Aims: Understanding genetic and functional heterogeneity in tumour cells allows us to gain insight on the mechanisms underscoring drug resistance and tumour aggressiveness. In contrast to invasive primary tumour sampling, liquid biopsy approach using circulating tumour cells (CTCs) provides accessible tumour material to assess the molecular and phenotypic changes of disseminated tumour cells. In this work, we developed a marker-free workflow to isolate CTCs from breast cancer patient for mRNA-seq analysis.
Methods: The CTCs were enriched from 7.5 ml of peripheral blood sample using ClearCell FX ®, a label- free spiral microfluidics- based system. The enriched cells were stained with Calcein AM (live-cell marker), CellTracker™ Orange (Universal marker), and Alexa 647 conjugated antibodies for CD45 and CD31. Further selection of CTCs by negative depletion of leukocytes and endothelial cells was accomplished using Fluidigm’s PolarisTM system. Following selection of CTC, the Polaris system generates full-length cDNA for mRNA-seq analysis. Sequencing libraries were constructed using Nextera kit and sequenced with Illumina MiSeq/NextSeq.
Results: CTCs were isolated and successfully extracted for RNA and cDNA generation. Gene expression data shows considerable heterogeneity as analyzed by unsupervised hierarchal clustering. However, PCA analysis of CTCs with gene expression data from various cancer type cell lines show that the CTCs cluster away from the other cancer type cell lines, with closest gene expression profile to that of a breast cancer cell line.
Conclusion: We have demonstrated a workflow to isolate CTCs and perform high quality single cell mRNA sequencing. The workflow captures viable CTCs individually and could potentially allow functional perturbation studies to be performed at the single cell level.