Single-cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT-related genes in metastatic prostate cancer.
TL;DR: It is postulate that CTCs expressing genes related to epithelial–mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer.
Abstract: BACKGROUND. Prostate tumors shed circulating tumor cells (CTCs) into the blood stream. Increased evidence shows that CTCs are often present in metastatic prostate cancer and can be alternative sources for disease profiling and prognostication. Here we postulate that CTCs expressing genes related to epithelial–mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer. METHODS. A microfiltration system was used to trap CTCs from peripheral blood based on size selection of large epithelial-like cells without CD45 leukocyte marker. These cells individually retrieved with a micromanipulator device were assessed for cell membrane physical properties using atomic force microscopy. Additionally, 38 CTCs from eight prostate cancer patients were used to determine expression profiles of 84 EMT-related and reference genes using a microfluidics-based PCR system. RESULTS. Increased cell elasticity and membrane smoothness were found in CTCs compared to noncancerous cells, highlighting their potential invasiveness and mobility in the
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TL;DR: How circulating tumour cells (CTCs), captured from a minimally invasive blood test—and readily amenable to serial sampling—have the potential to inform intratumour heterogeneity and tumour evolution is described, although it remains to be determined how useful this will be in the clinic.
Abstract: Growing evidence for intratumour heterogeneity informs us that single-site biopsies fall short of revealing the complete genomic landscape of a tumour. With an expanding repertoire of targeted agents entering the clinic, screening tumours for genomic aberrations is increasingly important, as is interrogating the tumours for resistance mechanisms upon disease progression. Multiple biopsies separated spatially and temporally are impractical, uncomfortable for the patient and not without risk. Here, we describe how circulating tumour cells (CTCs), captured from a minimally invasive blood test-and readily amenable to serial sampling-have the potential to inform intratumour heterogeneity and tumour evolution, although it remains to be determined how useful this will be in the clinic. Technologies for detecting and isolating CTCs include the validated CellSearch(®) system, but other technologies are gaining prominence. We also discuss how recent CTC discoveries map to mechanisms of haematological spread, previously described in preclinical models, including evidence for epithelial-mesenchymal transition, collective cell migration and cells with tumour-initiating capacity within the circulation. Advances in single-cell molecular analysis are enhancing our ability to explore mechanisms of metastasis, and the combination of CTC and cell-free DNA assays are anticipated to provide invaluable blood-borne biomarkers for real-time patient monitoring and treatment stratification.
541 citations
TL;DR: This review focuses on the opportunities as well as the challenges that should be addressed so that these tools may eventually be implemented into routine clinical care.
Abstract: Recent technological advances have enabled the detection and detailed characterization of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) in blood samples from patients with cancer. Often referred to as a "liquid biopsy," CTCs and ctDNA are expected to provide real-time monitoring of tumor evolution and therapeutic efficacy, with the potential for improved cancer diagnosis and treatment. In this review, we focus on these opportunities as well as the challenges that should be addressed so that these tools may eventually be implemented into routine clinical care.
297 citations
Cites background from "Single-cell analysis of circulating..."
...Similarly, prostate cancer also appears to be characterized by CTC heterogeneity, with distinct differences in single-cell expression of EMT-related genes between CTCs from castration-sensitive and castrate-resistant cancers (48)....
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TL;DR: A clinical perspective on IGF2 is provided and an update on recent research findings are provided, suggesting much novel clinical utility for its measurement.
Abstract: Insulin-like growth factor 2 (IGF2) is a 7.5 kDa mitogenic peptide hormone expressed by liver and many other tissues. It is three times more abundant in serum than IGF1, but our understanding of its physiological and pathological roles has lagged behind that of IGF1. Expression of the IGF2 gene is strictly regulated. Over-expression occurs in many cancers and is associated with a poor prognosis. Elevated serum IGF2 is also associated with increased risk of developing various cancers including colorectal, breast, prostate and lung. There is established clinical utility for IGF2 measurement in the diagnosis of non-islet cell tumour hypoglycaemia, a condition characterised by a molar IGF2:IGF1 ratio >10. Recent advances in understanding of the pathophysiology of IGF2 in cancer have suggested much novel clinical utility for its measurement. Measurement of IGF2 in blood and genetic and epigenetic tests of the IGF2 gene may help assess cancer risk and prognosis. Further studies will determine whether these tests enter clinical practice. New therapeutic approaches are being developed to target IGF2 action. This review provides a clinical perspective on IGF2 and an update on recent research findings.
233 citations
Cites background or methods from "Single-cell analysis of circulating..."
...…HBV infection Kim et al. (2006) tive recurrence of HCC Lee et al. (2012) Morcavallo et al. (2012) and Yoon et al. (2012) esis Hoshida (2011) lial ovarian cancer Pils et al. (2013) ncer Chen et al. (2013) r carcinoma; HCV, hepatitis C virus; IGF2, insulin-like growth factor 2; IGF2R, hypoglycaemia....
