Large Extracellular Vesicles-A New Frontier of Liquid Biopsy in Oncology

Abstract

Extracellular Vesicles (EVs) are emerging as pivotal elements in cancer. Many studies have focused on the role of Small- (S)-EVs but in recent years Large-(L)-EVs have progressively gained increasing interest due to their peculiar content and functions. Tumor-derived L-EVs carry a lot of oncogenic proteins, nucleic acids and lipids to recipient cells and are involved in the reshaping of the tumor microenvironment as well as in the metabolic rewiring and the promotion of the pro-metastatic attitude of cancer cells. Several techniques have been developed for the isolation of L-EVs and commercial kits are also available for efficient and easy recovery of these vesicles. Also, the improvement in DNA sequencing and "omics sciences" profoundly changed the way to analyze and explore the molecular content of L-EVs, thus providing novel and potentially useful cancer biomarkers. Herein, we review the most recent findings concerning the role of L-EVs in cancer and discuss their possible use in oncology as "liquid biopsy" tools as compared to the other classes of EVs.

Keywords: cancer; exosomes; large extracellular vesicles; microvesicles; oncosomes.

Figure 1

Principal mechanisms involved in the biogenesis of large extracellular vesicles (L-EVs). Many pathways are involved in L-EVs’ generation. (A) The Arrestin Domain-Containing Protein-1 (ARRDC1) induces the relocalization of TSG101 from the endosomal compartment to the plasma membrane, thus provoking shape changes in the cell membrane curvature that initiate the microvesicle gemmation. (B) Similar membrane plasticity modifications depend on the translocation of phosphatidylserine on the outer membrane layer or by the acid sphingomyelinase-mediated formation of ceramide. (C) The ADP-ribosylation factor 6 (ARF6) can influence the incorporation of integrins and the Major Histocompatibility Complexes type I (MHC-I) into the microvesicles. It also recruits the myosin light-chain kinase (MLCK) via ERK, thus initiating the outward budding of the plasma membrane. (D) Parallel, the generation of membrane blebs of 1–10 μm in diameter is mediated by the Diaphanous-related formin-3 (DIAPH3) inactivation, namely a cytoskeletal regulating protein often down-regulated in cancer.

Figure 2

BRAF mutational analysis of L-EV-DNA. Representative BRAFV600E mutational analysis completed on L-EVs isolated by ultracentrifugation from commercial SK-MEL28 melanoma cells. (A) As shown, L-EVs were characterized by flow-cytometry for size in relation to their disposition by FW/SSC scatter as well as expression of HSPA5 and CD81. (B) Following the DNA extraction, we demonstrated by Sanger sequencing the presence of the BRAFV600E mutation in both SK-Mel28 parental cells and relative L-EVs, as compared to BRAF wild type control cells (SK-MEL2). The triplet referred to the V600 position is shaded in blue, while the mutated base is contained in the purple box. (C) Similar findings on SK-MEL28 derived L-EVs were also obtained using the droplet digital-PCR. Blue dots represent the BRAFV^600E droplets, while gray dots represent the BRAF^WT droplets.