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Spectrum of Epithelial-Mesenchymal Transition Phenotypes in Circulating Tumour Cells from Early Breast Cancer Patients.

ABSTRACT: Circulating tumour cells (CTCs) can provide valuable prognostic information in a number of epithelial cancers. However, their detection is hampered due to their molecular heterogeneity, which can be induced by the epithelial-mesenchymal transition (EMT) process. Therefore, current knowledge about CTCs from clinical samples is often limited due to an inability to isolate wide spectrum of CTCs phenotypes. In the current work, we aimed at isolation and molecular characterization of CTCs with different EMT status in order to establish their clinical significance in early breast cancer patients. We have obtained CTCs-enriched blood fraction from 83 breast cancer patients in which we have tested the expression of epithelial, mesenchymal and general breast cancer CTCs markers (MGB1/HER2/CK19/CDH1/CDH2/VIM/PLS3), cancer stem cell markers (CD44, NANOG, ALDH1, OCT-4, CD133) and cluster formation gene (plakoglobin). We have shown that in the CTCs-positive patients, epithelial, epithelial-mesenchymal and mesenchymal CTCs markers were detected at a similar rate (in 28%, 24% and 24%, respectively). Mesenchymal CTCs were characterized by the most aggressive phenotype (significantly higher expression of CXCR4, uPAR, CD44, NANOG, p < 0.05 for all), presence of lymph node metastases (p = 0.043), larger tumour size (p = 0.023) and 7.33 higher risk of death in the multivariate analysis (95% CI 1.06?50.41, p = 0.04). Epithelial-mesenchymal subtype, believed to correspond to highly plastic and aggressive state, did not show significant impact on survival. Gene expression profile of samples with epithelial-mesenchymal CTCs group resembled pure epithelial or pure mesenchymal phenotypes, possibly underlining degree of EMT activation in particular patient's sample. Molecular profiling of CTCs EMT phenotype provides more detailed and clinically informative results, proving the role of EMT in malignant cancer progression in early breast cancer.

SUBMITTER: Markiewicz A

PROVIDER: S-EPMC6356662 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Spectrum of Epithelial-Mesenchymal Transition Phenotypes in Circulating Tumour Cells from Early Breast Cancer Patients.

Markiewicz Aleksandra A Topa Justyna J Nagel Anna A Skokowski Jaroslaw J Seroczynska Barbara B Stokowy Tomasz T Welnicka-Jaskiewicz Marzena M Zaczek Anna J AJ

Cancers 20190109 1

Circulating tumour cells (CTCs) can provide valuable prognostic information in a number of epithelial cancers. However, their detection is hampered due to their molecular heterogeneity, which can be induced by the epithelial-mesenchymal transition (EMT) process. Therefore, current knowledge about CTCs from clinical samples is often limited due to an inability to isolate wide spectrum of CTCs phenotypes. In the current work, we aimed at isolation and molecular characterization of CTCs with differ ...[more]

PMID: 30634453

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Project description: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.

Project description: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.

