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

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

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 inconve-

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

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

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 addi-

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

blood cells) [10–12]. The

(typically 1–10 CTCs per 10 ml of blood or ~1–100 CTCs per 109

potential to be used as a 'surrogate' to study the biology of cancer cells.

tion, where tissue is not easily accessible, CTCs may provide a diagnostic window.

metastasis [3, 4].

14 Liquid Biopsy

nience of collecting the biopsy sample.

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].

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

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.


**System Separation principle Strengths Weaknesses References**

• Cells remain intact and viable after isolation

• Unable to capture tumour cells that are smaller or similar in size to blood cells. • Unable to recover tumour cells with 100% purity

Profiling Circulating Tumour Cells for Clinical Applications

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

• Limited number of cell chambers for imaging • Unable to capture tumour cells that are smaller or similar in size to blood cells.

• Unable to recover tumour cells with 100% purity • Low CTC capture efficiency • Unable to capture tumour cells that are smaller or similar in size to blood cells.

• Unable to recover tumour cells with 100% purity

• Limited throughput • Large amount of sample loses

[11, 40]

17

[16, 17]

[57]

[35]

[58, 59]

• Has the potential to capture CTCs from various

(7.5 ml of blood in 3 h) • Allows single-cell isolation

• Able to isolate viable CTCs with 100% purity • Has the potential to capture CTCs from various

cancer types.

cancer types.

• Cost-effective

cancer types.

• Able to isolate viable CTCs with 100% purity • Has the potential to capture CTCs from various

cancer types.

Abbreviations used are: CTC, circulating tumour cell; EpCAM, epithelial cell adhesion molecule; CK, cytokeratin

isolation

• Cells remain intact and viable after isolation

• Has the potential to capture CTCs from various

(7.5 ml of blood in 20 min) • Cells remain intact and viable after isolation • Has the potential to capture CTCs from various

(3 ml/h)

• Cost-effective

cancer types.

Size-based • Fast processing time

Size-based • Fast processing time

Vortex Size-based • Fast processing time

Label-free capture ClearCell FX (spiral chip)

Microfluidic biochip

Microfluidic device for deformabilitybased cell classification

proteins.

Size and

deformability-based

DEPArray Electric charge-based • Allows single-cell

**Table 1.** Comparison of selected microfluidics systems used for enriching CTCs.


**System Separation principle Strengths Weaknesses References**

• Food and Drug administration (FDA)-approved for clinical use

pre-processing • Cells remain intact and viable after isolation

• Fast processing time (8 ml/h)

• Has the potential to capture CTCs from any

> detecting CTCs than CellSearch system

captured cells on a small

• Cells remain intact and viable after isolation • Reduces sample losses with minimal pre-processing

• Higher sensitivity of detecting CTCs than CellSearch system • Device geometry reduces capture of WBCs

area

cancer type. • Cells remain intact and viable after isolation

• Unable to capture tumour cells that lack EpCAM expression • Only applicable to cancers with epithelial origin • Cells are not viable after isolation

• Unable to capture tumour cells that lack EpCAM expression • Only applicable to cancers with epithelial origin • Unable to recover tumour cells with 100% purity

• Unable to capture tumour cells that are smaller or similar in size to blood cells. • Unable to recover tumour cells with 100% purity

• Unable to capture tumour cells that lack EpCAM expression • Only applicable to cancers with epithelial origin

• Unable to capture tumour cells that lack EpCAM expression • Only applicable to cancers with epithelial origin

• Only applicable to prostate cancer, breast cancer, gastric cancer • Unable to recover tumour cells with 100% purity

[28–30]

[33]

[51, 52]

[53]

[54]

[43, 55, 56]

Antibody-based capture

16 Liquid Biopsy

CellSearch Positive selection for

CTC-ichip Size-based separation

beads

GEDI microdevice followed by negative depletion of white blood cells with CD45 and CD66b magnetic

IsoFlux EpCAM-based • Higher sensitivity of

Magnetic Sifter EpCAM-based • Allows rapid imaging of

Prostate-specific membrane antigen (PSMA)/HER2-based

EpCAM, CK8/CK18/ CK19 and negative selection for CD45

CTC-chip EpCAM-based • Sample does not require

Abbreviations used are: CTC, circulating tumour cell; EpCAM, epithelial cell adhesion molecule; CK, cytokeratin proteins.

