Table 1.
List of the most relevant mesenchymal and stemness markers for CTCs detection.
| Marker | Mesenchymal (M)/Stem-Cell (SC) Marker | Functions and Clinical Relevance | Reference | Year |
|---|---|---|---|---|
| Breast Cancer | ||||
| Twist1, Akt2, PI3Kalpha, ALDH1 | M (Twist1, Akt2, PI3Kalpha), SC (ALDH1) | Higher expression rates of EMT markers and ALDH1 (62% and 44% of patients, respectively) in metastatic patient non-responders to therapies, compared to responder patients. | [49] | 2009 |
| CD44, CD24, ALDH1 | SC | Presence, in metastatic patients, of CTCs with stem-like/tumorigenic phenotype CD44+CD24−/low (35.2% of CTCs identified), and a less commonly observed population of ALDH1highCD24−/low (17.7% of CTCs analyzed in seven patients), thus identifying a subset of CTCs with putative stem cell progenitor phenotypes. | [58] | 2010 |
| CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC and SLC6A8 | M | 81% of advanced breast cancer patients with recurrence and 29% of breast cancer patients at initial diagnosis positive for at least one gene. | [114] | 2010 |
| ALDH1, vimentin, fibronectin | SC (ALDH1), M (vimentin, fibronectin) | Assessment of molecular profile of CTCs according to expression of ERa, HER2/neu, ALDH1, vimentin, and fibronectin. Detection of ALDH1+ CTCs in 28/61; vimentin+ CTCs in 17/61; and fibronectin+ CTCs in 11/61 of patients. Expression of ALDH1 on CTCs significantly correlated to stage of disease (p = 0.01) and to expression of vimentin and fibronectin (p = 0.001 for both). | [115] | 2011 |
| Twist, vimentin | M | Vimentin- and Twist-expressing CK(+) CTCs identified in 77% and 73%, respectively, of early and in 100% of metastatic breast cancer patients. Higher incidence of CK+vimentin+ and CK+Twist+ cells in patients with metastatic disease compared to early stage breast cancer. Significant correlation (p = 0.005) between the number of CTCs expressing Twist and vimentin within the same setting, with a reduction in an adjuvant chemotherapy setting in metastatic tumors. | [38] | 2011 |
| Fibronectin, vimentin | M | More accurate prediction of worse prognosis in metastatic patients due to the presence of mesenchymal markers, than expression of cytokeratins alone. | [50] | 2011 |
| MRP, ALDH1 | SC (ALDH1) | Shorter progression-free survival (PFS) in metastatic patients with a ‘drug resistance’ CTCs profile and expressing MRPs. Statistically significant correlation between the number of MRPs expressed in CTCs and ALDH1. The number of MRPs expressed on ALDH1+ CTC is predictive of poor response to treatment and significantly associated with shorter PFS. ALDH1/MRP-expressing CTCs have a greater tendency to intrinsic drug resistance. | [110] | 2011 |
| Vimentin, N-cadherin, O-cadherin, CD133 | M (Vimentin, N-cadherin, O-cadherin), SC (CD133) | More than 75% of CTCs in metastatic breast cancer co-express CK, vimentin, and N-cadherin. | [116] | 2011 |
| CD44 | SC | Clinical relevance of CTC (CD45-EpCAM+ cells) and CTSC (CD45-EpCAM+CD44+CD24− cells). Statistical differences between CTC < 50 and CTC ≥ 50 groups among TNM stages, histology stages, and ER and PR status (p < 0.05). Statistical differences between CTSC negative and positive groups within TNM stages and regional lymph node metastasis (RLNM) status (p < 0.05). | [117] | 2012 |
| Twist, Snail1, Slug, Zeb1, FoxC2 | M | Higher expression levels of EMT-inducing TF in patients receiving neoadjuvant therapy with respect to patients who received no neoadjuvant therapy (p = 0.003). | [39] | 2012 |
| Nanog, Oct3/4, Sox2, Nestin, and CD34 | SC | Linear relationship between gene expression of stemness markers and tumor stage (I-IV), as well as specific expression patterns by stage. | [118] | 2012 |
| CD44, CD47, MET | SC (CD44), M (CD47, MET) | Functional circulating tumor-initiating cells, with increased metastatic capacity. Correlation of EPCAMlowMEThighCD47highCD44high CTCs number and lower overall survival and increased number of metastases. | [36] | 2013 |
