title: miRNA in situ hybridization in circulating tumor cells--MishCTC.
abstract: Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.
authors.affiliation: GENYO. Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government PTS Granada. Avenida de la Ilustración, 114. 18016 GRANADA, Spain.
authors.forename: Francisco G
authors.initials: FG
authors.lastname: Ortega
authors.affiliation: 1] GENYO. Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government PTS Granada. Avenida de la Ilustración, 114. 18016 GRANADA, Spain [2] Laboratory of Genetic Identification, Department of Legal Medicine, University of Granada. Avda. Madrid 11, 18012 Granada, Spain.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Lorente
authors.affiliation: GENYO. Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government PTS Granada. Avenida de la Ilustración, 114. 18016 GRANADA, Spain.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Garcia Puche
authors.affiliation: GENYO. Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government PTS Granada. Avenida de la Ilustración, 114. 18016 GRANADA, Spain.
authors.forename: Maria P
authors.initials: MP
authors.lastname: Ruiz
authors.affiliation: GENYO. Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government PTS Granada. Avenida de la Ilustración, 114. 18016 GRANADA, Spain.
authors.forename: Rosario M
authors.initials: RM
authors.lastname: Sanchez-Martin
authors.affiliation: GENYO. Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government PTS Granada. Avenida de la Ilustración, 114. 18016 GRANADA, Spain.
authors.forename: Diego
authors.initials: D
authors.lastname: de Miguel-Pérez
authors.affiliation: GENYO. Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government PTS Granada. Avenida de la Ilustración, 114. 18016 GRANADA, Spain.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Diaz-Mochon
authors.affiliation: GENYO. Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government PTS Granada. Avenida de la Ilustración, 114. 18016 GRANADA, Spain.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Serrano
mesh_terms: D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000235:genetics*; D009369:Neoplasms / Q000097:blood / Q000235:genetics / Q000473:pathology; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs