INTERROGATING PROTEIN CARGOES OF MDSC-DERIVED EXOSOMES ON THE BASIS OF POST-TRANSLATIONAL MODIFICATIONS

Abstract

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells which accumulate in cancer patients and tumor-bearing mice. Their function in the tumor microenvironment is to inactivate the immune response to cancer by suppressing both the adaptive and innate immune system. Therefore, MDSC are a major obstacle in immunotherapeutic approaches designed to cure cancer. MDSC-derived from tumor bearing mice have been found to shed exosomes. Exosomes are nano-sized vesicles that carry biologically active molecules and play a role in intercellular communication. MDSC-derived exosomes have been reported to mediate the immunosuppressive functions of the parental cells by stimulating the accumulation of MDSC and also by converting macrophages to a tumor-promoting phenotype.

Recent developments in government policy have launched a goal of curing cancer using immune-based therapies (Cancer MoonShot 2020). The understanding of the mechanisms and functions of MDSC immune suppression will be crucial in the success of these therapeutic endeavors. Our current study focuses on interrogating the protein cargo carried by MDSC-derived exosomes based on differential post-translational modifications (PTMs). Post-translational modifications have important roles in functions, signaling, location and interactions of proteins. Selecting proteins based on a specific post-translational modification can aid in the identification of low-abundance proteins which may not be identified in a shotgun proteomics approach.

The first aim of this work was to successfully modify an existing surface chemistry method to use on exosomes. We then used a proteomic strategy to identify glycoproteins on the surface of MDSC-derived exosomes, and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. Furthermore, we also aimed at examining the ubiquitome of the MDSC-derived exosomes, using top-down and bottom-up proteomics. Since inflammation has been reported to enhance the tumor promoting activity of the MDSC, the bottom-up analysis focused on the effects of increased inflammation on the ubiquitination of the protein cargo of MDSC-derived exosomes. Spectral counting was used to estimate differences in abundance of proteins found with ubiquitinated proteoforms in high and basal levels of inflammation. The top-down analysis aimed at characterizing the length and topology of ubiquitin linkages on substrate proteins in MDSC-exosomes.