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Di Raimo et al. J Cancer Metastasis Treat 2018;4:54 I http://dx.doi.org/10.20517/2394-4722.2018.50 Page 3 of 14

row DTCs detection and enumeration have been included as important prognostic tools in both M0 and
[44]
M1 BC classes . The ability of CTCs to perform several functional and morphologic changes, conferring
them a high degree of heterogeneity and plasticity, lie behind their clinical and therapeutic attractiveness.
It has been deeply highlighted the important role of epithelial to mesenchymal transition (EMT) as an es-
sential trans-differentiation process in many physio/pathologic mechanisms, such as mesoderm formation
in embryonic development, tissue repair or fibrosis [45-47] . Generally, epithelial cells are defined as adherent
cells, expressing E-cadherin, a transmembrane glycoprotein involved in tight junctions’ formation between
adjacent cells, and CKs, such as CK8, CK18 and CK19, that exhibit an apicobasal polarity and a dense
network of intercellular adhesion complexes that prevent them from migrating. In contrast, mesenchymal
cells are single spindle-shaped cells that do not present intercellular junctions and, consequently, are able
to migrate. In addition, mesenchymal cells generally exhibit a specific proteins profile such as vimentin, fi-
[48]
bronectin and alpha-smooth muscle actin (α-SMA) . Therefore, considering the first part of the metastatic
process, in which cells loss their epithelial nature, acquire a mesenchymal-like expression profile and the
detachment from the primary tumor site occurs, CTCs undergo EMT [49,50] . This multiple complex signaling
system is triggered by the transforming growth factor-β (TGF-β) that enhanced cell migration, invasive-
[51]
ness and increased ability to counteract apoptosis . In fact, it has been demonstrated that TGF-β is able
to induce, in normal mammary epithelial cells, the phosphorylation of Smad2 and Smad3 and the activa-
tion of other EMT-related pathways, such as Notch, PI3K/AKT and Wnt [52,53] . This signal cascade activates
EMT transcriptional factors, such as ZEB1, ZEB2, Twist, Snail and Slug, that downregulate the expression
of E-cadherin [54-57] . Consequently, cell-cell adhesions are disintegrated, cytoskeleton fibers and extracellular
matrix (ECM) component undergo remodeling bringing a loss of cell basal-apical polarity and a strong
motile and invasive properties acquisition [58,59] . Together with E-cadherin, another epithelial-specific trans-
membrane protein, involved in EMT process, is the epithelial cell adhesion molecule (EpCAM). In normal
conditions, this protein is localized in the intercellular space, where it is able to promote tight junctions
formation and interact with E-cadherin, to maintain the epithelial integrity. On the contrary, in cancer tis-
sue, after EMT-related cell-cell adhesion disintegration, EpCAM becomes ubiquitously distributed on the
entire cancer cell surface and, for this reason, more easy to be detected with antibody-based assay. In view
of this, CTCs have long been traditionally defined positive for EpCAM and CK markers expression and
+
+
[60]
-
negative for the hematopoietic marker CD45 (EpCAM /CK /CD45 ). However, in 2014, Lustberg et al.
+
-
identified different circulating cell populations in MBC patients composed of EpCAM /CK cells expressing
mesenchymal markers, with few or no epithelial markers, and cells with both hematopoietic and epithe-
lial markers profile. This heterogenic nature of CTCs was also confirmed through several gene expression
profiling. In fact, whilst they supported the correlation between CTCs, metastatic process and patient’s
overall-survival, to date no consensus has been established regarding biological markers to be used to iden-
tify these cells [61-63] . Currently, putting together different studies, among all the analyzed genes related to
cell survival (IGFR1, FOXO3), the EMT process (TWIST1, SNAIL, SLUG, VIM) or tumor progression and
invasion (HER2, CXCR4, uPAR, VEGFA, VEGFR, Cathepsin D) only CK19, mucin 1 (MUC1) and EpCAM
result as the most accepted genes [61,64-68] . In addition, it has been demonstrated that metastasis exhibit, as
primary tumors, an epithelial phenotype instead of a mesenchymal one, and that, using mice models,
mammary tumors can promote an apparent EMT-independent lung metastatic process [69,70] . Considering
all these evidences, an epithelial-mesenchymal plasticity (EMP) model has been proposed as a hallmark
of CTCs in the metastatic process, in which circulating cells, during their migration to distant organs, are
able to switch between a hybrid phenotype along the epithelial to mesenchymal spectrum conferring them
the ability to adapt in different microenvironments [71-73] .

CTCs migration models
In support of the EMP model, several histopathological, intravital microscopy and in vitro studies demon-
strated that CTCs exhibit different invasion strategies (collective or individual) and are able to exchange to-
ward them according to the surrounding microenvironment [74-81] [Figure 1]. The classical migration model