Characterisation of circulating tumour cell phenotypes identifies a partial-EMT sub-population for clinical stratification of pancreatic cancer.

BACKGROUND: Limited accessibility of the tumour precludes longitudinal characterisation for therapy guidance in pancreatic ductal adenocarcinoma (PDAC). METHODS: We utilised dielectrophoresis-field flow fractionation (DEP-FFF) to isolate circulating tumour cells (CTCs) in 272 blood draws from 74 PDAC patients (41 localised, 33 metastatic) to non-invasively monitor disease progression. RESULTS: Analysis using multiplex imaging flow cytometry revealed four distinct sub-populations of CTCs: epithelial (E-CTC), mesenchymal (M-CTC), partial epithelial-mesenchymal transition (pEMT-CTC) and stem cell-like (SC-CTC). Overall, CTC detection rate was 76.8% (209/272 draws) and total CTC counts did not correlate with any clinicopathological variables. However, the proportion of pEMT-CTCs (prop-pEMT) was correlated with advanced disease, worse progression-free and overall survival in all patients, and earlier recurrence after resection. CONCLUSION: Our results underscore the importance of immunophenotyping and quantifying specific CTC sub-populations in PDAC.

Isolation of circulating tumor cells by dielectrophoresis.

Dielectrophoresis (DEP) is an electrokinetic method that allows intrinsic dielectric properties of suspended cells to be exploited for discrimination and separation. It has emerged as a promising method for isolating circulation tumor cells (CTCs) from blood. DEP-isolation of CTCs is independent of cell surface markers. Furthermore, isolated CTCs are viable and can be maintained in culture, suggesting that DEP methods should be more generally applicable than antibody-based approaches. The aim of this article is to review and synthesize for both oncologists and biomedical engineers interested in CTC isolation the pertinent characteristics of DEP and CTCs. The aim is to promote an understanding of the factors involved in realizing DEP-based instruments having both sufficient discrimination and throughput to allow routine analysis of CTCs in clinical practice. The article brings together: (a) the principles of DEP; (b) the biological basis for the dielectric differences between CTCs and blood cells; (c) why such differences are expected to be present for all types of tumors; and (d) instrumentation requirements to process 10 mL blood specimens in less than 1 h to enable routine clinical analysis. The force equilibrium method of dielectrophoretic field-flow fractionation (DEP-FFF) is shown to offer higher discrimination and throughput than earlier DEP trapping methods and to be applicable to clinical studies.

Correlations between the dielectric properties and exterior morphology of cells revealed by dielectrophoretic field-flow fractionation.

Although dielectrophoresis (DEP) has great potential for addressing clinical cell isolation problems based on cell dielectric differences, a biological basis for predicting the DEP behavior of cells has been lacking. Here, the dielectric properties of the NCI-60 panel of tumor cell types have been measured by dielectrophoretic (DEP) field-flow fractionation, correlated with the exterior morphologies of the cells during growth, and compared with the dielectric and morphological characteristics of the subpopulations of peripheral blood. In agreement with earlier findings, cell total capacitance varied with both cell size and plasma membrane folding and the dielectric properties of the NCI-60 cell types in suspension reflected the plasma membrane area and volume of the cells at their growth sites. Therefore, the behavior of cells in DEP-based manipulations is largely determined by their exterior morphological characteristics prior to release into suspension. As a consequence, DEP is able to discriminate between cells of similar size having different morphological origins, offering a significant advantage over size-based filtering for isolating circulating tumor cells, for example. The findings provide a framework for anticipating cell dielectric behavior on the basis of structure-function relationships and suggest that DEP should be widely applicable as a surface marker-independent method for sorting cells.

Antibody-independent isolation of circulating tumor cells by continuous-flow dielectrophoresis.

