Volume 247, Issue 3 p. 432-450

Reviews

Free Access

Epithelial–mesenchymal plasticity and circulating tumor cells: Travel companions to metastases

Marie-Emilie Francart,

Marie-Emilie Francart

GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium

Search for more papers by this author

Justine Lambert,

Justine Lambert

GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium

Search for more papers by this author

Aline M. Vanwynsberghe,

Aline M. Vanwynsberghe

GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium

Search for more papers by this author

Erik W. Thompson,

Erik W. Thompson

Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, and Translational Research Institute Brisbane, and University of Melbourne Department of Surgery, St Vincent's Hospital, Melbourne, Australia

Search for more papers by this author

Morgane Bourcy,

Morgane Bourcy

GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium

Search for more papers by this author

Myriam Polette,

Myriam Polette

Inserm UMR-S 903, University of Reims Champagne-Ardenne, Biopathology Laboratory, CHU of Reims, Reims, France

Search for more papers by this author

Christine Gilles,

Corresponding Author

Christine Gilles

[email protected]

orcid.org/0000-0002-6891-4920

GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium

Correspondence to: Christine Gilles, Laboratory of Tumor and Development Biology (LBTD), GIGA-Cancer, Pathology Tower, B23, CHU Sart-Tilman, University of Liège, Liège 4000, Belgium. E-mail: [email protected]Search for more papers by this author

Marie-Emilie Francart,

Marie-Emilie Francart

GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium

Search for more papers by this author

Justine Lambert,

Justine Lambert

GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium

Search for more papers by this author

Aline M. Vanwynsberghe,

Aline M. Vanwynsberghe

GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium

Search for more papers by this author

Erik W. Thompson,

Erik W. Thompson

Search for more papers by this author

Morgane Bourcy,

Morgane Bourcy

GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium

Search for more papers by this author

Myriam Polette,

Myriam Polette

Inserm UMR-S 903, University of Reims Champagne-Ardenne, Biopathology Laboratory, CHU of Reims, Reims, France

Search for more papers by this author

Christine Gilles,

Corresponding Author

Christine Gilles

[email protected]

orcid.org/0000-0002-6891-4920

GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, Liège, Belgium

First published: 13 April 2017

https://doi.org/10.1002/dvdy.24506

Citations: 77

Share a link

Email
Facebook
Twitter
LinkedIn
Reddit
Wechat

Abstract

Epithelial–mesenchymal transitions (EMTs) associated with metastatic progression may contribute to the generation of hybrid phenotypes capable of plasticity. This cellular plasticity would provide tumor cells with an increased potential to adapt to the different microenvironments encountered during metastatic spread. Understanding how EMT may functionally equip circulating tumor cells (CTCs) with an enhanced competence to survive in the bloodstream and niche in the colonized organs has thus become a major cancer research axis. We summarize here clinical data with CTC endpoints involving EMT. We then review the work functionally linking EMT programs to CTC biology and deciphering molecular EMT-driven mechanisms supporting their metastatic competence. Developmental Dynamics 247:432–450, 2018. © 2017 Wiley Periodicals, Inc.

Introduction

The hematogenous metastatic spread of epithelial tumors is a complex process involving the liberation of circulating tumor cells (CTCs) in the bloodstream, their survival in the circulation, the colonization of secondary organs as disseminated tumor cells (DTCs) and finally, after an eventual period of dormancy, growth at secondary sites and overt metastasis development.

Being easily accessible in the bloodstream, CTCs have attracted enormous attention for their potential clinical significance (Toss et al., 2014; Joosse et al., 2015; McInnes et al., 2015; Yu et al., 2015; Alix-Panabieres and Pantel, 2016; Lee et al., 2016; Masuda et al., 2016; Wang et al., 2017b). Detecting, enumerating, characterizing and understanding CTC biology may indeed help identify metastasis-predicting factors, guiding treatment decisions before the detection of overt metastases and assessing therapeutic efficacy (Krebs et al., 2014; Joosse et al., 2015; McInnes et al., 2015; Pantel and Speicher, 2015; Alix-Panabieres et al., 2017). CTCs are also considered as potential cellular therapeutic targets (Li and King, 2012; Li et al., 2015a, 2016).

The comprehension that CTCs constitute a genetically and phenotypically very heterogeneous population has further stimulated studies aiming to characterize the metastatic founders within the CTC population. It has thus become a major cancer research axis to phenotype CTCs, to identify premetastatic subsets and to unravel the molecular mechanisms enabling some of them to accomplish the early steps of the metastatic colonization (i.e., survival in the bloodstream and early seeding in colonized organs) (Nadal et al., 2013; Krebs et al., 2014; Pantel and Speicher, 2015).

We have reviewed here literature data on how epithelial–mesenchymal transitions (EMTs) may impact CTC biology by generating hybrid phenotypes with mesenchymal attributes (mesenchymally-shifted cells) that may favor their liberation and survival in the bloodstream, and their metastatic seeding.

Epithelial–Mesenchymal Plasticity/Epithelial-Mesenchymal Transition

Epithelial–mesenchymal plasticity (EMP) (Thompson et al., 2005; Thompson and Haviv, 2011; Savagner, 2015; Ye and Weinberg, 2015; Chaffer et al., 2016; Nieto et al., 2016) is today considered a central actor of the metastatic cascade, providing tumor cells with the ability to adapt to different microenvironments met during their translocation to colonized organs (i.e., adjacent stroma, blood, newly colonized organs). Timely and spatially regulated dynamic interconversions between epithelial states and states that are more mesenchymal indeed occur throughout the metastatic cascade, enabling tumor cells to survive/develop in successively encountered microenvironment. Schematically (Figs. 1 and 2), the classical view of EMP implication to the metastatic cascade involves sequential EMTs followed by mesenchymal–epithelial transitions (METs) at different body locations (Tsai and Yang, 2013; Jolly et al., 2015a; Liu et al., 2015; Beerling et al., 2016; Celia-Terrassa and Kang, 2016; Chaffer et al., 2016; Diepenbruck and Christofori, 2016; Kolbl et al., 2016).

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

**Hybrid phenotypes along the epithelial (E) -to-mesenchymal (M) spectrum**.

**Figure 2**
Open in figure viewer PowerPoint

**Schematic nonexclusive hypotheses of epithelial–mesenchymal transition's (EMT) contribution to the biology of CTC during the metastatic spread.** ❶ EMT occurs in the primary tumors providing tumor cells with enhanced invasive properties facilitating active intravasation and the liberation of mesenchymally-shifted circulating tumor cell (CTC) phenotypes. Displaying enhanced EMT-driven survival properties (i.e., activation of survival pathways, activation of local coagulation, microtentacle formation, or evasion from immune checkpoints), those mesenchymally-shifted CTCs are able to accomplish the early colonization phases. Among the EMT-derived CTCs that have niched in colonized organs, those belonging to the plasticity window will then be able to revert to a more epithelial state thought to be involved in the metastatic outgrowth. ❷ EMT occurs in the primary tumors, EMT-derived cells intravasate, bringing with them more epithelial intermediates through a cooperative process (which may eventually undergo a EMT within the blood stream, for instance by means of transforming growth factor-beta liberated from platelets). The more epithelial CTCs expressing low survival properties are submitted to shear stress, anoikis, or cytotoxic immune attack and are eliminated. As in scheme 1, mesenchymally-shifted CTCs survive and succeed in the early colonization phases. ❸ Clusters of CTC gain the circulation through corrupted blood vessels, minimizing the effect of anoikis, thus promoting the survival of more epithelial phenotypes in the bloodstream (the presence of mesenchymally-shifted cells in these clusters may further support the survival of CTCs within the clusters). Clusters of CTC may form niches, allowing those with metastatic competence to develop overt metastases. Platelets = , Fibrin = , Recruited stromal cells = .

Thus, a switch toward a more mesenchymal state (EMT) is considered to contribute to the first phases of the metastatic translocation, i.e., tumor invasion, intravasation, liberation of CTCs and survival in the bloodstream, and metastatic niche formation. Conversely, a reversed MET process has been associated with an enhanced ability to proliferate and develop overt metastases in secondary organs, thus contributing to the deadly late stages of metastatic development (Gunasinghe et al., 2012). Unlike developmental EMT, tumor-associated EMT has rarely been reported to involve a complete lineage switching, but rather the generation of intermediate states (hybrid phenotypes) that distribute along the epithelium (E) to mesenchymal (M) continuum (as schematically represented in Fig. 1). Phenotypic plasticity is believed to be restricted to certain hybrid phenotypes also endowed with stem cell characteristics [overlapping to some extent to the so-called cancer stem cell (CSC) population] (Jolly et al., 2015a).

Importantly, EMT programs have been shown to directly induce stem cell properties in epithelial tumor cells (Mani et al., 2008; Morel et al., 2008; Bhat-Nakshatri et al., 2010; Ansieau, 2013; Kotiyal and Bhattacharya, 2014; Mallini et al., 2014; Schmidt et al., 2015; Mladinich et al., 2016). Thus, EMT and CSC characteristics have been shown to overlap to some extent both in in vitro cell systems but also in tumors and CTCs from cancer patients (Aktas et al., 2009; Giordano et al., 2012; Kasimir-Bauer et al., 2012; Ksiazkiewicz et al., 2012; Barriere et al., 2014; Bock et al., 2014; Krawczyk et al., 2014; Tinhofer et al., 2014). Sometimes referred to as “metastable” phenotypes (Klymkowsky and Savagner, 2009; Savagner, 2015), these hybrid phenotypes with mesenchymal attributes and CSC markers would be more efficient for metastasis. Levine and co-workers have recently shown with elaborate modeling that the hybrid state may be quite stable (Jolly et al., 2015a,b, 2016).

Although the successive EMT/MET scheme of the metastatic spread presented above is generally accepted, it is still a subject of debate and discussions (Tsuji et al., 2009; Brabletz, 2012; Fischer et al., 2015; Zheng et al., 2015; Diepenbruck and Christofori, 2016). It indeed remains unclear if the same plastic tumor cell is able to overcome all obstacles of the metastatic cascade through phenotypic adaptation or whether further genetic alterations occur during the metastatic cascade that empower some tumor cells to form metastases. Whether different phenotypes along the E to M continuum cooperate throughout the metastatic process to protect cells with the highest ability to proliferate at secondary site is also a possibility. Two recent studies using transgenic models suggested that EMT is dispensable for metastasis but required for chemoresistance and further revived the debate (Fischer et al., 2015; Zheng et al., 2015). It is also very likely that all these mechanisms may coexist to different extents, depending on the model used or the cancer type analyzed, making our understanding of their roles in the metastatic process and the establishment of effective therapies more difficult.

EMP associated with metastatic progression is recognized to be commonly regulated by molecular actors of core EMT programs, although a full mesenchymal conversion is considered uncommon. These EMT core molecular actors have been reviewed elsewhere (Kalluri and Weinberg, 2009; De Craene and Berx, 2013; Tsai and Yang, 2013; Lamouille et al., 2014; Jolly et al., 2015b; Nieto et al., 2016) and will, therefore, not be detailed here. EMT associated with metastasis is nevertheless clearly not a well-defined unique molecular program driving a binary transformation from a fully epithelial cell to a mesenchymal cell, nor does it involve the generation of well-characterized sequential hybrid phenotypes. There is indeed a wide repertoire of epithelial plasticity and a multiplicity of potential hybrid phenotypes. There is thus a clear need to identify how specific molecular actors of EMT programs may functionally impact on specific stages of the metastatic cascade to further refine therapeutic strategies.

Examining the relationship between EMT and CTCs has gained a fast-growing interest in the past decade. EMT has indeed rapidly emerged as a process endowing tumor cells with properties that may functionally impact CTC life cycle (Bonnomet et al., 2010; Barriere et al., 2014; Krawczyk et al., 2014; Aceto et al., 2015; Jolly et al., 2015a; Liu et al., 2015; McInnes et al., 2015; Pantel and Speicher, 2015; Kolbl et al., 2016; Alix-Panabieres et al., 2017), including invasive/motile properties (Nieto et al., 2016), resistance to apoptosis/anoikis (Tiwari et al., 2012; Frisch et al., 2013; Cao et al., 2016), and stemness properties (Ombrato and Malanchi, 2014; Ye and Weinberg, 2015; Fabregat et al., 2016).

EMT Facilitates the Entry of CTCs into the Circulation

Because EMT has been extensively shown to confer migratory and invasive properties to epithelial tumor cells, it is generally considered that mesenchymally-shifted tumor cells display enhanced ability to actively enter the circulation and thus become CTCs (Bonnomet et al., 2010; Tsai and Yang, 2013; Chiang et al., 2016). Accordingly several experimental settings using transendothelial migration assays, chick chorioallantoic membrane (CAM) assays, and intravital imaging emphasized a contribution of EMT (mainly using cells with forced expression of EMT transcription factors such as Snail or ZEB1) in intravasation (Drake et al., 2009; Ota et al., 2009; Bonnomet et al., 2010). A crucial role of EMT-induced proteases has also been identified. Using intravital imaging on tumor cells xenografts, it has for instance been shown that the activation of the transforming growth factor-beta (TGF-β) pathway associates with a certain degree of trans-differentiation, promotes single cell motility, and enables invasion into blood vessels. The activation of the TGF-β pathway was shown to be a transient event and was not maintained at distant sites (Giampieri et al., 2009). This is also in agreement with several earlier studies associating EMT with a single-cell mode of migration (Friedl and Gilmour, 2009).

Other mechanisms of entry of tumor cells in the circulation have also been proposed. Thus, cooperative processes by which EMT shifted cells would “help” more epithelial tumor cell phenotypes (suggested to be more competent for metastasis) to gain the circulation may also occur and have also been reported in a syngenic tumor model (Tsuji et al., 2008, 2009). Indeed, Tsuji et al. demonstrated that EMT negative hamster keratinocytes (HPCP-1) were unable to metastasize after subcutaneous injection unless co-injected with EMT-transformed counterparts (overexpressing the downstream effector of the TGF-β pathway p12 ^CKD2-AP1) that enabled intravasation of the EMT-negative cells. Another study similarly reported a cooperation between EMT- and EMT + cells for metastasis in xenograft models of human prostate and bladder cancer cell lines in which the expression of Snail was modulated (Celia-Terrassa et al., 2012).

