Abstract 2081 Cancer Exosome Biogenesis Occurs Primarily by an Endocytosis-Independent Pathway

Exosomes are small extracellular vesicles (sEVs) that play major roles in cancer biology. Exosomes are though to arise by the formation and exocytosis of endosomal intralumenal vesicles (ILVs). Here we tested this model by interrogating the biogenesis of key exosome marker proteins CD63, CD81, and CD9. CD63 has an endocytosis signal, is constitutively endocytosed, requires syntenin for its exosomal secretion, but is loaded into exosomes at far lower efficiency than the exosome markers CD81 or CD9, which reside at the plasma membrane. We show here that syntenin drives the exosomal secretion of CD63 to levels that approach CD81 and CD9, but does so by selectively blocking CD63 endocytosis, allowing CD63 to accumulate at the plasma membrane. Other endocytosis inhibitors also selectively induced the plasma membrane accumulation of CD63, confirming that syntenin regulates CD63 endocytosis rather than exosome biogenesis per se, and that exosome cargo proteins bud primarily from the plasma membrane. We also investigated the fate of endocytosed exosomal proteins by introducing multiple endocytosis signals onto plasma membrane-associated exosomal proteins. Our results showed that the endocytosis of these cargo proteins reduced their exosomal secretion and triggered their destruction in lysosomes. These results have major implications for how cancer cells exploit the exosome biogenesis pathway and how cancers might be controlled through inhibition of exosome biogenesis. This work was supported by grants from the NIH (UG3 CA241687, R35 HL150807, UG3 TR002878), and institutional funds of Johns Hopkins University.