Circulating tumor cells (CTCs) are prognostic markers for the recurrence of cancer and may carry molecular information relevant to cancer diagnosis. Dielectrophoresis (DEP) has been proposed as a molecular marker-independent approach for isolating CTCs from blood and has been shown to be broadly applicable to different types of cancers. However, existing batch-mode microfluidic DEP methods have been unable to process 10 ml clinical blood specimens rapidly enough. To achieve the required processing rates of 10(6) nucleated cells/min, we describe a continuous flow microfluidic processing chamber into which the peripheral blood mononuclear cell fraction of a clinical specimen is slowly injected, deionized by diffusion, and then subjected to a balance of DEP, sedimentation and hydrodynamic lift forces. These forces cause tumor cells to be transported close to the floor of the chamber, while blood cells are carried about three cell diameters above them. The tumor cells are isolated by skimming them from the bottom of the chamber while the blood cells flow to waste. The principles, design, and modeling of the continuous-flow system are presented. To illustrate operation of the technology, we demonstrate the isolation of circulating colon tumor cells from clinical specimens and verify the tumor origin of these cells by molecular analysis.

Dielectrophoresis has broad applicability to marker-free isolation of tumor cells from blood by microfluidic systems.

The number of circulating tumor cells (CTCs) found in blood is known to be a prognostic marker for recurrence of primary tumors, however, most current methods for isolating CTCs rely on cell surface markers that are not universally expressed by CTCs. Dielectrophoresis (DEP) can discriminate and manipulate cancer cells in microfluidic systems and has been proposed as a molecular marker-independent approach for isolating CTCs from blood. To investigate the potential applicability of DEP to different cancer types, the dielectric and density properties of the NCI-60 panel of tumor cell types have been measured by dielectrophoretic field-flow fractionation (DEP-FFF) and compared with like properties of the subpopulations of normal peripheral blood cells. We show that all of the NCI-60 cell types, regardless of tissue of origin, exhibit dielectric properties that facilitate their isolation from blood by DEP. Cell types derived from solid tumors that grew in adherent cultures exhibited dielectric properties that were strikingly different from those of peripheral blood cell subpopulations while leukemia-derived lines that grew in non-adherent cultures exhibited dielectric properties that were closer to those of peripheral blood cell types. Our results suggest that DEP methods have wide applicability for the surface-marker independent isolation of viable CTCs from blood as well as for the concentration of leukemia cells from blood.

Dynamic physical properties of dissociated tumor cells revealed by dielectrophoretic field-flow fractionation.

Metastatic disease results from the shedding of cancer cells from a solid primary tumor, their transport through the cardiovascular system as circulating tumor cells (CTCs) and their engraftment and growth at distant sites. Little is known about the properties and fate of tumor cells as they leave their growth site and travel as single cells. We applied analytical dielectrophoretic field-flow fractionation (dFFF) to study the membrane capacitance, density and hydrodynamic properties together with the size and morphology of cultured tumor cells after they were harvested and placed into single cell suspensions. After detachment, the tumor cells exhibited biophysical properties that changed with time through a process of cytoplasmic shedding whereby membrane and cytoplasm were lost. This process appeared to be distinct from the cell death mechanisms of apoptosis, anoikis and necrosis and it may explain why multiple phenotypes are seen among CTCs isolated from patients and among the tumor cells obtained from ascitic fluid of patients. The implications of dynamic biophysical properties and cytoplasmic loss for CTC migration into small blood vessels in the circulatory system, survival and gene expression are discussed. Because the total capacitance of tumor cells remained higher than blood cells even after they had shed cytoplasm, dFFF offers a compelling, antibody-independent technology for isolating viable CTCs from blood even when they are no larger than peripheral blood mononuclear cells.

Endothelial and stem cell interactions on dielectrophoretically aligned fibrous silk fibroin-chitosan scaffolds.

Regenerative tissue engineering requires biomaterials that would mimic structure and composition of the extracellular matrix to facilitate cell infiltration, differentiation, and vascularization. Engineered scaffolds composed of natural biomaterials silk fibroin (SF) and chitosan (CS) blend were fabricated to achieve fibrillar nano-structures aligned in three-dimensions using the technique of dielectrophoresis. The effect of scaffold properties on adhesion and migration of human adipose-derived stem cells (hASC) and endothelial cells (HUVEC) was studied on SFCS (micro-structure, unaligned) and engineered SFCS (E-SFCS; nano-structure, aligned). E-SFCS constituted of a nano-featured fibrillar sheets, whereas SFCS sheets had a smooth morphology with unaligned micro-fibrillar extensions at the ends. Adhesion of hASC to either scaffolds occurred within 30 min and was higher than HUVEC adhesion. The percentage of moving cells and average speed was highest for hASC on SFCS scaffold as compared to hASC cocultured with HUVEC. HUVEC interactions with hASC appeared to slow the speed of hASC migration (in coculture) on both scaffolds. It is concluded that the guidance of cells for regenerative tissue engineering using SFCS scaffolds requires a fine balance between cell-cell interactions that affect the migration speed of cells and the surface characteristics that affects the overall adhesion and direction of migration.

