Detecting circulating tumor cells: current challenges and new trends

Abstract

Circulating tumor cells (CTCs) in the blood stream play a critical role in establishing metastases. The clinical value of CTCs as a biomarker for early cancer detection, diagnosis, prognosis, prediction, stratification, and pharmacodynamics have been widely explored in recent years. However, the clinical utility of current CTC tests is limited mainly due to methodological constraints. In this review, the pros and cons of the reported CTC assays are comprehensively discussed. In addition, the potential of tumor cell-derived materials as new targets for CTC detection, including circulating tumor microemboli, cell fragments, and circulating DNA, is evaluated. Finally, emerging approaches for CTC detection, including telomerase-based or aptamer-based assays and cell functional analysis, are also assessed. Expectantly, a thorough review of the current knowledge and technology of CTC detection will assist the scientific community in the development of more efficient CTC assay systems.

Keywords: CTC: Circulating tumor cell; CTDNA: Circulating tumor DNA; CTM: Circulating tumor microemboli; CTMat: Circulating tumor materials; POCT: Point-of-care test..

Figure 1

Schematic illustration of metastasis development via CTCs/CTM. Tumor cells and cell clusters are shed from the primary tumor and intravasate into the circulation, which might involve the process of epithelial-to-mesenchymal transition. The majority of the CTCs are, however, killed via apoptosis and necrosis, releasing debris, cell fragments and intracellular substances (CTMat and CTDNA). CTM, the even rarer species than CTCs in blood, undergo a dynamic life. Tumor cells can dissociate from CTM when subjected to shear force and/or frequent collisions in blood; they can also attach to other tumor or blood cells upon collision due to increased adhesion. The microenvironment established within CTM is unique, protecting the tumor cells inside from damage. CTM are, therefore, believed to be more aggressive than individual CTCs as they proliferate in the vessel and eventually rupture the vessel. Conversely, CTCs have to extravasate in order to form metastasis.

Figure 2

Schematic illustration of the three major steps of a CTC assay. Step 1. Sample preparation and tumor cell isolation: blood samples may be pre-treated to remove the majority of the erythrocytes and/or leukocytes, and therefore, rare CTCs can be detected at a higher frequency with low background interference. The isolation of CTCs from blood cells can be based on various ligand-receptor interactions, such as antibody-antigen, aptamer-receptor, or adhesion molecule-extracellular matrix, and unique physical properties of cancer cells, including cell size, density, deformability, and dielectric/charges. Step 2. Tumor cell staining or oncogene probing: CTCs can be labeled by antibodies or aptamers, or probed by DNA primers. Step 3. CTC detection via cytometry, microscopy, conductometry, fiber-optics, reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH).

Figure 3

Schematic representation of CTC enrichment, detection, and characterization. After blood collection, the whole blood is processed by a microdevice, for example, a herringbone-chip tethered with tumor-specific recognition molecules, e.g., aptamers, to isolate the CTCs from the majority of normal blood cells. Enriched CTCs are then identified, enumerated, and characterized via cytopathology, molecular biology, and other examinations. By means of morphology, tumor cells could be categorized into intact CTCs, circulating tumor microemboli (CTM), and circulating tumor materials (CTMat). Each of them may present particular metastatic potential as independent indicators for prediction. Captured CTCs could be cultured ex vivo, followed by additional systemic functional studies. For instance, cultured CTCs are injected into nude mice subcutaneously for a xenograft; and a heavier tumor is found than that induced by the parental cell line.