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However, a number of studies have shown that the levels of CTCs estimated by EpCAM-based methods
including CellSearch, is uncorrelated with prognosis in patients with some types of carcinomas. Most of the
evidence attributes this inconsistency to the large degree of heterogeneity in CTCs. Specifically, CTCs might
undergo full (or partial) EMT during dissemination, resulting in several phenotypes including epithelial,
mesenchymal or hybrid (epithelial/mesenchymal) CTCs. These subpopulations of cells may insufficiently
bind to antibodies, thereby evading detection [16,17] . Therefore, a lack of sensitive and specific biomarkers
still hinders the isolation and detection of CTCs. Recent studies provide some probabilities. Here are
some examples of successful markers. Glycan sialyl-Tn (STn) is often associated with cancer metastasis
and expressed in metastatic colorectal and bladder tumors. Neves et al. fabricated a STn affinity-based
[18]
microfluidic device for specifically isolating STn+ CTCs, following an enzyme-based method to recover viable
CTCs for downstream analyses. It showed greatly higher isolation efficiency from the blood of patients with
advanced bladder and colorectal cancers. Plastin3 (PLS3) is expressed in metastatic cancer cells but absent in
normal cells . Similarly, telomerase which is expressed at high levels in almost all the cancer cells, but not
[19]
in normal cells, plays an important role in cancer immortality by replenishing chromosome ends . Green
[20]
fluorescent protein (GFP) fused adenoviral was employed as a probe to target telomerase in cancer cells, and
this strategy was applied to detect and isolate CTCs in Non-Small Cell Lung Cancer (NSCLC) to evaluate
response to radiation therapy and to potentially detect recurrence and progression of disease. Oncofetal
chondroitin sulfate (ofCS) is expressed in both epithelial and mesenchymal tumor cells, as well as the cells
that have undergone EMT, suggesting that it may be an ideal candidate for isolating and analyzing CTCs [21,22] .
Agerbæk et al. employed recombinant VAR2CA (rVAR2) to efficiently target ofCS expressed CTCs from
[23]
patients with hepatic, prostate, pancreatic or lung cancer, allowing for isolation of a larger and more diverse
population of CTCs compared to anti-EpCAM-antibody approaches. More recently, Ding et al. detected
[24]
Folate receptor (FR) positive CTCs in peripheral blood from 200 patients with lung adenocarcinoma, and
further determined that FR+ CTC number could be used for screening solitary pulmonary nodules (SPNs) in
patients and diagnosing early-stage lung cancer with sensitivity of 70.2% and specificity of 79.3%. Meanwhile,
more specific biomarker for specific subgroup of CTCs is of interest. Cyclooxygenase-2 (COX-2) has been
implicated in transforming growth factor-β (TGF-β1) mediated EMT progress and has a higher level of
[25]
expression in subpopulations of mesenchymal CTCs correlated with distant metastases . These results
[26]
suggested FR might be a novel biomarker for isolation and therapy targets. A subpopulation of tumor cells
can express cluster of differentiation 146 (CD146) during EMT process, during which EpCAM expression
is reduced. Therefore, Huang et al. [Figure 2B] designed dual antibodies (anti-EpCAM and anti-CD146)
[27]
and biodegradable gelatin nanoparticle-coated microbeads to improve the capture of mesenchymal CTCs,
achieving high efficiency ( > 80%) and high cell viability (92.5%).

All aforementioned ex vivo detection systems require substantial quantities of blood. The GILUPI
CellCollector (NANOMEDIZIN), approved by Conformite Europeenne in 2012, is another commercial
EpCAM positive based selection device and is the first developed in vivo CTC isolation system to overcome
the limitations of blood sample volume [28-30] .

Except those EpCAM-based positive selection, CD45 negative selection is applied to deplete the CD45+ cells,
mostly using RosetteSep system (Stem Cell Technology, Vancouver), and to analyze the EpCAM-negative
CTCs in combination with EPISPOT (Epithelial Immunospot assay, France). Ramirez et al. first evaluated
[31]
the EPISPOT assay on a large cohort of metastatic breast cancer patients with a positive rate of 59% compared
with the 48% positive rate using CellSearch, demonstrating its clinical prognostic relevance.

Microfluidic and nanotechnology-based CTC devices
Microfluidic devices enable efficient processing of complex blood samples with minimal damage to target cells.
Owing to the synergistic benefits of the microfluidic devices and immunomagnetic separation, microchip-
based immunomagnetic technologies are also commonly used for CTC detection. The most representative