Re-engineering Tumor Cell-Derived Extracellular Vesicles with Exogenous Tags for Downstream Analysis

细胞外囊泡（EVs）是由细胞和载体持续分泌的磷脂和蛋白质结构体

Extracellular vesicles (EVs) are phospholipid and protein constructs continuously secreted by cells and carry parent cell-specific genetic materials, proteins, and lipids, which can be selectively taken up by neighboring or distant target cells far from their release. EVs derived from cells with abnormal conditions such as viral infections, neurodegenerative diseases, and cancer have specific cargos which can be exploited as a potential biomarker for the respective disease condition. Moreover, the reports suggest that tumor EVs play a role as metastatic site surveyors and are crucial to create a favorable microenvironment or niche for metastatic cells. Therefore, in order to elucidate the mode of action of EVs and their constituents, tracking EVs in vitro and in vivo is essential. Currently, EVs are detected by analyzing the endogenous marker proteins such as trans-membrane cluster of differentiation (CD) proteins (CD81, CD63, and CD9), and tumor susceptibility gene 101 protein (TSG101). However, these markers are present in nearly almost all cells at different level which results in analysis heterogeneity. Therefore, the exogenous tag is very important to study realistic cellular interaction and tracking of EVs. Within this background, the major goal of this proposal is to define the importance of reconstruction of EVs with exogenous fluorescent and metal tags as an analytical handle to elucidate its quantitative uptake in vital organs. Recently, the PI demonstrated delivery of water-soluble doxorubicin (DOX) to breast tumor xenografts using natural killer (NK) cell-derived EVs reconstructed liposome. Results showed the therapeutic advantage of reconstructed EVs with a tumor inhibition rate of 80% (free DOX = 65%). Similarly when macrophage-derived EVs reconstructed with liposome, a higher order of colloidal stability and drug loading were observed. Engineered EVs showed the differential targeting and uptake against normal and cancerous cells thereby putting itself in the group of potential tumor-targeted drug delivery candidates. These outstanding findings from re- engineered EVs validated our hypothesis that acquired properties from parent cells navigate to target recipient cell. Lesson learned from re-engineered system, our overarching hypothesis is to incorporate exogenous components such as near-infrared (NIR) dye, macrocyclic metal, and/or radiopharmaceuticals as a tag in EVs for downstream quantitative analysis. The related but independent aims are Aim 1. To re-engineer EVs with exogenous tags and study its physicochemical properties. Aim 2. To elucidate the cellular specificity and biocompatibility of re-engineered EVs. Aim 3. To evaluate the biodistribution of EVs using breast cancer models. We will incorporate exogenous tags into the EVs derived from human breast cancer cells (MCF-7 and MDA-MB- 231) to study its interaction and distribution in human breast cancer xenograft developed in humanized mice. These tags will be detected using state-of-art analytical techniques established in the PI’s lab. The research team anticipates the results will assist EVs system designs in accelerating the diagnosis and therapy of cancer.