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

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 common CTC definition (DAPI<sup>+</sup> , CK+ , CD45- ) but not in samples from healthy control indi‐ viduals, suggesting the presence of CTCs lacking CK in some patients [23].
