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INTRODUCTION
The main cause of cancer-related mortality is cancer metastasis. During this greatly complicated and multi-
stage disseminative process, tumor cells (the seeds) detach from primary roots, shed into blood and lymph
circulation, undergo the immune attack and shear stress, travel to preferable metastasis soil, and eventually
seed and proliferate to develop metastases. On their way to the potential organs, these circulating tumor
cells (CTCs) undergo epithelial-mesenchymal transition (EMT) , thereby resulting in enhanced motility
[1]
and migratory ability that facilitates vasculature invasion. Upon reaching a suitable niche, the CTCs undergo
mesenchymal-epithelial transition (MET), subsequently reacquiring the stem cell properties and reactivating
[2]
proliferative capability to colonize at metastatic sites . In order to prevent and surveil the development of
metastasis disease, especially metastasic carcinoma, the detection and characterization of CTCs are of great
interest to scientists. CTCs were first detected in cancer patient in 1869 by Australian physician named
Thomas Ashworth. In the past couple of decades, numerous studies have suggested that the presence of
CTCs in the blood of cancer patients has the clinical potential as a noninvasive diagnosis marker and a
prognosis indicator known as a “liquid biopsy” to replace traditional invasive biopsy, whilst also facilitating
technical advances for detection of CTCs.

CTC analysis has a variety of clinical applications, including real-time non-invasive monitoring of CTCs
as biomarkers for new cancer therapies as well as identifying new potential therapeutic targets that directly
inhibit cancer metastasis. Although the potential applications of CTC analysis appear to be very promising,
due to the rarity (one CTC per billion hematologic cells) and heterogeneity (e.g., differences in morphology
and gene expression) of CTCs in the blood of cancer patients, there are few commercially available techniques
for clinical use. High sensitivity and specificity of CTC detection methods thus have a great impact on
improving patient outcomes. Therefore, currently available technologies for CTC detection have become
increasingly more sensitive and reliable, with the goal of early cancer detection and thus successful cancer
treatment. An important new direction in this field is the development of devices and materials that provide
information beyond CTC enumeration. Integrated devices allow for the separation of heterogeneous CTCs
to facilitate a more in-depth characterization of these cells (e.g., phenotypic and molecular profiling) to
develop a personalized treatment plan. Nanomaterials and microfluidic-based nanotechnologies may be the
most promising strategies for implementing ideal CTC capture devices to replace traditional tools, primarily
relying on their small size, high throughput capacity and large surface-to-volume ratio to solve the problem
[3]
of CTC heterogeneity . In this review, we will provide an overview of current CTC isolation strategies and
molecular characterization with brief insights into the potential clinical implications of CTC capture and
characterization.

SENSITIVE CTC ISOLATION METHODS
CTCs may have the potential to predict the disease progression in patients with early-stage or advanced cancer,
even before the formation of primary tumor. However, the extreme rarity of CTCs in blood poses a challenge
for detecting CTCs from blood; for example, one study indicated that only 1.43% of 350 metastasis cancer
patients had ≥ 500 CTCs/7.5 mL blood . Inability to draw large volume of blood from patients highlights the
[4]
need for improved CTC isolation methods to achieve sensitive and specific CTC detection in small sample
volumes. CTC isolation methods have been developed based on either biological (surface antigen, cytoplasmic
protein, invasion capacity, et al.) or physical (size, density, deformability and charge, et al.) properties of tumor
cells. We discuss the most popular technologies and latest advances in the following sections [Figure 1].

Detection of CTCs based on their biological properties
Immunomagnetic beads-based isolation
The most widely used enrichment method is a positive selection method based on the epithelial cell
adhesion molecule (EpCAM) antibodies . So far, CellSearch System (Menarini Silicon Biosystems, Italy)
[5-7]
is the first and also the only one being up to the standard of US Food and Drug Administration (FDA),