| Fibronectin 1(FN1), cadherin 2 (CDH2), serpin peptidase inhibitor, clade E (SERPINE1) | M | Association of mesenchymal CTCs with disease progression: in an index patient, reversible shifts between mesenchymal and epithelial phenotypes accompanied each cycle of response to therapy and tumor progression. | [37] | 2013 |
| HER2, EGFR, NOTCH1, HPSE | Identification of a potential signature of brain metastases in CTCs comprising brain metastases selected markers HER2+EGFR+HPSE+NOTCH1+. CTC lines expressing this signature were highly invasive and competent in generating brain and lung metastases when xenografted in nude mice. | [112] | 2013 | |
| Plakoglobin | M | Intercellular tether that confers added metastatic potential. High levels in breast cancer patients denoting adverse outcomes, while selective knockdown inhibiting lung metastases in mouse model. | [25] | 2014 |
| UPAR, intβ1 | SC | Identification of DAPI−CD45−EpCAMnegativeCD24−CD44+uPARintβ1 CTC subsets with properties related to dormancy. Embryonic stem-cell gene expression profiling revealed high expression in uPAR+intβ1+ CTC subset, and in vitro assays confirmed the metastatic competency of uPARintβ1 CTCs. uPARintβ1 CTC subset may prospectively identify patients at high risk of brain metastases. | [21] | 2015 |
| IGFR1, UPA, VEGFA, VEGFR1 | M | Genes expressed exclusively in CTC-enriched samples, identified by profiling a panel of 55 breast cancer-associated genes. | [89] | 2016 |
| ALDH1, Twist1 | SC (ALDH1), M (Twist1) | Prognostic relevance in metastatic patients of single CSC+/partial-EMT+ CTCs (co-expressing cytokeratin, ALDH1, and nuclear Twist1). Evidence of CSC+/partial-EMT+ CTCs in 27.7% of patients at baseline, and correlation to lung metastases and decreased PFS. Detection of CSC+/partial-EMT+ CTCs as an independent factor predicting for increased risk of relapse. Additional association with reduced OS and increased risk of death in HER-2 negative patients. Significant increase in incidence of CSC+/partial-EMT+ CTCs due to chemotherapy confirmed in HER2-negative patients and in non-responders at the end of treatment. | [119] | 2019 |
| Ovarian, Cervical, Endometrial Cancers | ||||
| CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC and SLC6A8 | M | In the cervical, endometrial, and ovarian cancer groups, percentage of positive patients of 44%, 64%, and 19%, respectively. | [114] | 2010 |
| Vimentin | M | In ovarian cancers, greater percentage of tumor cells with very low EpCAM expression and high vimentin expression. EpCAM expression significantly lower in the vimentin high group (p = 0.0036). | [120] | 2010 |
| PPIC | M | Identification of a panel of 11 novel gene markers including PPIC for detection of CTCs with clinical impact in epithelial ovarian cancer (EOC) patients, both before primary therapy and during follow-up. Correlation of CTCs over-expressing PPIC, with an incomplete epithelial phenotype and a more aggressive potential, and resistant to chemotherapy, with an adverse outcome (DFS and OS), independently of clinicopathological parameters. | [121] | 2013 |
| ETV5, NOTCH1, Snai1, TGFB1, Zeb1, Zeb2, ALDH1, CD44 | M (ETV5, NOTCH1, Snai1, TGFB1, Zeb1, Zeb2), SC (ALDH1, CD44) | Remarkable plasticity phenotype in high-risk endometrial cancer CTCs defined by expression of the EMT markers ETV5, NOTCH1, SnaiI1, TGFB1, Zeb1, and Zeb2, further recapitulated through up-regulation of ETV5 in an EC cell line, and demonstrating an advantage in promoting metastasis in an in vivo mouse model. Expression of ALDH and CD44 in CTCs, pointing to an association with stemness. | [122] | 2014 |
| CD44, ALDH1A1, Nanog, Oct4, N-cadherin, Vimentin, Snai2, CD117, CD146 | SC (CD44, ALDH1A1, Nanog, Oct4), M (N-cadherin, Vimentin, Snai2, CD117, CD146) | Heterogeneous expression of stem cell- and EMT-associated transcripts in ovarian cancer CTCs. Co-expression of epithelial, mesenchymal, and stem cell transcripts on the same CTC was observed. | [123] | 2016 |
