title: Gene expression profiling of single circulating tumor cells in ovarian cancer - Establishment of a multi-marker gene panel.
abstract: The presence of circulating tumor cells (CTCs) in the blood of ovarian cancer patients was shown to correlate with decreased overall survival, whereby CTCs with epithelial-mesenchymal-transition (EMT) or stem-like traits are supposed to be involved in metastatic progression and recurrence. Thus, investigating the transcriptional profiles of CTCs might help to identify therapy resistant tumor cells and to overcome treatment failure. For this purpose, we established a multi-marker panel for the molecular characterization of single CTCs, detecting epithelial (EpCAM, Muc-1, CK5/7), EMT (N-cadherin, Vimentin, Snai1/2, CD117, CD146, CD49f) and stem cell (CD44, ALDH1A1, Nanog, SOX2, Notch1/4, Oct4, Lin28) associated transcripts. First primer specificity and PCR-performance of the multiplex-RT-PCRs were successfully validated on genomic DNA and cDNA isolated from OvCar3 cells. The assay sensitivity of the epithelial panel was evaluated by adding defined numbers of tumor cells into the blood of healthy donors and performing a subsequent immunomagnetic tumor cell enrichment (AdnaTest OvarianCancerSelect), resulting in a 100% concordance for the epithelial markers EpCAM and Muc-1 to the AdnaTest OvarianCancerDetect. Additionally, by processing blood from ovarian cancer patients, high assay sensitivity could be verified. In blood of healthy donors no signals for epithelial markers were detected, for EMT and stem cell markers, however, signals were obtained mainly originating from leukocytes which calls for single cell analysis. To that aim by using the ovarian cancer cell line OvCar3, we successfully established a workflow enabling the characterization of single CTCs. It consists of a density gradient-dependent enrichment for nucleated cells, a depletion of CD45-positive cells of hematopoietic origin followed by immunofluorescent labeling of CTCs by EpCAM and Muc-1. Single CTCs are then isolated by micromanipulation and processed for panel gene expression profiling. Finally, fifteen single CTCs from three ovarian cancer patients were analyzed and found to be positive for stem cell (CD44, ALDH1A1, Nanog, Oct4) and EMT markers (N-cadherin, Vimentin, Snai2, CD117, CD146). Albeit, inter-cellular and intra/inter-patient heterogeneity and co-expression of epithelial, mesenchymal and stem cell transcripts on the same CTC was observed. We have established a robust workflow to perform sensitive single cell panel gene expression analysis without the need of pre-amplification steps. Our data point towards a heterogeneous expression of stem cell and EMT associated transcripts in ovarian cancer CTCs.
authors.affiliation: Department of Obstetrics and Gynecology, Medical Faculty and University Hospital of the Heinrich-Heine University Duesseldorf, Life Science Center, Merowingerplatz 1A, 40225 Duesseldorf, Germany. Electronic address: Christina.Blassl@med.uni-duesseldorf.de.
authors.forename: Christina
authors.initials: C
authors.lastname: Blassl
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany; German Cancer Consortium (DKTK), Dresden and German Cancer Research Center (DKFZ), Heidelberg, Germany. Electronic address: Jan.Kuhlmann@uniklinikum-dresden.de.
authors.forename: Jan Dominik
authors.initials: JD
authors.lastname: Kuhlmann
authors.affiliation: Department of Obstetrics and Gynecology, Medical Faculty and University Hospital of the Heinrich-Heine University Duesseldorf, Life Science Center, Merowingerplatz 1A, 40225 Duesseldorf, Germany. Electronic address: al.c.webers@gmail.com.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Webers
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany; German Cancer Consortium (DKTK), Dresden and German Cancer Research Center (DKFZ), Heidelberg, Germany. Electronic address: Pauline.Wimberger@uniklinikum-dresden.de.
authors.forename: Pauline
authors.initials: P
authors.lastname: Wimberger
authors.affiliation: Department of Obstetrics and Gynecology, Medical Faculty and University Hospital of the Heinrich-Heine University Duesseldorf, Moorenstraße 5, 40225 Duesseldorf, Germany. Electronic address: Tanja.Fehm@med.uni-duesseldorf.de.
authors.forename: Tanja
authors.initials: T
authors.lastname: Fehm
authors.affiliation: Department of Obstetrics and Gynecology, Medical Faculty and University Hospital of the Heinrich-Heine University Duesseldorf, Life Science Center, Merowingerplatz 1A, 40225 Duesseldorf, Germany. Electronic address: Hans.Neubauer@med.uni-duesseldorf.de.
authors.forename: Hans
authors.initials: H
authors.lastname: Neubauer
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D004847:Epithelial Cells / Q000378:metabolism; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology*; D010051:Ovarian Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D059010:Single-Cell Analysis
chemical_list: D014408:Biomarkers, Tumor
keywords: Circulating tumor cells; Multiplex-RT-PCR; Ovarian cancer; Single cell expression analysis