The fractal dimension of cell membrane correlates with its capacitance: a new fractal single-shell model.

The scale-invariant property of the cytoplasmic membrane of biological cells is examined by applying the Minkowski-Bouligand method to digitized scanning electron microscopy images of the cell surface. The membrane is found to exhibit fractal behavior, and the derived fractal dimension gives a good description of its morphological complexity. Furthermore, we found that this fractal dimension correlates well with the specific membrane dielectric capacitance derived from the electrorotation measurements. Based on these findings, we propose a new fractal single-shell model to describe the dielectrics of mammalian cells, and compare it with the conventional single-shell model (SSM). We found that while both models fit with experimental data well, the new model is able to eliminate the discrepancy between the measured dielectric property of cells and that predicted by the SSM.

Dielectric characterization of complete mononuclear and polymorphonuclear blood cell subpopulations for label-free discrimination.

Dielectric spectroscopy is a powerful technique for the elucidation of a number of important cell biophysical properties, and it can provide information about cell morphology, physiological state, viability and identity. A high-impact application for dielectric cell analysis would be microfluidic flow-through impedance sensing to perform what is perhaps the most routinely ordered medical diagnostic assay, a complete blood count with white blood cell differential enumeration. To assess the biophysical feasibility of such an analysis, we obtained reference dielectric measurements of the complete complement of purified leukocyte subpopulations using the dielectrophoretic crossover frequency method. The sensitivity of this method can detect subtle changes in cell morphology and physiology, so we developed a leukocyte isolation protocol based on a suite of negative selection techniques to yield cell subpopulations that were minimally processed and in an as near native state as possible. This is the first reported study of the dielectric properties of all the major leukocyte subpopulations that includes separate analysis of the polymorphonuclear neutrophil, basophil and eosinophil cell types. We show that T-lymphocytes, B-lymphocytes, monocytes and granulocytes possess distinct membrane dielectric properties and that the morphologically similar granulocyte subpopulations can be identified via their dielectric and size properties. Finally, we discuss the application of our findings to label-free systems for the analysis of leukocytes.

Dielectrophoretic-field flow fractionation analysis of dielectric, density, and deformability characteristics of cells and particles.

Dielectrophoretic field-flow fractionation (DEP-FFF) has been used to discriminate between particles and cells based on their dielectric and density properties. However, hydrodynamic lift forces (HDLF) at flow rates needed for rapid separations were not accounted for in the previous theoretical treatment of the approach. Furthermore, no method was developed to isolate particle or cell physical characteristics directly from DEP-FFF elution data. An extended theory of DEP-FFF is presented that accounts for HDLF. With the use of DS19 erythroleukemia cells as model particles with frequency-dependent dielectric properties, it is shown that the revised theory accounts for DEP-FFF elution behavior over a wide range of conditions and is consistent with sedimentation-FFF when the DEP force is zero. Conducting four elution runs under specified conditions, the theory allows for the derivation of the cell density distribution and provides good estimates of the distributions of the dielectric properties of the cells and their deformability characteristics that affect HDLF. The approach allows for rapid profiling of the biophysical properties of cells, the identification and characterization of subpopulations, and the design of optimal DEP-FFF separation conditions. The extended DEP-FFF theory is widely applicable, and the parameter measurement methods may be adapted easily to other types of particles.

Isolation of rare cells from cell mixtures by dielectrophoresis.

