Abstract 2001: Engineering extracellular vesicle like liposomes with integrin αVβ5 to study its role in cancer metastasis
Authors: Chen Yunxi , López Rubén R. , Khyat Chaymaa Zouggari Ben El , Tsering Thupten , Nerguizian Vahé , Burnier Julia V. 
Published: April 2023

Abstract
Background: Extracellular vesicles (EVs) are key mediators of cancer metastasis. EV membrane-associated integrins show organotrophic properties by interacting with microenvironmental stromal cells and extracellular matrix. Specifically, integrin αVβ5 is associated with the metastasis of cancers with remarkable liver tropism, including pancreatic cancer, uveal melanoma, and colorectal cancer. Despite the importance of understanding the pathogenic roles of EVs, EV heterogeneity, lengthy isolation, and low yields make it difficult to study the function of EVs at the molecular level. Therefore, we explored engineering EV-like lipid nanoparticles with selected cargo, such as integrin αVβ5, as a tool to study EV biology.
Methods: EV-like liposomes (in terms of size and charge) have been produced by our group by nanoprecipitation. Aqueous solvent (proteins in Milli-Q water) and organic solvent (cholesterol and DMPC in ethanol) were mixed in a 3D-printed microfluidic chip. Recombinant integrin αVβ5 was purchased from R&D System and encapsulated into the liposomes in one step. Liposomes with recombinant GFP protein (Thermo Fisher) and liposomes with cellular proteins isolated from 92.1 and A431 cells were produced as proof of concept. Liposomes were produced with dye SP-DilC18 or labelled by PKH after production. Unencapsulated proteins and unbound dye was removed by ultracentrifugation or size exclusion chromatography (qEV, Izon). Stain-free gel (Biorad), flow cytometry, and Tecan Reader were used to detect encapsulated proteins. Hepatocyte uptake of liposomes was analyzed by Incucyte. The liposomes were characterized by transmission emission microscopy (TEM), nanoparticle tracking analysis, and dynamic light scattering.
Results: Liposomes with and without protein cargo presented a bi-layer lipid membrane by cryoTEM. 92.1 cellular protein, A431 cellular protein, GFP, and integrin αVβ5 encapsulated by liposomes were detected by stain-free gel. Positive signals from GFP encapsulated by liposomes were detected by flow cytometry and Tecan Reader, indicating encapsulated proteins can remain functional. By Incucyte fluorescence microscopy, we analyzed the uptake of liposomes by hepatocytes and found that different sizes and charges affect the uptake efficiency.
Conclusion: In this study, we produced EV-like liposomes with lipid bilayers in a controllable way in terms of size, charges, and functional cargo. We showed that EV-like liposomes could be a novel and easy-to-use model to study the role of specific EV cargo in cancer metastasis.
Citation Format: Yunxi Chen, Rubén R. López, Chaymaa Zouggari Ben El Khyat, Thupten Tsering, Vahé Nerguizian, Julia V. Burnier. Engineering extracellular vesicle like liposomes with integrin αVβ5 to study its role in cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2001.