**1. Introduction**

Cancer is a leading cause of death in many countries [1]. According to World Health Organisation, approximately one in six deaths is attributable to cancer. The development of cancer is a multistage process. Briefly, normal cells undergo transformation into tumour cells, which are defined by various hallmarks including the ability to sustain proliferative signals, evade growth suppressors, promote replicative immortality, avoid cell death and immune destruction, induce angiogenesis and activate invasion and metastasis [2]. This cellular transformation results in

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

uncontrolled proliferation and enables tumour cells to migrate from their primary organ to a distant organ, a process known as metastasis. In 90% of cancer patients, death occurs because of metastasis [3, 4].

cell separation. Furthermore, because of the small space and short flow distance in the microdevice, microfluidics-based technologies consume small amounts of reagents and greatly increase the speed and throughput of blood sample processing, allowing clinical adoption. The use of microfluidics facilitates the integration of downstream molecular characterisation of CTCs, which will enhance our understanding on the complexity of cancer development and enable clinicians to develop better therapeutic strategies to eradicate cancer cells and improve the overall survival of patients. In this chapter, we discuss the technologies for CTC isolation and the use of single-cell analysis in achieving early detection and prognosis of cancer, real-

Profiling Circulating Tumour Cells for Clinical Applications

http://dx.doi.org/10.5772/intechopen.79228

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Many technologies have been developed to enrich and isolate CTCs from the peripheral blood. In most methods, CTCs are separated from the blood cells based on their biological properties and/or physical properties such as size, deformability, density and electric charge. Conventional CTC enrichment systems such as fluorescence activated cell sorters (FACs) have been used to separate CTCs from whole blood based on the expression of cell surface protein markers [25]. Technologies that isolate CTCs based on physical properties also exist. For instance, ISET and ScreenCell use a filtration system to separate the slightly larger CTCs (12–25 μm) from the smaller WBCs (7–15 μm) and RBCs (8 μm) [9]. CTCs and mononuclear cells have a density (<1.077 g/ml) lower than other blood cells (>1.077 g/ml), allowing layered separation of CTCs [6]. CTCs and blood cells exhibit differences in deformability, allowing them to be separated [26]. The dielectric properties of CTCs are different from normal blood cells, allowing separation of CTCs when the cells are subjected to a non-uniform electric field [6, 27]. However, these conventional CTC enrichment methods suffer from limited ability to process large volumes of blood, limited detection sensitivity, inherent losses and poor recovery of viable CTCs, low throughput and insufficient purity due to contamination with WBCs [6].

To overcome these limitations, microfluidics technology offers an alternative platform for isolating CTCs with improved detection sensitivity, high recovery rate, high efficiency and throughput. Each microfluidics platform has its advantages and limitations (**Table 1**). In 2004, US Food and Drug Administration approved the clinical use of CellSearch system (Veridex) for CTC detection in epithelial cancer types such as breast [28], colorectal [29] and prostate cancer [30] for purposes of prognostication. This system uses immunomagnetic and fluorescence imaging technology to enrich and enumerate CTCs from 7.5 ml of whole blood based on the expression of specific proteins in CTCs [31, 32]. CTCs are first separated from other blood cells using magnetic iron nanoparticles coated with antibodies targeted against EpCAM (an epithelial cell adhesion molecule present on the cell surface of CTCs). Subsequently, cells are stained with antibodies targeted against cytokeratin (CK; a protein found in the cytoplasm of CTCs) and CD45 (a cell surface protein found exclusively on WBCs) to differentiate CTCs from contaminating WBCs. DAPI (4′,6-diamidino-2-phenylindole) is also used to stain the nuclei of CTCs and WBCs. Finally, a magnetic field is applied to collect the CTCs, which are identified by positive expression of EpCAM and CK and negative expression of CD45.

time monitoring of cancer therapy and tailoring of personalised treatments.

