Harnessing Extracellular Vesicles for Cancer Research: Dual Potential for Diagnostic and Therapeutic Applications

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles secreted by almost all type of cells that are involved in multiple mechanism of cellular communication and reflect the state of parental cells and their physiological and pathological conditions. Over recent years these vesicles have been extensively studied in cancer research as a potential tool to solve different unmet medical needs. Indeed, their unique properties position EVs both as promising biomarker sources for disease detection and as engineerable platforms for targeted drug delivery. This thesis, conducted as a part of my PhD program funded by the European Union, under the PNRR initiative regarding “Smart Lipid Nanocarriers”, explores both complementary roles of EVs. In the first project, we investigate the potential of EVs as biomarkers for pancreatic cancers. Pancreatic Cancer is one of the leading causes of cancer-related death, with unfavorable prognosis due to limited effective treatments and late diagnosis. Few biomarkers exist for non-invasive diagnosis, and those available lack sufficient specificity and sensitivity. Recognizing the promising diagnostic potential of extracellular vesicles, we ought to identify EVs surface protein markers capable of distinguishing cancer patients from healthy individuals. To this end, we first validated a robust EVs isolation method. Following isolation, we performed comprehensive qualitative and quantitative characterization using dynamic light scattering (DLS), transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). Once the isolation protocol was validated, we isolate EVs from thirteen patients representative of the different pancreatic cancer subtypes and analyzed different protein expression using a multiplex bead-based flow cytometry assay. We identify three differently expressed proteins that warrant further investigation explored in future studies. The second project conducted during 6 months visiting period at the Professor Wood’s laboratory at Institute of Developmental and Regenerative Medicine (IDRM), University of Oxford, focused on exploring EVs as therapeutics delivery vehicles. Extracellular Vesicles, being naturally released by cells, represent a low immunogenic, and effectively engineerable system. We transfected cells to produce bioengineered EVs displaying T-cell costimulatory ligands on their surface, including OX40L, CD40L, 4-1BBL, LIGHT, and GITRL. After assessing their in vitro activity, and identifying the lead candidate among these immune stimulatory ligands, we engineered dual-functionalized EVs to target hepatocellular carcinoma specifically by co- expressing an anti-GPC3 single-chain variable fragment (scFv). In conclusion, this thesis aims to explore the versatile applications of extracellular vesicles as natural and potent tools with dual utility: as diagnostic biomarkers for early cancer detection and as engineerable therapeutic platforms for targeted immunotherapy.