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Jayachandran et al. Hepatoma Res 2018;4:44 I http://dx.doi.org/10.20517/2394-5079.2018.59 Page 3 of 12

are distinguishable by their size and morphology (cup-shaped appearance) through electron microscopy.
Moreover, exosome surface profiling through flow cytometry and ELISAs allow classification of these
subcellular vesicles to an extent . Fourth, many detection and isolation techniques have been developed
[19]
for exosomes in research and therapy. Many commercial kits are available for high efficiency exosomes
isolation from small amounts of body fluids . Fifth, the lipo-proteinous architecture of exosomes
[10]
also protects the exosomal constituents from degradation. For example, microRNAs (miRs) within the
exosomes are resistant to RNases and are stable in the circulation and may be promising candidates as
novel biomarkers of cancers . Sixth, the routinely used serum HCC markers such as AFP and des-
[20]
gamma-carboxyprothrombin (DCP) are not accurate for the early detection of HCC as they lack adequate
sensitivity and specificity for effective HCC surveillance . Furthermore, several factors unrelated to
[21]
HCC such as obstructive jaundice, vitamin K deficiency, alcohol intake, or warfarin treatment may
elevate the serum DCP levels . A recent study has highlighted that exosomal serum miRs are promising
[22]
biomarkers to improve sensitivity, specificity, early detection and prognostic prediction of HCC . Thus,
[20]
exosome-based diagnostics may improve the detection of early HCC and prove to be more superior to the
frequently used HCC biomarkers such as AFP and DCP.

Finally, exosome-based liquid biopsy is preferred as a robust standalone diagnostic and prognostic method
compared with other liquid biopsy-based biomarkers such as circulating tumor DNA (ctDNA) and
circulating tumor cells (CTCs). Both ctDNA and CTCs have limitations biologically and technically and
appear unsuited for clinical practice at the present moment. ctDNA is a single stranded or double stranded
[23]
DNA, shed by either living or dying tumor into the blood . Clinical use of ctDNA levels alone as cancer
biomarker is currently not recommended as it is not a cancer-specific biomarker and elevated ctDNA levels
have been detected in healthy controls with infections. Moreover, increased ctDNA levels are associated
with pathological conditions unrelated to cancer such as chronic inflammation and autoimmune disease .
[24]
ctDNA are less stable as they have a short half-life .
[25]
CTCs are cancer cells that have detached from tumor tissue and are present in the bloodstream. They have
the potential to seed the cancer to other sites . CTC application is confronted with many challenges. A
[26]
major challenge with CTCs is to obtain tumor cells in adequate numbers for evaluation, as CTCs are rare
in blood (1-10 CTCs per 10 mL). CTCs also lack cancer-specific surface markers, making detection and
[25]
isolation difficult . In summary, the many benefits of exosome-based liquid biopsy application render it a
useful method for both diagnosis and prognosis of cancers including HCC and it also appears promising in
providing a new dimension to personalized cancer care.

ISOLATION AND IDENTIFICATION METHODS OF HCC-DERIVED EXOSOMES
Differential ultracentrifugation is considered the gold standard method for purification of exosomes and
most of the studies have applied this technique for isolating HCC-derived exosomes [27,28] . However, depending
on the starting material and downstream applications, other methods for the purification and enrichment
of HCC-derived exosomes have also been used such as ultrafiltration, size exclusion chromatography (SEC)
or the ExoQuick TC method [19,29] .

Majority of HCC-derived exosomes have been identified by a combination of different methods including,
round or cup shaped morphology by transmission electron microscopy, size of 50-100 nm in diameter by
nanoparticle tracking analysis and exosomal surface profiling for markers such the tetraspanins (CD9,
CD63, and CD81), heat shock proteins (HSP90 and HSP60), Alix and Tsg101 by immunoblot and flow
cytometry [19,20,29-32] . Studies have demonstrated high expression of glypican-3 (GPC-3) and AFP, traditional
markers of HCC, within the HCC-derived exosomes, thereby confirming the hepatoma-based origin of
these exosomes .
[31]