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...This technique was used in patients with prostate cancer to examine expression of multiple epithelial–mesenchymal transition genes including IGF2 (Chen et al. 2013)....
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TL;DR: Preclinical studies have demonstrated the potential of inhibitors that target WNT receptor complexes at the cell membrane or that block the interaction of β-catenin with lymphoid enhancer-binding factor 1 and the androgen receptor, in preventing prostate cancer progression.
Abstract: Genome sequencing and gene expression analyses of prostate tumours have highlighted the potential importance of genetic and epigenetic changes observed in WNT signalling pathway components in prostate tumours - particularly in the development of castration-resistant prostate cancer. WNT signalling is also important in the prostate tumour microenvironment, in which WNT proteins secreted by the tumour stroma promote resistance to therapy, and in prostate cancer stem or progenitor cells, in which WNT-β-catenin signals promote self-renewal or expansion. Preclinical studies have demonstrated the potential of inhibitors that target WNT receptor complexes at the cell membrane or that block the interaction of β-catenin with lymphoid enhancer-binding factor 1 and the androgen receptor, in preventing prostate cancer progression. Some WNT signalling inhibitors are in phase I trials, but they have yet to be tested in patients with prostate cancer.
215 citations
TL;DR: This work extends the evidence that mesenchymal and epithelial states in cancer cells contribute differentially to their capacities for tumor initiation and metastatic seeding, respectively, and that EMT tumor cells exist with plasticity that can contribute to multiple stages of the metastatic cascade.
Abstract: The epithelial-mesenchymal transition (EMT) has been postulated as a mechanism by which cancer cells acquire the invasive and stem-like traits necessary for distant metastasis. However, direct in vivo evidence for the role of EMT in the formation of cancer stem-like cells (CSC) and the metastatic cascade remains lacking. Here we report the first isolation and characterization of mesenchymal-like and EMT tumor cells, which harbor both epithelial and mesenchymal characteristics, in an autochthonous murine model of prostate cancer. By crossing the established Pb-Cre(+/-);Pten(L/L);Kras(G12D) (/+) prostate cancer model with a vimentin-GFP reporter strain, generating CPKV mice, we were able to isolate epithelial, EMT, and mesenchymal-like cancer cells based on expression of vimentin and EpCAM. CPKV mice (but not mice with Pten deletion alone) exhibited expansion of cells with EMT (EpCAM(+)/Vim-GFP(+)) and mesenchymal-like (EpCAM(-)/Vim-GFP(+)) characteristics at the primary tumor site and in circulation. These EMT and mesenchymal-like tumor cells displayed enhanced stemness and invasive character compared with epithelial tumor cells. Moreover, they displayed an enriched tumor-initiating capacity and could regenerate epithelial glandular structures in vivo, indicative of epithelia-mesenchyme plasticity. Interestingly, while mesenchymal-like tumor cells could persist in circulation and survive in the lung following intravenous injection, only epithelial and EMT tumor cells could form macrometastases. Our work extends the evidence that mesenchymal and epithelial states in cancer cells contribute differentially to their capacities for tumor initiation and metastatic seeding, respectively, and that EMT tumor cells exist with plasticity that can contribute to multiple stages of the metastatic cascade.
168 citations
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TL;DR: The 2-Delta Delta C(T) method as mentioned in this paper was proposed to analyze the relative changes in gene expression from real-time quantitative PCR experiments, and it has been shown to be useful in the analysis of realtime, quantitative PCR data.
139,407 citations
TL;DR: Processes similar to the EMTs associated with embryo implantation, embryogenesis, and organ development are appropriated and subverted by chronically inflamed tissues and neoplasias and the identification of the signaling pathways that lead to activation of EMT programs during these disease processes is providing new insights into the plasticity of cellular phenotypes.
Abstract: The origins of the mesenchymal cells participating in tissue repair and pathological processes, notably tissue fibrosis, tumor invasiveness, and metastasis, are poorly understood. However, emerging evidence suggests that epithelial-mesenchymal transitions (EMTs) represent one important source of these cells. As we discuss here, processes similar to the EMTs associated with embryo implantation, embryogenesis, and organ development are appropriated and subverted by chronically inflamed tissues and neoplasias. The identification of the signaling pathways that lead to activation of EMT programs during these disease processes is providing new insights into the plasticity of cellular phenotypes and possible therapeutic interventions.
8,587 citations
"Single-cell analysis of circulating..." refers background in this paper
...and SNAIL gene families that repress epithelial cell adhesion and induce other mesenchymal proteins [16]....
