Extracellular vesicles, tumor growth, and the metastatic process

Exosomes

Exosomes range from 30 to 150 nm in diameter and form as intraluminal vesicles (ILVs) packaged within larger multivesicular bodies (MVBs). Fusion of MVBs with the cell plasma membrane results in the release of exosomes into the extracellular space [ ]. MVB biogenesis is regulated by two major mechanisms, endosomal sorting complex required for transport (ESCRT) dependent and ESCRT independent, both of which influence the resulting exosomes.

The ESCRT machinery is used to package ubiquitinated proteins in ILVs. The process is initiated by ESCRT-0 which sorts ubiquitinated proteins in the late endosomal membrane, while ESCRT-I/II and ESCRT-III trigger the involution of the membrane to form the lumen of the MVB within which the ILVs accumulate [ ]. It still remains unclear if the major destination of ESCRT-dependent MVBs is lysosomal degradation, or if fusion with the plasma membrane and extracellular release is a common occurrence. Ubiquitin-independent protein sorting into ILVs also occurs, the regulation of which is linked to an interaction between heparin sulfate proteoglycan syndecan, cytosolic adaptor protein syntenin, and ESCRT-III protein ALIX. Clustering of this protein complex stimulates the loading of CD-63, but not CD-81, flotillin, or CD-9, indicating there are distinct subsets of ESCRT-dependent exosomes [ , ].

MVBs are also formed by ESCRT-independent pathways, namely at sites of lipid raft microdomains. The creation of ceramide by neutral sphingomyelinase 2 (nSMase2) creates a negative curvature of the membrane. This cone-shaped structure allows ILV accumulation into forming MVB [ ], as well as causing differences in the lipid and membrane composition. The potential influence of exosome biogenesis on loading of cargo molecules raises important questions on whether different sorting mechanisms play the most predominant role in determining an exosome's cargo (including proteins and nucleic acids), or whether exosome subpopulations resulting from differing biogenesis is the critical step. Defining the contribution of each factor may be critical to understanding the role of EVs in cancer, determining if it is the preferential loading of a particular cargo composition into all exosomes or an exosome subpopulation released by cancer cells that mediates tumor–microenvironment communication.

Microvesicles

EVs that can directly bud from the plasma membrane are commonly referred to as forming microvesicles (MVs), but are also termed shedding vesicles, ectosomes, shedding bodies, and microparticles. MVs cover a wide range of sizes, spanning 50 nm to 1000 nm . Multiple mechanisms govern the production of EVs, some of which overlap with components of exosome biogenesis. For example, the ESCRT machinery is involved in the production of MVs enriched in cell surface proteins, while nSMase2 as well as being involved in ILV formation, is also involved in ceramide-dependent MV production [ ].

An alternative mechanism, and one frequently seen in aggressive tumors, is nonapoptotic plasma membrane blebbing. The cell surface membrane continuously expands and contracts in focused areas, causing a bleb-like appearance, and contributes to an ameboid-like cell motility [ ]. Rearrangement of the actin cytoskeleton at the focus of these blebs causes their release as MVs [ ]. Many tumor cells are seen to adopt this process of cell motility during migration, indicating MVs may be being secreted in larger numbers during invasion and metastasis.