Abstract
Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.
Highlights
Circulating tumor cells (CTC) are cells that part from a primary tumor or metastasis and enter the blood stream via the leaky vasculature that arises around a growing tumor
These improvements combined with a multi-marker staining protocol allow the user to distinguish CTCs by fluorescent staining of cell surface markers
Validation of MagSweeper performance revealed that the mean capture of live LNCaP cells spiked into blood is 81%616% which is comparable with the capture reported for high EpCAM expressing epithelial cancer cell lines spiked into blood on other CTC capture platforms [32]
Summary
Circulating tumor cells (CTC) are cells that part from a primary tumor or metastasis and enter the blood stream via the leaky vasculature that arises around a growing tumor. A small number of CTCs survive and extravasate into surrounding tissues to seed metastasis or reseed the primary tumor [3]. Described over a century ago [4], CTCs can be enumerated using the FDA approved CellSearch platform to provide prognostic information regarding survival for metastatic breast, colon and prostate cancer patients [5,6,7]. Several groups have suggested that genetic and transcriptional analysis of individual CTCs might be leveraged to make personalized medical decisions for cancer therapy and provide insights into the biological processes involved in metastasis [8,9,10]. Of the platforms currently in use for isolating CTCs, the MagSweeper technology provides great ease of use and access to highly pure, intact, individual CTCs suitable for genetic and proteomic analysis [22,23]
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