**4. EMT phenotype in CTCs isolated with alternate strategies**

The inability of EpCAM-based CTC isolation to optimally account for EMT-CTCs with EpCAM loss has led to the targeting of alternative, EMT-associated cell-surface markers, for CTC enrichment, or by avoiding these methods altogether and focussing on CTC enrichment due to physical cancer cell properties, mainly size exclusion. Not surprisingly, when EpCAM- based immunomagnetic CTC isolation was directly compared with size exclusion CTC enrichment of parallel blood samples from 40 NSCLC patients, CTCs were isolated from a higher proportion of patients (80 vs 23%) by size exclusion, and as expected the isolated cells tended to lack EpCAM; however, they expressed CK and had elevated levels of the EMTassociated epithelial growth factor receptor (EGFR) and thus likely were tumour cells [24].

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].

common CTC definition (DAPI<sup>+</sup>

244 Tumor Metastasis

**3. EMT phenotypes in CTCs isolated by EpCAM targeting**

, CK+

viduals, suggesting the presence of CTCs lacking CK in some patients [23].

**4. EMT phenotype in CTCs isolated with alternate strategies**

, CD45-

The inability of EpCAM-based CTC isolation to optimally account for EMT-CTCs with EpCAM loss has led to the targeting of alternative, EMT-associated cell-surface markers, for CTC enrichment, or by avoiding these methods altogether and focussing on CTC enrichment due to physical cancer cell properties, mainly size exclusion. Not surprisingly, when EpCAM-

) but not in samples from healthy control indi‐

It is important to emphasise that, despite the epithelial nature of EpCAM, CTCs isolated by EpCAM targeting can display markers of EMT. This is due to the fact that changes between epithelial and mesenchymal phenotypes are dynamic, and cells can be found in intermittent stages and express mesenchymal as well as epithelial markers at the same time. Accordingly, mesenchymal markers have been successfully detected in CTCs after EpCAM-based isolation. For instance, intermittent EMT phenotype characterised by co-expression of mesenchymal proteins vimentin, N-cadherin and CD133 with epithelial markers EpCAM, CK and E-cadherin was shown in breast cancer and prostate cancer CTCs isolated by EpCAM targeting [21]. EpCAM-based breast cancer CTC isolation also yielded cells with common gene expression of the EMT markers TGFβ1, FOXC1, CXCR4, NFKB1, VIM and ZEB2 [22]. Moreover, higher breast cancer staging correlated with mesenchymal vimentin and fibronectin expression in EpCAM-enriched CTC samples. Interestingly, vimentin and fibronectin expression was also detected in 31 of 92 (34%) of patient samples, which were CTC negative according to the An elegant approach to account for the CTCs missed during EpCAM-based capture in HER2 positive breast cancer patients utilised CD45 immunomagnetic depletion of blood cells after an initial EpCAM-based CTC capture, to further enrich the remaining EpCAM-negative CTCs. The EMT-linked transcription factors SNAI1 and ZEB1 were more commonly expressed in these EpCAM-negative cells that were likely tumour-derived cells compared to the EpCAMisolated counterparts [25]. Vimentin, best known for its functions as a cytoskeletal support protein, can also be present on the cell surface of mesenchymal cells and has been successfully targeted in immunomagnetic isolation of CTCs from colorectal cancer patients and breast cancer patients. After CD45 immunodepletion, CTCs were positively selected with cell-surface vimentin (CSV) targeting. The authors suggest that CSV expression is restricted to cancer cells, and CSV targeting isolates significantly more CTCs from colorectal cancer patients with progressive disease than those with stable disease; moreover, higher CSV-CTC counts were more commonly found in therapy-resistant patients. In a direct comparison of CSV- and EpCAM-based CTC isolation (CellSearch CTC platform) in breast cancer patients, CTCs isolated with CSV targeting were a more reliable marker for progressive disease compared to stable disease. In both CSV-isolated breast and colorectal cancer CTCs, the EMT markers FOXC2, SNAIL, Twist-1 and Slug tended to be highly expressed while E-cadherin and EpCAM levels were low. The CSV antibody is currently not commercially available, thus limiting its

**Figure 2.** EMT analysis in isolated CTCs. Quadruple staining to detect CTCs with EMT phenotype according to levels of the EMT marker vimentin. (A) CTCs were identified by nuclear Hoechst staining 'Ncl' (blue, Fluxion enumeration kit) and cytokeratin (CK) staining (green; FITC-conjugated anti-cytokeratin antibody: clone c-11, Sigma-Aldrich) as well as exclusion of CD45 expression (red, antibodies: CTC enumeration kit, Fluxion). Vimentin staining, 'Vim' (or‐ ange, AF647-conjugated anti-vimentin antibody: clone V9, Abcam), in CTCs, was scored as indicated: Staining of a rep‐ resentative cell for each category, negative (−), weak (+), positive (++) and strongly positive (+++), is depicted. (B) Cell counts for vimentin-positive EMT-CTCs isolated from a representative patient sample using EpCAM- versus N-cad‐ herin- 'N-cad'-based isolation of CTCs using the quadruple staining assay. CTCs were isolated with the IsoFlux CTC isolation platform using EpCAM-based or N-cadherin-based immunomagnetic isolation with the Rare Cell Isolation Kit, Fluxion.


OS: overall survival, PFS: progression-free survival. Dependent on their nature, EMT markers follow protein or gene nomenclature

**Table 1.** Detection of EMT biomarkers in CTCs.

use and confirmation of the data by others [26, 27]. A study that investigated gene expression in ovarian cancer CTCs showed the expression of EMT markers in most individual CTCs from three patients, while only 30% of these cells also expressed epithelial CK5 or CK7. However, all CTCs expressed epithelial MUC1 [28]. In a patient with non-small cell lung cancer who was CTC-negative according to EpCAM-based (CellSearch) enrichment, it is worth noting that CTCs enriched by CD45 depletion were tumourigenic in mice and CTCs isolated from the same patient by size exclusion showed predominant EMT or intermittent phenotype [29].

In our laboratory, we embarked on the isolation of CTCs from advanced ovarian cancer patients using N-cadherin-based immunomagnetic isolation and captured approximately three times more CTCs when using N-cadherin-based versus EpCAM-based CTC isolation (data not shown). We also developed an assay to probe CTCs for vimentin as marker of EMT, which showed that N-cadherin-based CTC isolation from advanced ovarian cancer patients increased the capture of EMT-CTCs (**Figure 2**). Studies that investigated EMT markers in CTCs, isolated by various strategies, are compiled in **Table 1**.
