miRNA21 as a specific marker for the detection of circulating tumor cell.

e22025 Background: To develop clinical applications of Circulating Tumor Cells (CTC), their phenotypic and genetic characterization is required. Here, we present the first protocol to detect miRNAs in CTCs by in situ hybridization (ISH) combined with both immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Methods: LNA probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA in CTCs. This protocol was optimized using both tumor and non-tumor epithelial cell lines, MDA-MB468 and MCF-10, respectively , and miRNA-21 was chosen as the target miRNA due to its known role as an onco-miRNA. Results: Hematopoietic cells did not express miRNA-21, making it a perfect marker for detecting CTCs. The peripheral blood of 25 cancer patients was analyzed, with 11 of the 25 patients presenting CTCs. In every case, isolated CTCs expressed both cytokeratin (CK) and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, a tumor epithelial cell line. While CK was lost in those cells, miRNA-21 was still expressed. Conclusions: Our results suggest that miRNA-21 might be a good marker for detection of different subpopulations of CTCs including CTCs with EMT phenotype.