This application addresses the broad Research Area: 06, Enabling Technologies And Challenge Topic: 06-HG-102* Technologies for obtaining genomic, proteomic, and metabolomic data from individual viable cells in complex tissues. Methods will be developed to analyze gene expression from single cancer cells enabling studies at an unprecedented level of sensitivity. We propose techniques to measure RNA transcription from individual circulating tumor cells (CTCs) isolated from the blood of cancer patients. By accessing confirmed cancer cells, separated from the large background of normal cells, it will be possible to gain unexplored knowledge about the disease process and its spread through the body by metastasis. Recent advances in several key technologies have made this approach feasible: 1) Researchers at the Scripps Research Institute have developed the means to fluorescently label CTCs so that they can be identified from human blood samples, 2) Technologies for isolating single cells have been combined with nucleic acid amplification methods at the J. Craig Venter institute enabling the first demonstration of DNA sequencing from single cells, and 3) Life Technologies (Invitrogen/Applied Biosystems) have combined methods for cDNA synthesis from single cells and whole genome expression profiling with their next generation SOLiD sequencing platform. These three institutions will collaborate to isolate single cancer cells, synthesize cDNA and sequence it by the SOLiD method. The goal is to observe cancer at the most basic level of the single cell.
We propose development of a method to measure gene expression in individual cancer cells obtained from human blood. These circulating cancer cells (CTCs) are very rare but can be responsible for the spread of cancer throughout the body by metastasis. With new strategies to fluorescently label CTCs it is possible to isolate them. Other new technology enables determination of mRNA expression levels from a single cell giving an understanding of the alterations to gene expression. A new understanding of the physiology of CTCs and how they lead to metastasis will be possible.
| Krishnaswami, Suguna Rani; Grindberg, Rashel V; Novotny, Mark et al. (2016) Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nat Protoc 11:499-524 |
| Grindberg, Rashel V; Yee-Greenbaum, Joyclyn L; McConnell, Michael J et al. (2013) RNA-sequencing from single nuclei. Proc Natl Acad Sci U S A 110:19802-7 |