The application of dielectrophoretic field-flow fractionation (depFFF) to the isolation of circulating tumor cells (CTCs) from clinical blood specimens was studied using simulated cell mixtures of three different cultured tumor cell types with peripheral blood. The depFFF method can not only exploit intrinsic tumor cell properties so that labeling is unnecessary but can also deliver unmodified, viable tumor cells for culture and/or all types of molecular analysis. We investigated tumor cell recovery efficiency as a function of cell loading for a 25 mm wide x 300 mm long depFFF chamber. More than 90% of tumor cells were recovered for small samples but a larger chamber will be required if similarly high recovery efficiencies are to be realized for 10 mL blood specimens used CTC analysis in clinics. We show that the factor limiting isolation efficiency is cell-cell dielectric interactions and that isolation protocols should be completed within approximately 15 min in order to avoid changes in cell dielectric properties associated with ion leakage.

Dielectric cell separation of fine needle aspirates from tumor xenografts.

As an approach to isolating tumor cells from fine needle biopsy specimens, we investigated a dielectric cell preparation method using an in vivo xenographic tumor model. Cultured human MDA-MB-435 tumor cells were grown as solid tumors in nude mice and fine needle aspiration biopsies were conducted. Biopsied cells were suspended in sucrose medium and collected on slides patterned with microelectrode arrays (electrosmears) energized by electrical signals in the range 10 to 960 kHz. The unlabeled cells adhered to characteristic regions of the slides in accordance with their morphology as a result of dielectric forces. Tumor cells were trapped between 40 and 60 kHz and were separated according to whether they were mitotic, large and complex, or small. Damaged tumor cells were captured at between 60 and 120 kHz; granulocytes between 70 and 90 kHz; lymphocytes between 85 and 105 kHz; healthy erythrocytes between 140 and 180 kHz, and damaged erythrocytes above 180 kHz. Using intrinsic cell characteristics, the electrosmear presented cell subpopulations from fine needle aspiration biopsy specimens in a manner that is compatible with automated slide-based analysis systems. The approach has the potential to facilitate the analysis of the role of cell subpopulations in disease.

Dielectrophoretic field-flow fractionation system for detection of aquatic toxicants.

Dielectrophoretic field-flow fractionation (dFFF) was applied as a contact-free way to sense changes in the plasma membrane capacitances and conductivities of cultured human HL-60 cells in response to toxicant exposure. A micropatterned electrode imposed electric forces on cells in suspension in a parabolic flow profile as they moved through a thin chamber. Relative changes in the dFFF peak elution time, reflecting changes in cell membrane area and ion permeability, were measured as indices of response during the first 150 min of exposure to eight toxicants having different single or mixed modes of action (acrylonitrile, actinomycin D, carbon tetrachloride, endosulfan, N-nitroso- N-methylurea (NMU), paraquat dichloride, puromycin, and styrene oxide). The dFFF method was compared with the cell viability assay for all toxicants and with the mitochondrial potentiometric dye assay or DNA alkaline comet assay according to the mode of action of the specific agents. Except for low doses of nucleic acid-targeting agents (actinomycin D and NMU), the dFFF method detected all toxicants more sensitively than other assays, in some cases up to 10 (5) times more sensitively than the viability approach. The results suggest the dFFF method merits additional study for possible applicability in toxicology.

Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation.

We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations.

A Continuous-Flow Polymerase Chain Reaction Microchip With Regional Velocity Control.

This paper presents a continuous-flow polymerase chain reaction (PCR) microchip with a serpentine microchannel of varying width for "regional velocity control." Varying the channel width by incorporating expanding and contracting conduits made it possible to control DNA sample velocities for the optimization of the exposure times of the sample to each temperature phase while minimizing the transitional periods during temperature transitions. A finite element analysis (FEA) and semi-analytical heat transfer model was used to determine the distances between the three heating assemblies that are responsible for creating the denaturation (96 degrees C), hybridization (60 degrees C), and extension (72 degrees C) temperature zones within the microchip. Predictions from the thermal FEA and semi-analytical model were compared with temperature measurements obtained from an infrared (IR) camera. Flow-field FEAs were also performed to predict the velocity distributions in the regions of the expanding and contracting conduits to study the effects of the microchannel geometry on flow recirculation and bubble nucleation. The flow fields were empirically studied using micro particle image velocimetry (mu-PIV) to validate the flow-field FEA's and to determine experimental velocities in each of the regions of different width. Successful amplification of a 90 base pair (bp) bacillus anthracis DNA fragment was achieved.

