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Wang et al. Hepatoma Res 2020;6:61 I http://dx.doi.org/10.20517/2394-5079.2020.55 Page 5 of 15
[41]
antigen, and so on . EpCAM is the most frequently used antigen in CTC recognition, as the only FDA-
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approved semi-automated CTC detection device, CellSearch system, is based on the expression of surface
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EpCAM. CellSearch system utilizes anti-EpCAM-coated magnetic beads for CTC sorting in 7.5 mL
of blood, and the extracted CTCs are then fixed, stained by antibodies against EpCAM and cytokeratin,
and counted. EpCAM and cytokeratin also have been regarded as a clinical standard in CTC labeling
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among other markers [16,42] . CellSearch system can retain morphological and immunological characters
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of isolated cells, thus allowing the following fluorescence-based assays. However, CellSearch system is
incompatible with direct downstream single-cell molecular analysis since these cells have been fixed, which
[43]
limits their clinical utility in CTC-based comprehensive analysis . Fluorescence-activated cell sorting is
another widely used CTC detecting method that combines conventional flow cytometry technique and
immunoaffinity sorting, while this strategy is limited by the makers’ selection [44-46] .

Biophysical properties-based method
The biophysical property-based enrichment utilizes various physical properties including density, size,
[47]
shape, inertia, and electrical property of CTCs to distinguish them from other blood cells . These
so-called “label-free” methods are gaining increasing attention, as they avoid cell loss when choosing
specific antigens targeting CTCs. In addition, unlabeled CTCs are generally compatible with a variety of
downstream analyses .
[48]

Among these strategies, density-based gradient centrifugation is the most commonly used method,
which utilizes the differences in specific densities CTCs to separate the target tumor cells and blood
cells ; this is also a common pre-processing step integrated with many methods for CTC detection.
[49]
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Ficoll-Paque is a cell separation medium used for the isolation of CTCs in patients with various types
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of cancer, including liver cancer and colorectal cancer [39,50] . Oncoquick has made improvements in the
centrifuge tubes that combines filtration and centrifugation and has superior CTC recovery rate and less
blood cell contamination . RossetteSep CTC enrichment cocktail is as an example of label-free CTC
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[51]
enrichment, where a mixture of antibodies is used to target and cross-link unwanted blood cells to form
immunorosettes, and then density gradient centrifugation is performed to deplete the unwanted cells [52,53] .

Another common method that utilizes the biophysical properties of CTC is size-based filtration. The
diameter of tumor cells is generally larger than blood cells, and CTCs would be retained in the filter,
while smaller blood cells pass through. Isolation by size of epithelial tumor cells (ISET) was developed as
[54]
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a microfilter to isolate tumor cells by a polycarbonate membrane with calibrated pores . ISET system
[55]
can visualize and count CTCs and CTC clusters in blood samples obtained from HCC patients . More
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recently, ScreenCell device was developed as an advanced microfilter to isolate viable CTCs with a
[56]
high recovery rate. Immunocytochemistry assays for CTCs can be performed directly on the filter . To
conclude, the filtration method is one of the simplest and most widely studied methods for capturing CTCs.
However, this method is limited by its low specificity that the products may be contaminated by other cells
[34]
owing to natural variation in size of leukocytes, and small CTCs may be lost during the filtration .
Alternatively, microfluidic techniques are now increasingly being exploited in CTC isolation, which allows
for precise control of fluids in a small volume and rapid sample processing at relatively low cost and high
[57]
sensitivity . Microfluidic platforms enable on-chip CTC isolation, identification, and even culturing.
The first microfluidic device named “CTC-chip” was developed to capture rare CTCs in 2007, which
successfully identified CTCs in the peripheral blood of patients with metastatic lung, prostate, pancreatic,
[58]
breast, and colon cancer . In 2010, an improved herringbone-chip was developed. The herringbone-
shaped grooves of this chip can generate a microvortex when blood is pumped, which enhances the contact
between the chip surface and tumor cell. Its clinical utility was demonstrated in specimens from patients
[59]
with prostate cancer . Another novel CTC-iChip platform utilizes the distinct differences between cancer