The analysis of circulating tumor cells (CTCs) is an important capability that may lead to new approaches for cancer management. CTC capture devices developed to date isolate a bulk population of CTCs and do not differentiate subpopulations that may have varying phenotypes with different levels of clinical relevance. Here, we present a new device for CTC spatial sorting and profiling that sequesters blood-borne tumor cells with different phenotypes into discrete spatial bins. Validation data are presented showing that cancer cell lines with varying surface expression generate different binning profiles within the device. Working with patient blood samples, we obtain profiles that elucidate the heterogeneity of CTC populations present in cancer patients and also report on the status of CTCs within the epithelial-to-mesenchymal transition (EMT).
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.
We have developed an integrated platform for rolling circle amplification (RCA) and circle-to-circle amplification (C2CA) of circular probe (padlock probe) and subsequent microchip electrophoretic detection of a specific gene on a poly(methyl methacrylate) microchip. RCA and C2CA were successfully carried out at a steady temperature of 37 degrees C in the sample well of the microchip, and their respective product was detected on the same channel of the microchip, which was prefilled with a polymer separation matrix and fluorescent dye. Using a species-specific padlock probe for bacterial pathogen V. cholerae, a 25-ng bacterial genomic DNA could be detected in less than 65 min (including RCA and microchip electrophoresis) by this platform. Stable dsDNA C2CA product of genomic DNA for V. cholerae can be detected with the introduced integrated platform. Furthermore, the usefulness of this technique for the monitoring of RCA was demonstrated. This integrated platform provides a sensitive, fast, high-throughput, and reproducible method for signal amplification and detection of the padlock probes in the same microchip and is a promising tool for highly specific gene detection strategies.
Isolating subpopulations of heterogeneous cancer cells is an important capability for the meaningful characterization of circulating tumor cells at different stages of tumor progression and during the epithelial-to-mesenchymal transition. Here, we present a microfluidic device that can separate phenotypically distinct subpopulations of cancer cells. Magnetic nanoparticles coated with antibodies against the epithelial cell adhesion molecule (EpCAM) are used to separate breast cancer cells in the microfluidic platform. Cells are sorted into different zones on the basis of the levels of EpCAM expression, which enables the detection of cells that are losing epithelial character and becoming more mesenchymal. The phenotypic properties of the isolated cells with low and high EpCAM are then assessed using matrix-coated surfaces for collagen uptake analysis, and an NAD(P)H assay that assesses metabolic activity. We show that low-EpCAM expressing cells have higher collagen uptake and higher folate-induced NAD(P)H responses compared to those of high-EpCAM expressing cells. In addition, we tested SKBR3 cancer cells undergoing chemically induced hypoxia. The induced cells have reduced expression of EpCAM, and we find that these cells have higher collagen uptake and NAD(P)H metabolism relative to noninduced cells. This work demonstrates that nanoparticle-mediated binning facilitates the isolation of functionally distinct cell subpopulations and allows surface marker expression to be associated with invasiveness, including collagen uptake and metabolic activity.
The analysis of circulating tumor cells (CTCs) is an important capability that may lead to new approaches for cancer management. CTC capture devices developed to date isolate a bulk population of CTCs and do not differentiate subpopulations that may have varying phenotypes with different levels of clinical relevance. Here, we present a new device for CTC spatial sorting and profiling that sequesters bloodborne tumor cells with different phenotypes into discrete spatial bins. Validation data are presented showing that cancer cell lines with varying surface expression generate different binning profiles within the device. Working with patient blood samples, we obtain profiles that elucidate the heterogeneity of CTC populations present in cancer patients and also report on the status of CTCs within the epithelial-to-mesenchymal transition (EMT).
A dynamic coating using methylcellulose (MC) and a nonionic detergent (polysorbate 20) was developed, which controlled protein adsorption onto the surface of microchannels on a microchip made of poly(methyl methacrylate) (PMMA). Optimum concentration of polysorbate 20 in combination with the range of MC concentrations controlled the protein adsorption onto the microchannel surface, and increased the solubility of the protein samples while facilitating the injection of high concentrations of MC solutions into the microchannels. Higher concentrations of nonionic detergent increased the EOF mobility as opposed to the electrophoretic mobility and caused the electrophoresis to fail. Nondenaturing microchip electrophoresis of protein samples with molecular masses ranging from 20 to 100 kDa were completed in 100 s. Also, successful separation of a BSA sample and its complex with anti-BSA mAb ( 220 kDa) was achieved on a PMMA microchip. The separation exhibited high reproducibility in both migration time (RSD = 1%) and peak area (RSD = 10-15%).
Biomarkers such as proteins and nucleic acids released from human cells, bacteria, and viruses offer a wealth of information pertinent to diagnosis and treatment ranging from cancer to infectious disease. The release of these molecules from within cells is a crucial step in biomarker analysis. Here we show that purely electric-field-driven lysis can be achieved, inline, within a microfluidic channel; that it can produce highly efficient lysis and biomarker release; and, further, that it can do so with minimal degradation of the released biomarkers. Central to this new technology is the use of three-dimensional sharp-tipped electrodes (3DSTEs) in lysis, which we prove using experiment and finite-element modeling produce the electric field concentration necessary for efficient cell wall rupture.
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