Expulsion of iron-rich ferritin via CD63-mediated exosome drives ferroptosis resistance in ovarian cancer cells

Anna Martina Battaglia ¹ Alessandro Sacco ¹ Emanuele Giorgio ¹ Lavinia Petriaggi ¹ Julia Elzanowska ² Ana Rita Cruz ² Luis Rocha ² Catarina Esteves Pereira ² Maria Carolina Strano Moraes ² Bruno Costa-Silva ^2* Flavia Biamonte ^1*

• ¹ Department of Clinical and Experimental Medicine, Magna Græcia University of Catanzaro, Catanzaro, Italy
• ² Champalimaud Research, Champalimaud Centre for the Unknown, Champalimaud Foundation, Lisbon, Portugal

Ferroptosis is a promising new target for ovarian cancer (OVCA) treatment. However, some OVCA cell types resist the induction of ferroptosis by limiting the intracellular accumulation of the labile iron pool (LIP). In this study, we demonstrate that erastin treatment (8µM, 8h) is accompanied by the release of iron-rich ferritin via exosome (EV) pathway in COV318 and PEO4 OVCA cells, thus failing to exert cytotoxic effects. Mechanistically, erastin causes the upregulation of CD63, a tetraspanin involved in forming multivesicular bodies (MVBs) and EVs, and the increase of MBVs assessed by transmission electron microscopy. Consistent with these findings, EV isolation followed by nanoparticle tracking analysis revealed a significant increase in EVs/cell in erastin-treated COV318 and PEO4 cells. Notably, EVs harvested from these cells contained CD63 and ferritin heavy chain (FtH), a major iron-storage protein. Inhibition of EV biogenesis with GW4869 prevented FtH release and restored LIP accumulation, lipid peroxidation, and ferroptosis sensitivity in COV318 and PEO4 cells. Overall, our results indicate that OVCA cells can utilize CD63 + EVs to secrete iron-rich ferritin as a mechanism to evade erastin-induced ferroptosis. These findings suggest that combining erastin with EV inhibitors could offer promising strategy for overcoming ferroptosis resistance in OVCA.