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Jayachandran et al. Hepatoma Res 2018;4:44 I http://dx.doi.org/10.20517/2394-5079.2018.59 Page 5 of 12

and at an advanced clinical stage (stage III/IV) of HCC . Another study found decreased expression of
[40]
serum exosomal miR-638 in HCC patients . High miR-1247-3p in serum exosomal levels correlated with
[41]
lung metastasis, poor overall survival and poor disease-free survival in HCC patients .
[19]
A study identified a panel of miRs including miR-221, miR-191, let-7a, miR-181a, and miR-26a to be an
optimal gene reference set for normalising the expression of liver-specific miRNAs . The serum levels of
[42]
a panel of exosomal miRs including miR-18a, miR-221, miR-222 and miR-224 were significantly higher in
patients with HCC than those with Hepatitis B and cirrhosis . The serum levels of exosomal miR-101, miR-
[43]
106b, miR-122 and miR-195 were lower in patients with HCC than in patients with hepatitis B . Circulating
[43]
miRNAs, miR-939, miR-595, miR-519d and miR-494 could identify cirrhotic patients with HCC. Upon
comparison of serum and tissue miR levels in 14 patients surgically treated for HCC, a correlation between
circulating and tissue levels of miR-519d, miR-494 and miR-21 was found in HCC patients . A whole micro-
[17]
RNAome microarray analysis was applied to explore dysregulated expression of miRNAs in patients with
cirrhosis, early, intermediate and advanced HCC. This study identified exosome-mediated dysregulation of
circulatory miRNAs, miR-519d, miR-21, miR-221 and miR- 1228 . A significant reduction in miR-320a level
[17]
was detected by miRNA sequencing of exosomes derived from cancer-associated stromal fibroblasts (CAFs)
when compared to corresponding paracancer fibroblasts (PAFs) of 6 HCC patients . By using nanoparticle
[44]
tracking analysis, the serum exosome concentration in HCC patients was found to be higher than in
cholangiocarcinoma (CCA) and primary sclerosing cholangitis (PSC) patients . Long noncoding ribonucleic
[30]
acid (lncRNA) X-inactive-specific transcript (Xist) was upregulated in peripheral blood of HCC patients .
[45]
Exosomal protein biomarkers
Furthermore, when the protein content of serum exosomes was characterised in 29 HCC patients and 32
healthy individuals, Galectin-3-binding protein (LG3BP) and polymeric immunoglobulin receptor (PIGR)
was found to be abundant in HCC patients. In particular LG3BP could distinguish patients with HCC from
CCA and PSC patients . Together these studies suggest exosomal miRNAs, lncRNA and proteins may
[30]
serve as novel diagnostic and prognostic biomarkers of HCC.

Exosomes as delivery vehicles for HCC therapeutics
Emerging studies demonstrate the importance of exosomes as potential targets for therapeutic intervention.
Exosomes can be used as biological delivery vehicles for incorporating specific cargo into target cells.
One study used exosomes to horizontally transfer therapeutic miRNAs into HCC cells . A recent study
[46]
demonstrated the inhibitory effects of mesenchymal stem cells on HCC. In this study, rats models of HCC
treated with adipose-derived mesenchymal stem cell (ADMSC) exosomes harboured significantly smaller
tumours and more intratumoural invariant (CD8α+) natural killer T (NKT)-cells and low-grade HCC than the
controls . As ADMSCs produce large amounts of exosomes, these cells are well suited for the mass production
[47]
of exosomes . Another study utilised ADMSCs derived exosomes for miR-122 delivery into HCC xenograft
[48]
models . This study also demonstrated that miR-122 promoted cheomsensitivity of HCC cells . Furthermore,
[49]
[49]
exosomes isolated from human hepatic stellate (LX2) cells were loaded with miR-335-5p and these exosomes
were taken up by HCC cells in vitro and in vivo. This preclinical study showed an inhibition of HCC cell
proliferation and invasion in vitro and also demonstrated HCC tumour shrinkage in vivo upon uptake of
these engineered exosomes . There are several advantages of using exosome-based therapy, as exosomes
[50]
show low immunogenicity, toxicity and are stable in tissue and in circulation. Together, this information
suggests that exosomes have great translational potential as therapeutics or delivery vehicles for targeted
therapy. Therefore, further studies must identify the optimal delivery method of exosomes to HCC patients.

HCC-derived exosome functions in preclinical studies
HCC-derived exosomes have pleiotropic biological functions, including roles in tumour growth, metastasis,
immune response, intercellular communication, and drug resistance. In this section, we will dissect