In line with the observation of corrupted blood vessels in tumors, a passive mode of entry of tumor cells in the circulation, that could thus be EMT-dependent or EMT-independent, has also been suggested (Alpaugh et al., 2002; Bockhorn et al., 2007; Bednarz-Knoll et al., 2012). Such a passive mode of entry has been more particularly advocated to explain the detection of clusters of CTCs in the blood of cancer patients, although a collective migration process during intravasation cannot be excluded (Giampieri et al., 2010; Aceto et al., 2015). Clusters of CTCs had actually been observed as early as in the 1970s (Fidler, 1973; Liotta et al., 1976). The possibility that these clusters might actually form during the processing of the blood samples during the CTC assay has been debated (Bednarz-Knoll et al., 2012), although they are much less prevalent than isolated CTCs. These clusters are today recognized as functional entities and have been detected in various types of cancers including breast, lung, pancreas, prostate, or kidney cancers (Molnar et al., 2001; Stott et al., 2010; Cho et al., 2012; Hou et al., 2012; Krebs et al., 2012; Yu et al., 2013; Aceto et al., 2014; Mu et al., 2015; Paoletti et al., 2015; Wang et al., 2017a).

Adding to this, a shift toward a more mesenchymal phenotype may also be gained within the circulation. For instance, Labelle et al. suggested that TGF-β liberated from activated platelets may enhance EMT in TGF-β-responsive tumor cells and promote metastasis formation (Labelle et al., 2011; Pantel and Speicher, 2015).

CTCs may thus enter the circulation either as mesenchymally-shifted cells or not but may also acquire mesenchymal attributes within the circulation (Fig. 2). Reflecting the likelihood of coexisting mechanisms used by tumor cells to enter the bloodstream, and adding genomic heterogeneity, CTCs are indeed a phenotypically heterogeneous population. Although the active contribution of EMT implication to the different processes of entry of CTCs in the circulation may vary, the repetitive observation of CTCs expressing mesenchymal attributes both in the blood of tumor animal models or in the blood of cancer patients (Table 1) clearly support a contribution of EMT to CTC phenotypical heterogeneity (Bednarz-Knoll et al., 2012; Krawczyk et al., 2014; Liu et al., 2015; Pantel and Speicher, 2015; Kolbl et al., 2016; Alix-Panabieres et al., 2017).

Table 1. Detection of EMT and Stem Cell Markers in CTCs from Cancer Patients

EMT and stemness markers (+ others associated)	Type of tumor	Method of separation/characterization	Method of detection	Nr of patients	Correlation between EMT markers and clinical parameters	Ref
EGFR, pEGFR, HER2, pAkt, pPI3K	Breast cancer	Ficoll density gradient/EpCAM positive CELLection beads/IF CK	IF	38	Metastases [EGFR and pPI3K]	(Kallergi et al., 2008)
Twist, PI3Kα, Akt2, ALDH1	Beast cancer	AdnaTest BreastCancer	RT PCR	39	Therapy resist [CTC^EMT+/ALDH1]	(Aktas et al., 2009)
ALDH1, CD44	Breast cancer (M₁)	Ficoll density gradient/IF CK-CD24	IF	30	ND	(Theodoropoulos et al., 2010)
Vim, FN	Breast cancer (M₁)	EpCAM positive CELLection beads/RT PCR CD45-CK	RT PCR	55	↓ FPS	(Gradilone et al., 2011)
Vim, E-cad, N-cad, CKs	Lung cancer (M₀ and M₁)	ISET (filter-based size exclusion approach)/IHC CD45	IHC	7	ND	(Hou et al., 2011)
Vim, Twist	Breast cancer (M₀ and M₁)	Ficoll density gradient/CD45 negative Dynal CELLection beads/IF CK	IF	50	Metastases	(Kallergi et al., 2008)
Vim, CKs	NSCLC (M₁)	ISET (filter-based size exclusion approach)/Identification by a cytopathologist	IF	6	ND	(Lecharpentier et al., 2011)
Vim, FN, ALDH1	Breast cancer (multi stages)	EpCAM positive CELLection Dynabeads/RT PCR CD45-CK	RT PCR	92	Stage disease	(Raimondi et al., 2011)
Twist, Snail, Slug, ZEB1, FoxC2	Breast cancer (M₀)	CellSearch, AdnaTest Breast Cancer, Ficoll density gradient + CD45 magnetic beads depletion	RT PCR	52	Neoadjuvant therapies resist [CTC^EMT+]	(Mego et al., 2011)
Vim, E-cad, N-cad, O-cad, CD133	CRPC (M₁) Breast cancer (M₁)	CellSearch	IF	57	ND	(Armstrong et al., 2011)
Twist, PI3K-α, Akt2, ALDH1	Breast cancer (M₀)	AdnaTest Breast Cancer	RT PCR	130	No correlation found	(Barriere et al., 2012bb)
Twist, PI3K-α, Akt2, ALDH1, Bmi1, CD44	Breast cancer	AdnaTest EMT-1/Stem Cell	RT PCR	61	Negative metastases lymph node status [ddCTC]	(Barriere et al., 2012aa)
Twist, Snail, ZEB1, TG2	Breast cancer (M₁ HER2+)	CellSearch	RT PCR	28	ND	(Giordano et al., 2012)
Twist, PI3K-α, Akt2, ALDH1	Breast cancer	AdnaTest Breast Cancer	RT PCR	502	No correlation found	(Kasimir-Bauer et al., 2012)
CD44, CD47, c-MET	Breast cancer (M₀ luminal)	CellSearch RosetteSep selection kit hematopoietic cells depletion/FACS EpCAM -CD45-PI	FACS	350	↓ OS [CTCs^{CD44+MET+CD47+}] Metastatic [CTCs^{CD44+MET+CD47+}] Disease progression	(Baccelli et al., 2013)
84 EMT-related genes tested	Prostate cancer	ScreenCell Selection (filter-based size exclusion approach)/IF CD45 + micromanipulator	qRT PCR chip assay	8	Castration treatment resist [markers CTC^EMT+]	(Chen et al., 2013)
Vim, Twist	Hepatocellular carcinoma	Ficoll density gradient/Asialofetuin biotinylation magnetic beads positive selection/IF CD45-Hepatocyte-Specific Antigen	IF	60	Tumor size, TNM, Milan criteria [vim] Portal vein tumor trombosis [vim, Twist]	(Li et al., 2013)
Plastin3	Colorectal cancer	Ficoll density gradient/CD45 magnetic beads depletion/EpCAM magnetic beads positive selection/IF CK-vim	IF	771	↓ OS	(Yokobori et al., 2013)
CKs 8,19, TFF1/TFF3, PAI-1, FN1, ECM, WNT, integrin, interleukin receptors, TGF-β, interleukin-6, mammaglobins	Breast cancer	Herringbone–chip EpCAM, HER2, EGFR positive selection	FISH	41	Disease progression [CTC^M+] Therapy response [reversible shifts between CTC^E+/CTC ^M+]	(Yu et al., 2013)
Snail, ZEB1, ZEB2, ETV5, Notch1, TGFβ1, ALDH1, CD44	Endometrial cancer	CELLection epithelial enrich/qRT PCR CD45	qRT PCR	34	High risk endometrial cancer [CTC ^{high plasticity}]	(Alonso-Alconada et al., 2014)
ZEB2, LOXL3, VIL1 TIMP1, CLU	Colorectal cancer	CELLection epithelial enrich/qRT PCR CD45	qRT PCR	50	↓ PFS/OS	(Barbazan et al., 2014)
Twist, Snail, Slug, ZEB1, CK19	Breast cancer (M0)	RossetteSep selection kit CD45 negative selection	qRT PCR	149	High grade tumor ↑ KI67	(Cierna et al., 2014)
Vim, CD44, seprase, methyl group to CpG sites	CRPC (M1)	Vitatex's rare cell enrichment technology: hematologic lineage markers depletion, EpCAM, PSMA, tumor/stem cell markers positive selection or CellSearch	IHC, FACS, DNA methylation array	23	ND	(Friedlander et al., 2014)
Vim, Snail, uPAR	Breast cancer (M0)	Density gradient centrifugation/CD45 magnetic particles depletion	qRT PCR IF	117	Lymph nodes metastases	(Markiewicz et al., 2014)
Twist, ALDH1	Breast cancer (M0 and M1)	Ficoll density gradient/IF CD45, CKs	IF	130	Metastases [ALDH1^high/Twist^nuclear]	(Papadaki et al., 2014)
Vim, Slug, EGFR	Breast cancer (M0)	Carcinoma Cell Enrichment and Detection kit, MACS technology CKs magnetic beads positive selection/IF CD45	IF	78	↓ PFS after receiving systemic treatment [CTCs ^{CK- EGFR+}] Tumor size [vim, Slug]	(Serrano et al., 2014)
Ratio Vim/CKs	Breast cancer (M0 and M1)	CellSearch	IF	61	↓ OS [CTCs ^{Epcam+ CK low}]	(Polioudaki et al., 2015)
EpCAM, CKs 8, 18, 19, Vim, Twist	Gastric cancer	CanPatrol system (filtration system)/FISH CD45	FISH	44	Therapy response [CTC^M+]	(Li et al., 2015b)
Cell surface vimentin (84–1 vimentin Ab)	Colorectal cancer (M1)	CD45 depletion/84–1 positive cell selection	FISH	101	Therapy response	(Satelli et al., 2014)
EpCAM, CKs 8,18,19, Vim, Twist	NSCLC, liver, breast, gastric and nasopharyngeal cancer	CD45 depletion/CanPatrol (filtration system)/IF-FISH CD45	FISH IF	164	Metastases [CTC^M+]	(Wu et al., 2015)
EpCAM, E-Cad, N-Cad, CD44, CD146	Breast cancer (M1)	CD45 MicroBeads depletion/DEPArray sorting CD45 negative cells	RT PCR	56	↓ PFS/OS [CTC ^EM+] Tumor type, disease progression, proliferation, metastases site [CTC^all]	(Bulfoni et al., 2016aa)
Vim, c-MET, HER3, ERCC1, JAG1	NSCLC	CellSearch system, Adna-EMT-2 test + multiplex RT-PCR vs qRT PCR, Ficoll density gradient + IF CK CD45 ALDH1	qRT PCR	62	ND	(Hanssen et al., 2016)
55 breast cancer associated genes	Breast cancer (M1)	AdnaTest BreastCancer	qRT PCR	147	↓ RFS [RAD51] ↓ OS [CD24, HDAC2, KRAS, PIK3CA, RAD51]	(Hensler et al., 2016)
EpCAM, CKs7, 8	Breast cancer (M1)	Parallel multi-orifice flow fractionation chip (p-MOFF system)/IF CD45-CK	IF	32	ND	(Hyun et al., 2016)
Ki67, Vim	CRPC (M1)	CellSearch	IF	93	↓ OS	(Lindsay et al., 2016)
EpCAM, CKs 8, 18, 19, Vim, Twist	Hepato cellular carcinoma	CD45 depletion/CanPatrol CTC enrichment technique (filtration system)/FISCH CD45	FISH	33	Extra hepatic metastases [CTC ^M+] Intra hepatic metastases [CTC ^EMT+] Tumor size [CTC ^E+]	(Liu et al., 2016)
Twist, PI3Kα, Akt2, ALDH1	Colorectal cancer (M1)	CD45 Dynabeads M-450 depletion/Dynabeads Epithelial Enrich/qRT PCR survivin-CK	qRT PCR	78	↓ PFS/OS [Akt2]	(Ning et al., 2016)
Twist, PI3Kα, Akt2, ALDH1	Bladder Cancer	AdnaTest EMT2/SC system	PCR	83	Clinical stage	(Todenhofer et al., 2016)
EpCAM, CK8,18,19, Vim, Twist, Akt2 and Snail	Colorectal cancer	CD45 depletion/CanPatrol CTC enrichment technique (filtration system)/FISCH CD45	FISH	1203	Clinical stage, lymph node and distant metastases [CTC^{M+ and E+/M+}]	(Zhao et al., 2016aa)

Vim, vimentin; FN, fibronectin; CKs, cytokeratins; Cad, cadherin; Ab, antibody; M₀, non metastatic; M₁, metastatic; NSCLC, non small cell lung cancer; CRPC, castro resistant prostate cancer; PSMA, prostate specific membrane antigen; FACS, fluorescence activating cells sorting; IF, immunofluorescence; HIS, immunohistochemistry; FISH, fluorescence in situ hybridization; PI, propidium iodide; (q)RT PCR, (quantitative) reverse transcription polymerase chain polymerization; OS, overall survival; PFS, progression free survival; RFS, relapse free survival; TNM, tumor nodes metastases; ddCTC, dedifferentiated circulating tumor cells; CTSC, circulating tumor stem cells; ND, not determined; *The Cancer Genome Atlas data set.

Regarding animal models, Rhim and coworkers detected CTCs expressing the EMT transcription factor ZEB2 at the premalignant stage of tumor progression in a K-Ras-driven mouse pancreatic tumor model (Rhim et al., 2012). Using a transgenic mouse model expressing Twist under an inducible keratin 5 promoter, Tsai and colleagues showed that Twist induction increased the number of CTCs and that these CTCs presented an EMT phenotype with a loss of E-cadherin and a gain of vimentin expression (Tsai et al., 2012). Using a xenograft system of human breast tumor MDA-MB-468 cells, we also reported dynamic EMT changes in the primary tumors and the liberation of CTCs expressing EMT markers, including Snail, Slug, and vimentin (Bonnomet et al., 2012). In these different studies, the liberation of EMT-shifted CTCs correlated with the appearance of metastatic lesions, supporting the idea that these CTCs expressing mesenchymal traits could be metastatic founders.

Similarly, CTCs expressing common EMT actors such as EMT transcription factors (ZEB1, Twist, Snail, or Slug), vimentin, or N-cadherin have been observed in different types of cancers and particularly in breast cancer patients but EMT-shifted CTCs were also identified in patients with lung, colorectal, prostate, bladder, or endometrial cancers (Table 1).

