Abstract

CANCER CELL TARGETING VIA Small EXTRACELLULAR VESICLES

Lucia R. Languino, Professor of Cancer Biology, Thomas Jefferson University

Cancer cells crosstalk with the tumor microenvironment by releasing small extracellular vesicles (sEVs). sEVs, isolated from cancer cell culture media, express the epithelial-specific alphaVbeta6 integrin.  After confirming the fidelity of the sEV preparations by electron microscopy, density gradient, and immunoblotting, we determined that the alphaVbeta6 integrin is actively packaged into sEVs isolated from cancer cells.  sEVs mediate protein transfer of alphaVbeta6 integrin to microvascular endothelial cells and increase the number of their junctions and tubules.  We also show that alphaVbeta6 is transferred from cancer cells to monocytes by sEVs and that sEVs, purified via density gradients, promote M2 polarization.  In addition, as evaluated by our proteomic analysis, alphaVbeta6 down-regulation causes a significant increase in donor cancer cells, and their sEVs, of two molecules that have a tumor suppressive role, STAT1 and MX1/2.  De novo alphaVbeta6 expression in an alphaVbeta6-negative recipient cell is not a result of a change in mRNA levels but is a consequence of sEV-mediated transfer of this integrin.

We have now selected alphaVbeta6, which is expressed on the cell surface in many cancers but absent in normal tissues, as candidate for therapeutic targeting via sEVs.  The benefits of sEV-mediated cancer therapy are: low immunogenicity, ability to infiltrate biological barriers and ‘targetability’.  We demonstrate an efficient strategy to therapeutically target alphaVbeta6 by using short interfering RNA (siRNA) loaded into sEVs.  We first demonstrate that fluorescently labeled siRNAs can be efficiently loaded into sEVs by electroporation.  By confocal microscopy, we show efficient internalization of these siRNA-loaded sEVs into recipient cells.  We then show that sEV-mediated delivery of alphaVbeta6 -targeting siRNA into cancer cells specifically downregulates expression and function of alphaVbeta6.  Overall, this study shows that sEVs from cancer cells may contribute to a horizontal propagation of integrin-associated phenotypes from cancer cells to the tumor microenvironment.