**1. Introduction**

Circulating tumour cells (CTCs) were first discovered by the Australian pathologist Thomas Ashworth in 1869, who described single cells and cell clusters in a patient's blood and proposed a role for CTCs in the metastatic process [1]. Recently, due to improved CTC detection techniques, these cells, together with circulating tumour nucleic acids (ctNA), are emerging as attractive, accessible, non-invasive biopsies to guide the best therapy for a

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patient's cancer. CTC counts are closely related to cancer progression and stage, and there is mounting evidence from studies on prostate-, breast-, colorectal- and other cancers that CTCs have prognostic value (reviewed by Caixeiro et al. [2]).

In essence, CTCs are very rare cells, and usually only between 0 and 30 CTCs can be isolated from a 5–10-ml blood sample of a cancer patient; although for some patients, CTC counts can be considerably higher. Isolation technologies allow enrichment and separation of CTCs from the millions of surrounding blood cells by initial gradient centrifugation or red blood cell lysis followed by further enrichment of CTCs due to their physical properties or by employing antibody-based negative or positive enrichment techniques (reviewed by Yu et al. [3]). Enrichment steps are followed by CTC identification primarily by immunocytostaining. The most common CTC identification pattern relies on positive staining for nucleated cells (4',6 diamidino-2-phenylindole (DAPI) or Hoechst staining) and cytokeratin (CK; positive CTC marker) associated with a lack of CD45 staining (negative CTC marker, expressed on leuco‐ cytes). Advances in single cell analysis technology have contributed to maximise the informa‐ tion that can be gained from CTCs isolated from a single blood sample. Tumour biomarkers such as gene amplification, mutation, rearrangement and expression can be successfully analysed while CTC protein levels can be determined. There are high expectations that CTCbased assays will find utility for clinical testing, guiding therapy and monitoring treatment in the not-too-distant future (reviewed by Becker et al. [4]). However, cancer cells, including CTCs, are extremely heterogeneous, and therefore, isolating a representative range of CTCs remains difficult.

A particular challenge is the capture of CTCs that have undergone epithelial-to-mesenchymal transition (EMT) [5, 6]. EMT and its reverse, the mesenchymal-to-epithelial transition (MET), are reversible phenotypical changes that allow a cell to form either dense epithelial structures with tight interaction to neighbouring epithelial cells or, by undergoing EMT, to loosen interactions with other cells and become more mesenchymal and migratory. The ability to undergo these changes is important for cells during development to allow the migration of cells and the formation of different tissues. Cancer cells that are able to take advantage of these processes and undergo EMT are proposed to be more motile and consequently are more likely to become CTCs by entering the blood stream [7]. Not surprisingly, EMT-phenotype cancer cells are linked to the presence of metastases. Additionally, cancer cells that have undergone EMT tend to be distinctly more resistant to chemo and radiation therapy [8]. Consequently, the detection and analysis of EMT-phenotype CTCs appear necessary to fully harness CTC information about a given cancer and monitor disease evolution; yet, we are still poorly equipped to detect these cells. Currently, most methods to isolate CTCs, and nearly all current approaches to identify CTCs, rely on the presence of epithelial cell markers. CTC isolation predominantly relies on immunomagnetic targeting of the epithelial cell adhesion molecule (EpCAM), but this epithelial glycoprotein diminishes during EMT, thereby compromising the effectiveness of this strategy [5, 6]. The identification of CTCs usually involves immunocytos‐ taining for epithelial proteins of the cytokeratin protein family, which are similarly downre‐ gulated during EMT [9]. Equally problematic is the method of probing for EpCAM, which is frequently used to identify CTCs after size exclusion enrichment [10].

In this chapter, we summarise the current understanding of EMT in CTC formation, detection of EMT markers in CTCs isolated by common methods and their limitations, and new approaches to better isolate and identify EMT-phenotype CTCs (EMT-CTCs). The clinical relevance of detecting EMT-CTCs is also discussed.
