Circulating tumor cell isolation, culture, and downstream molecular analysis

Abstract

Circulating tumor cells (CTCs) are a major contributor of cancer metastases and hold a promising prognostic significance in cancer detection. Performing functional and molecular characterization of CTCs provides an in-depth knowledge about this lethal disease. Researchers are making efforts to design devices and develop assays for enumeration of CTCs with a high capture and detection efficiency from whole blood of cancer patients. The existing and on-going research on CTC isolation methods has revealed cell characteristics which are helpful in cancer monitoring and designing of targeted cancer treatments. In this review paper, a brief summary of existing CTC isolation methods is presented. We also discuss methods of detaching CTC from functionalized surfaces (functional assays/devices) and their further use for ex-vivo culturing that aid in studies regarding molecular properties that encourage metastatic seeding. In the clinical applications section, we discuss a number of cases that CTCs can play a key role for monitoring metastases, drug treatment response, and heterogeneity profiling regarding biomarkers and gene expression studies that bring treatment design further towards personalized medicine.

Keywords: Cell enrichment; Circulating tumor cells (CTCs); Liquid biopsy; Personalized therapies; Point of care.

Figure 1

Outline of existing isolation, detection and characterization techniques and promising future clinical utilities.

Figure 2

(A) (i) Insertion of the functionalized structured medical Seldinger guidewire (FSMW) into the vein through a conventional cylindrical (instrument is also known as GILUPI GmbH CellCollector). (ii) Wire (anti-EpCAM-antibody-functionalized) is slowly inserted inside the cannula till surface is in contact with the blood flow in vein lumen. (B) Seldinger guidewire-Gold-plated with a size of 200 nm- coated with polycarboxylate hydrogel (functionalized with anti-EpCAM-antibodies) which capture circulating cells expressing EpCAM antigen on surface. (C) Image illustrating breast cancer cells (SK-BR-3) captured by functionalized guidewire. Reprinted with permissions from (Saucedo-Zeni et al., 2012).

Figure 3

(A) Aptamer-mediated CTC capture and release via complementary oligonucleotide sequences (CS= complementary sequences) (Zhang et al., 2012). (i) explain about the basic structure of the of the hydrogel immobilized on the glass slide (ii) illustration of the aptamer sequence hybridized with complementary sequences (CSs) (iii) represents the process of cell release. The stable aptamer hybridized with triggering CSs, forming a new hybridized state that leads to rapid dissociation of the aptamer from the hydrogel hence releasing the cells from the hydrogel. (B) Illustrating the cell binding and aptamer formed by SAPE of anti-EGFR aptamer – DNA and biotin, and addition of RELease particles. After washing, the fluorescence cells (caused by selective binding with aptamer) completely hybridizes with anti-EGFR aptamer because of the presences of RELease RNA. This leads to opening up its herpin sturucture which releases 76% of anti-EGFR aptamer from the cellular surface. (Wan et al., 2012)

Figure 4

Ex-vivo CTC (breast cancer) culture (A) Nonadherent CTC cultures (B) mouse xenografts derived from cultured CTCs implanted in mammary fat pad. (C) Orignal breast tumors and corresponding CTCs culture and two different CTC lines derived from mouse xenografts. (Yu et al., 2014)