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Panfoli et al. J Cancer Metastasis Treat 2020;6:35 I http://dx.doi.org/10.20517/2394-4722.2020.50 Page 3 of 8

(ILVs) in MVBs [6,35] . Exo acquire their luminal and membrane contents through both direct and stochastic
[6]
sorting . If nascent ILVs are not degraded by fusion with lysosomes, they fuse with the plasma membrane
[36]
and are released into the extracellular space . Once in the circulation, Exo are directed to other cells via
an unclear mechanism, probably involving members of the tetraspannin family (CD9, CD63 and CD81),
where they are taken up by the target tissues through membrane fusion, endocytosis, or receptor-ligand
interaction [2,34,37,38] .

Exo isolation involves a variety of techniques [39,40] . Several commercial exosome isolation kits are
[41]
in use, although their ability to yield pure and functional Exo is uncertain . To date, differential
ultracentrifugation is considered the gold standard, and it is much preferred when pure Exo are required.
Low-speed centrifugation subsequent to polyethylene glycol precipitation has been utilized to enrich
Exo from large sample volumes, but there is the possibility of polymer contamination . The other Exo
[42]
isolation methods are size-based, antibody capture-based, microfluidics-based techniques or precipitation
by altering Exo solubility with water-excluding polymers such as polyethylene glycol, all of which do not
[40]
require high-speed centrifugation . Fluidic techniques such as exosome total isolation chip, can sort Exo
from a heterogeneous population of EVs based on their size, with the advantage of allowing the handling
[43]
of small sample volumes . Label-free efficient separation of Exo from human blood has been reported,
using an integrated continuous-flow acoustofluidic device . This last automated isolation technique is
[44]
[44]
useful in case of biohazard, and for integration with downstream Exo analysis systems . Notably, most
novel isolation methods are limited by the requirement of sophisticated instrumentation or costly reagents.
Therefore, notwithstanding their promising potential, a concern about the clinical applications of Exo
appears to be the standardization of isolation techniques.

THE GOOD SIDE OF EXOSOMES
Exo are emerging as promising and sensitive cancer biomarkers for disease diagnosis [21,23,24,26] . Once released
from cancer cells, Exo enter the circulation and are transported in biological fluids carrying their functional
[1]
cargoes . Profiling Exo cancer-derived nucleic acids by ultrasensitive next-generation sequencing and
proteins by large-scale high-throughput proteomics could allow early cancer detection, therapeutic
stratification and response to therapy monitoring [17,18,38,45,46] .

Liquid biopsy has drawn attention as a minimally invasive and cost-effective method for sampling of
[47]
genetic, proteomic and metabolic material from different types of cancer . While traditional cancer liquid
biopsies utilize cell free DNA or CTC , Exo are emerging as a novel tool in the field of precision medicine
[48]
in the early diagnostic and recurrence assessment applications [24,49] . This has proven true especially for
urologic tumours [29,50] . The advantage of Exo over existing approaches is their stability in body fluids
[1,4]
allowing the specific cancer-derived proteins and nucleic acids to be preserved . For example, a cell
membrane-anchored proteoglycan, glypican-1 (GPC1), overexpressed in a variety of cancers such as breast
and pancreatic cancer, was also identified in the circulating Exo from the serum of pancreatic cancer-
+
bearing patients. Detection of GPC1 Exo in the serum can be utilized to distinguish healthy subjects from
pancreatic cancer patients with very high specificity and sensitivity, findings that can then be correlated with
[51]
survival rates . Considering its cited role in the progression of human pancreatic cancer, miR-301a-3p
[52]
was proposed as a biomarker in the diagnosis of this type of cancer . An Exo-specific “melanoma
[33]
signature” with prognostic and therapeutic potential has been proposed .
[23]
Exo can also become stable and reliable engineered nanocarriers . In fact, the benefits of the use of
Exo as therapeutic agents include their biocompatibility and ability to deliver their content to specific
target cells, an unfavourable feature per se, that can however be of advantage. The characteristic exosomal
membrane stability, which is enriched in cholesterol, sphingomyelin and ceramide, along with its tumour-
targeting capacity, has suggested the use of Exo in anticancer therapy delivering therapeutic miRNAs