Richard J SIMPSON | GIR Open Seminar October-15, 2019
Title: Proteomic & Transcriptomic Landscape of Extracellular vesicles (EVs):
functional insights and diagnostic potential

Presentation Summary
Two broad classes of EVs, exosomes and shed microvesicles (sMVs), which differ in size distribution
and protein/RNA profiles have been described. Accruing evidence reveals subtypes within each EV
class. This presentation discusses strategies for isolating and characterising EVs from both cell culture
medium and body fluids.

Abstract
Secretion and exchange of EVs by most cell types is emerging as a central paradigm for intercellularcommunication. Although much is known about EVs there is still lack of definition as to how many
naturally-occurring EV classes/subtypes there are and how their properties and functionalities might
differ. An understanding of this problem is critical if EVs are to be fully harnessed for therapeutic
applications such as regenerative medicine, vaccination against infectious disease, and EV vaccines
for cancer therapeutics.
EVs modulate recipient cell behaviour by transfer of intrinsic cargo constituents such as
oncogenic proteins, cytokines, infectious proteins (amyloid-β proteins, prions, malarial proteins),
miRNAs, mRNAs, DNA, lipids, and metabolites. There is now an increasing awareness that EVs playa critical role in the development of diverse pathologies such as cancer (e.g., pre-metastatic nicheformation), neurodegenerative disorders, and infectious diseases. Collectively, these studies haveengendered significant interest in harvesting EVs for therapeutic applications - leading to several
clinical and pre-clinical investigations of EV-based therapies. Additionally, many EV-containing
proteins (e.g., GPC1, MIF, EGFVIII,) and RNA species (e.g., miRNA signatures) are currently being
investigated for their diagnostic potential for cancer stratification.
To date, two major EV classes have been described: 30-1000 nm diameter microvesicles
referred to as shed microvesicles (sMVs) or membrane blebs) and 30-150 nm diameter EVs, referred
to as exosomes. Accruing evidence reveals subtypes within each EV class. sMVs are generated bydirectly outward budding from the plasma membrane. By contrast, exosome biogenesis involves theearly/late endosomal pathway and the inward budding of multivesicular body (MVB) luminal
membranes to form intraluminal vesicles (ILVs); MVBs then traffic to and fuse with the plasma
membrane whereupon they release their ILV contents into extracellular space (now referred to asexosomes). Given that most EV functional studies are performed using ill-defined EV preparations, it
is very difficult to ascribe function to a specific EV subtype – this has potential ramifications when
designing EV therapeutics such as possible EV-subtype side-effects in clinical investigations.
In this lecture I will provide a brief overview of EV classification, discuss methods used fortheir isolation and characterization, and outline the diagnostic potential of EV-protein biomarkers in
disease. Our recent findings show that human colon cancer-derived EVs have specific protein andRNA (mRNA, splice-variant mRNA, miRNA, and lncRNA) profiles that allow their discrimination.
New advances in the role of exosomes in the tumour microenvironment (especially, exosomedependent tumour-stromal communication), pre- metastatic niche formation, epithelial-mesenchymaltransition (EMT), and mapping of vesicle surface proteins (surfaceome) will be discussed
References:Extracellular vesicles in cancer—implications for future improvements in cancer care R Xu et al., RJ
Simpson 2018 Nature Reviews Clinical Oncology (PMID: 29795272); R Xu et al. RJ Simpson 2016 J
Clinical Investigation (PMID: 27035807)
Proteomic profiling reveals key cancer progression modulators in shed microvesicles released fromisogenic human primary and metastatic colorectal cancer cell lines Suwakulsiri W et al., & Simpson
RJ (2018) BBA -Proteins and proteomics https://doi.org/10.1016/j.bbapap.2018.11.008
Surfaceome of exosomes secreted from the colorectal cancer cell line SW480: Peripheral and integral
membrane proteins analyzed by proteolysis and TX114 R Xu, DW Greening, M Chen, A Rai, H Ji, NTakahashi &, RJ Simpson Proteomics, 2019
Exosomes derived from human primary and metastatic colorectal cancer cells contribute to functional
heterogeneity of activated fibroblasts by reprogramming their proteome A Rai, DW Greening, M
Chen, R Xu, H Ji, RJ Simpson - Proteomics, 2019
Distinct shed microvesicle and exosome microRNA signatures reveal diagnostic markers forcolorectal cancer M Chen, R Xu, A Rai, W Suwakulsiri, K Izumikawa & RJ Simpson PloS one 14(1), e0210003
Understanding extracellular vesicle diversity–current status DW Greening & RJ.
Simpson (2018) Expert Review of Proteomics, 15:11, 887910, DOI: 10.1080/14789450.2018.1537788