In light of these observations, it has been proposed that examining mesenchymal markers should be more systematically included in the detection of CTCs (Bednarz-Knoll et al., 2012; Barriere et al., 2014; Bulfoni et al., 2016b). Although CTC isolation techniques have been extensively reviewed elsewhere and will not be detailed here, it is important to perceive that the potential contribution of EMT to CTC biology largely complicates their purification and characterization (Bednarz-Knoll et al., 2012; Alix-Panabières and Pantel, 2014; Joosse et al., 2015; Pantel and Speicher, 2015; Hyun et al., 2016; Alix-Panabieres et al., 2017). Indeed, CTC isolation and identification techniques are most commonly based on the detection of epithelial markers (i.e., EpCAM is frequently used in antibody-based purification procedure and cytokeratins are often examined to discriminate epithelial CTCs from blood cells). It has thus been recognized that certain mesenchymally-shifted CTCs could be excluded from different CTC assays. This has stimulated the development of technologies to include the detection of EMT-shifted CTCs and their more systematic examination (Bednarz-Knoll et al., 2012; Barriere et al., 2014; Bulfoni et al., 2016b). Strategies, essentially based on physical properties of CTCs (mostly size-based technologies) and using filtration or microfluidic device, and/or negative selection of blood cells, are thus being developed that do not rely on epithelial marker for CTC isolation, thus enriching untagged CTCs (Aceto et al., 2015; Alix-Panabieres et al., 2017).

EMT Enhances CTC Metastatic Competence

Considering that CTCs expressing mesenchymal attributes are commonly found in cancer patients in the light of the pro-metastatic properties provided by EMT programs, it has been suggested that these cells exhibiting hybrid phenotypes within the CTC population might be metastatic founders, so called MICs (metastasis initiating cells).

In accordance with this hypothesis, the presence of mesenchymally-shifted CTCs has been associated with poor clinical parameters in several studies as detailed in Table 1. Among the most frequently examined EMT markers in CTCs are EMT transcription factors (Twist, ZEB1, Snail, or Slug), vimentin, fibronectin, N-cadherin, PAI-1 (plasminogen activator inhibitor-1), c-MET (HGF receptor), or molecular actors of survival pathways (epidermal growth factor receptor [EGFR], Akt, PI3K). Regarding epithelial markers, E-cadherin and cytokeratins are commonly investigated. In the light of the finding that EMT features often correlate or even induce the expression of stem cell markers, CD44, ALDH1 or CD133 (promin 1) are also frequently examined. Thus, it was for instance shown that CTCs expressing the functional stem cell marker ALDH1A1 together with the EMT transcription factor Twist were more frequently detected in patients with metastatic breast cancer (Papadaki et al., 2014). Another study identified Plastin 3 as a good marker of EMT-shifted CTCs, which were shown to harbor a prognostic relevance (Yokobori et al., 2013). Bulfoni and coworkers also associated EMT traits in CTCs with poor prognosis in metastatic breast cancer patients (Bulfoni et al., 2016a).

The prevalence of mesenchymally-shifted CTCs is, however, hard to establish, varying with the tumor type, the stage of the disease and the markers analyzed. In an elegant study, Yu et al. (2013), examined several epithelial (Keratins 5, 7, 8, 18, 19, EpCAM, E-cadherin) and mesenchymal (N-cadherin, fibronectin, PAI-1) markers as probe pools in CTCs from breast cancer patients. They provided evidence of EMT both in rare cells within primary tumors and more abundantly in CTCs and further arranged CTC phenotypes along the E to M spectrum into five categories. In 17 breast cancer patients analyzed, 12 displayed more than 50% of CTCs with mesenchymal attributes. Only 4 patients had a higher percentage of epithelial CTCs than mesenchymal CTCs. The percentage of mesenchymal CTCs was the highest (between 80 and 100%) in the aggressive triple negative subtype of breast cancers. Serial monitoring of 11 patients also revealed an increase in the percentage of mesenchymal CTCs, leading the authors to suggest an association between these CTCs and disease progression. Comparing blood samples performed in cancer patients before and after therapy, the authors further revealed that patients who had progressive disease while on therapy displayed an increased number of mesenchymal CTCs in the post-treatment sample (Yu et al., 2013).

Although it is feasible that the hybrid state has become stably encoded through genetic/epigenetic selection, it seems likely that the frequent detection of hybrid CTC phenotypes associated with poor clinical parameters also reflects dynamic plasticity and higher ability of hybrid E/M phenotypes to better adapt to a selective environmental context. In support of this, although the authors did not specifically examine EMT markers, phenotypical interconversion between HER2 + and HER2- CTC subpopulations isolated from breast cancer patients, demonstrate the existence of a dynamic plasticity within the CTC population (Jordan et al., 2016).

In support of these data, functional observations further suggest that specific mesenchymal attributes may support CTC metastatic competence. Thus, adding to EMT-driven invasive properties that may contribute to CTC liberation from tumor masses, EMT is also likely to enhance CTC survival in the bloodstream and in colonized organs, and also to favor early metastatic niche formation.

EMT Increases CTC Survival and Early Colonization

During translocation in the bloodstream and the initial hours of metastatic colonization, CTCs are confronted to harsh selective pressure: they have to resist shear stress, modifications of cell–cell and cell–matrix contacts (inducing the specialized cell death program anoikis), and cytotoxic immune attack (particularly implicating natural killers, NK cells). Accordingly, several works identified a large number of apoptotic CTCs in cancer patients (Larson et al., 2004; Rossi et al., 2010) and, in some studies, a low percentage of apoptotic CTCs has been associated with poorer clinical parameters (Kallergi et al., 2013; Spiliotaki et al., 2014). For instance, Kallergi et al. detected apoptotic CTCs in patients with breast cancer irrespective of their clinical status, although the incidence of detection was higher in early compared with metastatic patients (Kallergi et al., 2013).

The mechanisms used by certain CTCs to survive in the bloodstream and during initial metastatic colonization are being better understood, and a role of EMT in conveying these specific properties is being recognized. EMT-associated properties endowing CTCs with enhanced survival/early colonizing potential may include the activation of survival pathways, the activation of the coagulation cascade, the formation of particular cytoskeletal structures (microtentacles), the evasion of particular immune checkpoints and cluster formation (Fig. 3). The contribution of EMT in supporting these mechanisms is discussed below.

Induction of Survival Pathways

An EMT-driven gain of resistance to apoptosis/anoikis has been widely reported in many cellular tumor cell systems (Bonnomet et al., 2010; Tiwari et al., 2012; Bonavida et al., 2013; Cao et al., 2016). For instance, silencing or enhancing the core transcription factors of EMT Snail, Slug, or Twist, have been shown to enhance or reduce resistance to apoptosis induced by various means (e.g. irradiation, cytotoxic drugs, serum deprivation,…) and in many tumor cell types (Kajita et al., 2004; Zhang et al., 2007; Escriva et al., 2008; Haddad et al., 2009; Kurrey et al., 2009; Vichalkovski et al., 2010; Zhang et al., 2010; Lim et al., 2013; Mariano et al., 2015; Roberts et al., 2016). In several studies, the positive impact of EMT on resistance to anoikis was more particularly examined (Onder et al., 2008; Smit et al., 2009; Howe et al., 2011; Kumar et al., 2011; Bao et al., 2013; Huang et al., 2013). Among others, the observed effects of EMT-induced resistance to apoptosis/anoikis include the activation of the Akt survival pathway, a diminution of caspase activity, an induction of Bcl2 and antagonizing of p53 activity. Along these lines, molecular actors of survival pathways (e.g., EGFR, Akt, PI3K,…) have been detected in CTCs, sometimes in association with more classical EMT markers such as EMT transcription factors or stem cell markers (Aktas et al., 2009; Kallergi et al., 2011; Barriere et al., 2012a, 2012b; Kasimir-Bauer et al., 2012; Hanssen et al., 2016; Todenhofer et al., 2016) (Table 1).

Expectedly and interestingly, the ability of EMT to induce tumor resistance to chemo or targeted therapies (resistance to EGFR inhibitors has been most often examined) has become a major research axis and this mechanism has been demonstrated in a variety of in vitro (Zhang et al., 2007, 2010; Haddad et al., 2009; Lim et al., 2013; Mariano et al., 2015; Roberts et al., 2016; Zhao et al., 2016b) and in vivo models (Fischer et al., 2015; Zheng et al., 2015), and also clinically (Thomson et al., 2005; Yauch et al., 2005; Du and Shim, 2016). More particularly, CTCs expressing mesenchymal markers have been associated with a resistance to chemo or targeted therapies (Mitra et al., 2015). This has been illustrated in different animal models (Fischer et al., 2015; Zheng et al., 2015) but also in cancer patients. For instance, Mego et al. reported that CTCs expressing EMT transcription factors (Twist1, Snail, Slug, ZEB1, and FOXC2 were examined in their study) in breast cancers are more resistant to neoadjuvant therapy (Mego et al., 2011). EMT and the stem cell marker ALDH1 on CTCs might also serve as an indication for therapy resistant tumor cell population (Aktas et al., 2009). An incremental expression of EMT-related genes in CTCs has also been associated with metastatic castration-resistant prostate cancers (Chen et al., 2013). It has thus been proposed that CTCs with EMT attributes could better resist chemo- or targeted therapy and would thus be related to relapse occurring after a period of remission.

Activation of Coagulation

Interestingly, increasing data also suggest an implication of coagulation actors in enhancing the ability of CTCs to survive and accomplish early metastatic colonization. The activation of coagulation has indeed long been correlated with malignancy and the beneficial impact of anticoagulants on cancer progression has been demonstrated in animal models and evaluated in clinical studies (Lee, 2010; Degen and Palumbo, 2012; Gil-Bernabe et al., 2013). The presence of CTCs has also been associated with increased risk of thromboembolism in metastatic breast cancer (Mego et al., 2009). More specifically, tissue factor (TF), a cell-associated activator of the coagulation cascade expressed by a variety of tumor cells, has emerged as a central player linking coagulation and cancer (Palumbo, 2008; Garnier et al., 2010; Ruf, 2012; Williams and Mackman, 2012; Cole and Bromberg, 2013; Gil-Bernabe et al., 2013; Ünlü and Versteeg, 2014).

Coagulation-dependent effects of TF on early survival and early metastasis have been demonstrated by the laboratory of Pr. Degen, who injected intravenously tumor cells modified for TF expression in mice with genetic defects in distal hemostatic factors (prothrombin and fibrinogen). They thus revealed that TF supports early metastasis through mechanisms dependent on these distal hemostatic factors (Palumbo and Degen, 2007; Palumbo et al., 2007; Palumbo, 2008; Degen and Palumbo, 2012). Recently, we linked TF expression and EMT and emphasized the importance of this regulatory axis on CTC survival (Bourcy et al., 2016). We indeed observed that growth factor induced-EMT triggers TF expression in several cell systems along with increased coagulant properties. Another group also reported enhanced TF expression in A431 cervical cancer cells induced to EMT through EGFR activation or E-cadherin blockade (Milsom et al., 2008). Further strengthening the link between EMT programs and coagulation, we reported that silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity. EMT-positive cells also exhibited a higher survival/persistence in lungs of mice colonized 24 hrs after intravenous injection, which was diminished by silencing of TF or ZEB1. Conversely, triggering de novo expression of Snail in MDA-MB-468 breast tumor cells was shown to increase TF, coagulant properties and early metastasis in mice. Tumor cells that persisted and stopped in the lungs were shown to be surrounded by fibrin and platelets. Supportively, using three-dimensional visualization of direct infusion of fluorescence labeled antibody to observe the interaction of tumor cells with platelets and fibrin(ogen) in isolated lung preparations, Im and co-workers also observed that tumor cells arrested in the pulmonary vasculature were associated with a clot composed of both platelets and fibrin(ogen) (Im et al., 2004). Thus, EMT-driven local coagulation around EMT-positive CTCs could thus generate a fibrin/platelet rich microclot around these CTCs. This fibrin-rich network could constitute a protective shield enhancing CTC survival by physically protecting the CTCs against shear stress or NK-mediated clearance, or by providing an anchoring matrix that would minimize anoikis.

Accordingly, a mechanism involving a protective role of fibrin and/or platelets in NK-induced cytotoxicity against tumor cells has been proposed based on in vitro observations and on preclinical experiments in mice using a variety of mouse and human carcinoma cell lines (Palumbo et al., 2000, 2005; Stegner et al., 2014; Lou et al., 2015; Leblanc and Peyruchaud, 2016; Li et al., 2016). Although platelet binding to CTCs may involve direct interactions, fibrin has also been shown to enhance adherence between platelets and tumor cells and to inhibit cytotoxicity against tumor cells (Biggerstaff et al., 1999, 2008). Palumbo et al. further showed that fibrin(ogen)-dependent and platelet-dependent evasion of NK cell-mediated clearance of early micrometastases rely on TF expression by tumor cells, although they clearly demonstrated that NK-independent mechanisms are also involved in TF-mediated metastasis (Palumbo et al., 2007). Considering our findings linking TF expression, coagulation and EMT with these observations, it is conceivable that EMT-shifted CTCs may display enhanced potential to resist immune clearance.

Along these lines and providing clinical relevance for the involvement of an EMT-driven local activation of the coagulation cascade around the EMT-shifted phenotypes, our group identified a subpopulation of CTCs expressing vimentin and TF in the blood of metastatic breast cancer patients (Bourcy et al., 2016).

Formation of Specific Protective Cytoskeletal Structures (Microtentacles)

Another mechanism conveyed by EMT that may facilitate resistance to anoikis and the initial colonization of distant organs involves the formation of particular cytoskeletal structures (Matrone et al., 2010). Thus, it has been proposed that free-floating CTCs display cytoskeletal reorganization compared with tumor cells attached to a matrix, which help them resist to shear stress and anoikis. This mechanism involves the formation of so-called microtentacles. These structures are built of Glu-tubulin (generating more stable detyrosinated microtubules) and are enhanced by vimentin, one of the most best-known markers of EMT (Whipple et al., 2008). Further implicating EMT in the process, Snail and Twist were also shown to regulate these structures in tumor cells (Whipple et al., 2010). Additionally, microtentacles have been reported to infiltrate the junctions between endothelial cells, suggesting that they may also facilitate early colonization by enhancing the reattachment of CTCs to the vascular endothelium (Whipple et al., 2010).

