Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment of most cancer patients. They are a major obstacle to immunotherapy because they suppress both adaptive and innate immune responses. MDSCs collected from tumor-bearing mice release nano-sized vesicles, called exosomes, which carry biologically active molecules and participate in intercellular communication. Exosomes released by MDSC stimulate migration of other MDSC towards the tumor microenvironment and convert macrophages to a tumor-promoting phenotype. Among the proteins identified in MDSC-released exosomes, S100A8 and S100A9 are low-mass, highly abundant, pro-inflammatory mediators already known to contribute directly to the immune suppressive functions of MDSC.

The aim of this work was to successfully interrogate the exosomal intact protein cargo using top-down proteomics, a strategy for protein analysis that has not previously been applied to exosomes of any kind. Several protein forms (proteoforms) were fully

characterized, which is critical as post-translational modifications regulate protein functions, cellular location and protein interactions. Additionally, since the tumor promoting activity of MDSC is enhanced by inflammation, we focused on evaluating the effect of increased inflammation on the proteoforms relative abundance using current top-down label-free quantitation techniques (peak intensities and peak areas), and comparing them to our recently validated spectral counting approach. Using spectral counting we were able to estimate differences in abundances of both S100A8 and S100A9 proteoforms.

Furthermore, it has been previously reported that exosomes can carry micro RNAs and messenger RNAs. In order to investigate if MDSC-derived exosomes also contain RNAs, a collaborative study was carried out entailing the qualitative and quantitative analysis of miRNAs, mRNA and proteins present in MDSC and their exosomes, and evaluate their changes due to heightened inflammation. The MDSC and exosome protein cargo was analysed by bottom-up proteomics in this case, and the RNA cargo by next generation sequencing. A large number of mRNA and miRNA species were found to be carried by MDSC-derived exosomes and, strikingly, their putative functions were associated to MDSC expansion and suppressive function, and cancer development.