**2. Technologies for enrichment and isolation of CTCs**

**2.1. Use of microfluidics in CTC enrichment**

According to Cancer Research UK, 46% of patients in England are diagnosed in the advance stages of disease, leading to less effective treatment outcomes. To reduce the number of deaths caused by cancer, detection at an early stage of disease development is critical so that clinical intervention can come in place to improve the chances of survival for cancer patients. The diagnosis of cancer involves multiple tests including tissue biopsy, liquid biopsy, imaging scans, genetic tests and an examination of medical history. Tissue biopsy is regarded as the gold standard for the clinical diagnosis of cancer [5, 6], despite the invasiveness and inconvenience of collecting the biopsy sample.

In recent years, liquid biopsy is increasing being used for the detection of cancer because it only requires a routine draw of blood and is less invasive compared to tissue biopsy, which may also not be repeatedly done safely or feasibly [7]. Liquid biopsy includes the analysis of circulating tumour cells (CTCs), circulating cell-free DNA (cfDNA) or exosomes present in the patient's blood [8]. CTCs, in particular, have garnered much attention for its potential clinical utility. CTCs are cells disseminated from the primary or secondary tumour into the peripheral blood and are associated with the development of metastasis. They are precursors of secondary tumour formation and may carry key information relating to the mechanism of metastasis. They are approximately 12–25 μm [9] and present in extremely low numbers (typically 1–10 CTCs per 10 ml of blood or ~1–100 CTCs per 109 blood cells) [10–12]. The number of CTCs found in the blood varies with the type and stage of cancer and the treatment provided [13]. Typically, patients in the advanced stages of cancers have higher number of CTCs [14]. CTCs of different cellular morphology may exist in the blood and these include the epithelial CTCs, epithelial-to-mesenchymal (EMT) CTCs and mesenchymal CTCs. Moreover, the mutations found in CTCs are often concordant with the primary tumour [15–17], suggesting that the genetic composition of CTCs is similar to the primary tumour. Thus, CTCs has the potential to be used as a 'surrogate' to study the biology of cancer cells.

In cancer treatment, drug resistance is a major concern. The failure of chemotherapeutic drugs to work in patients lies in the heterogeneity and complexity of cancer cells [18, 19]. Cancer stem cells are resistant to chemotherapy and contribute to the intra-tumoural heterogeneity [20]. Therefore, there is a need for molecular profiling of tumour cells at the single-cell level to better address the intra- and inter-cellular differences in cancer cells and enable clinicians to have a better picture of the disease complexity. While paired tumour/normal tissues is the gold standard for molecular analyses of tumour [21], CTCs may provide information on the dynamic changes in tumour cells when blood is extracted at different times, which cannot be achieved in tissue biopsy. In addition, where tissue is not easily accessible, CTCs may provide a diagnostic window.

However, the main challenges of CTC research are that these cells are extremely rare and the population of isolated CTCs may not be pure due to contamination with white blood cells (WBCs). Therefore, highly sensitive and specific technologies are required to isolate CTCs efficiently. Over the past decade, microfluidics technology has greatly advanced the enrichment and isolation of CTCs from whole blood containing red blood cells (RBCs) and WBCs [22]. Microfluidics deals with the behaviour of fluid passing through the microchannels [23, 24]. It makes use of the laminar flow of fluid in the microchannels to manipulate the fluid to achieve cell separation. Furthermore, because of the small space and short flow distance in the microdevice, microfluidics-based technologies consume small amounts of reagents and greatly increase the speed and throughput of blood sample processing, allowing clinical adoption. The use of microfluidics facilitates the integration of downstream molecular characterisation of CTCs, which will enhance our understanding on the complexity of cancer development and enable clinicians to develop better therapeutic strategies to eradicate cancer cells and improve the overall survival of patients. In this chapter, we discuss the technologies for CTC isolation and the use of single-cell analysis in achieving early detection and prognosis of cancer, realtime monitoring of cancer therapy and tailoring of personalised treatments.