| Lung Cancers | ||||
| Vimentin | M | Greater percentage of tumor cells with very low EpCAM expression and high vimentin expression. EpCAM expression significantly lower in the vimentin high group (p = 0.012). | [120] | 2010 |
| Vimentin | M | In non-small-cell lung carcinoma (NSCLC), correlation of the presence of CTCs detected by both CellSearch and ISET (vimentin-positive cells with cytological criteria of malignancy) with shorter DFS. Complementary methods for detection of CTCs in preoperative surgery. | [124] | 2011 |
| Vimentin | M | Detection of isolated or clusters of dual CTCs strongly co-expressing vimentin and keratin in all metastatic NSCLC patients analyzed, confirming the existence of hybrid CTCs with an epithelial/mesenchymal phenotype. | [125] | 2011 |
| Vimentin | M | Expression of vimentin in the majority of cells within circulating tumor microemboli and only in some CTCs; no homogeneous EMT transition in tumor cells within the circulation, both in small cell lung cancer and NSCLC. | [90] | 2011 |
| Vimentin, N-cadherin | M | Detection of ALK rearrangement in CTCs of patients with ALK-positive NSCLC, enabling both diagnostic testing and monitoring of crizotinib treatment. CTCs with a unique ALK rearrangement and mesenchymal phenotype may arise from clonal selection of ALK-positive tumor cells which acquired metastatic potential. | [126] | 2013 |
| CD133 | SC | In advanced metastatic tumors, identification of CD133+ cells, a marker of stem-like behavior in highly tumorigenic cells, in subsets of CTCs isolated using centrifugal forces. | [127] | 2013 |
| BMI1, Twist1, CD133, ALDH1A1 | M (BMI1, Twist1), SC (CD133, ALDH1A1) | Overexpression in CTCs of ALDH1A1 (10/10 of patients), CD133 (3/10 of patients), BMI1 (7/10 of patients), and Twist1 (3/10 of patients), thus confirming the presence of CSCs in NSCLC, complying with the recent demonstration of tumor initiation capabilities in lung CTCs. | [101] | 2016 |
| Hepatocellular Carcinoma | ||||
| CD90, CD44 | SC | Presence of CD45-CD90+ CTCs in 91.6% of patients, generating tumor nodules in immunodeficient mice. The CD90+CD44+ cells demonstrated a more aggressive phenotype than the CD90+CD44- counterpart and generated lung metastatic lesions in mice. CD44 blockade prevented local and metastatic tumor nodules by CD90+ cells. | [128] | 2008 |
| ICAM-1 | SC | Expressed on a minor cell population in HCC CTCs (0.3%), acting as a marker of HCC stem cells. ICAM-1 inhibitors slow tumor formation and metastasis in mice. Increased numbers of CD45-ICAM-1+ CTCs in patients with HCC correlated with worse clinical outcomes. | [129] | 2013 |
| N-cadherin, Vimentin | M | Identification of different individual cell type profiles in CTCs, with distinct clinical implications. The presence of mesenchymal cells correlated to survival, while an increase in epithelial cells was associated with a worse treatment outcome. The shift from mesenchymal to epithelial cell profiles was significantly correlated with shortened TTP in both N-cadherin and vimentin to cytokeratins ratios, respectively. | [130] | 2013 |
| Twist, Vimentin, E-cadherin | M | Detection of Twist and vimentin in CTCs from 84.8% and 80.4% of patients, respectively. Significant correlation of co-expression of both in CTCs (in 69.6% of patients) with portal vein tumor thrombus, TNM classification, and tumor size, as promising biomarkers for evaluating metastasis and prognosis. Significant association of E-cadherin, vimentin, and Twist levels in HCC tumors with CTCs. | [131] | 2013 |
| CD90, CXCR4 | SC | Generation of tumors after serial adoptive transplantations of CD90+CXCR4+ HCC cells into NOD/SCID mice, and more frequent detection of released CTCs. | [105] | 2015 |
| CD133 | SC | Detectable CD133+ CTC positively correlated with serum ANXA3 level (r = 0.601, p < 0.001), which is associated with a higher risk of recurrence and shorter overall survival: ANXA3 could stimulate and maintain the stem cell-like traits of CD133+ CTCs to promote tumor recurrence and metastasis. | [111] | 2018 |
