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Abstract

A major challenge in monitoring and managing metastatic cancer is the frequent inability to use invasive biopsies as a means of obtaining information regarding tumour progression. Circulating tumour cell (CTC) liquid biopsies—that is, collecting and analyzing CTCs from a blood sample—represent an opportunity for non-invasive cancer monitoring. The challenges of obtaining rare CTCs and sequencing single cell-amounts of miRNA have thus far made it impossible to accurately assess miRNA from CTCs. Here we present an integrated method combining negative-selection, high-throughput microscopy, micromanipulation, and microfluidics to measure the genome-wide expression of microRNAs (miRNAs) in single CTCs. Using our method, we generated single-cell miRNA expression profiles for 258 CTCs from 14 late-stage metastatic prostate cancer patients (mean 18 per patient). The total miRNA reads per cell was 27,423—accounting for 39% of aligned reads—while the mean detected miRNA species per cell was 155. Individual CTCs displayed significant interpatient heterogeneity, while intrapatient heterogeneity was comparatively low. Retrospective analysis of CTCs from castration-resistant prostate cancer patients provides preliminary evidence that miR-200b and miR-200a expression is negatively correlated with more aggressive tumour phenotypes; this demonstrates the potential of this method for monitoring and predicting disease progression in prostate cancer. This study establishes a method for obtaining single-cell miRNA profiles of CTCs, thereby enabling the ability to assess the important roles miRNAs play in cancer development, progression and response to treatment.