title: Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines.
abstract: BACKGROUND
  To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.
  
  
  RESULTS
  We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.
  
  
  CONCLUSIONS
  For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery.
authors.affiliation: Department of Surgery, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Ashley A
authors.initials: AA
authors.lastname: Powell
authors.forename: Amirali H
authors.initials: AH
authors.lastname: Talasaz
authors.forename: Haiyu
authors.initials: H
authors.lastname: Zhang
authors.forename: Marc A
authors.initials: MA
authors.lastname: Coram
authors.forename: Anupama
authors.initials: A
authors.lastname: Reddy
authors.forename: Glenn
authors.initials: G
authors.lastname: Deng
authors.forename: Melinda L
authors.initials: ML
authors.lastname: Telli
authors.forename: Ranjana H
authors.initials: RH
authors.lastname: Advani
authors.forename: Robert W
authors.initials: RW
authors.lastname: Carlson
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Mollick
authors.forename: Shruti
authors.initials: S
authors.lastname: Sheth
authors.forename: Allison W
authors.initials: AW
authors.lastname: Kurian
authors.forename: James M
authors.initials: JM
authors.lastname: Ford
authors.forename: Frank E
authors.initials: FE
authors.lastname: Stockdale
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Quake
authors.forename: R Fabian
authors.initials: RF
authors.lastname: Pease
authors.forename: Michael N
authors.initials: MN
authors.lastname: Mindrinos
authors.forename: Gyan
authors.initials: G
authors.lastname: Bhanot
authors.forename: Shanaz H
authors.initials: SH
authors.lastname: Dairkee
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.forename: Stefanie S
authors.initials: SS
authors.lastname: Jeffrey
mesh_terms: D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008223:Lymphoma / Q000097:blood; D046228:Microarray Analysis / Q000379:methods; D046210:Microfluidic Analytical Techniques; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating / Q000378:metabolism; D059010:Single-Cell Analysis / Q000295:instrumentation* / Q000379:methods