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Page 6 of 15 Wang et al. Hepatoma Res 2020;6:61 I http://dx.doi.org/10.20517/2394-5079.2020.55
[60]
cells and blood cells in size and deformability, reaching a 97% yield of rare tumor cells . The Cluster-
[61]
Chip is a unique 3D microfiltration system, designed specifically to capture CTC clusters . One obvious
advantage of microfluidic chip is that it can isolate CTCs from whole blood in a high-throughput fashion
without complicated initial preparation step, thus decreasing the possibility of destruction and loss of
[62]
CTCs .

Technology developed for detecting CTC in HCC
CTCs in patients with HCC are a highly heterogeneous population; currently, there are no widely accepted
TM
antigens specifically targeting HCC CTCs [27,63] . Although CellSearch system is commonly used in CTC
detection, it is reported that the EpCAM-based CTC-sorting strategy could only identify approximately
10%-35% of the total amount of tumor cells in blood due to the EMT process and heterogeneous CTC
molecular phenotypes [64,65] . Thus, a clinically relevant subset of CTCs may be missed by singular epithelial
markers-based sorting strategies [29,66] . Some research groups have broadened target epitopes to include
[67]
alternative candidates specific to hepatocytes. For example, Xu et al. developed a novel CTC enumeration
system for HCC by taking advantage of a “biotin and anti-biotin antibody” combination, in which
circulating HCC cells were bound by biotinylated asialofetuin, an asialoglycoprotein receptor (ASGPR)
ligand and subsequently magnetically labeled by anti-biotin antibody-coated magnetic beads, followed by
magnetic separation. This system was able to detect CTCs in 69 out of 85 (81%) HCC patients. The same
group used an anti-ASGPR antibody instead of ASGPR ligand with successful CTC detection in 89% of
HCC patients . Glypican-3 (GPC3) is an oncofetal heparan sulfate proteoglycan that is currently used as
[68]
[69]
a pathologic biomarker for HCC diagnosis. It can also be used for the detection of HCC-specific CTCs .
Recently, the novel NanoVelcro CTC assay uses an antibody cocktail targeting the cell-surface markers
ASGPR, GPC3, and EpCAM to detect CTCs. It has been showed that multi-marker capture detected
[70]
greater numbers of CTCs than any individual antibody alone in both cell line and HCC patient samples .

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“Label-free” strategies have also exhibited great utilities in HCC CTC enrichment. RosetteSep and
quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were combined for the optimized
[71]
[72]
CTC detection in HCC patients, yielding a 41.2% positive rate of EpCAM mRNA+ CTCs . Kalinich et al.
integrated a microfluidic chip device and RNA-based digital PCR to detect molecular signatures derived
from HCC CTCs. Using the identified 10 liver-specific transcripts, 9 out of 16 (56%) untreated HCC
TM
cases had detectable CTCs. The CanPatrol CTC analysis platform used a two-step technique including
microfiltration and subsequent RNA in situ hybridization (ISH) assay, to characterize epithelial (EpCAM
and cytokeratin 8/9/19) and mesenchymal (Vimentin and Twist) markers of CTCs from patients with
[73]
HCC . Wan et al. used a labyrinth microfluidic device to detect CTCs in patients with HCC. They
[74]
showed that 71.4% of the HCC patients had CTCs positive for cancer stem cell marker CD44, while 55% of
the patients had the presence of CTM, which was correlated with advanced HCC stage.

EARLY DETECTION AND PREDICTION OF TUMOR PROGRESSION IN HCC
Research focusing on clinical implications of CTCs in HCC patients has flourished over the past decade.
[14]
CTCs have shown great potential for the early diagnosis and prognostication of HCC patients . The
clinical applications of CTC detection in patients with HCC are summarized in Table 2.

Currently, EpCAM CTCs have been extensively investigated in HCC, as immunoaffinity-based CTC
+
+
TM
enrichment techniques such as CellSearch have been widely used to capture EpCAM CTCs. In 2013,
+
TM
[17]
Sun et al. used CellSearch to detect EpCAM CTCs in HCC patients undergoing tumor resection.
+
+
They found that 66.7% of HCC patients had detectable EpCAM CTCs preoperatively; moreover, EpCAM
CTCs ≥ 2 per 7.5 mL of blood was the strongest predictor of HCC recurrence. The prognostic value of
CTCs was retained in patient subgroups with minor recurrence risk by traditional evaluation. They also
found that CTC numbers were significantly correlated to the systemic immune-inflammation index