Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells.
Bottom Line: To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing.Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness.The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.
Affiliation: Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA; Department of Medicine, Harvard Medical School, Boston, MA 02114, USA.Show MeSH
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Mentions: Single-cell RNA-seq performance was comparable for all samples analyzed, with a mean 4.4–8.5 million reads, of which a mean 46%–61% uniquely aligned to the mouse genome (Figure S1). Genome-aligned reads were annotated and counted using UCSC Known Gene transcriptome reference and normalized in reads per million (rpm). Normalized reads were then analyzed by unsupervised hierarchical clustering (Figure 2A). Single-cell transcriptomes from MEFs, the NB508 pancreatic cancer cell line, and normal WBCs clustered tightly, supporting the analytic reliability of the RNA-seq strategy. Three distinct clustering patterns of candidate CTCs were identified, all of which were distinct from matched primary tumor sequences and cancer cell lines. Principal component analysis shows the clustering and interrelationships of these different groups (Figure 2B).
Affiliation: Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA; Department of Medicine, Harvard Medical School, Boston, MA 02114, USA.