Emanuela Fina1, Maurizio Callari1, Carolina Reduzzi1, Francesca D'Aiuto1, Gabriella Mariani2, Daniele Generali3, Marco A Pierotti4, Maria G Daidone5, Vera Cappelletti1. 1. Biomarkers Unit, Department of Experimental Oncology and Molecular Medicine. 2. Medical Oncology Unit, and. 3. U.O. Multidisciplinare di Patologia Mammaria, U.S. Terapia Molecolare e Farmacogenomica, A.O. Istituti Ospitalieri di Cremona, Cremona, Italy. 4. Scientific Directorate, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 5. Biomarkers Unit, Department of Experimental Oncology and Molecular Medicine, mariagrazia.daidone@istitutotumori.mi.it.
Abstract
BACKGROUND: Determining the transcriptional profile of circulating tumor cells (CTCs) may allow the acquisition of clinically relevant information while overcoming tumor heterogeneity-related biases associated with use of tissue samples for biomarker assessment. However, such molecular characterization is challenging because CTCs are rare and outnumbered by blood cells. METHODS: Here, we describe a technical protocol to measure the expression of >29 000 genes in CTCs captured from whole blood with magnetic beads linked with antibodies against epithelial cell adhesion molecule (EpCAM) and the carcinoma-associated mucin, MUC1, designed to be used for CTC characterization in clinical samples. Low numbers of cells (5-200) from the MCF7 and MDA-MB-468 breast cancer cell lines were spiked in healthy donor blood samples and isolated with the AdnaTest EMT-1/Stem CellSelect kit. Gene expression profiles (GEPs) were obtained with the WG-DASL HT assay and compared with GEPs obtained from RNA isolated from cultured cell lines and unspiked samples. RESULTS: GEPs from samples containing 25 or more spiked cells correlated (r = 0.95) with cognate 100-ng RNA input samples, clustered separately from blood control samples, and allowed MCF7 and MDA-MB-468 cells to be distinguished. GEPs with comparable technical quality were also obtained in a preliminary series of clinical samples. CONCLUSIONS: Our approach allows technically reliable GEPs to be obtained from isolated CTCs for the acquisition of biologically useful information. It is reproducible and suitable for application in prospective studies to assess the clinical utility of CTC GEPs, provided that >25 CTCs can be isolated.
BACKGROUND: Determining the transcriptional profile of circulating tumor cells (CTCs) may allow the acquisition of clinically relevant information while overcoming tumor heterogeneity-related biases associated with use of tissue samples for biomarker assessment. However, such molecular characterization is challenging because CTCs are rare and outnumbered by blood cells. METHODS: Here, we describe a technical protocol to measure the expression of >29 000 genes in CTCs captured from whole blood with magnetic beads linked with antibodies against epithelial cell adhesion molecule (EpCAM) and the carcinoma-associated mucin, MUC1, designed to be used for CTC characterization in clinical samples. Low numbers of cells (5-200) from the MCF7 and MDA-MB-468 breast cancer cell lines were spiked in healthy donor blood samples and isolated with the AdnaTest EMT-1/Stem CellSelect kit. Gene expression profiles (GEPs) were obtained with the WG-DASL HT assay and compared with GEPs obtained from RNA isolated from cultured cell lines and unspiked samples. RESULTS: GEPs from samples containing 25 or more spiked cells correlated (r = 0.95) with cognate 100-ng RNA input samples, clustered separately from blood control samples, and allowed MCF7 and MDA-MB-468 cells to be distinguished. GEPs with comparable technical quality were also obtained in a preliminary series of clinical samples. CONCLUSIONS: Our approach allows technically reliable GEPs to be obtained from isolated CTCs for the acquisition of biologically useful information. It is reproducible and suitable for application in prospective studies to assess the clinical utility of CTC GEPs, provided that >25 CTCs can be isolated.
Authors: Sven Kruspe; David D Dickey; Kevin T Urak; Giselle N Blanco; Matthew J Miller; Karen C Clark; Elliot Burghardt; Wade R Gutierrez; Sneha D Phadke; Sukriti Kamboj; Timothy Ginader; Brian J Smith; Sarah K Grimm; James Schappet; Howard Ozer; Alexandra Thomas; James O McNamara; Carlos H Chan; Paloma H Giangrande Journal: Mol Ther Nucleic Acids Date: 2017-08-12 Impact factor: 8.886
Authors: Danylo Rafhael Costa-Silva; Maria da Conceição Barros-Oliveira; Francisco Adelton Alves-Ribeiro; Larysse Cardoso Campos-Verdes; Elmo de Jesus Nery Junior; Samara Fernanda Vieira-Valença; Rodrigo Jose de Vasconcelos-Valença; Veronica Mendes Soares; André Luiz Pinho-Sobral; Emerson Brandão Sousa; Pedro Vitor Lopes-Costa; Alesse Ribeiro Dos Santos; Jackeline Lopes Viana; Arquimedes Cavalcante Cardoso; Victoria Maria Luz-Borges; Renato de Oliveira Pereira; Cleciton Braga Tavares; Vladimir Costa Silva; Dorival Mendes Rodrigues-Junior; Luiz Henrique Gebrim; Benedito Borges da Silva Journal: Medicine (Baltimore) Date: 2020-10-23 Impact factor: 1.817