Analysis of circulating tumor cells (CTCs) in a mouse model of pancreatic cancer identified distinct patterns of gene expression in several groups of CTCs, including significant differences from the primary tumor that may contribute to the ability to generate metastases. This study identified several different classes of pancreatic CTCs and found unexpected factors that may prove to be targets for improved treatment of the deadly tumor.
“Our ability to combine a novel microfluidic CTC isolation device with single-cell RNA sequencing has given us new biological insights into these cells and revealed novel avenues to try and block the spread of cancer,” said lead author David T. Ting, MD, of the Massachusetts General Hospital (MGH) Cancer Center in Boston. The study was published in Cell Reports (2014; doi:10.1016/j.celrep.2014.08.029).
Pancreatic cancer is among the most deadly of tumors because it spreads rapidly via CTCs in the bloodstream. The earliest technologies for isolating CTCs from blood samples relied on interactions with known tumor-specific marker proteins, potentially missing cells that did not express those particular markers.
The device used in the current study, called the CTC-iChip, enables the isolation of all CTCs in a blood sample, regardless of the proteins they express on their surface, by removing all other components. Since the CTCs collected are in solution, unlike with previous CTC capture devices, they are suitable for advanced RNA sequencing techniques to reveal the gene expression patterns of each individual cell.
Using a well-known mouse model of pancreatic cancer, the researchers first isolated 168 single CTCs from the bloodstreams of five individual mice. Analysis of the RNA transcripts of each CTC revealed several different subsets of CTCs, based on gene expression patterns that were different from each other and from the primary tumor.
The largest subset, which the authors call ‘classic CTCs,’ was found to have elevated expression of a stem cell gene called Aldh1 a2. It also expressed genes characteristic of two basic cell types, epithelial and mesenchymal. Transitioning between these cell types has been associated with tumor metastasis.
Another gene expressed by almost all classic CTCs, Igfbp5, is only expressed in primary tumor at locations where epithelial cancer cells interface with the supporting stromal cells that provide a nurturing microenvironment, an observation that suggests that those regions may be the source of CTCs.
The research team was most surprised to observe that extracellular matrix (ECM) genes in general, which are usually expressed primarily in stromal cells, were highly expressed in all classic CTCs. Expression of ECM genes by CTCs themselves suggests that the blood-borne cells may provide or help prepare their own ‘soil.’
Analysis of CTCs from blood samples of human patients with pancreatic, breast, or prostate cancer also found elevated expression of several ECM genes.