Immune Escape

The expression of programmed death-ligand 1 (PD-L1) at the surface of tumor cells is considered to play a pivotal role in the ability of tumor cells to escape elimination by the immune system. Indeed, PD-L1 and its programmed death receptor (PD-1) constitute a physiological immune checkpoint system. Thus, PD-L1 is expressed on antigen-presenting cells and PD-1 is expressed at the surface of activated T-cells. Upon binding of the ligand to the receptor, a strong proliferative inhibitory signal is sent to the T-cells. By expressing PD-L1, tumor cells have been shown to hijack this recognition mechanism to escape elimination by the immune system. Recently, PD-L1 has been detected on the surface of CTCs of breast cancer patients (Mazel et al., 2015), non small cell lung cancer patients (Nicolazzo et al., 2016), or bladder cancer patients (Anantharaman et al., 2016) and has been proposed as a mechanism enhancing CTC immune escape and survival (Wang et al., 2016; Alix-Panabieres et al., 2017). Independent studies have revealed that EMT pathways, such as the miR200/ZEB1 axis or the EMT-induced PI3K/Akt pathway, regulate PD-L1 in vitro (Chen et al., 2014; Alsuliman et al., 2015). An association between PD-L1 expression and EMT markers has also been observed in breast cancer or head and neck squamous cell carcinoma (Alsuliman et al., 2015; Ock et al., 2016). It is thus conceivable that CTCs with an EMT-shifted phenotype would be more efficient in hijacking this PD-1/PD-L1 immune escape system.

Although it has not been specifically examined in CTCs, it is interesting to note that other molecular mechanisms may be used by EMT-shifted cells to resist immune attack (Akalay et al., 2013; Tripathi et al., 2016). For instance, a depleted repertoire of HLA class I-bound peptides has been reported in mesenchymally-shifted non small cell lung carcinoma cells, thereby increasing their resistance to cytotoxic T lymphocytes (Tripathi et al., 2016).

Formation of Clusters of CTCs

The ability of CTCs to form and/or travel as homotypic or heterotypic (together with platelets, blood cells, or normal cells from the primary site such as fibroblasts and/or endothelial cells,…) clusters has also been proposed as a mechanism enhancing their metastatic competence (Duda et al., 2010; Aceto et al., 2015; Cheung et al., 2016; Pothula et al., 2016), although the prevalence of hybrid E/M phenotypes in these structures is unclear. Such aggregates could indeed enhance physical protection of CTCs during their translocation in the circulation but also facilitate the attachment of CTCs to blood vessels and niche formation. Although much less prevalent than isolated CTCs, CTC clusters have been suggested to harbor high metastatic potential (Aceto et al., 2015). Accordingly, their presence has been associated with a poor prognosis in several cancer types including breast, lung, and prostate cancers (Hou et al., 2012; Aceto et al., 2014; Paoletti et al., 2015; Wang et al., 2017a).

An early report by Glinsky and co-workers (Glinsky et al., 2003) showed in vitro, ex vivo, and in vivo that metastatic breast and prostate carcinoma cells form multicellular homotypic aggregates at the sites of their primary attachment to the endothelium. Alternatively, a study using fluorescently tagged tumor cell xenograft models, suggests that CTC clusters do not form within the circulation but are derived from grouping tumor cells that enter the circulation together (Aceto et al., 2014). These authors also revealed that ex vivo formed clusters are highly metastatic when injected intravenously in mice and that their metastatic potential relies on the expression of the cell–cell adhesion molecule plakoglobin. Plakoglobin was accordingly observed by these authors in CTC clusters in metastatic breast cancer patients (Aceto et al., 2014). Although the authors report that cell–cell contacts are present in these clusters, the EMT status in these entities is still underexplored (Aceto et al., 2015).

In a model of cell tracing in a MMTV-PyMT mouse background, Cheung et al. (2016) also observed polyclonal tumor cell clusters at different stages of metastasis including circulating tumor cell clusters. They further reported that cells in the clusters frequently expressed the epithelial marker keratin 14 and further demonstrated a functional role of K14 expression in metastasis. Interestingly, transcriptomic analyses revealed that the K14 + population not only expressed epithelial markers (several cell–cell adhesion molecules) but was also enriched for EMT/stemness markers such as Tenascin C or Jagged1 (Cheung et al., 2016). Along the same lines, an in vitro study has shown that the Notch-Jagged1 signaling pathway is involved in the formation of clusters exhibiting a hybrid E/M phenotype (Boareto et al., 2016).

Directly supporting the idea that circulating tumor emboli are made of cells expressing hybrid E/M phenotypes, the study of Yu et al. (2013) identified cells expressing both epithelial (such as EpCAM or cytokeratins) and mesenchymal markers (including fibronectin, N-cadherin or PAI-1) in isolated CTCs but also in CTC clusters from breast cancer patients. Vimentin expression has also been observed in circulating tumor microemboli detected in lung cancer patients (Hou et al., 2011). Because, as we discussed above, EMT contributes through diverse mechanisms to enhance CTC survival (activation of survival pathway, microtentacles, coagulation), the presence of EMT-shifted CTCs in clusters combined with the survival advantage of cell–cell interactions, would very likely overall enhance the survival of the clustered tumor cells (Aceto et al., 2015). Also, the possibility that activation of the coagulation cascade may contribute to the formation or the survival of the clusters cannot be excluded. Supportively, CTC clusters identified in breast cancer patients showed an abundance of attached platelets (Yu et al., 2013).

EMT Supports Metastatic niche Formation

Regardless of the suspected involvement of a MET in the final metastatic outgrowth, data suggest that CTCs expressing mesenchymal and cancer stem cell attributes harbor enhanced competence to accomplish the very early colonization steps and the formation of metastatic niches. CTC homing in colonized organs (thus becoming disseminated tumor cells, DTCs) involves interactions with the endothelium and the establishment of a favorable microenvironment, a metastatic niche, that will sustain their growth after a possible period of dormancy. The molecular and cellular entities implicated, and the timing of metastatic niche formation, are still poorly understood but are being characterized. Although niche formation is likely to implicate a repertoire of cellular and molecular actors varying upon the context (Descot and Oskarsson, 2013), it is today clear that a particular matrix is created in the niche and that host cells, including immune cells and other stromal cells (activated fibroblasts, mesenchymal stem cells, endothelial cells,…) are recruited and are essential to the formation of the niche (Ombrato and Malanchi, 2014; Plaks et al., 2015). Interactions between the CTCs/DTCs and the niche microenvironment are determinant for the survival and the adaptation of the CTCs.

Thus, the EMT-associated cancer stem cell attributes and activation of the cell surviving mechanisms listed above are likely to cooperate to sustain self-renewal, survival, possible dormancy and therapeutic resistance of DTCs. Adding to this, some of these mechanisms may also directly contribute to the establishment of the niche and the recruitment of host cells. Therefore, if coagulation is suspected to protect CTCs during their travel in the bloodstream and during early hours of colonization, published literature also implicates coagulation in early phases of niche formation (Palumbo and Degen, 2007; Palumbo, 2008; Degen and Palumbo, 2012; Gil-Bernabe et al., 2013). Thus, both fibrin(ogen) and platelets may guide the formation of early metastatic niche by regulating CTC interactions with the endothelial wall, and platelets were also shown to enhance the recruitment of inflammatory cells into the niche (Im et al., 2004; Labelle and Hynes, 2012; Labelle et al., 2014; Lou et al., 2015; Leblanc and Peyruchaud, 2016; Tesfamariam, 2016). Importantly, TF was shown to enhance tumor cell survival after arrest in the lung during experimental lung metastasis and to stimulate metastatic niche formation by recruiting macrophages (Gil-Bernabe et al., 2012). Although the studies mentioned above have not directly examined a potential contribution of EMT, it is conceivable that EMT, by enhancing TF expression and the formation of microclots around CTCs, could directly modulate these processes.

Directly evidencing the importance of specific mesenchymal attributes of MICs in the establishment of the metastatic niche, an EMT axis modulated by the Axl receptor tyrosine kinase (RTK), has been shown to play a key role in the early establishment of metastatic niches by activating fibroblasts in the colonized organs as a result of thrombospondin 2 secretion (Ombrato and Malanchi, 2014; Del Pozo Martin et al., 2015). The authors demonstrated that the cancer cell acquired Axl-driven mesenchymal features facilitate fibroblast activation and the first phase of a metastatic niche formation. They then identified a second phase of metastatic colonization in which the activated stroma modulates the EMT of cancer cells toward a more epithelial state with loss of Axl and EMT markers expression. Additionally, using a model of MMTV-Her2 mice, Aguirre-Ghiso's group showed that HER2 + DTCs activate a Wnt-dependent EMT dissemination program without the complete loss of an epithelial phenotype. They further reported that these early disseminated cancer cells expressing Twist1 and a low level of E-cadherin are capable of forming metastasis after a dormancy phase (Harper et al., 2016). In agreement with other studies (Ocana et al., 2012; Tsai et al., 2012), these findings are consistent with the concept that mesenchymal attributes of hybrid MIC phenotypes are required for metastatic niche initiation and an eventual dormancy phase while a dynamic EMT inhibition (MET) at the secondary site occurs within the second phase of metastatic colonization (Bednarz-Knoll et al., 2012; Brabletz, 2012; Tsai and Yang, 2013; Ombrato and Malanchi, 2014).

In line with these experimental observations, few reports have analyzed EMT markers in DTC from the bone marrow of cancer patients to examine their potential hybrid phenotype. So far, data have been essentially collected from cell lines established from DTCs isolated from bone marrow of cancer patients demonstrating the co-existence of epithelial (keratins, EpCAM) and mesenchymal/stem cell markers (vimentin, PAI-1, Twist, CD-44) (Willipinski-Stapelfeldt et al., 2005; Balic et al., 2006; Watson et al., 2007; Bartkowiak et al., 2009).

Functional Assays Reveal that MICs are Present in the Blood of Cancer Patients

The data reported so far thus support the idea that CTCs displaying hybrid phenotypes with mesenchymal and stem cell attributes would be those with enhanced metastatic competence. Ultimately, demonstrating whether MICs are present in CTCs of cancer patients relies on the development of assays allowing examination of the functional metastatic competence of CTCs (Alix-Panabieres et al., 2016). Thus, several in vitro cultures and assays have been developed to examine viability, proliferative, or invasive properties of CTCs isolated from cancer patients (Gao et al., 2014; Martin et al., 2014; Khoo et al., 2015, 2016; Alix-Panabieres et al., 2016). Nevertheless, the most obvious assay with which to examine the tumorigenic and metastatic competence of CTCs is the CTC-derived xenograft assay (CDX), consisting in injecting different subpopulations of CTCs isolated from cancer patients in immunodeficient mice (Hodgkinson et al., 2014; Yu et al., 2014; Cayrefourcq et al., 2015; Alix-Panabieres et al., 2016). Some authors also focused on examining CTC metastatic competence after intravenous injection of CTCs. Another variation consists in culturing and expanding the CTCs in vitro before injection in the animal. Using such in vivo assays, the presence of MICs within the CTC population of different types of cancers has been demonstrated (Rossi et al., 2014; Sullivan et al., 2014; Alix-Panabieres et al., 2016). Although further investigations and more extensive phenotyping are required, few studies more particularly support a potential relationship between hybrid E/M CTC phenotypes and metastatic competence. Thus, Zhang et al. established cell lines from breast cancer-derived, EpCAM-negative CTCs that expressed hybrid phenotypes with the expression of epithelial, mesenchymal and cancer stem cell markers (cytokeratins 8 and 18, vimentin, CD44) and that displayed metastatic competence after tail-vein or intracardiac injection (Zhang et al., 2013). They further established a molecular signature (HER2+/EGFR+/HPSE+/Notch1+) that accompanied enhanced metastatic competency in the brain. Injecting CTCs isolated from patients with metastatic breast cancer into the femur of immunocompromised mice, Baccelli et al. also demonstrated the existence of MICs that gave rise to bone, lung, and liver metastases. These MICs were found to express EpCAM, CD44, CD47, and c-MET (HGF receptor) (Baccelli et al., 2013). Additionally, the authors reported that the levels of CD44+/c-MET+/CD47 + CTCs, but not bulk CTCs, correlated with lower overall survival and increased number of metastatic sites.

Concluding Remarks

CTCs with hybrid epithelial/mesenchymal phenotypes and expressing stem cell markers are present in the blood of cancer patients harboring epithelial tumors. The detection of mesenchymally-shifted CTCs has further been associated with poor clinical parameters in several studies, stimulating their more systematic detection for predictive/prognostic perspectives. Adding to this, literature accumulates that functionally implicates EMT in the biology of CTCs and in the early phases of the metastatic spread. EMT may thus contribute to the liberation of CTCs into the bloodstream and provide CTCs with enhanced survival properties and increased ability to initiate metastatic niches. It has been suggested that the CTCs harboring mesenchymal attributes are those with an enhanced metastatic competence, so called MICs. Nevertheless, there is a repertoire of epithelial plasticity and a multiplicity of potentially metastable hybrid phenotypes. There is thus a clear need to identify specific molecular mediators of EMP that participate functionally in specific stages of the metastatic spread, so as to further refine therapeutic strategies.

Acknowledgments

M-E. Francart, J. Lambert, and A.M. Vanwynsberghe are supported by Télévie grants (Belgium). E.W. Thompson was supported in part by the National Breast Cancer Foundation (Australia). C. Gilles is a Senior Research Associates at the F.R.S-FNRS (Belgium). The Translational Research Institute is supported by a grant from the Australian government.

