Abstract PO-009: Genetically engineered exosomes enable active pancreatic cancer targeting and evading mononuclear phagocytic system
Authors: Liu Shi-He , Creeden Justin , Brunicardi F. Charles , Zhang JianTing 
Published: November 2020

Abstract
Introduction: Exosomes are emerging as promising nanocarriers for genes and drugs; however, major challenges exist including the absence of active tumor targeting and a high proportion of clearance by mononuclear phagocytic system (MPS) in liver and spleen. To overcome these obstacles currently preventing the effective application of nanodelivery technology in the field of cancer biology, we generate a novel exosome that recognizes both pancreatic ductal adenocarcinoma (PDAC) specific cell surface marker avb3 and the signal regulatory protein (SIRPa) on macrophages. Using this novel exosome, we have achieved active PDAC targeting and MPS evasion. Methods: We engineered the CD9, an exosome marker, to display a HA tagged RGD peptide at the E174 site of CD9 (CD9-HA-RGD). We overexpressed CD9-HA-RGD and a Flag tagged CD47 (CD47-Flag) in HEK 293 cells. CD47, a “don’t eat me” protein signal, mediates inhibition of phagocytosis through interacting with SIRPa on macrophages. Exosomes overexpressing CD9-HA-RGD and CD47-Flag were harvested from HEK 293 cell culture media by ultracentrifugation followed by purification with size exclusion chromatography (SEC). Exosomes displaying RGD and CD47 (Exo-RGD/CD47) were characterized in vitro using ELISA, western blot (WB), flow cytometry (FCM), PDAC cell uptake, and macrophage phagocytosis assays. Exo-RGD/CD47 was also evaluated in vivo in patient-derived PDAC cell line (PDCL5) orthotopic xenograft mouse model. Results: RGD and CD47 were demonstrated to be successfully displayed on exosome surface by analysis of HA and Flag expression using ELISA and FCM. Engineered Exo-RGD/CD47 exosomes achieved enhanced cell binding and increased uptake capacities in an avb3 expression dependent manner in PDAC cells. This was confirmed using excess RDG peptide in a competition assay. Control exosomes demonstrated low uptake efficiency by PDAC cells. Exo-RGD/CD47 resulted in 71.5% reduction of macrophage phagocytosis when compared to control exosomes as evaluated by THP1 cells phagocytosis assay. Using DiR stained exosomes, we demonstrated that Exo-RGD/CD47 systemic delivery significantly reduced liver accumulation up to 80.6% and 65.8% after 1h and 6h after injection, respectively, as compared to control group (p&lt;0.05). Similarly, Exo-RGD/CD47 delivery resulted in 70.1% reduction of splenic accumulation as compared to control at 6h after application (p&lt;0.05). Exo-RGD/CD47 systemic delivery also showed significant tumor accumulation in the PDCL5 xenograft tumor model as compared to control (p&lt;0.05). Conclusions: These data demonstrate that CD9 is an editable exosome biomarker by which peptides can be effectively displayed on the surface of an exosome. Displaying RGD and CD47 on the exosome surface results in a novel exosome that enables active PDAC targeting and evasion of phagocytosis by MPS. These findings suggest the newly designed exosome may have a better translational potential in PDAC diagnosis and therapy than traditional exosomes.
Citation Format: Shi-He Liu, Justin Creeden, F. Charles Brunicardi, JianTing Zhang. Genetically engineered exosomes enable active pancreatic cancer targeting and evading mononuclear phagocytic system [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-009.