**Table 1.** Comparison of selected microfluidics systems used for enriching CTCs.

In 2007, a microfluidics chip developed for CTC enrichment and isolation, known as CTC-chip, was introduced [33]. In this system, CTCs are captured as blood flows through the microchannel containing EpCAM antibody-coated microposts [33]. CTCs are captured with ~50% purity and sample processing takes 1–2 ml/h. Following CTC-chip, a broad range of microfluidic devices were generated to isolate CTCs based on physical size, density, deformability [34–41] or antibody-mediated CTC capture in surface functionalised microchannels [33, 42–47].

large population of WBCs [17]. The separation efficiency is high because as few as 1 CTC in 20,000 WBCs can be recovered. Prior to starting the run, the cell suspension is stained with specific fluorophore-conjugated antibodies such as CK or CD45 to facilitate the differentiation of CTCs from WBCs. The device works on the principle of hydrodynamic focusing to restrict cells to flow in a single stream and hold them passively in active control cell chambers that are positioned along the outer curvature of the channel. CTCs and other blood cells flowing through the channel will experience a slight centrifugal force that facilitates their entry into the cell chambers. Because the microfluidics biochip is integrated into a microscope, the cell sitting in the chambers can be observed under the microscope to determine whether it is a CTC or WBC based on size or staining outcome. Each chamber holds 1 cell at a time and each is connected to a control line that can be activated to eject the selected cell back into the main channel and into the collection well. In this manner, the cells isolated in the chambers can be ejected sequentially so that each cell can be recovered individually. To maximise the recovery of rare CTCs, the effluent of each run is recycled back into the device for three times. The device is able to process 7.5–8 ml of blood in 3 h [16], facilitating the enumeration of CTCs in clinical blood samples in a short period of time. This method also allows the collection of

Profiling Circulating Tumour Cells for Clinical Applications

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

19

Given the convenience and ease of obtaining blood samples from cancer patients, CTCs are currently being adopted for clinical practice. This is especially the case when tissue biopsy samples are not readily available. With current technologies, CTCs can be isolated in bulk or individually for studies on disease progression and therapeutic treatment. Single-cell analysis offers several advantages over pooled cell analysis. First, single-cell analysis is able to detect critical driver mutations for drug response that are present in low frequency, which may be masked in pooled cell analysis since the mutations may only be present in a subset of clones. For instance, in stage IV non-small cell lung cancer (NSCLC) patients who developed resistance after tyrosine kinase inhibitor (TKI) treatment, sequencing of single CTC revealed the presence of epidermal growth factor receptor (EGFR) mutation T790 M that confers resistance in a subset of CTCs isolated from each patient (0–3 CTCs carry the mutation among <10 CTCs isolated per patient) [17]. Determining the mutation status of EGFR in NSCLC patients is important for patient stratification and treatment (see Section 3.4). Second, single-cell analysis reveals the heterogeneity in gene mutations and chromosomal copy number aberrations (CNA) among tumour cells while bulk cell analysis would have average out the signals. In metastatic breast cancer, a sequencing analysis of 40 single CTCs from five patients demonstrated heterogeneous mutations in four genes, phosphatidylinositol 3-kinase catalytic subunit alpha (*PIK3CA*), tumour protein P53 (*TP53*), oestrogen receptor 1 (*ESR1*) and *KRAS* [63]. Within the same metastatic breast cancer patient, not all CTCs harbour the particular mutation and different patients harbour different gene mutations [63]. In small cell lung cancer (SCLC) patients, chromosomal CNA profiling of individual CTCs obtained at pre-treatment can predict whether the patient is sensitive or refractory to subsequent chemotherapy at an accuracy of 83.3% [64], facilitating clinical decision-making. Third, single-cell analysis facilitates the study of clonal diversity and mutation evolution over the course of chemotherapy, which is difficult to achieve in bulk cell

single CTCs for downstream molecular profiling.