References

Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA, Yu M, Pely A, Engstrom A, Zhu H, Brannigan BW, Kapur R, Stott SL, Shioda T, Ramaswamy S, Ting DT, Lin CP, Toner M, Haber DA, Maheswaran S. 2014. Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158: 1110–1122.
Aceto N, Toner M, Maheswaran S, Haber DA. 2015. En route to metastasis: circulating tumor cell clusters and epithelial-to-mesenchymal transition. Trends Cancer 1: 44–52.
Akalay I, Janji B, Hasmim M, Noman MZ, Andre F, De Cremoux P, Bertheau P, Badoual C, Vielh P, Larsen AK, Sabbah M, Tan TZ, Keira JH, Hung NT, Thiery JP, Mami-Chouaib F, Chouaib S. 2013. Epithelial-to-mesenchymal transition and autophagy induction in breast carcinoma promote escape from T-cell-mediated lysis. Cancer Res 73: 2418–2427.
Aktas B, Tewes M, Fehm T, Hauch S, Kimmig R, Kasimir-Bauer S. 2009. Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients. Breast Cancer Res 11: R46.
Alix-Panabieres C, Bartkowiak K, Pantel K. 2016. Functional studies on circulating and disseminated tumor cells in carcinoma patients. Mol Oncol 10: 443–449.
Alix-Panabieres C, Mader S, Pantel K. 2017. Epithelial-mesenchymal plasticity in circulating tumor cells. J Mol Med (Berl) 95: 133–142.
Alix-Panabieres C, Pantel K. 2016. Clinical applications of circulating tumor cells and circulating tumor DNA as liquid biopsy. Cancer Discov 6: 479–491.
Alix-Panabières C, Pantel K. 2014. Technologies for detection of circulating tumor cells: facts and vision. Lab Chip 14: 57–62.
Alonso-Alconada L, Muinelo-Romay L, Madissoo K, Diaz-Lopez A, Krakstad C, Trovik J, Wik E, Hapangama D, Coenegrachts L, Cano A, Gil-Moreno A, Chiva L, Cueva J, Vieito M, Ortega E, Mariscal J, Colas E, Castellvi J, Cusido M, Dolcet X, Nijman HW, Bosse T, Green JA, Romano A, Reventos J, Lopez-Lopez R, Salvesen HB, Amant F, Matias-Guiu X, Moreno-Bueno G, Abal M. 2014. Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer. Mol Cancer 13: 223.
Alpaugh ML, Tomlinson JS, Kasraeian S, Barsky SH. 2002. Cooperative role of E-cadherin and sialyl-Lewis X/A-deficient MUC1 in the passive dissemination of tumor emboli in inflammatory breast carcinoma. Oncogene 21: 3631–3643.
Alsuliman A, Colak D, Al-Harazi O, Fitwi H, Tulbah A, Al-Tweigeri T, Al-Alwan M, Ghebeh H. 2015. Bidirectional crosstalk between PD-L1 expression and epithelial to mesenchymal transition: significance in claudin-low breast cancer cells. Mol Cancer 14: 149.
Anantharaman A, Friedlander T, Lu D, Krupa R, Premasekharan G, Hough J, Edwards M, Paz R, Lindquist K, Graf R, Jendrisak A, Louw J, Dugan L, Baird S, Wang Y, Dittamore R, Paris PL. 2016. Programmed death-ligand 1 (PD-L1) characterization of circulating tumor cells (CTCs) in muscle invasive and metastatic bladder cancer patients. BMC Cancer 16: 744.
Ansieau S. 2013. EMT in breast cancer stem cell generation. Cancer Lett 338: 63–68.
Armstrong AJ, Marengo MS, Oltean S, Kemeny G, Bitting RL, Turnbull JD, Herold CI, Marcom PK, George DJ, Garcia-Blanco MA. 2011. Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers. Mol.Cancer Res 9: 997–1007.
Baccelli I, Schneeweiss A, Riethdorf S, Stenzinger A, Schillert A, Vogel V, Klein C, Saini M, Bäuerle T, Wallwiener M, Holland-Letz T, Höfner T, Sprick M, Scharpff M, Marmé F, Sinn HP, Pantel K, Weichert W, Trumpp A. 2013. Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nat Biotechnol 31: 539–544.
Balic M, Lin H, Young L, Hawes D, Giuliano A, McNamara G, Datar RH, Cote RJ. 2006. Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin Cancer Res 12: 5615–5621.
Bao W, Qiu H, Yang T, Luo X, Zhang H, Wan X. 2013. Upregulation of TrkB promotes epithelial-mesenchymal transition and anoikis resistance in endometrial carcinoma. PLoS One 8: e70616.
Barbazan J, Muinelo-Romay L, Vieito M, Candamio S, Diaz-Lopez A, Cano A, Gomez-Tato A, Casares de Cal Mde L, Abal M, Lopez-Lopez R. 2014. A multimarker panel for circulating tumor cells detection predicts patient outcome and therapy response in metastatic colorectal cancer. Int J Cancer 135: 2633–2643.
Barriere G, Fici P, Gallerani G, Fabbri F, Zoli W, Rigaud M. 2014. Circulating tumor cells and epithelial, mesenchymal and stemness markers: characterization of cell subpopulations. Ann Transl Med 2: 109.
Barriere G, Riouallon A, Renaudie J, Tartary M, Rigaud M. 2012a. Mesenchymal and stemness circulating tumor cells in early breast cancer diagnosis. BMC Cancer 12: 114.
Barriere G, Riouallon A, Renaudie J, Tartary M, Rigaud M. 2012b. Mesenchymal characterization: alternative to simple CTC detection in two clinical trials. Anticancer Res 32: 3363–3369.
Bartkowiak K, Wieczorek M, Buck F, Harder S, Moldenhauer J, Effenberger KE, Pantel K, Peter-Katalinic J, Brandt BH. 2009. Two-dimensional differential gel electrophoresis of a cell line derived from a breast cancer micrometastasis revealed a stem/ progenitor cell protein profile. J Proteome Res 8: 2004–2014.
Bednarz-Knoll N, Alix-Panabieres C, Pantel K. 2012. Plasticity of disseminating cancer cells in patients with epithelial malignancies. Cancer Metastasis Rev 31: 673–687.
Beerling E, Seinstra D, de Wit E, Kester L, van der Velden D, Maynard C, Schafer R, van Diest P, Voest E, van Oudenaarden A, Vrisekoop N, van Rheenen J. 2016. Plasticity between epithelial and mesenchymal states unlinks EMT from metastasis-enhancing stem cell capacity. Cell Rep 14: 2281–2288.
Bhat-Nakshatri P, Appaiah H, Ballas C, Pick-Franke P, Goulet R, Jr., Badve S, Srour EF, Nakshatri H. 2010. SLUG/SNAI2 and tumor necrosis factor generate breast cells with CD44+/CD24- phenotype. BMC Cancer 10: 411.
Biggerstaff JP, Seth N, Amirkhosravi A, Amaya M, Fogarty S, Meyer TV, Siddiqui F, Francis JL. 1999. Soluble fibrin augments platelet/tumor cell adherence in vitro and in vivo, and enhances experimental metastasis. Clin Exp Metastasis 17: 723–730.
Biggerstaff JP, Weidow B, Dexheimer J, Warnes G, Vidosh J, Patel S, Newman M, Patel P. 2008. Soluble fibrin inhibits lymphocyte adherence and cytotoxicity against tumor cells: implications for cancer metastasis and immunotherapy. Clin Appl Thromb Hemost 14: 193–202.
Boareto M, Jolly MK, Goldman A, Pietila M, Mani SA, Sengupta S, Ben-Jacob E, Levine H, Onuchic JN. 2016. Notch-Jagged signalling can give rise to clusters of cells exhibiting a hybrid epithelial/mesenchymal phenotype. J R Soc Interface 13: 20151106.
Bock C, Rack B, Huober J, Andergassen U, Jeschke U, Doisneau-Sixou S. 2014. Distinct expression of cytokeratin, N-cadherin and CD133 in circulating tumor cells of metastatic breast cancer patients. Future Oncol 10: 1751–1765.
Bockhorn M, Jain RK, Munn LL. 2007. Active versus passive mechanisms in metastasis: do cancer cells crawl into vessels, or are they pushed? Lancet Oncol 8: 444–448.
Bonavida B, Jazirehi A, Vega MI, Huerta-Yepez S, Baritaki S. 2013. Roles each of Snail, Yin Yang 1 and RKIP in the regulation of tumor cells chemo-immuno-resistance to apoptosis. For Immunopathol Dis Therap 4: 79–92.
Bonnomet A, Brysse A, Tachsidis A, Waltham M, Thompson EW, Polette M, Gilles C. 2010. Epithelial-to-mesenchymal transitions and circulating tumor cells. J Mammary Gland Biol Neoplasia 15: 261–273.
Bonnomet A, Syne L, Brysse A, Feyereisen E, Thompson EW, Noel A, Foidart JM, Birembaut P, Polette M, Gilles C. 2012. A dynamic in vivo model of epithelial-to-mesenchymal transitions in circulating tumor cells and metastases of breast cancer. Oncogene 31: 3741–3753.
Bourcy M, Suarez-Carmona M, Lambert J, Francart ME, Schroeder H, Delierneux C, Skrypek N, Thompson EW, Jerusalem G, Berx G, Thiry M, Blacher S, Hollier BG, Noel A, Oury C, Polette M, Gilles C. 2016. Tissue factor induced by epithelial-mesenchymal transition triggers a pro-coagulant state that drives metastasis of circulating tumor cells. Cancer Res 76: 4270–4282.
Brabletz T. 2012. To differentiate or not — routes towards metastasis. Nat Rev Cancer 12: 425–436.
Bulfoni M, Gerratana L, Del Ben F, Marzinotto S, Sorrentino M, Turetta M, Scoles G, Toffoletto B, Isola M, Beltrami CA, Di Loreto C, Beltrami AP, Puglisi F, Cesselli D. 2016a. In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis. Breast Cancer Res 18: 30.
Bulfoni M, Turetta M, Del Ben F, Di Loreto C, Beltrami AP, Cesselli D. 2016b. Dissecting the heterogeneity of circulating tumor cells in metastatic breast cancer: going far beyond the needle in the haystack. Int J Mol Sci 17:pii: E1775.
Cao Z, Livas T, Kyprianou N. 2016. Anoikis and EMT: lethal “liaisons” during cancer progression. Crit Rev Oncog 21: 155–168.
Cayrefourcq L, Mazard T, Joosse S, Solassol J, Ramos J, Assenat E, Schumacher U, Costes V, Maudelonde T, Pantel K, Alix-Panabieres C. 2015. Establishment and characterization of a cell line from human circulating colon cancer cells. Cancer Res 75: 892–901.
Celia-Terrassa T, Kang Y. 2016. Distinctive properties of metastasis-initiating cells. Genes Dev 30: 892–908.
Celia-Terrassa T, Meca-Cortes O, Mateo F, Martinez de Paz A, Rubio N, Arnal-Estape A, Ell BJ, Bermudo R, Diaz A, Guerra-Rebollo M, Lozano JJ, Estaras C, Ulloa C, Alvarez-Simon D, Mila J, Vilella R, Paciucci R, Martinez-Balbas M, de Herreros AG, Gomis RR, Kang Y, Blanco J, Fernandez PL, Thomson TM. 2012. Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells. J Clin Invest 122: 1849–1868.
Chaffer CL, San Juan BP, Lim E, Weinberg RA. 2016. EMT, cell plasticity and metastasis. Cancer Metastasis Rev 35: 645–654.
Chen CL, Mahalingam D, Osmulski P, Jadhav RR, Wang CM, Leach RJ, Chang TC, Weitman SD, Kumar AP, Sun L, Gaczynska ME, Thompson IM, Huang TH. 2013. Single-cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT-related genes in metastatic prostate cancer. Prostate 73: 813–826.
Chen L, Gibbons DL, Goswami S, Cortez MA, Ahn YH, Byers LA, Zhang X, Yi X, Dwyer D, Lin W, Diao L, Wang J, Roybal JD, Patel M, Ungewiss C, Peng D, Antonia S, Mediavilla-Varela M, Robertson G, Jones S, Suraokar M, Welsh JW, Erez B, Wistuba II, Chen L, Peng D, Wang S, Ullrich SE, Heymach JV, Kurie JM, Qin FX. 2014. Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression. Nat Commun 5: 5241.
Cheung KJ, Padmanaban V, Silvestri V, Schipper K, Cohen JD, Fairchild AN, Gorin MA, Verdone JE, Pienta KJ, Bader JS, Ewald AJ. 2016. Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters. Proc Natl Acad Sci U S A 113: E854–E863.
Chiang SP, Cabrera RM, Segall JE. 2016. Tumor cell intravasation. Am J Physiol Cell Physiol 311: C1–C14.
Cho EH, Wendel M, Luttgen M, Yoshioka C, Marrinucci D, Lazar D, Schram E, Nieva J, Bazhenova L, Morgan A, Ko AH, Korn WM, Kolatkar A, Bethel K, Kuhn P. 2012. Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors. Phys Biol 9: 016001.
Cierna Z, Mego M, Janega P, Karaba M, Minarik G, Benca J, Sedlackova T, Cingelova S, Gronesova P, Manasova D, Pindak D, Sufliarsky J, Danihel L, Reuben JM, Mardiak J. 2014. Matrix metalloproteinase 1 and circulating tumor cells in early breast cancer. BMC Cancer 14: 472.
Cole M, Bromberg M. 2013. Tissue factor as a novel target for treatment of breast cancer. Oncologist 18: 14–18.
De Craene B, Berx G. 2013. Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer 13: 97–110.
Degen JL, Palumbo JS. 2012. Hemostatic factors, innate immunity and malignancy. Thromb Res 129(suppl 1): S1–S5.
Del Pozo Martin Y, Park D, Ramachandran A, Ombrato L, Calvo F, Chakravarty P, Spencer-Dene B, Derzsi S, Hill CS, Sahai E, Malanchi I. 2015. Mesenchymal cancer cell-stroma crosstalk promotes niche activation, epithelial reversion, and metastatic colonization. Cell Rep 13: 2456–2469.
Descot A, Oskarsson T. 2013. The molecular composition of the metastatic niche. Exp Cell Res 319: 1679–1686.
Diepenbruck M, Christofori G. 2016. Epithelial-mesenchymal transition (EMT) and metastasis: yes, no, maybe? Curr Opin Cell Biol 43: 7–13.
Drake JM, Strohbehn G, Bair TB, Moreland JG, Henry MD. 2009. ZEB1 enhances transendothelial migration and represses the epithelial phenotype of prostate cancer cells. Mol Biol Cell 20: 2207–2217.
Du B, Shim JS. 2016. Targeting epithelial-mesenchymal transition (EMT) to overcome drug resistance in cancer. Molecules 21: 965.
Duda DG, Duyverman AM, Kohno M, Snuderl M, Steller EJ, Fukumura D, Jain RK. 2010. Malignant cells facilitate lung metastasis by bringing their own soil. Proc Natl Acad Sci U S A 107: 21677–21682.
Escriva M, Peiro S, Herranz N, Villagrasa P, Dave N, Montserrat-Sentis B, Murray SA, Franci C, Gridley T, Virtanen I, Garcia de Herreros A. 2008. Repression of PTEN phosphatase by Snail1 transcriptional factor during gamma radiation-induced apoptosis. Mol Cell Biol 28: 1528–1540.
Fabregat I, Malfettone A, Soukupova J. 2016. New insights into the crossroads between EMT and stemness in the context of cancer. J Clin Med 5: 37.
Fidler IJ. 1973. The relationship of embolic homogeneity, number, size and viability to the incidence of experimental metastasis. Eur J Cancer 9: 223–227.
Fischer KR, Durrans A, Lee S, Sheng J, Li F, Wong ST, Choi H, El Rayes T, Ryu S, Troeger J, Schwabe RF, Vahdat LT, Altorki NK, Mittal V, Gao D. 2015. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature 527: 472–476.
Friedl P, Gilmour D. 2009. Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol 10: 445–457.
Friedlander TW, Ngo VT, Dong H, Premasekharan G, Weinberg V, Doty S, Zhao Q, Gilbert EG, Ryan CJ, Chen WT, Paris PL. 2014. Detection and characterization of invasive circulating tumor cells derived from men with metastatic castration-resistant prostate cancer. Int J Cancer 134: 2284–2293.
Frisch SM, Schaller M, Cieply B. 2013. Mechanisms that link the oncogenic epithelial-mesenchymal transition to suppression of anoikis. J Cell Sci 126: 21–29.
Gao D, Vela I, Sboner A, Iaquinta PJ, Karthaus WR, Gopalan A, Dowling C, Wanjala JN, Undvall EA, Arora VK, Wongvipat J, Kossai M, Ramazanoglu S, Barboza LP, Di W, Cao Z, Zhang QF, Sirota I, Ran L, MacDonald TY, Beltran H, Mosquera JM, Touijer KA, Scardino PT, Laudone VP, Curtis KR, Rathkopf DE, Morris MJ, Danila DC, Slovin SF, Solomon SB, Eastham JA, Chi P, Carver B, Rubin MA, Scher HI, Clevers H, Sawyers CL, Chen Y. 2014. Organoid cultures derived from patients with advanced prostate cancer. Cell 159: 176–187.
Garnier D, Milsom C, Magnus N, Meehan B, Weitz J, Yu J, Rak J. 2010. Role of the tissue factor pathway in the biology of tumor initiating cells. Thromb Res 125(suppl 2): S44–S50.
Giampieri S, Manning C, Hooper S, Jones L, Hill CS, Sahai E. 2009. Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility. Nat Cell Biol 11: 1287–1296.
Giampieri S, Pinner S, Sahai E. 2010. Intravital imaging illuminates transforming growth factor beta signaling switches during metastasis. Cancer Res 70: 3435–3439.
Gil-Bernabe AM, Ferjancic S, Tlalka M, Zhao L, Allen PD, Im JH, Watson K, Hill SA, Amirkhosravi A, Francis JL, Pollard JW, Ruf W, Muschel RJ. 2012. Recruitment of monocytes/macrophages by tissue factor-mediated coagulation is essential for metastatic cell survival and premetastatic niche establishment in mice. Blood 119: 3164–3175.
Gil-Bernabe AM, Lucotti S, Muschel RJ. 2013. Coagulation and metastasis: what does the experimental literature tell us? Br J Haematol 162: 433–441.
Giordano A, Gao H, Anfossi S, Cohen E, Mego M, Lee BN, Tin S, De LM, Parker CA, Alvarez RH, Valero V, Ueno NT, De PS, Mani SA, Esteva FJ, Cristofanilli M, Reuben JM. 2012. Epithelial-mesenchymal transition and stem cell markers in patients with HER2-positive metastatic breast cancer. Mol Cancer Ther 11: 2526–2534.
