miRNA in situ hybridization in circulating tumor cells--MishCTC

Scientific Reports
Francisco G OrtegaMaria J Serrano

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,...Continue Reading

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Citations

Jun 13, 2015·International Journal of Molecular Sciences·Martyna O UrbanekWlodzimierz J Krzyzosiak
Jan 14, 2016·Tumour Biology : the Journal of the International Society for Oncodevelopmental Biology and Medicine·Yupeng Hong, Qi Zhang
Aug 17, 2016·International Journal of Molecular Sciences·Alexandra C KölblUlrich Andergassen
Oct 27, 2018·Clinical & Experimental Metastasis·Kirsten Nguyen KnudsenBoye Schnack Nielsen
Nov 20, 2016·Frontiers in Genetics·Jinpao HouS C Cesar Wong
Mar 7, 2017·Frontiers in Physiology·Shukuan LingYingxian Li
Mar 21, 2020·Genome Medicine·Francesc Castro-Giner, Nicola Aceto
Mar 31, 2018·The Cancer Journal·Camila D M CamposSteven A Soper
Jan 27, 2021·Biochimica Et Biophysica Acta. Reviews on Cancer·Sai Shyam VasantharajanAniruddha Chatterjee
Feb 23, 2021·Frontiers in Cell and Developmental Biology·Aitor Rodriguez-CasanovaAngel Diaz-Lagares
May 1, 2021·Journal of Personalized Medicine·Katalin BalázsKatalin Lumniczky
Jul 3, 2021·Cancers·Cora Palanca-BallesterAlfonso Calvo

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Methods Mentioned

BETA
biopsy
fluorescence microscopy
density gradient centrifugation

Software Mentioned

MishCTC
CellSearch
GILUPI
Zen

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