**2. EMT in CTC formation**

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

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

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].

have prognostic value (reviewed by Caixeiro et al. [2]).

remains difficult.

242 Tumor Metastasis

The role of EMT in the metastatic process has been controversial mainly because cells in metastatic tumours often display epithelial rather than mesenchymal characteristics, despite the presence of cells with mesenchymal features in the primary cancer. The recognition that EMT is a reversible process has led to a model adaptation, which postulates that EMT reversal, termed MET, has to occur after extravasation to allow motile cancer cells to resettle and form metastases [11] (**Figure 1**). Regardless of robust *in vivo* data that show increased metastasis associated with an EMT phenotype in the primary tumour, experimentally tracking EMT or MET in the metastatic process remains challenging [12].

**Figure 1.** EMT in CTC formation. Simplified illustration of cells in a primary tumour undergoing EMT changes, which enable them to disseminate from the primary cancer, intravasate into the blood stream and travel as CTCs before ex‐ travasating the vascular system and, by undergoing MET, regain the ability to form a metastatic tumour.

Several elegant studies and *in vivo* evidence (mainly from mouse models) show that EMT aids tumour cell dissemination and promotes intravasation into the vascular system (CTC forma‐ tion). MDA-MB-468 breast cancer cells that can be driven to undergo EMT by epidermal growth factor (EGF) exposure were used in a severely compromised immunodeficient (SCID) mouse xenograft model. Xenografts in this model lead to lung and liver metastases, and a peak in CTC counts coincided with the appearance of cells strongly staining for the EMT marker vimentin in the initial xenograft. Vimentin was also expressed in CTCs and CTC clusters, suggesting that EMT promoted CTC formation [13]. Another study, in which KRAS-pancreatic tumour model mice were treated with cerulein to induce pancreatitis and EMT changes in the cancer cells, showed significant increases in CTC counts [14]; however, this finding was not corroborated in a more recent study involving a similar model [15]. A role for EMT in CTC formation was further substantiated when a squamous cell carcinoma prone mouse model with targeted transcription factor Twist1 induction confirmed that Twist1 caused tumour cells to undergo EMT, and this was associated with a doubling of CTC counts as well as increased metastasis [16]. Recently, an innovative model of endothelial cells that form vascular-like structures *in vitro* was used to show that SW620 colorectal cancer cells could migrate into these 'vessels', especially when hypoxia-induced EMT was triggered [17]. A concept supporting the notion that mesenchymal properties afford cancer cells some protection in circulation suggests that any cells shedding from a tumour without undergoing EMT might undergo stressful, traumatic events required for the interruption of the strong epithelial cell-cell interactions resulting in reduced viability [18]. Additionally, EMT-phenotype changes are generally thought to reduce sensitivity to stress signals that would normally lead to apoptosis [8]. Taken together, these observations underpin the emerging opinion that EMT-CTCs may comprise a more viable, aggressive tumour cell population than epithelial CTCs, and go some way to explain the association of EMT-CTCs and increased metastasis. In that regard, it is worth noting that transforming growth factor β (TGFβ), which is commonly released by platelets, may promote or maintain EMT in CTCs while in the circulation and promote extravasation [19]. The current understanding of the role of CTCs in establishing distant metastatic sites was recently reviewed and is beyond the scope of this chapter [2, 20].
