and metastasis. Indeed, sEVs can mediate both cell-cell and cell-matrix communication by transferring oncogenic molecules that promote proliferation, migration, invasion, and metastatic spreading. Here we focus on the inter-relation between extracellular matrix and sEVs, highlighting the unexplored aspect of the influence of the ECM stiffness on sEVs release. Methods: Here, we use Triple Negative Breast Cancer (TNBC) cells as a clinically relevant model to study the sEV release from cells plated on substrates at different stiffness. We plated MDA-MB-231 cells on two collagen coated Polydimethylsiloxane (PDMS) substrates at different stiffness (0.2 and 3.6 MPa), comparing them with the glass substrate, and then we isolated sEVs by differential ultracentrifugation. After a careful control of the cell growth conditions (vitality, morphology by immunofluorescence microscopy, stiffness by Atomic Force Microscopy (AFM)) we performed a multi-parametric analysis based on complementary techniques (AFM, Nanoparticle Tracking Analysis, and Asymmetric Flow Field Flow Fractionation with a Multi-Angle Light Scattering detector) for the TNBC-derived sEV characterization. Results: We observe that soft substrates induce TNBC cell softening and rounding. This effect promotes the release of a high number of larger sEVs, Conclusion: Here we show the role of ECM physical properties in the regulation of sEV release in a TNBC model. The molecular mechanisms regulating this effect need further investigation, however our report represents a step towards an improved understanding of ECM-cell-sEVs crosstalk.

SENIGAGLIESI Beatrice;  GEISS Otmar;  VALENTE Stefano;  VONDRACEK Hendrik;  CEFARIN Nicola;  CECCONE Giacomo;  CALZOLAI Luigi;  BALLERINI Laura;  PARISSE Davide;  CASALIS Loredana; 

2024-10-09

OAE PUBLISHING INC.

JRC138626

2767-6641 (online),   

https://www.oaepublish.com/articles/evcna.2024.47,    https://publications.jrc.ec.europa.eu/repository/handle/JRC138626,   

10.20517/evcna.2024.47 (online),