Heterogeneity of miR-10b expression in circulating tumor cells

Circulating tumor cells (CTCs) have been demonstrated to be an independent prognostic marker of clinical outcome in patients with solid tumors1,2,3. Numerous assays for the enrichment and detection of CTCs have been developed and used, however the semi-automated CellSearch® system (Veridex, Raritan, NJ, USA) remains the only device cleared by the Food and Drug Administration (FDA) for clinical use in metastatic breast, prostate and colon cancer4,5. To date, little is known about phenotypic characteristics of clinically significant CTCs capable of establishing metastasis at secondary sites, or the functional significance of CTC heterogeneity6,7. As a link between the primary tumor and the formation of metastasis throughout the body, CTCs are not only a promising target for future anti-metastasis therapeutic strategies, but can also be analyzed via a ‘liquid biopsy’ that can be obtained by a simple blood draw4,8. Furthermore, CTC analysis enables continuous monitoring of patient condition during the clinical course of their disease allowing early detection of recurrence and monitoring response to treatment5,8. However, the number of CTCs can be very small; as few as one CTC in 109 peripheral blood mononuclear cells (PBMNCs). This rarity makes the enrichment, detection and analysis of CTCs challenging4,5,8. Molecular analysis of cell fractions enriched for CTCs [e.g. after epithelial cell adhesion molecule (EpCAM) enrichment] often face the problem of an over-representation of leukocytes, making data obtained by bulk CTC isolation protocols difficult to interpret8,9. Moreover, small but clinically significant subpopulations are often missed during routine analysis of primary tumor biopsies4,6,10,11, and only a few CTCs are capable of extravasating into surrounding tissue and growing as a metastasis at a secondary site6,7. These cells give important clues as to the genotypic and phenotypic changes that enable metastasis and ultimately patient death. Thus, collective analysis of individual CTCs is the most promising way of overcoming these critical issues and improving the utility of CTC analysis. MiRNAs function as post-transcriptional regulators of gene expression and mediate a range of pathways linked to disease and development12,13. Several miRNAs, such as miR-10b have been directly linked to the epithelial-mesenchymal transition (EMT)12,13,14 and metastasis in breast and prostate cancer15,16,17,18. Furthermore, increased expression of miR-10b has been demonstrated in glioma, as well as metastatic pancreatic cancers19,20. Notably, increased miR-10b blood serum levels have been described in gastric cancer, non-small lung cancer and metastatic breast cancer18,21,22. However, as there is no association between miR-10b levels in the primary tumor and metastasis the link between miR-10b and metastasis remains controversial. It has been suggested that miR-10b may have a biological effect on a few cells at the leading edge of the tumor, or a small number of cancer cells shed into the blood23. For analysis of cell-based miRNA expression ISH protocols using locked-nucleic acid (LNA) probes have been developed and applied to cell preparations, fresh frozen tissue and paraffin embedded tissues24,25,26. To enable the detection of miR-10b in distinct cell types present in a complex cellular environment such as CTCs in peripheral blood we have adapted current ISH protocols, combined with antibody based immunofluorescence (IF), to be used with the CellSearch® CTC detection system (Fig. 1). Using this protocol we demonstrate miR-10b heterogeneity in CTCs isolated from the blood of metastatic cancer patients for the first time. Figure 1 Schematic representation of the combined ISH/IF procedure.