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DEPArray TM cartridge (Menarini Silicon Biosystems, Italy) that contains an array of electrodes embedded
with detection sensors is based on the same principle for isolating target cells for subsequent analysis.

MOLECULAR CHARACTERIZATION OF CTCS
Not all the CTCs are detectable and not all detected CTCs have the potential for metastases, indicating
that CTC enumeration alone may not be an effective marker of progressive disease. A commonly used
chemotherapy agent, isosfamide, was reported to decrease the number of lung cancer nodules but also increase
CTC frequency in a pre-clinical model of osteosarcoma . Although a large number of clinical trials have
[58]
suggested that CTC presence is associated with poor survival in patients with some metastatic cancers .
[59]
Their characterization, including phenotyping and genotyping, could lead to a better understanding of
heterogeneity of metastatic tumor and further facilitate the management of patients for individualized
treatment.

Protein analysis of CTCs
Most CTC detection assays are compatible with identification systems for numeration and follow-up
characterization. The most common procedure consists of morphological analysis (size, shape, nuclear
cytoplasmic ratio, cell shrinkage), immunohistochemical analysis [Figure 3A], and fluorescence
[4]
immunocytochemistry (ICC). There are various markers that are useful for ICC analysis. For instance, DAPI
(nuclear counterstaining), pan-keratin (positive marker), and CD45 (negative marker) are applied in the
CellSearch system. Fluorescence channels and specific antibodies are now accessible for users to define the
detection of more established markers, for example, epidermal growth factor receptor (EGFR) , androgen
[60]
receptor (AR) , folate receptor (FR) , and several EMT-related markers (such as vimentin) etc., which are
[62]
[61]
not only the identification markers but also therapy-associated targets. Furthermore, the ELISPOT assay
combines cell culture and an immunospot test to quantitatively and qualitatively detect single viable cells
expressing-cancer associated marker proteins.

One of the more reliable analytical approaches is protein profiling of captured CTCs, which can shed light on
the roles of CTCs in tumor metastases and disease progression. At present, proteomic analyses mostly rely
on mass spectrometry and the sensitivity of this method is improved with appropriate sample preparation
and targeted cell enrichment. High-resolution porous layer open tube based liquid chromatograph-mass
spectrometry (PLOT-LC-MS) lead to identification of approximately 4000 proteins of 100-200 MCF-7 cells
with zeptomole detection sensitivity. Recently, Zhu et al. incorporated the nanodroplet processing platform
[63]
(nanoPOTS) with ultrasensitive LC-MS, allowing identification of 1500-3000 proteins from 10-140 cells. He
subsequently combined CD45 negative selection and laser capture microdissection-based purification with
nanoPOTS-LC-MS for studying protein expression of rare or single CTCs, identifying 164 and 607 protein
groups of 1 and 5 spiked LNCaP cells, respectively .
[64]
Because of these analytical advances, a wealth of information regarding disease metastasis and progression
is being discovered. For example, a recent report about profiling of single live CTC protease activity
demonstrated the increased expression of matrix metalloproteases (MMPs) secreted by CTCs relative to
normal cells is capable of triggering proteolytic processes that assist in invasion and immune evasion .
[65]

Gene analysis of CTCs
Fluorescence in situ hybridization (FISH) technology is highly commercial and easily accessible for analysis
of genetic alterations (deletion, insertion, translocation and rearrangement) at the chromosomal level, and
is widely used in CTC identification and characterization. For example, RNA-ISH has been utilized for
detection of onco-miRNA (such as miRNA-21) , while DNA-ISH has been used for quanitifying copy
[66]
number of cancer-related genes (such as HER2/neu gene) . Based on an optimized FISH method, Frithiof
[13]
et al. [Figure 3B] performed a CellSearch-based CTC separation assay to quantitatively measure HER-2
[67]