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Page 6 of 18 Huang et al. J Cancer Metastasis Treat 2019;5:34 I http://dx.doi.org/10.20517/2394-4722.2018.94

microfluidic device based on anti-EpCAM for CTCs isolation is a microscope slide sized CTC-Chip with a
mass of geometrically distributed microposts coated with anti-EpCAM . A 98% viability of captured CTCs
[32]
was reported with minimal preprocessing and low flow stress . Ozkumur et al. developed an automated
[34]
[33]
platform, termed “CTC-iChip”, combining the strengths of microfluidics and the benefits of magnetic-based
cell isolation for single-cell separation. This CTC-iChip was able to detect the EML4-ALK gene fusion in
lung cancer, suggesting that it could be a promising tool for clinical diagnosis. Then Stott et al. developed
[35]
a microvortex-generating herringbone (HB)-Chip for effective capture of CTCs. The micromixer device was
fabricated to enhance the cell-surface interaction. Subsequently, improvement was achieved by employing
nanostructured substrates and chaotic micromixers, increasing the recovery rate up to 95% .
[36]

For the sake of increasing the sensitivity of capturing exceedingly rare CTCs, many efforts have been made
to fabricate nanostructures into the microfluidics to increase the interaction between ligands and cells;
such devices include electropolymerized polymer nanodots , electrospun TiO2 nanofiber , and silicon
[37]
[38]
nanowires [39-41] . More recently, Dong et al. [Figure 2C] utilized a nanotopographical surface ( HGMS),
NS
[42]
based on the CTC isolation technology of anti-EpCAM antibody modified Self-floating hollow glass
microspheres (HGMS), to achieve excellent capture performance (93.6% ± 4.9% efficiency and 30 cells/mL
detection limit in 20 min). A preferable method was based on a combination of advantages of different
approaches. Wu et al. [Figure 2D] tactfully fabricated a 3D bionic cytosensor with electrospun polymers
[43]
(PLGA) nanofibers crosswise stacked on Ni micropillars for better CTC filopodia climbing, subsequently
coupled with immuno-selection by anti-EpCAM quantum dots, demonstrating a sensitive detection range
5
1
and limit of 10 -10 cells/mL and 8 cells/mL, respectively, as well as a recovery range of 93.5%-105%.
However, fabricating these nanoscale substrates is time-consuming. Sheng et al. developed a microfluidic
[44]
device combined with DNA aptamer modified gold nanoparticles (AuNPs) to enhance the capture
performance without elaborate establishment for nanostructure. When compared with aptamer on the
surface alone, the binding efficiency of AuNPs-aptamer showed a 39-fold increase and the capture efficiency
rose from 49% to 92%. In order to profile the dynamic phenotypes of rare CTCs, Poudineh et al. [Figure 2E]
[45]
fabricated a magnetic nanoparticles-enabled ranking cytometry (MagRC) approach to separate and in-line
profile heterogeneous CTCs based on the longitudinal profile of magnetic field gradients. They demonstrated
that this device was capable of profiling CTCs with higher sensitivity at a single-cell resolution in unprocessed
blood from cancer patients compared to other previously developed magnetic sorting techniques. Similarly,
an immunomagnetic nanoparticle-mediated binning and profiling approach [Figure 2F] was developed to
separate CTCs with different phenotypes based on the differential expression of surface markers . The CTC
[46]
subpopulations could be spatially sorted in different compartments of a fluidic chip, providing a powerful
means to sort heterogeneous CTCs and investigate EMT in patient CTCs.

Detection of CTCs based on their physical properties
CTCs undergo cellular processes (EMT, MET, et al.) during dissemination, resulting in a number of
phenotypes. Thus, it is important to determine which CTC fractions possess greater metastatic potential
and/or stronger resistance to immune surveillance and medical treatment. In this case, CTCs would have
better prognostic and therapeutic values. The aforementioned methods depend on specific markers of interest
for isolation; however, subpopulations of CTCs lacking the markers may be unintentionally overlooked.
Therefore, additional methods that could serve as complements to protein markers are urgently needed.

Size-based CTC isolation
Alternative methods that isolate CTCs dependent on physical properties have been developed to replace
or complement the antibody-based isolation methods. Most of the CTCs are believed to be larger than
normal blood cells (leukocytes, erythrocytes). And pores with ≈ 8 μm in diameter have been shown to be
appropriate for CTC detainment.