Aptamer and Antisense-Mediated Two-Dimensional Isolation of Specific Cancer Cell Subpopulations

Abstract

Cancer cells, and in particular those found circulating in blood, can have widely varying phenotypes and molecular profiles despite a common origin. New methods are needed that can deconvolute the heterogeneity of cancer cells and sort small numbers of cells to aid in the characterization of cancer cell subpopulations. Here, we describe a new molecular approach to capturing cancer cells that isolates subpopulations using two-dimensional sorting. Using aptamer-mediated capture and antisense-triggered release, the new strategy sorts cells according to levels of two different markers and thereby separates them into their corresponding subpopulations. Using a phenotypic assay, we demonstrate that the subpopulations isolated have markedly different properties. This system provides an important new tool for identifying circulating tumor cell subtypes.

Enrichment and single-cell analysis of circulating tumor cells

“…5C). 62 Cells were first identified by MNPs modified with an EpCAM aptamer to sort the isolated cells into four subpopulations according to their level of epithelial marker expression. The four subpopulations were then released using a complementary oligonucleotide and subsequently tagged with HER2 aptamer functionalized MNPs to divide them into 16 subpopulations.…”

Materials and microfluidics: enabling the efficient isolation and analysis of circulating tumour cells

“…3D) isolate magnetic affinity-labelled CTCs in different zones of the device depending on antigen (e.g., EpCAM 114–119 HER2, 114,117 EGFR, 117 , MUC1, 114 N-Cadherin 118 ) expression level, thus correlating disease progression with CTC phenotype, 114,118 similar to CTC immunophenotyping 29 and RNA-ISH analysis. 28 …”

“…Whole blood, 114–118 RBC lysed blood, 117,118 or RBC lysed and WBC-depleted blood 117,119 was spiked with Ab-coated 114–116,118 or aptamer-coated 117,119 (discussed below) magnetic nanoparticles. The sample was infused through a series of microfluidic chambers, and an external magnetic field pulled both free magnetic nanoparticles and labelled CTCs onto the device surface.…”

“…The sample was infused through a series of microfluidic chambers, and an external magnetic field pulled both free magnetic nanoparticles and labelled CTCs onto the device surface. 117 To accomplish efficient CTC recovery by this method, 114 magnetic forces must overcome strong fluid forces, which could be achieved by pumping sample at volumetric flow rate of 0.05 mL/h. 114 To increase volumetric throughput, X-shaped microstructures were fabricated to locally create a fluid velocity of 3–30 μm/s within the X-microstructure crevices while maintaining overall throughput of 0.6 mL/h 115–119 to 2 mL/h.…”

“…117 The benefit of phenotypic ranking for EpCAM expressing CTC was fully apparent in cell line xenografts, which showed EpCAM downregulation after implantation, 115 but the identification of EpCAM(−) CTCs in addition to low EpCAM expressing CTCs would require simultaneous phenotypic ranking against additional mesenchymal markers.…”