Glinsky VV, Glinsky GV, Glinskii OV, Huxley VH, Turk JR, Mossine VV, Deutscher SL, Pienta KJ, Quinn TP. 2003. Intravascular metastatic cancer cell homotypic aggregation at the sites of primary attachment to the endothelium. Cancer Res 63: 3805–3811.
Gradilone A, Raimondi C, Nicolazzo C, Petracca A, Gandini O, Vincenzi B, Naso G, Agliano AM, Cortesi E, Gazzaniga P. 2011. Circulating tumor cells lacking cytokeratin in breast cancer: the importance of being mesenchymal. J Cell Mol Med 15: 1066–1070.
Gunasinghe NP, Wells A, Thompson EW, Hugo HJ. 2012. Mesenchymal-epithelial transition (MET) as a mechanism for metastatic colonisation in breast cancer. Cancer Metastasis Rev 31: 469–478.
Haddad Y, Choi W, McConkey DJ. 2009. Delta-crystallin enhancer binding factor 1 controls the epithelial to mesenchymal transition phenotype and resistance to the epidermal growth factor receptor inhibitor erlotinib in human head and neck squamous cell carcinoma lines. Clin Cancer Res 15: 532–542.
Hanssen A, Wagner J, Gorges TM, Taenzer A, Uzunoglu FG, Driemel C, Stoecklein NH, Knoefel WT, Angenendt S, Hauch S, Atanackovic D, Loges S, Riethdorf S, Pantel K, Wikman H. 2016. Characterization of different CTC subpopulations in non-small cell lung cancer. Sci Rep 6: 28010.
Harper KL, Sosa MS, Entenberg D, Hosseini H, Cheung JF, Nobre R, Avivar-Valderas A, Nagi C, Girnius N, Davis RJ, Farias EF, Condeelis J, Klein CA, Aguirre-Ghiso JA. 2016. Mechanism of early dissemination and metastasis in Her2 + mammary cancer. Nature 540: 588–592.
Hensler M, Vancurova I, Becht E, Palata O, Strnad P, Tesarova P, Cabinakova M, Svec D, Kubista M, Bartunkova J, Spisek R, Sojka L. 2016. Gene expression profiling of circulating tumor cells and peripheral blood mononuclear cells from breast cancer patients. Oncoimmunology 5: e1102827.
Hodgkinson CL, Morrow CJ, Li Y, Metcalf RL, Rothwell DG, Trapani F, Polanski R, Burt DJ, Simpson KL, Morris K, Pepper SD, Nonaka D, Greystoke A, Kelly P, Bola B, Krebs MG, Antonello J, Ayub M, Faulkner S, Priest L, Carter L, Tate C, Miller CJ, Blackhall F, Brady G, Dive C. 2014. Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer. Nat Med 20: 897–903.
Hou JM, Krebs M, Ward T, Sloane R, Priest L, Hughes A, Clack G, Ranson M, Blackhall F, Dive C. 2011. Circulating tumor cells as a window on metastasis biology in lung cancer. Am J Pathol 178: 989–996.
Hou JM, Krebs MG, Lancashire L, Sloane R, Backen A, Swain RK, Priest LJ, Greystoke A, Zhou C, Morris K, Ward T, Blackhall FH, Dive C. 2012. Clinical significance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small-cell lung cancer. J Clin Oncol 30: 525–532.
Howe EN, Cochrane DR, Richer JK. 2011. Targets of miR-200c mediate suppression of cell motility and anoikis resistance. Breast Cancer Res 13: R45.
Huang RY, Wong MK, Tan TZ, Kuay KT, Ng AH, Chung VY, Chu YS, Matsumura N, Lai HC, Lee YF, Sim WJ, Chai C, Pietschmann E, Mori S, Low JJ, Choolani M, Thiery JP. 2013. An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor, saracatinib (AZD0530). Cell Death Dis 4: e915.
Hyun KA, Koo GB, Han H, Sohn J, Choi W, Kim SI, Jung HI, Kim YS. 2016. Epithelial-to-mesenchymal transition leads to loss of EpCAM and different physical properties in circulating tumor cells from metastatic breast cancer. Oncotarget 7: 24677–24687.
Im JH, Fu W, Wang H, Bhatia SK, Hammer DA, Kowalska MA, Muschel RJ. 2004. Coagulation facilitates tumor cell spreading in the pulmonary vasculature during early metastatic colony formation. Cancer Res 64: 8613–8619.
Jolly MK, Boareto M, Huang B, Jia D, Lu M, Ben-Jacob E, Onuchic JN, Levine H. 2015a. Implications of the hybrid epithelial/mesenchymal phenotype in metastasis. Front Oncol 5: 155.
Jolly MK, Jia D, Boareto M, Mani SA, Pienta KJ, Ben-Jacob E, Levine H. 2015b. Coupling the modules of EMT and stemness: a tunable ‘stemness window’ model. Oncotarget 6: 25161–25174.
Jolly MK, Tripathi SC, Jia D, Mooney SM, Celiktas M, Hanash SM, Mani SA, Pienta KJ, Ben-Jacob E, Levine H. 2016. Stability of the hybrid epithelial/mesenchymal phenotype. Oncotarget 7: 27067–27084.
Joosse SA, Gorges TM, Pantel K. 2015. Biology, detection, and clinical implications of circulating tumor cells. EMBO Mol Med 7: 1–11.
Jordan NV, Bardia A, Wittner BS, Benes C, Ligorio M, Zheng Y, Yu M, Sundaresan TK, Licausi JA, Desai R, O'Keefe RM, Ebright RY, Boukhali M, Sil S, Onozato ML, Iafrate AJ, Kapur R, Sgroi D, Ting DT, Toner M, Ramaswamy S, Haas W, Maheswaran S, Haber DA. 2016. HER2 expression identifies dynamic functional states within circulating breast cancer cells. Nature 537: 102–106.
Kajita M, McClinic KN, Wade PA. 2004. Aberrant expression of the transcription factors snail and slug alters the response to genotoxic stress. Mol Cell Biol 24: 7559–7566.
Kallergi G, Agelaki S, Kalykaki A, Stournaras C, Mavroudis D, Georgoulias V. 2008. Phosphorylated EGFR and PI3K/Akt signaling kinases are expressed in circulating tumor cells of breast cancer patients. Breast Cancer Res 10: R80.
Kallergi G, Konstantinidis G, Markomanolaki H, Papadaki MA, Mavroudis D, Stournaras C, Georgoulias V, Agelaki S. 2013. Apoptotic circulating tumor cells in early and metastatic breast cancer patients. Mol Cancer Ther 12: 1886–1895.
Kallergi G, Papadaki MA, Politaki E, Mavroudis D, Georgoulias V, Agelaki S. 2011. Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients. Breast Cancer Res 13: R59.
Kalluri R, Weinberg RA. 2009. The basics of epithelial-mesenchymal transition. J. Clin. Invest 119: 1420–1428.
Kasimir-Bauer S, Hoffmann O, Wallwiener D, Kimmig R, Fehm T. 2012. Expression of stem cell and epithelial-mesenchymal transition markers in primary breast cancer patients with circulating tumor cells. Breast Cancer Res 14: R15.
Khoo BL, Grenci G, Jing T, Lim YB, Lee SC, Thiery JP, Han J, Lim CT. 2016. Liquid biopsy and therapeutic response: circulating tumor cell cultures for evaluation of anticancer treatment. Sci Adv 2: e1600274.
Khoo BL, Lee SC, Kumar P, Tan TZ, Warkiani ME, Ow SG, Nandi S, Lim CT, Thiery JP. 2015. Short-term expansion of breast circulating cancer cells predicts response to anti-cancer therapy. Oncotarget 6: 15578–15593.
Klymkowsky MW, Savagner P. 2009. Epithelial-mesenchymal transition: a cancer researcher's conceptual friend and foe. Am J Pathol 174: 1588–1593.
Kolbl AC, Jeschke U, Andergassen U. 2016. The Significance of Epithelial-to-Mesenchymal Transition for Circulating Tumor Cells. Int J Mol Sci 17: 1308.
Kotiyal S, Bhattacharya S. 2014. Breast cancer stem cells, EMT and therapeutic targets. Biochem Biophys Res Commun 453: 112–116.
Krawczyk N, Meier-Stiegen F, Banys M, Neubauer H, Ruckhaeberle E, Fehm T. 2014. Expression of stem cell and epithelial-mesenchymal transition markers in circulating tumor cells of breast cancer patients. Biomed Res Int 2014: 415721.
Krebs MG, Hou JM, Sloane R, Lancashire L, Priest L, Nonaka D, Ward TH, Backen A, Clack G, Hughes A, Ranson M, Blackhall FH, Dive C. 2012. Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches. J Thorac Oncol 7: 306–315.
Krebs MG, Metcalf RL, Carter L, Brady G, Blackhall FH, Dive C. 2014. Molecular analysis of circulating tumour cells-biology and biomarkers. Nat Rev Clin Oncol 11: 129–144.
Ksiazkiewicz M, Markiewicz A, Zaczek AJ. 2012. Epithelial-mesenchymal transition: a hallmark in metastasis formation linking circulating tumor cells and cancer stem cells. Pathobiology 79: 195–208.
Kumar S, Park SH, Cieply B, Schupp J, Killiam E, Zhang F, Rimm DL, Frisch SM. 2011. A pathway for the control of anoikis sensitivity by E-cadherin and epithelial-to-mesenchymal transition. Mol Cell Biol 31: 4036–4051.
Kurrey NK, Jalgaonkar SP, Joglekar AV, Ghanate AD, Chaskar PD, Doiphode RY, Bapat SA. 2009. Snail and slug mediate radioresistance and chemoresistance by antagonizing p53-mediated apoptosis and acquiring a stem-like phenotype in ovarian cancer cells. Stem Cells 27: 2059–2068.
Labelle M, Begum S, Hynes RO. 2011. Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell 20: 576–590.
Labelle M, Begum S, Hynes RO. 2014. Platelets guide the formation of early metastatic niches. Proc Natl Acad Sci U S A 111: E3053–E3061.
Labelle M, Hynes RO. 2012. The initial hours of metastasis: the importance of cooperative host-tumor cell interactions during hematogenous dissemination. Cancer Discov 2: 1091–1099.
Lamouille S, Xu J, Derynck R. 2014. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 15: 178–196.
Larson CJ, Moreno JG, Pienta KJ, Gross S, Repollet M, O'Hara S M, Russell T, Terstappen LW. 2004. Apoptosis of circulating tumor cells in prostate cancer patients. Cytometry A 62: 46–53.
Leblanc R, Peyruchaud O. 2016. Metastasis: new functional implications of platelets and megakaryocytes. Blood 128: 24–31.
Lecharpentier A, Vielh P, Perez-Moreno P, Planchard D, Soria JC, Farace F. 2011. Detection of circulating tumour cells with a hybrid (epithelial/mesenchymal) phenotype in patients with metastatic non-small cell lung cancer. Br J Cancer 105: 1338–1341.
Lee AY. 2010. Thrombosis in cancer: an update on prevention, treatment, and survival benefits of anticoagulants. Hematology Am Soc Hematol Educ Program 2010: 144–149.
Lee JS, Magbanua MJ, Park JW. 2016. Circulating tumor cells in breast cancer: applications in personalized medicine. Breast Cancer Res Treat 160: 411–424.
Li J, Ai Y, Wang L, Bu P, Sharkey CC, Wu Q, Wun B, Roy S, Shen X, King MR. 2016. Targeted drug delivery to circulating tumor cells via platelet membrane-functionalized particles. Biomaterials 76: 52–65.
Li J, King MR. 2012. Adhesion receptors as therapeutic targets for circulating tumor cells. Front Oncol 2: 79.
Li J, Sharkey CC, Huang D, King MR. 2015a. Nanobiotechnology for the therapeutic targeting of cancer cells in blood. Cell Mol Bioeng 8: 137–150.
Li TT, Liu H, Li FP, Hu YF, Mou TY, Lin T, Yu J, Zheng L, Li GX. 2015b. Evaluation of epithelial–mesenchymal transitioned circulating tumor cells in patients with resectable gastric cancer: relevance to therapy response. World J Gastroenterol 21: 13259–13267.
Li YM, Xu SC, Li J, Han KQ, Pi HF, Zheng L, Zuo GH, Huang XB, Li HY, Zhao HZ, Yu ZP, Zhou Z, Liang P. 2013. Epithelial-mesenchymal transition markers expressed in circulating tumor cells in hepatocellular carcinoma patients with different stages of disease. Cell Death Dis 4: e831.
Lim S, Becker A, Zimmer A, Lu J, Buettner R, Kirfel J. 2013. SNAI1-mediated epithelial-mesenchymal transition confers chemoresistance and cellular plasticity by regulating genes involved in cell death and stem cell maintenance. PLoS One 8: e66558.
Lindsay CR, Le Moulec S, Billiot F, Loriot Y, Ngo-Camus M, Vielh P, Fizazi K, Massard C, Farace F. 2016. Vimentin and Ki67 expression in circulating tumour cells derived from castrate-resistant prostate cancer. BMC Cancer 16: 168.
Liotta LA, Kleinerman J, Saidel GM. 1976. The significance of hematogenous tumor cell clumps in the metastatic process. Cancer Research 36: 889–894.
Liu H, Zhang X, Li J, Sun B, Qian H, Yin Z. 2015. The biological and clinical importance of epithelial-mesenchymal transition in circulating tumor cells. J Cancer Res Clin Oncol 141: 189–201.
Liu YK, Hu BS, Li ZL, He X, Li Y, Lu LG. 2016. An improved strategy to detect the epithelial-mesenchymal transition process in circulating tumor cells in hepatocellular carcinoma patients. Hepatol Int 10: 640–646.
Lou XL, Sun J, Gong SQ, Yu XF, Gong R, Deng H. 2015. Interaction between circulating cancer cells and platelets: clinical implication. Chin J Cancer Res 27: 450–460.
Mallini P, Lennard T, Kirby J, Meeson A. 2014. Epithelial-to-mesenchymal transition: what is the impact on breast cancer stem cells and drug resistance. Cancer Treat Rev 40: 341–348.
Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA. 2008. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133: 704–715.
Mariano G, Ricciardi MR, Trisciuoglio D, Zampieri M, Ciccarone F, Guastafierro T, Calabrese R, Valentini E, Tafuri A, Del Bufalo D, Caiafa P, Reale A. 2015. PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting Snail expression. Oncotarget 6: 15008–15021.
Markiewicz A, Ksiazkiewicz M, Welnicka-Jaskiewicz M, Seroczynska B, Skokowski J, Szade J, Zaczek AJ. 2014. Mesenchymal phenotype of CTC-enriched blood fraction and lymph node metastasis formation potential. PLoS One 9: e93901.
Martin OA, Anderson RL, Russell PA, Cox RA, Ivashkevich A, Swierczak A, Doherty JP, Jacobs DH, Smith J, Siva S, Daly PE, Ball DL, Martin RF, MacManus MP. 2014. Mobilization of viable tumor cells into the circulation during radiation therapy. Int J Radiat Oncol Biol Phys 88: 395–403.
Masuda T, Hayashi N, Iguchi T, Ito S, Eguchi H, Mimori K. 2016. Clinical and biological significance of circulating tumor cells in cancer. Mol Oncol 10: 408–417.
Matrone MA, Whipple RA, Balzer EM, Martin SS. 2010. Microtentacles tip the balance of cytoskeletal forces in circulating tumor cells. Cancer Res 70: 7737–7741.
Mazel M, Jacot W, Pantel K, Bartkowiak K, Topart D, Cayrefourcq L, Rossille D, Maudelonde T, Fest T, Alix-Panabieres C. 2015. Frequent expression of PD-L1 on circulating breast cancer cells. Mol Oncol 9: 1773–1782.
McInnes LM, Jacobson N, Redfern A, Dowling A, Thompson EW, Saunders CM. 2015. Clinical implications of circulating tumor cells of breast cancer patients: role of epithelial mesenchymal-plasticity. Front Oncol 5: 42–42.
Mego M, De Giorgi U, Broglio K, Dawood S, Valero V, Andreopoulou E, Handy B, Reuben JM, Cristofanilli M. 2009. Circulating tumour cells are associated with increased risk of venous thromboembolism in metastatic breast cancer patients. Br J Cancer 101: 1813–1816.
Mego M, Mani SA, Lee BN, Li C, Evans KW, Cohen EN, Gao H, Jackson SA, Giordano A, Hortobagyi GN, Cristofanilli M, Lucci A, Reuben JM. 2011. Expression of epithelial-mesenchymal transition-inducing transcription factors in primary breast cancer: the effect of neoadjuvant therapy. Int J Cancer 130: 808–816.
Milsom CC, Yu JL, Mackman N, Micallef J, Anderson GM, Guha A, Rak JW. 2008. Tissue factor regulation by epidermal growth factor receptor and epithelial-to-mesenchymal transitions: effect on tumor initiation and angiogenesis. Cancer Res 68: 10068–10076.
Mitra A, Mishra L, Li S. 2015. EMT, CTCs and CSCs in tumor relapse and drug-resistance. Oncotarget 6: 10697–10711.
Mladinich M, Ruan D, Chan CH. 2016. Tackling cancer stem cells via inhibition of EMT transcription factors. Stem Cells Int 2016: 5285892.
Molnar B, Ladanyi A, Tanko L, Sreter L, Tulassay Z. 2001. Circulating tumor cell clusters in the peripheral blood of colorectal cancer patients. Clin Cancer Res 7: 4080–4085.
Morel AP, Lievre M, Thomas C, Hinkal G, Ansieau S, Puisieux A. 2008. Generation of breast cancer stem cells through epithelial-mesenchymal transition. PLoS One 3: e2888.
Mu Z, Wang C, Ye Z, Austin L, Civan J, Hyslop T, Palazzo JP, Jaslow R, Li B, Myers RE, Jiang J, Xing J, Yang H, Cristofanilli M. 2015. Prospective assessment of the prognostic value of circulating tumor cells and their clusters in patients with advanced-stage breast cancer. Breast Cancer Res Treat 154: 563–571.
Nadal R, Lorente JA, Rosell R, Serrano MJ. 2013. Relevance of molecular characterization of circulating tumor cells in breast cancer in the era of targeted therapies. Expert Rev Mol Diagn 13: 295–307.