**3. Clinical applications of CTCs**

Although the affinity binding methods (e.g. CellSearch, CTC-chip) may isolate CTCs of better purity than the physical methods, the strong antibody-antigen interaction in the microdevice may affect the recovery of viable CTCs [11, 48] and hinder subsequent downstream analysis. Additionally, affinity binding methods will lose out on the subpopulation of CTCs that have down-regulated expression of epithelial markers (e.g. EpCAM) such as the mesenchymal CTCs, resulting in an underrepresentation of the actual CTC count in the blood [11, 49]. Therefore, the limitations imposed by affinity binding methods prompt the development of label-free microfluidic devices that can isolate CTCs with increased purity and viability.

To efficiently separate CTCs from the large pool of RBCs and WBCs, a spiral microfluidics chip was introduced in 2013 [11, 50]. The spiral microchannel (500 μm width × 160 μm height) consists of two inlets and two outlets over a length of ~10 cm. Blood (diluted 2–2.5×) is pumped into the outer inlet while sheath fluid is pumped through the inner inlet. The additional sheath fluid in the spiral chip facilitates the Dean migration of large volume of RBCs in a well-controlled manner, thus allowing high haematocrit samples (20–25%) to be processed. In spiral microchannel, CTCs and other blood cells experience Dean drag forces in addition to inertial lift forces and the combined effects cause hydrodynamic focusing of the cells to specific region of the microchannel. The larger CTCs are focused near the inner wall of the channel while smaller WBCs and RBCs are focused along the outer wall, leading to efficient size-based isolation of CTCs [50]. Furthermore, the spiral chip is able to recover >85% spiked cancer cells and deplete almost 100% of WBCs from the blood sample [11, 50]. Trypan blue staining showed that most of the recovered cells (>98%) are viable [11]. Therefore, the spiral chip allows continuous isolation of CTCs in a single step with high sensitivity, recovery and throughput (3 ml of blood can be processed in an hour) [11]. The spiral chip is also designed to have large microchannel dimensions and high flow rate to prevent non-specific binding of CTCs to the walls and eliminate any potential clogging issues [11].

#### **2.2. Microfluidics and single CTC isolation**

Most microfluidics systems isolate CTCs in bulk rather than individually. The bulk analysis of CTCs may mask the presence of key mutations that are critical to disease progression, indicating the need for single-cell analysis. To isolate and study CTCs at the single-cell level, approaches such as micropipette aspiration [60, 61] and laser microdissection [62] have been used to manually select the individual CTCs. However, these methods are laborious and suffer from low throughput, making them less suitable for clinical use. Commercial platforms such as DEPArray (Silicon Biosystems), which rely on the dielectric properties of CTC for single-cell isolation, suffer from large amount of sample losses [58].

To address these limitations, Yeo et al. developed a microfluidics device capable of performing high throughput, selective isolation of individual viable CTCs with 100% purity amidst a large population of WBCs [17]. The separation efficiency is high because as few as 1 CTC in 20,000 WBCs can be recovered. Prior to starting the run, the cell suspension is stained with specific fluorophore-conjugated antibodies such as CK or CD45 to facilitate the differentiation of CTCs from WBCs. The device works on the principle of hydrodynamic focusing to restrict cells to flow in a single stream and hold them passively in active control cell chambers that are positioned along the outer curvature of the channel. CTCs and other blood cells flowing through the channel will experience a slight centrifugal force that facilitates their entry into the cell chambers. Because the microfluidics biochip is integrated into a microscope, the cell sitting in the chambers can be observed under the microscope to determine whether it is a CTC or WBC based on size or staining outcome. Each chamber holds 1 cell at a time and each is connected to a control line that can be activated to eject the selected cell back into the main channel and into the collection well. In this manner, the cells isolated in the chambers can be ejected sequentially so that each cell can be recovered individually. To maximise the recovery of rare CTCs, the effluent of each run is recycled back into the device for three times. The device is able to process 7.5–8 ml of blood in 3 h [16], facilitating the enumeration of CTCs in clinical blood samples in a short period of time. This method also allows the collection of single CTCs for downstream molecular profiling.
