MCF-7 Extracellular Vesicle Characterisation and Hydrogen Peroxide Dependent Vesicle Release

Dash, Ayusman (2016) MCF-7 Extracellular Vesicle Characterisation and Hydrogen Peroxide Dependent Vesicle Release. Masters thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Cells in a multicellular organism maintain a state of dynamic interaction. Cellular level interaction is achieved by employing various kinds of intercellular communication. One of the ways by which cells mediate this is by shedding membranous vesicles. Based on their origin, from endosomes or the plasma membrane, these vesicles are named exosomes and microvesicles, respectively, and are broadly classified as extracellular vesicles (EVs). EVs act as vehicles carrying lipids and proteins and are also known to transfer miRNAs, and thereby modulate gene expression. As they constitute the naturally targeting vesicles released by cells, EVs can be engineered to ferry drugs and/or photosensitizers to desired locations for therapeutic and imaging purposes, with minimal off-target side effects. Unlike liposomes and polymeric nanoparticles, theranostic EVs are completely physiological, biocompatible, capable of carrying multiple therapeutic payloads and inherently targeted towards specific tissues, thus obviating the need for exogenous targeting molecules on the surface. The study is aimed at characterising EVs released by MCF-7 (breast cancer) cells and the effect hydrogen peroxide (H₂O₂) has on EV release. This is based on the hypothesis that H₂O₂ causes extensive oxidation of membrane lipids, membrane proteins and cytosolic proteins which destabilises cytoskeleton. As EV shedding is attributable to disruption of cytoskeleton, H₂O₂ treatment should enhance EV secretion by MCF-7 cells. EVs, in the size range of 50-300 nm primarily, were detected in fetal bovine serum (FBS) using scanning electron microscopy (SEM). This finding suggests the need to use EV free FBS for culturing cells, which might be achieved by eliminating EVs by ultracentrifugation or by heat inactivation. Subsequently, through flow cytometric analysis fluorescence generated from Annexin V-FITC binding to EV surface was determined, thereby, confirming EV secretion by MCF-7 cells. EV release was found to be proportional to the number of cells seeded. These cells, additionally, responded to H2O2 treatment by generating reactive oxygen species (ROS) in a dose dependent manner, which were detected by fluorescence microscopy using 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) staining. Finally, it was confirmed via flow cytometric analysis that EV secretion was attributable to disruption of cytoskeleton caused by ROS generation upon H2O2 treatment and was proportional to the concentration of H₂O₂ used. This study, thus, presents H2O2 as one of the factors responsible for promoting EV secretion.

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