Dielectrophoretic segregation of different human cell types on microscope slides.

A new method for preparing cells for microscopic examination is presented in which cell mixtures are fractionated by dielectrophoretic forces and simultaneously collected into characteristic zones on slides. The method traps cells directly from the suspending medium onto the slide, reducing cell loss. Furthermore, it exploits differences in the dielectric properties of the cells, which sensitively reflect their morphology. Because different cell types are trapped in characteristic zones on the slide, the technique represents an advance over existing methods for slide preparation, such as centrifugation and smears where cells are randomly distributed. In particular, the new method should aid in the detection of rare and anomalous cell subpopulations that might otherwise go unnoticed against a high background of normal cells. As well as being suitable for traditional microscopic examination and automated slide scanning approaches, it is compatible with histochemical and immunochemical techniques, as well as emerging molecular and proteomic methods. This paper describes the rationale and design of this so-called electrosmear instrumentation and shows experimental results that verify the theory and applicability of the method with model cell lines and normal peripheral blood subpopulations.

A High-Voltage Integrated Circuit Engine for a Dielectrophoresis-based Programmable Micro-Fluidic Processor.

A high-voltage (HV) integrated circuit has been demonstrated to transport droplets on programmable paths across its coated surface. This chip is the engine for a dielectrophoresis (DEP)-based micro-fluidic lab-on-a-chip system. This chip creates DEP forces that move and help inject droplets. Electrode excitation voltage and frequency are variable. With the electrodes driven with a 100V peak-to-peak periodic waveform, the maximum high-voltage electrode waveform frequency is about 200Hz. Data communication rate is variable up to 250kHz. This demonstration chip has a 32×32 array of nominally 100V electrode drivers. It is fabricated in a 130V SOI CMOS fabrication technology, dissipates a maximum of 1.87W, and is about 10.4 mm × 8.2 mm.

Dielectrophoresis-based programmable fluidic processors.

Droplet-based programmable processors promise to offer solutions to a wide range of applications in which chemical and biological analysis and/or small-scale synthesis are required, suggesting they will become the microfluidic equivalents of microprocessors by offering off-the-shelf solutions for almost any fluid based analysis or small scale synthesis problem. A general purpose droplet processor should be able to manipulate droplets of different compositions (including those that are electrically conductive or insulating and those of polar or non-polar nature), to control reagent titrations accurately, and to remain free of contamination and carry over on its reaction surfaces. In this article we discuss the application of dielectrophoresis to droplet based processors and demonstrate that it can provide the means for accurately titrating, moving and mixing polar or non-polar droplets whether they are electrically conductive or not. DEP does not require contact with control surfaces and several strategies for minimizing surface contact are presented. As an example of a DEP actuated general purpose droplet processor, we show an embodiment based on a scaleable CMOS architecture that uses DEP manipulation on a 32 x 32 electrode array having built-in control and switching circuitry. Lastly, we demonstrate the concept of a general-purpose programming environment that facilitates droplet software development for any type of droplet processor.

Droplet-based chemistry on a programmable micro-chip.

We describe the manipulation of aqueous droplets in an immiscible, low-permittivity suspending medium. Such droplets may serve as carriers for not only air- and water-borne samples, contaminants, chemical reagents, viral and gene products, and cells, but also the reagents to process and characterise these samples. We present proofs-of-concept for droplet manipulation through dielectrophoresis by: (1). moving droplets on a two-dimensional array of electrodes, (2). achieving dielectrically-activated droplet injection, (3). fusing and reacting droplets, and (4). conducting a basic biological assay through a combination of these steps. A long-term goal of this research is to provide a platform fluidic processor technology that can form the core of versatile, automated, micro-scale devices to perform chemical and biological assays at or near the point of care, which will increase the availability of modern medicine to people who do not have ready access to modern medical institutions, and decrease the cost and delays associated with that lack of access.