Abstract
BackgroundSingle-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer.MethodsSingle circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns.ResultsCirculating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells.ConclusionThe analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.
Highlights
Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process
The isolated single cells were subjected to mRNA profiling with a multi-marker mRNA panel (Table 1) designed to capture inherent heterogeneity in pancreatic Circulating tumour cell (CTC)
The panel consisted of sequences that identified specific epithelial markers (KRT8, KRT19, EPCAM, E-Cadherin); mesenchymal/epithelialmesenchymal transition (EMT) markers (Vimentin, N-Cadherin, ZEB1); cancer stem cell (CSC) markers (CD24, CD44, and ALDH1A1); an extracellular matrix (ECM) marker (SPARC), a reference marker (HPRT1), and a leucocyte marker (CD45)
Summary
Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process Such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. The small number of studies on the clinical relevance of CTCs in pancreatic cancer have produced ambiguous results (reviewed in [6, 7]) They have been limited to analysing CTCs with only one or a few markers, which is insufficient to elucidate the complexity. Heterogeneous expression of RNA transcripts was demonstrated in CTCs with single-cell mRNA profiling in samples from a small cohort of patients with pancreatic cancer [11]. No other study has described transcriptional heterogeneity among single pancreatic CTCs and its potential clinical relevance
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