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GENYO Team Reports In Situ Hybridization Method for microRNA Detection in CTCs

NEW YORK (GenomeWeb) – Amid growing evidence pointing to the prognostic potential of microRNAs in circulating tumor cells (CTCs), researchers from GENYO — the Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research in Spain — reported this month a technique for detection of the small, non-coding RNAs in CTCs using in situ hybridization.

CTCs are generated by primary tumors and shed into circulation where they colonize other tissues and organs leading to metastatic disease. Seeing an opportunity to use these epithelial cells to monitor cancer progression and track a patient's response to treatment, researchers have made significant advances with technologies for their detection.

Still, most CTC assays have low sensitivity, mainly because only a few epithelial biomarkers — primarily epithelial cell adhesion molecule and cytokeratins (CKs) — are used to identify and isolate the cells from whole blood by most existing devices, the GENYO team wrote in Science Reports.

Further complicating CTC detection are recent data pointing to the existence of a subset of CTCs that don't express EpCAM or CKs. Instead, these cells exhibit features of epithelial to mesenchymal transition (EMT), a process linked to tumor metastasis whereby epithelial cells take on mesenchymal characteristics that boost mobility, invasiveness, and resistance to apoptosis.

A reliance on epithelial biomarkers also leaves open the possibility of detecting epithelial cells within hematopoietic cell populations that are not derived from tumors but are instead from other epithelial tissues, the researchers added in their paper.

As a result, efforts to explore the biomarker potential of miRNAs in the context of CTCs have been limited, despite the increased attention miRNAs are receiving in cancer and other diseases.

To address this, the GENYO scientists developed an approach dubbed miRNA in situ hybridization in CTC, or MishCTC, which combines existing in situ hybridization protocols with methodological steps to isolate and identify CTCs in patient blood.

To demonstrate the technique, the team focused on miR-21, an miRNA known to target numerous tumor suppressor genes and, importantly, one that is expressed in cancer cells but not hematopoietic cells.

The researchers obtained peripheral blood samples from 25 patients with metastastic cancer, finding that 11 of these samples contained CTCs expressing both CK and miR-21. MishCTC was also used to successfully track miR-21 expression in an EMT-induced epithelial tumor cell line wherein CK expression was lost.

According to the study's authors, the data represent the first demonstration of an in situ hybridization method for miRNA identification in CTCs. They are currently recruiting a larger cohort of cancer patients in order to study CTC heterogeneity based on miRNA expression using MishCTC, they noted.