Doctoral defence: Olavi Reinsalu “Cancer-testis antigen MAGE-A4 is incorporated into extracellular vesicles and is exposed to the surface”

On 16 August at 14:15 Olavi Reinsalu will defend his doctoral thesis “Cancer-testis antigen MAGE-A4 is incorporated into extracellular vesicles and is exposed to the surface”.

Supervisor:
Professor Reet Kurg, University of Tartu

Opponent:
Adjunct Professor (Docent) Pia Riitta-Maria Siljander, University of Helsinki (Finland)

Summary
Cancer-testis antigens (CTAs) are proteins that are normally expressed mainly in testis but aberrantly in various tumours. It has been shown that CTAs contribute to tumorigenic processes and, as antigens, these proteins are known to induce anticancer immune responses. MAGE-A4, a known CTA, is a soluble cytoplasmic or nuclear protein with a partially disordered structure. Although its exact cellular function has largely remained elusive, MAGE-A4 has been shown to have tumorigenic and antitumorigenic properties. Extracellular vesicles (EVs) are membrane-bound nanoscale vesicles released into extracellular space by cells. EVs are the mediators of intercellular communication as they carry bioactive cargoes from one cell to another. EVs are essentially involved in most physiological processes and pathological conditions, including cancer. EVs are considered potential candidates for novel anticancer therapy and diagnostic technologies. In this dissertation, MAGE-A4 is incorporated into virus-like particles (VLPs) of Moloney murine leukaemia virus and native EVs released by cells that express it. The artificially induced VLPs and natively emerged EVs expose MAGE-A4 to their outer surface, which is considered a fascinating phenomenon considering the intracellular localization of MAGE-A4. MAGE-A4 was found in EVs of different sizes, indicating its incorporation into different EV subtypes. The MAGE-A4 carrying EVs (MAGE-A4-EVs) were found to be very stable in common storage conditions for at least 21 days, but subjecting them to more than two cycles of freeze-thawing damages the vesicles, as a decrease in the MAGE-A4 concentration was detected. Chemical manipulations of the MAGE-A4-EVs indicated that MAGE-A4 is not bound to the surface of EVs by electrostatic forces but rather by hydrophobic properties. MAGE-A4 was shown to have biochemical properties that allow it to bind to EVs and VLPs in vitro. This means that the cellular pathways for the MAGE-A4 incorporation into vesicles are not entirely essential. MAGE-A4 was also recombined with EGFP and expressed as a fusion protein that was found to be incorporated into the EVs, showing the ability of MAGE-A4 to populate EVs with additional cargo. Moreover, as a fusion protein, MAGE-A4 was still exposed on the outer surface of the vesicles, and it sustained its EV membrane binding ability in vitro conditions. The results indicated that MAGE-A4 might be incorporated into the EVs and VLPs and exposed to their surface by cellular mechanisms or its biochemical properties. The features of MAGE-A4 described in the dissertation make it appealing for EV-based noninvasive cancer diagnostics and anticancer therapeutic applications.