Circulating Tumor Cells (CTCs)
- Detection and isolation technologies
Circulating Tumor Cells
-
During the progression of metastasis, cancer cells detach from the solid
primary tumor, enter the blood stream, and travel to different tissues of the
body.  Breakaway cancer cells in the peripheral blood: Circulating tumor
cells (CTCs).
- Extremely rare, comprising as few as one cell per 109 hematologic cells in the blood of
patients with metastatic cancer.  Tremendous technical challenge.
- A real-time “liquid biopsy” in cancer patients.
Probability of CTC survival
Danila et al. (2007) Clin Cancer Res
Longitudinal correlation of CTCs with disease course
Why study CTCs?
 Noninvasive sampling
- Continuous monitoring of patients
- Improving patient compliance
 Early detection of cancer
- Metastatic cancer, vascular invasion
 Biological insights for cancer
- Understanding of cancer metastasis
Current Methods for CTCs Isolation
CD45: protein tyrosine phosphatase (PTP) located in
hematopoietic cells except ethrocytes and platelets.
Leukocyte antigen
Pantel et al., (2010) Trends in Molecular Med
Size exclusion methods of CTCs isolation
Erythrocyte : 3-5㎛
Leukocyte : 6-8㎛
Platelet : 2-3㎛
Epithelial tumor cells: 10-15㎛
Zheng et al., (2007) J Chromatography A
- Isolation by size of Epithelial Tumor cells (ISET by ScreenCell):direct enrichment of epithelial cells
using filtration and size exclusion, thereby releasing the dependence for detection on the
expression of a selected epithelial marker(s) potential to uncover CTC heterogeneity.
- Principles: the majority of leukocytes are the smallest cells in the body and that most flow
through the pores whereas larger tumor cells are captured on the membrane or particular
channels.
Size exclusion methods of CTCs isolation
Mohamed et al., (2009) J Chromatography A
Density gradient isolation of CTCs
- Ficoll (Amersham, Upsala, Sweden), Lymphoprep (Nycomed, Oslo, Norway) or other similar
density gradient liquids.
- Whole blood is directly layered on the density gradient. After centrifugation, from bottom to top
are found: erythrocytes, neutrophils, mononuclear cells (lymphocytes, monocytes, epithelial cells,
tumor cells), and plasma which is the upper layer. Tumor cells can also migrate in the plasma
fraction.
Immunomagnetic isolation
Anti-EpCAM
CTC
Magnetic particles
(approx. 200nm)
- The CellSearchTM technology (FDA approved for prognosis in breast, prostate and colorectal cancer) :
Ferrofluid consisting of nanoparticles with a magnetic core surrounded by a polymeric layer coated
with antibodies targeting epithelial cell adhesion molecule (EpCAM)
-
After immunomagnetic capture and enrichment, cells are fixed, permeabilized and labeled with
fluorescent probes to identify and enumerate CTCs.
The standard probe set is comprised of anti-Cytokeratin, (characteristic of epithelial cells), and
anti-CD45, allowing negative selection of leukocytes.
Microfluidic isolation: micropost
Shyamala et al 2008, NEJM
Nagrath et al 2007, Nature
- The CTC-chip : an array of microposts that are made chemically functional with anti-EpCAM Ab
Microfluidic isolation: herringbone chip
Scott et al., (2010) PNAS
- (C) Herringbone-Chip, and (D) a traditional flat-walled microfluidic device. Flow visualization
studies using two paired streams of the same viscosity (one stream is green, the other clear)
demonstrate (E) the chaotic microvortices generated by the herringbone grooves, and the lack of
mixing in (F) traditional flat-walled devices.
- The HB-Chip design : passive mixing of blood cells through the generation of microvortices
significantly increase the interactions between target CTCs and the Ab-coated chip surface.
Nanopillar (fly paper) approach
Wang et al., (2009) Angew. Chem. Int. Ed.
- The use of 3D nanostructured substrates specifically, a siliconnanopillar (SiNP) array which allows for enhanced local topographic
interactions
Micromachine approach
Balasubramanian et al., (2011) Angew. Chem. Int. Ed.
-
Microrockets for capture and isolation of cancer cells.
Upon encountering the cells, the anti-CEA mAb-modified microrockets recognize the CEA surface
antigens on the target cancer cells, allowing their selective pickup and transport.
The top-right and bottom-left insets illustrate the preparation of the Ab-modified microrockets
and their functionalization, respectively.
Mei et al., (2008) Advanced Materials
Solovev et al., (2009) Small
Identification of CTCs
DAPI
Cytoketatin
CD45
Cancer cell
White blood cell
- After immunomagnetic capture and enrichment, cells are fixed, permeabilized and labeled with
fluorescent probes to identify and enumerate CTCs. The standard probe set is comprised of antiCytokeratin, (characteristic of epithelial cells), and anti-CD45 allowing negative selection of
leukocytes.
Analysis of CTCs
Yu et al. (2011) J Cell Biol