Project description:Metastasis is the leading cause of cancer-related deaths worldwide. The epithelial-mesenchymal plasticity (EMP) status of primary tumours has relevance to metastatic potential and therapy resistance. Circulating tumour cells (CTCs) provide a window into the metastatic process, and molecular characterisation of CTCs in comparison to their primary tumours could lead to a better understanding of the mechanisms involved in the metastatic cascade. In this study, paired blood and tumour samples were collected from four prostate cancer patient-derived xenograft (PDX) models (BM18, LuCaP70, LuCaP96, LuCaP105) and assessed using an EMP-focused, 42 gene human-specific, nested quantitative RT-PCR assay. CTC burden varied amongst the various xenograft models with LuCaP96 having the highest number of CTCs per mouse (mean: 704; median: 31) followed by BM18 (mean: 101; median: 21), LuCaP70 (mean: 73; median: 16) and LuCaP105 (mean: 57; median: 6). A significant relationship was observed between tumour size and CTC number (p = 0.0058). Decreased levels of kallikrein-related peptidase 3 (KLK3) mRNA (which encodes prostate-specific antigen; PSA) were observed in CTC samples from all four models compared to their primary tumours. Both epithelial- and mesenchymal-associated genes were commonly expressed at higher levels in CTCs compared to the bulk primary tumour, although some common EMT-associated genes (CDH1, VIM, EGFR, EPCAM) remained unchanged. Immunofluorescence co-staining for pan-cytokeratin (KRT) and vimentin (VIM) indicated variable proportions of CTCs across the full EMP axis, even in the same model. EMP hybrids predominated in the BM18 and LuCaP96 models, but were not detected in the LuCaP105 model, and variable numbers of KRT+ and human VIM+ cells were observed in each model. SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), was enriched at the RNA level in CTCs compared to primary tumours and was the most commonly expressed mesenchymal gene in the CTCs. Co-staining for SERPINE1 and KRT revealed SERPINE1+ cells in 7/11 samples, six of which had SERPINE+KRT+ CTCs. Cell size variation was observed in CTCs. The majority of samples (8/11) contained larger CTCs ranging from 15.3 to 37.8 µm, whilst smaller cells (10.7 ± 4.1 µm, similar in size to peripheral blood mononuclear cells (PBMCs)) were identified in 6 of 11 samples. CTC clusters were also identified in 9/11 samples, containing 2-100 CTCs per cluster. Where CTC heterogeneity was observed in the clusters, epithelial-like cells (KRT+VIM-) were located on the periphery of the cluster, forming a layer around hybrid (KRT+VIM+) or mesenchymal-like (KRT-VIM+) cells. The CTC heterogeneity observed in these models emphasises the complexity in CTC isolation and classification and supports the increasingly recognised importance of the epithelial-mesenchymal hybrid state in cancer progression and metastasis.

Project description:The presence of circulating tumor cells (CTC) in breast cancer might be associated with stem cell-like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, to be able to disseminate and metastasize, CTC must be able to perform epithelial-mesenchymal transition (EMT). We studied the expression of three EMT markers and the stem cell marker ALDH1 in CTC from 502 primary breast cancer patients. Data were correlated with the presence of disseminated tumor cells (DTC) in the bone marrow (BM) and with clinicopathological data of the patients.A total of 2 × 5 ml of blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1, HER2 and beta-Actin transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [TWIST1, Akt2, phosphoinositide kinase-3 (PI3K?)] and separately for the tumor stem cell marker ALDH1. The identification of EMT markers was considered positive if at least one marker was detected in the sample. Two BM aspirates from all patients were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.Ninety-seven percent of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts, respectively. CTC were detected in 97/502 (19%) patients. At least one of the EMT markers was expressed in 29% and ALDH1 was present in 14% of the samples, respectively. Interestingly, 5% of the ALDH1-positive and 18% of the EMT-positive patients were CTC-negative based on the cut-off level determined for CTC-positivity applying the AdnaTest BreastCancer. DTC in the BM were detected in 107/502 (21%) patients and no correlation was found between BM status and CTC positivity (P = 0.41). The presence of CTC, EMT and ALDH1 expression was not correlated to any of the prognostic clinical markers.Our data indicate that (1) a subset of primary breast cancer patients shows EMT and stem cell characteristics and (2) the currently used detection methods for CTC are not efficient to identify a subtype of CTC which underwent EMT. (3) The clinical relevance on prognosis and therapy response has to be further evaluated in a prospective trial.

Dataset Information

Spectrum of Epithelial-Mesenchymal Transition Phenotypes in Circulating Tumour Cells from Early Breast Cancer Patients.

Publications

Spectrum of Epithelial-Mesenchymal Transition Phenotypes in Circulating Tumour Cells from Early Breast Cancer Patients.

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