IntroductionEpithelial to mesenchymal transition (EMT) is considered an essential process in the metastatic cascade. EMT is characterised by upregulation of vimentin, Twist, Snail, Slug and Sip1 among others. Metastasis is also associated with the presence of circulating tumour cells (CTCs) and disseminated tumour cells in the blood and bone marrow, respectively, of breast cancer patients, but the expression of EMT markers in these cells has not been reported so far.MethodsThe expression of Twist and vimentin in CTCs of 25 metastatic and 25 early breast cancer patients was investigated by using double-immunofluorescence experiments in isolated peripheral blood mononuclear cell cytospins using anti-cytokeratin (anti-CK) anti-mouse (A45-B/B3) and anti-Twist or anti-vimentin anti-rabbit antibodies.ResultsAmong early breast cancer patients, vimentin-and Twist-expressing CK+ CTCs were identified in 77% and 73% of the patients, respectively, and in 100% of the patients with metastatic breast cancer for both markers (P = 0.004 and P = 0.037, respectively). Among patients with early disease, 56% and 53% of the CK+ CTCs were double-stained with vimentin and Twist, and the corresponding values for metastatic patients were 74% and 97%, respectively (P = 0.005 and P = 0.0001, respectively). The median expression of CK+vimentin+ and CK+Twist+ cells per patient in metastatic patients was 98% and 100%, and in an adjuvant chemotherapy setting the corresponding numbers were 56% and 40.6%, respectively. Triple-staining experiments revealed that all CK+Twist+ or CK+vimentin+ cells were also CD45-, confirming their epithelial origin. Immunomagnetic separation of CTCs and triple-immunofluorescence with anti-CK/anti-Twist/anti-vimentin antibodies demonstrated that both mesenchymal markers could be coexpressed in the same CK+ cell, since 64% of the total identified CTCs were triple-stained. There was a significant correlation (P = 0.005) between the number of CTCs expressing Twist and vimentin within the same setting.ConclusionsCTCs expressing Twist and vimentin, suggestive of EMT, are identified in patients with breast cancer. The high incidence of these cells in patients with metastatic disease compared to early stage breast cancer strongly supports the notion that EMT is involved in the metastatic potential of CTCs.

Circulating tumor cells (CTCs) bearing phenotypes related to cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) have been identified in breast cancer; however, their clinical significance is not clear. In the current study, we investigated the prognostic relevance of single CSC þ /partial-EMT þ CTCs in patients with metastatic breast cancer and the effect of first-line chemotherapy on their incidence. For this purpose, triple immunofluorescence against cytokeratin, ALDH1, and TWIST1 was performed in peripheral blood mononuclear cell (PBMC) cytospins from 130 patients before and after first-line chemotherapy. CSC þ / partial-EMT þ CTCs were characterized as cells co-expressing cytokeratin, high levels of ALDH1, and nuclear TWIST1. CSC þ /partial-EMT þ CTCs were evident in 27.7% of patients at baseline and were correlated to lung metastases (P ¼ 0.010) and decreased progression-free survival [PFS; median 10.2 (8.9-11.6) vs. 13.5 (11.3-15.7) months; P ¼ 0.024].Their detection was an independent factor predicting for increased risk of relapse [multivariate analysis; HR (95% confidence interval (CI)): 1.785 (1.171-2.720); P ¼ 0.007]. In HER-2-negative patients, CSC þ /partial-EMT þ CTCs were additionally associated with reduced overall survival (OS) [median 39 (26.2-51.9) vs. 51 (15.7-86.4) months; P ¼ 0.020] and increased risk of death [multivariate analysis; HR (95% CI): 2.228 (1.066-4.655); P ¼ 0.033]. Chemotherapy resulted in a significant increase in the incidence of CSC þ /partial-EMT þ CTCs (mean CTC% per patient: 59.4% post vs. 39.5% pre; P ¼ 0.018), which was subsequently confirmed only in HER2-negative patients (P ¼ 0.040) and in non-responders at the end of treatment (P ¼ 0.020). In conclusion, CSC þ /partial-EMT þ CTCs represent a chemoresistant subpopulation, which independently predicts for unfavorable outcome in metastatic breast cancer. Efficient targeting of these CTCs could potentially increase patient survival.