Nicolazzo C, Raimondi C, Mancini M, Caponnetto S, Gradilone A, Gandini O, Mastromartino M, Del Bene G, Prete A, Longo F, Cortesi E, Gazzaniga P. 2016. Monitoring PD-L1 positive circulating tumor cells in non-small cell lung cancer patients treated with the PD-1 inhibitor Nivolumab. Sci Rep 6: 31726.
Nieto MA, Huang RY, Jackson RA, Thiery JP. 2016. EMT: 2016. Cell 166: 21–45.
Ning Y, Zhang W, Hanna DL, Yang D, Okazaki S, Berger MD, Miyamoto Y, Suenaga M, Schirripa M, El-Khoueiry A, Lenz HJ. 2016. Clinical relevance of EMT and stem-like gene expression in circulating tumor cells of metastatic colorectal cancer patients. Pharmacogenomics J [Epub ahead of print].
Ocana OH, Corcoles R, Fabra A, Moreno-Bueno G, Acloque H, Vega S, Barrallo-Gimeno A, Cano A, Nieto MA. 2012. Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1. Cancer Cell 22: 709–724.
Ock CY, Kim S, Keam B, Kim M, Kim TM, Kim JH, Jeon YK, Lee JS, Kwon SK, Hah JH, Kwon TK, Kim DW, Wu HG, Sung MW, Heo DS. 2016. PD-L1 expression is associated with epithelial-mesenchymal transition in head and neck squamous cell carcinoma. Oncotarget 7: 15901–15914.
Ombrato L, Malanchi I. 2014. The EMT universe: space between cancer cell dissemination and metastasis initiation. Crit Rev Oncog 19: 349–361.
Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA. 2008. Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 68: 3645–3654.
Ota I, Li XY, Hu Y, Weiss SJ. 2009. Induction of a MT1-MMP and MT2-MMP-dependent basement membrane transmigration program in cancer cells by Snail1. Proc Natl Acad Sci U S A 106: 20318–20323.
Palumbo JS. 2008. Mechanisms linking tumor cell-associated procoagulant function to tumor dissemination. Semin Thromb Hemost 34: 154–160.
Palumbo JS, Degen JL. 2007. Mechanisms linking tumor cell-associated procoagulant function to tumor metastasis. Thromb Res 120(suppl 2): S22–S28.
Palumbo JS, Kombrinck KW, Drew AF, Grimes TS, Kiser JH, Degen JL, Bugge TH. 2000. Fibrinogen is an important determinant of the metastatic potential of circulating tumor cells. Blood 96: 3302–3309.
Palumbo JS, Talmage KE, Massari JV, La Jeunesse CM, Flick MJ, Kombrinck KW, Hu Z, Barney KA, Degen JL. 2007. Tumor cell-associated tissue factor and circulating hemostatic factors cooperate to increase metastatic potential through natural killer cell-dependent and-independent mechanisms. Blood 110: 133–141.
Palumbo JS, Talmage KE, Massari JV, La Jeunesse CM, Flick MJ, Kombrinck KW, Jirouskova M, Degen JL. 2005. Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells. Blood 105: 178–185.
Pantel K, Speicher MR. 2015. The biology of circulating tumor cells. Oncogene 35: 1216–1224.
Paoletti C, Li Y, Muniz MC, Kidwell KM, Aung K, Thomas DG, Brown ME, Abramson VG, Irvin WJ, Jr., Lin NU, Liu MC, Nanda R, Nangia JR, Storniolo AM, Traina TA, Vaklavas C, Van Poznak CH, Wolff AC, Forero-Torres A, Hayes DF. 2015. Significance of circulating tumor cells in metastatic triple-negative breast cancer patients within a randomized, phase II trial: TBCRC 019. Clin Cancer Res 21: 2771–2779.
Papadaki MA, Kallergi G, Zafeiriou Z, Manouras L, Theodoropoulos PA, Mavroudis D, Georgoulias V, Agelaki S. 2014. Co-expression of putative stemness and epithelial-to-mesenchymal transition markers on single circulating tumour cells from patients with early and metastatic breast cancer. BMC Cancer 14: 651.
Plaks V, Kong N, Werb Z. 2015. The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells? Cell Stem Cell 16: 225–238.
Polioudaki H, Agelaki S, Chiotaki R, Politaki E, Mavroudis D, Matikas A, Georgoulias V, Theodoropoulos PA. 2015. Variable expression levels of keratin and vimentin reveal differential EMT status of circulating tumor cells and correlation with clinical characteristics and outcome of patients with metastatic breast cancer. BMC Cancer 15: 399.
Pothula SP, Xu Z, Goldstein D, Pirola RC, Wilson JS, Apte MV. 2016. Key role of pancreatic stellate cells in pancreatic cancer. Cancer Lett 381: 194–200.
Raimondi C, Gradilone A, Naso G, Vincenzi B, Petracca A, Nicolazzo C, Palazzo A, Saltarelli R, Spremberg F, Cortesi E, Gazzaniga P. 2011. Epithelial-mesenchymal transition and stemness features in circulating tumor cells from breast cancer patients. Breast Cancer Res Treat 130: 449–455.
Rhim AD, Mirek ET, Aiello NM, Maitra A, Bailey JM, McAllister F, Reichert M, Beatty GL, Rustgi AK, Vonderheide RH, Leach SD, Stanger BZ. 2012. EMT and dissemination precede pancreatic tumor formation. Cell 148: 349–361.
Roberts CM, Tran MA, Pitruzzello MC, Wen W, Loeza J, Dellinger TH, Mor G, Glackin CA. 2016. TWIST1 drives cisplatin resistance and cell survival in an ovarian cancer model, via upregulation of GAS6, L1CAM, and Akt signalling. Sci Rep 6: 37652.
Rossi E, Basso U, Celadin R, Zilio F, Pucciarelli S, Aieta M, Barile C, Sava T, Bonciarelli G, Tumolo S, Ghiotto C, Magro C, Jirillo A, Indraccolo S, Amadori A, Zamarchi R. 2010. M30 neoepitope expression in epithelial cancer: quantification of apoptosis in circulating tumor cells by CellSearch analysis. Clin Cancer Res 16: 5233–5243.
Rossi E, Rugge M, Facchinetti A, Pizzi M, Nardo G, Barbieri V, Manicone M, De Faveri S, Chiara Scaini M, Basso U, Amadori A, Zamarchi R. 2014. Retaining the long-survive capacity of Circulating Tumor Cells (CTCs) followed by xeno-transplantation: not only from metastatic cancer of the breast but also of prostate cancer patients. Oncoscience 1: 49–56.
Ruf W. 2012. Tissue factor and cancer. Thromb Res 130: S84–S87.
Satelli A, Mitra A, Brownlee Z, Xia X, Bellister S, Overman MJ, Kopetz S, Ellis LM, Meng QH, Li S. 2014. Epithelial-mesenchymal transitioned circulating tumor cells capture for detecting tumor progression. Clin Cancer Res 21: 899–907.
Savagner P. 2015. Epithelial-mesenchymal transitions: from cell plasticity to concept elasticity. Curr Top Dev Biol 112: 273–300.
Schmidt JM, Panzilius E, Bartsch HS, Irmler M, Beckers J, Kari V, Linnemann JR, Dragoi D, Hirschi B, Kloos UJ, Sass S, Theis F, Kahlert S, Johnsen SA, Sotlar K, Scheel CH. 2015. Stem-cell-like properties and epithelial plasticity arise as stable traits after transient Twist1 activation. Cell Rep 10: 131–139.
Serrano MJ, Ortega FG, Alvarez-Cubero MJ, Nadal R, Sanchez-Rovira P, Salido M, Rodriguez M, Garcia-Puche JL, Delgado-Rodriguez M, Sole F, Garcia MA, Peran M, Rosell R, Marchal JA, Lorente JA. 2014. EMT and EGFR in CTCs cytokeratin negative non-metastatic breast cancer. Oncotarget 5: 7486–7497.
Smit MA, Geiger TR, Song JY, Gitelman I, Peeper DS. 2009. A Twist-Snail axis critical for TrkB-induced epithelial-mesenchymal transition-like transformation, anoikis resistance, and metastasis. Mol Cell Biol 29: 3722–3737.
Spiliotaki M, Mavroudis D, Kapranou K, Markomanolaki H, Kallergi G, Koinis F, Kalbakis K, Georgoulias V, Agelaki S. 2014. Evaluation of proliferation and apoptosis markers in circulating tumor cells of women with early breast cancer who are candidates for tumor dormancy. Breast Cancer Res 16: 485.
Stegner D, Dutting S, Nieswandt B. 2014. Mechanistic explanation for platelet contribution to cancer metastasis. Thromb Res 133(suppl 2): S149–S157.
Stott SL, Hsu CHC-H, Tsukrov DI, Yu M, Miyamoto DT, Waltman BA, Rothenberg SM, Shah AM, Smas ME, Korir GK, Floyd FP, Gilman AJ, Lord JB, Winokur D, Springer S, Irimia D, Nagrath S, Sequist LV, Lee RJ, Isselbacher KJ, Maheswaran S, Haber DA, Toner M. 2010. Isolation of circulating tumor cells using a a microvortex-generating herringbone-chip. Proc Natl Acad Sci U S A 107: 18392–18397.
Sullivan JP, Nahed BV, Madden MW, Oliveira SM, Springer S, Bhere D, Chi AS, Wakimoto H, Rothenberg SM, Sequist LV, Kapur R, Shah K, Iafrate AJ, Curry WT, Loeffler JS, Batchelor TT, Louis DN, Toner M, Maheswaran S, Haber DA. 2014. Brain tumor cells in circulation are enriched for mesenchymal gene expression. Cancer Discov 4: 1299–1309.
Tesfamariam B. 2016. Involvement of platelets in tumor cell metastasis. Pharmacol Ther 157: 112–119.
Theodoropoulos PA, Polioudaki H, Agelaki S, Kallergi G, Saridaki Z, Mavroudis D, Georgoulias V. 2010. Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer. Cancer Lett 288: 99–106.
Thompson EW, Haviv I. 2011. The social aspects of EMT-MET plasticity. Nat Med 17: 1048–1049.
Thompson EW, Newgreen DF, Tarin D. 2005. Carcinoma invasion and metastasis: a role for epithelial-mesenchymal transition? Cancer Res 65: 5991–5995.
Thomson S, Buck E, Petti F, Griffin G, Brown E, Ramnarine N, Iwata KK, Gibson N, Haley JD. 2005. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res 65: 9455–9462.
Tinhofer I, Saki M, Niehr F, Keilholz U, Budach V. 2014. Cancer stem cell characteristics of circulating tumor cells. Int J Radiat Biol 90: 622–627.
Tiwari N, Gheldof A, Tatari M, Christofori G. 2012. EMT as the ultimate survival mechanism of cancer cells. Semin Cancer Biol 22: 194–207.
Todenhofer T, Hennenlotter J, Dorner N, Kuhs U, Aufderklamm S, Rausch S, Bier S, Mischinger J, Schellbach D, Hauch S, Feniuk N, Bedke J, Gakis G, Stenzl A, Schwentner C. 2016. Transcripts of circulating tumor cells detected by a breast cancer-specific platform correlate with clinical stage in bladder cancer patients. J Cancer Res Clin Oncol 142: 1013–1020.
Toss A, Mu Z, Fernandez S, Cristofanilli M. 2014. CTC enumeration and characterization: moving toward personalized medicine. Ann Transl Med 2: 108.
Tripathi SC, Peters HL, Taguchi A, Katayama H, Wang H, Momin A, Jolly MK, Celiktas M, Rodriguez-Canales J, Liu H, Behrens C, Wistuba II, Ben-Jacob E, Levine H, Molldrem JJ, Hanash SM, Ostrin EJ. 2016. Immunoproteasome deficiency is a feature of non-small cell lung cancer with a mesenchymal phenotype and is associated with a poor outcome. Proc Natl Acad Sci U S A 113: E1555–E1564.
Tsai JH, Donaher JL, Murphy DA, Chau S, Yang J. 2012. Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis. Cancer Cell 22: 725–736.
Tsai JH, Yang J. 2013. Epithelial-mesenchymal plasticity in carcinoma metastasis. Genes Dev 27: 2192–2206.
Tsuji T, Ibaragi S, Hu GF. 2009. Epithelial-mesenchymal transition and cell cooperativity in metastasis. Cancer Res 69: 7135–7139.
Tsuji T, Ibaragi S, Shima K, Hu MG, Katsurano M, Sasaki A, Hu GF. 2008. Epithelial-mesenchymal transition induced by growth suppressor p12CDK2-AP1 promotes tumor cell local invasion but suppresses distant colony growth. Cancer Res 68: 10377–10386.
Ünlü B, Versteeg HH. 2014. Effects of tumor-expressed coagulation factors on cancer progression and venous thrombosis: is there a key factor? Thromb Res 133(suppl): S76–S84.
Vichalkovski A, Gresko E, Hess D, Restuccia DF, Hemmings BA. 2010. PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage. Oncogene 29: 3554–3565.
Wang C, Mu Z, Chervoneva I, Austin L, Ye Z, Rossi G, Palazzo JP, Sun C, Abu-Khalaf M, Myers RE, Zhu Z, Ba Y, Li B, Hou L, Cristofanilli M, Yang H. 2017a. Longitudinally collected CTCs and CTC-clusters and clinical outcomes of metastatic breast cancer. Breast Cancer Res Treat 161: 83–94.
Wang H, Stoecklein NH, Lin PP, Gires O. 2017b. Circulating and disseminated tumor cells: diagnostic tools and therapeutic targets in motion. Oncotarget 8: 1884–1912.
Wang X, Sun Q, Liu Q, Wang C, Yao R, Wang Y. 2016. CTC immune escape mediated by PD-L1. Med Hypotheses 93: 138–139.
Watson MA, Ylagan LR, Trinkaus KM, Gillanders WE, Naughton MJ, Weilbaecher KN, Fleming TP, Aft RL. 2007. Isolation and molecular profiling of bone marrow micrometastases identifies TWIST1 as a marker of early tumor relapse in breast cancer patients. Clin Cancer Res 13: 5001–5009.
Whipple RA, Balzer EM, Cho EH, Matrone MA, Yoon JR, Martin SS. 2008. Vimentin filaments support extension of tubulin-based microtentacles in detached breast tumor cells. Cancer Res 68: 5678–5688.
Whipple RA, Matrone MA, Cho EH, Balzer EM, Vitolo MI, Yoon JR, Ioffe OB, Tuttle KC, Yang J, Martin SS. 2010. Epithelial-to-mesenchymal transition promotes tubulin detyrosination and microtentacles that enhance endothelial engagement. Cancer Res 70: 8127–8137.
Williams JC, Mackman N. 2012. Tissue factor in health and disease. Front Biosci (Elite Ed) 4: 358–372.
Willipinski-Stapelfeldt B, Riethdorf S, Assmann V, Woelfle U, Rau T, Sauter G, Heukeshoven J, Pantel K. 2005. Changes in cytoskeletal protein composition indicative of an epithelial-mesenchymal transition in human micrometastatic and primary breast carcinoma cells. Clin Cancer Res 11: 8006–8014.
Wu S, Liu S, Liu Z, Huang J, Pu X, Li J, Yang D, Deng H, Yang N, Xu J. 2015. Classification of circulating tumor cells by epithelial-mesenchymal transition markers. PLoS One 10: e0123976.
Yauch RL, Januario T, Eberhard DA, Cavet G, Zhu W, Fu L, Pham TQ, Soriano R, Stinson J, Seshagiri S, Modrusan Z, Lin CY, O'Neill V, Amler LC. 2005. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res 11: 8686–8698.
Ye X, Weinberg RA. 2015. Epithelial-mesenchymal plasticity: a central regulator of cancer progression. Trends Cell Biol 25: 675–686.
Yokobori T, Iinuma H, Shimamura T, Imoto S, Sugimachi K, Ishii H, Iwatsuki M, Ota D, Ohkuma M, Iwaya T, Nishida N, Kogo R, Sudo T, Tanaka F, Shibata K, Toh H, Sato T, Barnard GF, Fukagawa T, Yamamoto S, Nakanishi H, Sasaki S, Miyano S, Watanabe T, Kuwano H, Mimori K, Pantel K, Mori M. 2013. Plastin3 is a novel marker for circulating tumor cells undergoing the epithelial-mesenchymal transition and is associated with colorectal cancer prognosis. Cancer Res 73: 2059–2069.
Yu M, Bardia A, Aceto N, Bersani F, Madden MW, Donaldson MC, Desai R, Zhu H, Comaills V, Zheng Z, Wittner BS, Stojanov P, Brachtel E, Sgroi D, Kapur R, Shioda T, Ting DT, Ramaswamy S, Getz G, Iafrate aJ, Benes C, Toner M, Maheswaran S, Haber Da. 2014. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility. Science 345: 216–220.
Yu M, Bardia A, Wittner BS, Stott SL, Smas ME, Ting DT, Isakoff SJ, Ciciliano JC, Wells MN, Shah AM, Concannon KF, Donaldson MC, Sequist LV, Brachtel E, Sgroi D, Baselga J, Ramaswamy S, Toner M, Haber DA, Maheswaran S. 2013. Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science 339: 580–584.
Yu N, Zhou J, Cui F, Tang X. 2015. Circulating tumor cells in lung cancer: detection methods and clinical applications. Lung 193: 157–171.
Zhang K, Jiao X, Liu X, Zhang B, Wang J, Wang Q, Tao Y, Zhang D. 2010. Knockdown of snail sensitizes pancreatic cancer cells to chemotherapeutic agents and irradiation. Int J Mol Sci 11: 4891–4892.
Zhang L, Ridgway LD, Wetzel MD, Ngo J, Yin W, Kumar D, Goodman JC, Groves MD, Marchetti D. 2013. The identification and characterization of breast cancer CTCs competent for brain metastasis. Sci Transl Med 5: 180ra148.
Zhang X, Wang Q, Ling MT, Wong YC, Leung SC, Wang X. 2007. Anti-apoptotic role of TWIST and its association with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells. Int J Cancer 120: 1891–1898.
Zhao R, Cai Z, Li S, Cheng Y, Gao H, Liu F, Wu S, Liu S, Dong Y, Zheng L, Zhang W, Wu X, Yao X. 2016a. Expression and clinical relevance of epithelial and mesenchymal markers in circulating tumor cells from colorectal cancer. Oncotarget 8: 9293–9302.
Zhao XY, Li L, Wang XB, Fu RJ, Lv YP, Jin W, Meng C, Chen GQ, Huang L, Zhao KW. 2016b. Inhibition of Snail Family Transcriptional Repressor 2 (SNAI2) enhances multidrug resistance of hepatocellular carcinoma cells. PLoS One 11: e0164752.
Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, Wu CC, LeBleu VS, Kalluri R. 2015. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 527: 525–530.

