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.
Highlights
Correspondence and requests for materials should be addressed to Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications
Detection was performed via an enzyme-labeled fluorescence (ELF) signal amplification approach[17] using miRCURY technology, which is based on Locked-Nucleic Acid (LNA) probes[18]
We present the first protocol for detecting miRNAs in CTCs using an in situ hybridization (ISH) technique called miRNA in situ hybridization in CTC (MishCTC)
Summary
Correspondence and requests for materials should be addressed to Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Hematopoietic cells do not express miRNA-21; 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. Metastasis is responsible for the vast majority of cancer-related deaths[1] During this process, circulating tumor cells (CTCs) are generated and shed from the primary tumor, colonize distant organs and lead to overt metastatic disease. EpCAM and cytokeratins (CKs) are the two main epithelial biomarkers that are used in most of the devices that have been utilized to date[4,5,6] Among these devices, CellSearch and GILUPI, which have been approved as medical devices by the FDA and the EU, respectively, can detect only EpCAM in circulating cells in the blood[7,8].
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