BackgroundThe detection of circulating tumor cells (CTCs) in peripheral blood (PB) of patients with breast cancer predicts poor clinical outcome. Cancer cells with stemness and epithelial-to-mesenchymal transition (EMT) features display enhanced malignant and metastatic potential. A new methodology was developed in order to investigate the co-expression of a stemness and an EMT marker (ALDH1 and TWIST, respectively) on single CTCs of patients with early and metastatic breast cancer.MethodsTriple immunofluorescence using anti-pancytokeratin (A45-B/B3), anti-ALDH1 and anti-TWIST antibodies was performed in cytospins prepared from hepatocellular carcinoma HepG2 cells and SKBR-3, MCF-7 and MDA.MB.231 breast cancer cell lines. Evaluation of ALDH1 expression levels (high, low or absent) and TWIST subcellular localization (nuclear, cytoplasmic or absent) was performed using the ARIOL system. Cytospins prepared from peripheral blood of patients with early (n = 80) and metastatic (n = 50) breast cancer were analyzed for CTC detection (based on pan-cytokeratin expression and cytomorphological criteria) and characterized according to ALDH1 and TWIST.ResultsCTCs were detected in 13 (16%) and 25 (50%) patients with early and metastatic disease, respectively. High ALDH1 expression (ALDH1high) and nuclear TWIST localization (TWISTnuc) on CTCs was confirmed in more patients with metastatic than early breast cancer (80% vs. 30.8%, respectively; p = 0.009). In early disease, ALDH1low/neg CTCs (p = 0.006) and TWISTcyt/neg CTCs (p = 0.040) were mainly observed. Regarding co-expression of these markers, ALDH1high/TWISTnuc CTCs were more frequently evident in the metastatic setting (76% vs. 15.4% of patients, p = 0.001; 61.5% vs. 12.9% of total CTCs), whereas in early disease ALDH1low/neg/TWISTcyt/neg CTCs were mainly detected (61.5% vs. 20% of patients, p = 0.078; 41.9% vs. 7.7% of total CTCs).ConclusionsA new assay is provided for the evaluation of ALDH1 and TWIST co-expression at the single CTC-level in patients with breast cancer. A differential expression pattern for these markers was observed both in early and metastatic disease. CTCs expressing high ALDH1, along with nuclear TWIST were more frequently detected in patients with metastatic breast cancer, suggesting that these cells may prevail during disease progression.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-651) contains supplementary material, which is available to authorized users.

IntroductionThe detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs.MethodsThe expression of cytokeratins (CK), VEGF, vascular endothelial growth factor receptor-2 (VEGF2), HIF-1α and phosphorylated-focal adhesion kinase (pFAK) in CTCs from 34 patients with metastatic breast cancer who had detectable CK-19 mRNA-positive CTCs was assessed using double staining experiments and confocal laser scanning microscopy. Peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 pancytokeratin antibody in combination with either VEGF or VEGFR2 or HIF-1α or pFAK antibodies, respectively.ResultspFAK expression in circulating tumor cells was detected in 92% of patients whereas expression of VEGF, VEGF2 and HIF-1α was observed in 62%, 47% and 76% of patients, respectively. VEGF, VEGF2, HIF-1α and pFAK were expressed in 73%, 71%, 56% and 81%, respectively, of all the detected CTCs. Vascular endothelial growth mRNA was also detected by quantitative real-time RT-PCR in immunomagnetically-separated CTCs. Double and triple staining experiments in cytospins of immunomagnetically-isolated CTCs showed that VEGF co-expressed with HIF-1α and VEGF2.ConclusionsThe expression of pFAK, HIF-1α, VEGF and VEGF2 in CTCs of patients with metastatic breast cancer could explain the metastatic potential of these cells and may provide a therapeutic target for their elimination.

The detection of circulating tumor cells (CTC) in breast cancer is strongly associated with disease relapse. Since it is unclear whether all CTCs are capable of generating metastasis, we investigated their apoptotic and proliferative status in 56 CTC-positive (29 early and 27 metastatic) patients with breast cancer. Double-staining immunofluorescence experiments were carried out in peripheral blood mononuclear cells (PBMC) cytospins, using the pancytokeratin A45-B/B3 antibody and either M30 (apoptotic marker) or Ki67 (proliferation marker) antibodies. Apoptosis was also evaluated using a polycaspase detection kit. Patients with metastatic disease had significantly lower numbers of apoptotic CTCs compared with patients with early breast cancer (polycaspase kit: 8.1% vs. 47.4% of the total CTC number; P ¼ 0.0001; M30-antibody: 32.1% vs. 76.63%; P ¼ 0.002). The median percentage of apoptotic CTCs per patient was also lower in patients with advanced compared with those with early disease (polycaspase kit: 0% vs. 53.6%; M30-antibody: 15% vs. 80%). Ki67-positive CTCs were identified in 51.7% and 44% of patients with early and metastatic disease, respectively. Adjuvant chemotherapy reduced both the number of CTCs per patient and the number of proliferating CTCs (63.9% vs. 30%). In conclusion, apoptotic CTCs could be detected in patients with breast cancer irrespective of their clinical status, though the incidence of detection is higher in early compared with metastatic patients. The detection of CTCs that survive despite adjuvant therapy implies that CTC elimination should be attempted using agents targeting their distinctive molecular characteristics. Mol Cancer Ther; 12(9); 1886-95. Ó2013 AACR.