Circulating tumor cells: approaches to isolation and characterization

Abstract

Circulating tumor cells (CTCs) shed from primary and metastatic cancers are admixed with blood components and are thus rare, making their isolation and characterization a major technological challenge. CTCs hold the key to understanding the biology of metastasis and provide a biomarker to noninvasively measure the evolution of tumor genotypes during treatment and disease progression. Improvements in technologies to yield purer CTC populations amenable to better cellular and molecular characterization will enable a broad range of clinical applications, including early detection of disease and the discovery of biomarkers to predict treatment responses and disease progression.

Figure 1.

Micrographs of CTCs captured from patients’ blood using an anti-EpCAM–coated CTC-chip. (A–C) Combined fluorescent and reflected light micrographs of a cytokeratin 7/8 (green)–stained CTC captured from breast cancer patient blood and a contaminating CD45-positive (red) white blood cell (A) and cytokeratin 7/8 (green, B)– and PSA (green, C)-stained CTCs captured from a prostate cancer patient. (D and E) HER2 (green, D)- and cytokeratin 7/8 (green, E)–stained CTCs captured from a breast cancer patient. (F and G) Individual and merged fluorescent micrographs of CTCs captured from prostate cancer patients’ blood stained positive for PSA (green) and Ki-67 (red, proliferative marker, F), and PSA (green) and M30 (red, apoptotic marker) demonstrated the heterogeneity in CTCs (G). In all panels, the nuclei are stained with DAPI (blue). CK7/8, cytokeratin 7/8. Bars, 10 µm.

Figure 2.

Illustration of current and potential applications of CTC technologies. The peripheral blood of a cancer patient is collected and processed through various CTC isolation technologies. CTCs are captured along with contaminating leukocytes. Immunostaining for specific markers and FISH for genomic amplification and translocation can be applied to CTCs. DNA or RNA can be extracted from the CTCs and subjected to sequencing, quantitative RT-PCR (qRT-PCR), and potential expression profile analysis. Viable cells can be released and propagated in cell culture.

Figure 3.

CTC clusters captured from lung cancer patients’ blood using an anti-EpCAM–coated HB-chip. (A and B) Fluorescent micrographs of cytokeratin 7/8 (CK; green)–, CD45 (red)-, and DAPI (blue)-stained CTC clusters taken at different focal planes and corresponding hematoxylin and eosin (H&E) stains are shown. Bars, 10 µm.