**6. EMT-phenotype CTCs, do they have clinical relevance?**

Circumstantial evidence linking EMT changes to advanced disease and increased metastasis is strong. EMT phenotype in patient tumour tissue is often prognostic and correlates negatively to overall survival and disease progression. Most notably, a switch from the expression of the epithelial cell-cell adhesion molecule E-cadherin to the mesenchymal N-cadherin is thought to be central to EMT, and it is commonly found in association with disease progression in various cancers including melanoma, pancreatic-, bladder- and colorectal cancer (reviewed by Cavallaro et al. [56]). Other studies found elevated expression of a number of E-cadherin transcriptional repressors such as Twist, Slug, ZEB1/2 and Snail1 linked to poorer prognosis in endometrial-, colorectal-, hepatocellular-, bladder-, gastric- and lung cancer [57–63]. Equally, increased expression of vimentin was associated with poorer outcomes for patients with gastric-, colorectal-, bladder- and breast cancer [58, 64–66]. However, the correlation of EMT with poorer overall or disease-free survival is not universal. A recent study, which established a comprehensive EMT gene expression signature in tumour tissue, found that poorer disease-free survival was associated with an EMT gene expression pattern in ovarian and colorectal cancer but not in breast cancer; therefore, it will be important to better define the context in which EMT gives cancer cells a selective advantage [67]. As detailed above, EMT marker gene or protein expression has also been studied in CTCs, and the overall emerging evidence suggests that increased EMT-phenotype detection in CTCs correlates with more advanced disease stages and is the predominant phenotype found in the blood of patients with metastatic disease (see **Table 1**).

Cancer cells frequently undergo EMT when exposed to stress, and this makes them signifi‐ cantly more resistant to a variety of therapies. However, it is not well understood whether the range of phenotypic EMT changes that cause increased mobility and metastasis is instrumental in resistance or merely associated with it. Mounting *in vitro* evidence suggesting that EMT confers drug resistance has been thoroughly reviewed previously [8]. More recently, *in vivo* data reaffirm the link between EMT and therapy resistance. For instance, the loss of E-cadherin expression in erlotinib-treated non-small cell lung cancer tumour tissues correlated with poorer progression-free patient survival [68], and EMT gene expression signatures in ovarian or prostate cancer patient tissue were associated with resistance to platinum therapy or docetaxel and androgen deprivation, respectively [69, 70]. Interestingly, two recent studies suggested that in breast- and pancreatic cancer mouse models, the majority of cells that metastasised to the lungs did not undergo EMT. Nevertheless, EMT was involved in drug resistance and conditional metastatic outgrowth when mice were treated with the drugs cyclophosphamide and gemcitabine, respectively [15, 71]. These data add to the controversy regarding the role of EMT in the metastatic process and warrant further research. The data do support the survival/therapy resistance functions associated with EMT and it is plausible that cancer cells may 'escape into EMT' to render themselves resistant to drug treatment. Data are starting to emerge suggesting these changes might be detectable by CTC analysis as subtyping CTCs as possessing epithelial, intermittent or mesenchymal characteristics showed that gastric cancer CTCs of a patient progressing on therapy were all of EMT phenotype. However, and confusingly, any remaining CTCs detected in gastric cancer patients that responded to therapy were more epithelial in nature [39].

Regardless, it is possible that therapy inadvertently induces cells that survive drug exposure to change into more mobile, viable and aggressive clones. Due to their EMT phenotype, these cells may be ideally equipped to leave the primary tumour, become CTCs, prevent anoikis and potentially form distant metastases sites. Additionally, the survival advantage of EMT cancer cells might allow time to acquire alternative resistance mechanisms such as mutations. In turn, that would allow EMT-CTCs to undergo MET after extravasation to enable the resettling and formation of proliferating metastases. The underlying mechanisms need to be more thorough‐ ly investigated, and the ability to accurately isolate EMT-CTCs will prove central to clarifying the role of EMT in therapy resistance, disease relapse and metastatic processes. Efficient EMT-CTC isolation and identification may also allow the development of diagnostic tests that monitor escape into EMT as part of therapy response to inform improved patient management.
