Characterization of Single Extracellular Vesicles by Atomic-Force Microscopy and Fluorescence Microscopy for Cancer Diagnosis

Small extracellular vesicles (sEVs) of sizes between 30 and 200 nm are released from almost all the cells of the human body in the extracellular space (i.e. outside the cell of origin). By traveling through body fluids (blood, urine, saliva, etc.) these nano-sized vesicles transport biological information between distant cells in the form of lipids, proteins, RNAs and DNAs among others. This novel way of cell-to-cell communication interaction has been discovered to be important in the development of several diseases, such as cancer development and metastasis. Hence, the study of sEVs is crucial for the early non-invasive diagnosis and therapy of different forms of cancer (e.g. lung cancer). Although they are nano-objects, sEVs feature a high heterogeneity both in size and in their molecular cargo (e.g. they can contain different types of surface proteins as well as different amounts of those). Consequently, studies aiming at the characterisation of sEVs at the single-vesicle level are very interesting since they can shed light on their biological nature and they can eventually drive their application potential. In this thesis, we present the characterization of sEVs at the single-vesicle level by performing at the same time size-characterization using an atomic-force microscope (AFM) and surface molecular profiling (markers CD63 and CD9) by means of fluorescence microscopy. The results contained in this work relate to sEVs of the human embryonic kidney engineered cell line (HEK293) but could be applied in the future to clinical samples as well. By correlating the size and molecular profile measurements of single sEVs, we were able to study their correlation which can be important from the biomedical point-of-view.