Citing Literature

Volume247, Issue3

Special Issue: Epithelial-Mesenchymal Transition and Plasticity in Development, Cancer & Fibrosis

March 2018

Pages 432-450

Epithelial–mesenchymal plasticity and circulating tumor cells: Travel companions to metastases

Abstract

Introduction

Epithelial–Mesenchymal Plasticity/Epithelial-Mesenchymal Transition

EMT Facilitates the Entry of CTCs into the Circulation

EMT Enhances CTC Metastatic Competence

EMT Increases CTC Survival and Early Colonization

Induction of Survival Pathways

Activation of Coagulation

Formation of Specific Protective Cytoskeletal Structures (Microtentacles)

Immune Escape

Formation of Clusters of CTCs

EMT Supports Metastatic niche Formation

Functional Assays Reveal that MICs are Present in the Blood of Cancer Patients

Concluding Remarks

Acknowledgments

References

Citing Literature

Figures

References

Information

About Wiley Online Library

Help & Support

Opportunities

Connect with Wiley

Epithelial–mesenchymal plasticity and circulating tumor cells: Travel companions to metastases

Abstract

Introduction

Epithelial–Mesenchymal Plasticity/Epithelial-Mesenchymal Transition

EMT Facilitates the Entry of CTCs into the Circulation

EMT Enhances CTC Metastatic Competence

EMT Increases CTC Survival and Early Colonization

Induction of Survival Pathways

Activation of Coagulation

Formation of Specific Protective Cytoskeletal Structures (Microtentacles)

Immune Escape

Formation of Clusters of CTCs

EMT Supports Metastatic niche Formation

Functional Assays Reveal that MICs are Present in the Blood of Cancer Patients

Concluding Remarks

Acknowledgments

References

Citing Literature

Figures

References

Related

Information