Abstract

Unique sEV sub-Populations Derived from alphaVbeta3 Integrin+ Cancer Cells

Cecilia Verrillo, Master's Student, Thomas Jefferson University

It is known that transmembrane cell adhesion receptors, such as integrins, are deregulated in cancer. The alphaVbeta3 integrin is upregulated in many cancers, is a promoter of metastasis, and is found in small extracellular vesicles (sEVs) isolated from prostate cancer (PrCa) cells.  We demonstrate that the exogenous expression of alphaVbeta3 in sEVs isolated from the PrCa cell line C4-2B results in the formation of unique, low density sEVs.  These sEVs were characterized by NTA and western blot (WB) analysis of sEV markers (Alix, TSG101, and the tetraspanins (TSPs) CD9 and CD81) and by the absence of calnexin.  These sEVs also express Kindlin-2 (K2), a positive regulator of alphaVbeta3 activity. Downregulation of K2 via CRISPR/Cas 9 in PC3 PrCa cells shows that endogenous expression of alphaVbeta3 in sEVs is unaffected when compared to CRISPR/Cas 9 control sEVs.  This result indicates that there is an alphaVbeta3 loading mechanism into sEVs that is independent of K2.  We investigated the activity of alphaVbeta3 in PC3 sEVs by utilizing fibrinogen, a molecule that specifically binds to the active conformation of alphaVbeta3.  This binding was visualized using ExoView R100, which is a platform that allows single sEV analysis.  A monoclonal antibody (mAB) to alphaVbeta3 was used to detect alphaVbeta3 levels on the surface of PC3 sEVs.  Our results show that both fibrinogen and the mAB to alphaVbeta3 display similar binding patterns; alphaVbeta3 is mainly present in sEVs captured by the CD9 TSP antibody and is less abundant in sEVs captured by CD63 and CD81 TSP antibodies.  We conclude that alphaVbeta3 may be present in its active conformation in specific CD9-positive populations of sEVs. We finally show, through WB and ExoView analysis, that programmed death-ligand 1 (PD-L1), a protein upregulated in PrCa and key player in the immune evasion of tumor cells, is present in PrCa sEVs.  Since alphaVbeta3 is known to upregulate cellular expression of PD-L1, our data suggest that alphaVbeta3 may influence the levels of PD-L1 in sEVs derived from PrCa cells.