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...Although we cannot rule out technical limitations of detecting some prostate cancer-related genes at the single-cell level, our initial results suggest a dramatic shift of gene expression occurring in CTCs that escaped from their primary tumor sites [16]....
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...EMT is a gradual process, and gene markers specific for mesenchymal and stem-like cells can be detected in CTCs [15,16]....
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TL;DR: The use of sipuleucel-T prolonged overall survival among men with metastatic castration-resistant prostate cancer and immune responses to the immunizing antigen were observed in patients who received sipuleUcel- T.
Abstract: Background Sipuleucel-T, an autologous active cellular immunotherapy, has shown evidence of efficacy in reducing the risk of death among men with metastatic castration-resistant prostate cancer. Methods In this double-blind, placebo-controlled, multicenter phase 3 trial, we randomly assigned 512 patients in a 2:1 ratio to receive either sipuleucel-T (341 patients) or placebo (171 patients) administered intravenously every 2 weeks, for a total of three infusions. The primary end point was overall survival, analyzed by means of a stratified Cox regression model adjusted for baseline levels of serum prostate-specific antigen (PSA) and lactate dehydrogenase. Results In the sipuleucel-T group, there was a relative reduction of 22% in the risk of death as compared with the placebo group (hazard ratio, 0.78; 95% confidence interval [CI], 0.61 to 0.98; P = 0.03). This reduction represented a 4.1-month improvement in median survival (25.8 months in the sipuleucel-T group vs. 21.7 months in the placebo group). The 36-month survival probability was 31.7% in the sipuleucel-T group versus 23.0% in the placebo group. The treatment effect was also observed with the use of an unadjusted Cox model and a log-rank test (hazard ratio, 0.77; 95% CI, 0.61 to 0.97; P = 0.02) and after adjustment for use of docetaxel after the study therapy (hazard ratio, 0.78; 95% CI, 0.62 to 0.98; P = 0.03). The time to objective disease progression was similar in the two study groups. Immune responses to the immunizing antigen were observed in patients who received sipuleucel-T. Adverse events that were more frequently reported in the sipuleucel-T group than in the placebo group included chills, fever, and headache. Conclusions The use of sipuleucel-T prolonged overall survival among men with metastatic castration-resistant prostate cancer. No effect on the time to disease progression was observed. (Funded by Dendreon; ClinicalTrials.gov number, NCT00065442.)
4,840 citations
"Single-cell analysis of circulating..." refers background in this paper
...Despite improvement in median overall survival, most patients did not achieve PSA response to therapy [34]....
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TL;DR: The affy package is an R package of functions and classes for the analysis of oligonucleotide arrays manufactured by Affymetrix that provides the user with extreme flexibility when carrying out an analysis and make it possible to access and manipulate probe intensity data.
Abstract: Motivation: The processing of the Affymetrix GeneChip data has been a recent focus for data analysts. Alternatives to the original procedure have been proposed and some of these new methods are widely used.
Results: The affy package is an R package of functions and classes for the analysis of oligonucleotide arrays manufactured by Affymetrix. The package is currently in its second release, affy provides the user with extreme flexibility when carrying out an analysis and make it possible to access and manipulate probe intensity data. In this paper, we present the main classes and functions in the package and demonstrate how they can be used to process probe-level data. We also demonstrate the importance of probe-level analysis when using the Affymetrix GeneChip platform.
4,822 citations
TL;DR: The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs, which are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.
Abstract: Purpose: The purpose of this study was to determine the accuracy, precision, and linearity of the CellSearch system and evaluate the number of circulating tumor cells (CTCs) per 7.5 mL of blood in healthy subjects, patients with nonmalignant diseases, and patients with a variety of metastatic carcinomas. Experimental Design: The CellSearch system was used to enumerate CTCs in 7.5 mL of blood. Blood samples spiked with cells from tumor cell lines were used to establish analytical accuracy, reproducibility, and linearity. Prevalence of CTCs was determined in blood from 199 patients with nonmalignant diseases, 964 patients with metastatic carcinomas, and 145 healthy donors. Results: Enumeration of spiked tumor cells was linear over the range of 5 to 1,142 cells, with an average recovery of ≥85% at each spike level. Only 1 of the 344 (0.3%) healthy and nonmalignant disease subjects had ≥2 CTCs per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTCs ranged from 0 to 23,618 CTCs per 7.5 mL (mean, 60 ± 693 CTCs per 7.5 mL), and 36% (781 of 2,183) of the specimens had ≥2 CTCs. Detection of ≥2 CTCs occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers. Conclusions: The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs. CTCs are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.
2,343 citations
"Single-cell analysis of circulating..." refers background in this paper
...The FDA-approved CellSearch(1) system has to date been considered the gold standard for CTC detection in the clinical setting [6,7]....
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