| Colorectal Cancer | ||||
| ALDH1, CD44, CD133 | SC | Analysis of prognostic value of drug resistance and stemness markers in CTCs from metastatic patients treated with oxaliplatin- and 5-fluoruracil-based regimens. No correlation between the expression of CD44 or CD133 in CTCs and patient outcome; statistically significantly shorter PFS in patients with CTCs positive for ALDH1, survivin, and MRP5, for selection of patients resistant to chemotherapy. | [132] | 2010 |
| Plastin3 (PLS3) | M | PLS3-positive CTC independently associated with prognosis in metastatic tumors, particularly in patients with Dukes B and Dukes C CRC. | [83] | 2013 |
| CD44 variant exon 9 (CD44v9) | SC | Positive expression of CD44v9 in CTCs in 40% of patients. CD44v9 in CTCs as a factor predicting recurrence, prognosis, and treatment efficacy, both in patients with stage III and stage IV disease. | [103] | 2015 |
| CD133, CD54, CD44 | SC | Higher survival in patients who underwent resection of the primary tumor and surgical treatment for liver metastasis (p < 0.001). Worse survival in patients with high expression of CD133+CD54+ (p < 0.001), CD133−CD54+ (p = 0.004), and CD133+CD44+CD54+ (p = 0.003) CTCs subsets. Prognostic value, especially in survival of CRC patients who did not undergo surgical treatment for metastasis. Carcinoembryonic antigen levels, treatment strategy and CD133+CD44+CD54+ CTCs as independent prognostic factors. | [133] | 2017 |
| Pancreatic Adenocarcinoma | ||||
| Vimentin | M | Vimentin-positive CTCs identified in patients who did not have detectable CTCs by CellSearch, isolated by using the ISET (isolation by size of epithelial tumor cells) filtration device. | [134] | 2012 |
| ALDH1A2, IGFBP5 | M (IGFBP5), SC (ALDH1A2) | Presence in CTCs of low-proliferative signatures, enrichment of the stem-cell-associated gene ALDH1A2, biphenotypic expression of epithelial and mesenchymal markers, IGFBP5, a gene transcript enriched at the epithelial-stromal interface, and high expression of stromal-derived extracellular matrix proteins. | [135] | 2014 |
| Prostate Cancer | ||||
| ALDH | SC | Elevated clonogenicity and migratory behavior of ALDH-high cells. Competence in generation of distant metastasis and enhanced tumor progression of ALDH-high in preclinical models. Expression of several ALDH isoforms in clinical specimens of primary tumors with matched bone metastases. | [136] | 2010 |
| Vimentin | M | Identification of vimentin positive CTCs with BRCA losses, correlated with advanced tumor, invasiveness and recurrence, and shortened DFS. | [51] | 2010 |
| Vimentin, N-cadherin, O-cadherin, CD133 | M (Vimentin, N-cadherin, O-cadherin), SC (CD133) | In metastatic castration-resistant prostate cancer (CRPC), the majority (>80%) of CTCs co-express epithelial proteins (EpCAM, cytokeratins, E-cadherin), with mesenchymal proteins (vimentin, N-cadherin, O-cadherin), and the stem cell marker CD133. | [116] | 2011 |
| IGF1, IGF2, EGFR, FOXP3, TGFB3 | M | Commonly observed in CTCs in metastatic tumors, despite heterogeneous expression patterns of individual CTCs. An additional subset of EMT-related genes (PTPRN2, ALDH1, ESR2, and WNT5A) were expressed in CTCs of metastatic CRPC, but less frequently in castration-sensitive cancer. | [137] | 2013 |
| Gastric Cancer | ||||
| CD44 | SC | Prognostic significance of CD44-positive CTCs. Patients with higher CD44-positive CTCs were more likely to develop metastasis and recurrence than patients with CD44-negative CTCs. The presence of CD44-positive CTCs and TNM stage were independent predictors of recurrence. | [104] | 2014 |
| Melanoma | ||||
| MLANA, ABCB5, TGFβ2, PAX3d, MCAM | M | Detection of CTCs at all stages and after surgical resection of tumor. Significant prognostic value of expression of ABCB5 and MLANA in inferring disease recurrence. Correlation of MCAM expression with poor patient outcome after therapeutic nonsurgical treatment. | [138] | 2013 |