Circulating Tumor Cells (CTCs)

Circulating Tumor Cells (CTCs). - Detection and isolation technologies. Circulating Tumor Cells.

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Circulating Tumor Cells (CTCs)

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1. Circulating Tumor Cells (CTCs) - Detection and isolation technologies
2. 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.
3. Probability of CTC survival Danila et al. (2007) Clin Cancer Res
4. Longitudinal correlation of CTCs with disease course
5. 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
6. 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
7. 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.
8. Size exclusion methods of CTCs isolation Mohamed et al., (2009) J Chromatography A
9. 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.
10. CTC Immunomagnetic isolation Anti-EpCAM 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.
11. 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
12. 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.
13. Nanopillar (fly paper) approach Wang et al., (2009) Angew. Chem. Int. Ed. - The use of 3D nanostructured substrates specifically, a silicon-nanopillar (SiNP) array which allows for enhanced local topographic interactions
14. 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.
15. Mei et al., (2008) Advanced Materials Solovev et al., (2009) Small
16. 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 anti-Cytokeratin, (characteristic of epithelial cells), and anti-CD45 allowing negative selection of leukocytes.
17. Analysis of CTCs Yu et al. (2011) J Cell Biol

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