DEPArray™ system: An automatic image-based sorter for isolation of pure circulating tumor cells.

Circulating tumor cells (CTCs) are rare cells shed into the bloodstream by invasive tumors and their analysis offers a promising noninvasive tool to predict and monitor therapeutic responses. CTCs can be isolated from patient blood and their characterization at single-cell level can inform on the genomic landscape of a tumor. All CTC enrichment methods bear a burden of contaminating normal cells, which mandate a further step of purification to enable reliable downstream genetic analysis. Here, we describe the DEPArray™ technology, a microchip-based digital sorter, which combines precise microfluidic and microelectronic enabling precise, image-based isolation of single CTCs, which can then be analyzed by Next Generation Sequencing (NGS) methods. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products.

Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.

Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing.

Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.

Molecular profiling of single circulating tumor cells with diagnostic intention.

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

Lysis-on-chip of single target cells following forced interaction with CTLs or NK cells on a dielectrophoresis-based array.

Guiding the interaction of single cells acting as partners in heterotypic interactions (e.g., effectors and targets of immune lysis) and monitoring the outcome of these interactions are regarded as crucial biomedical achievements. In this study, taking advantage of a dielectrophoresis (DEP)-based Laboratory-on-a-chip platform (the DEPArray), we show that it is possible to generate closed DEP cages entrapping CTLs and NK cells as either single cells or clusters; reversibly immobilize a single virus-presenting or tumor cell within the chip at a selected position; move cages and their content to predetermined spatial coordinates by software-guided routing; force a cytotoxic effector to physically interact with a putative target within a secluded area by merging their respective cages; generate cages containing effector and target cells at predetermined E:T ratios; accurately assess cytotoxicity by real-time quantitation of the release kinetics of the fluorescent dye calcein from target cells (>50 lytic events may be tested simultaneously); estimate end points of calcein release within 16 min of initial E:T cell contact; simultaneously deliver Ab-based phenotyping and on-chip lysis assessment; and identify lytic and nonlytic E:T combinations and discriminate nonlytic effector phenotypes from target refractoriness to immune lysis. The proof of principle is provided that DEPArray technology, previously used to levitate and move single cells, can be used to identify highly lytic antiviral CTLs and tumor cells that are particularly refractory to NK cell lysis. These findings are of primary interest in targeted immunotherapy.

Programmable interactions of functionalized single bioparticles in a dielectrophoresis-based microarray chip.

Manipulating single biological objects is a major unmet challenge of biomedicine. Herein, we describe a lab-on-a-chip platform based on dielectrophoresis (DEP). The DEParray is a prototypal version consisting of 320 × 320 arrayed electrodes generating >10,000 spherical DEP cages. It allows the capture and software-guided movement to predetermined spatial coordinates of single biological objects. With the DEParray we demonstrate (a) forced interaction between a single, preselected target cell and a programmable number of either microspheres or natural killer (NK) cells, (b) on-chip immunophenotypic discrimination of individual cells based on differential rosetting with microspheres functionalized with monoclonal antibodies to an inhibitory NK cell ligand (HLA-G), (c) on-chip, real-time (few minutes) assessment of immune lysis by either visual inspection or semiautomated, time-lapse reading of a fluorescent dye released from NK cell-sensitive targets, and (d) manipulation and immunophenotyping with limiting amounts (about 500) cells. To our knowledge, this is the first report describing a DEP-based lab-on-a-chip platform for the quick, arrayed, software-guided binding of individually moved biological objects, the targeting of single cells with microspheres, and the real-time characterization of immunophenotypes. The DEParray candidates as a discovery tool for novel cell:cell interactions with no prior (immuno)phenotypic knowledge.

Electronic sorting and recovery of single live cells from microlitre sized samples.

Sorting and recovering specific live cells from samples containing less than a few thousand cells have become major hurdles in rare cell exploration such as stem cell research, cell therapy and cell based diagnostics. We describe here a new technology based on a microelectronic chip integrating an array of over 100,000 independent electrodes and sensors which allow individual and parallel single cell manipulation of up to 10,000 cells while maintaining viability and proliferation capabilities. Manipulation is carried out using dynamic dielectrophoretic traps controlled by an electronic interface. We also demonstrate the capabilities of the chip by sorting and recovering individual live fluorescent cells from an unlabeled population.

Dielectrophoresis-based 'Lab-on-a-chip' devices for programmable binding of microspheres to target cells.

There is a general agreement on the fact that the Laboratory on chip (Lab-on-a-chip) technology will enable laboratory testing to move from laboratories employing complex equipments into non-laboratory settings. In this respect, dielectrophoresis (DEP) is a very valuable approach to design and produce Lab-on-a-chip devices able to manipulate microparticles and cells. In this study, we report the application of DEP-based devices for facilitating programmable interactions between microspheres and target tumor cells. We used two Lab-on-a-chip devices, one (the SmartSlide) carrying 193 parallel electrodes and generating up to 50 cylinder-shaped DEP cages, the other (the DEP array) carrying 102,400 arrayed electrodes and generating more than 10,000 spherical DEP cages. We determined whether these devices can be used to levitate and move microspheres and cells in order to obtain a forced interaction between microspheres and target cells. The first major point of this manuscript is that the DEP-based SmartSlide can be used for transfection experiments in which microspheres and target cells are forced to share the same DEP cage, leading to efficient binding of the microspheres to target cells. The data obtained using the DEP array show that this system allows the sequential, software-controlled binding of individually and independently moved single microspheres to a single target tumor cell. To our knowledge, this is the first report on the possible use of a DEP-based Lab-on-a-chip device for guided multiple binding of singularly moved microspheres to a single tumor cell. This approach can be of interest in the field of drug discovery, delivery and diagnosis.

Separation of white blood cells from erythrocytes on a dielectrophoresis (DEP) based 'Lab-on-a-chip' device.

The 'Lab-on-a-chip technology' involves miniaturization of complex analytical procedures and is expected to enable laboratory testing to move from the central laboratory employing complex equipment into non-laboratory settings. We report the application of a printed circuit board (PCB)-based chip, generating dielectrophoretic (DEP)-based cylinder-shaped cages for separation and recovery of white blood cells from erythrocytes. This possibility is of interest to develop low-cost Lab-on-a-chip devices for diagnostic purposes. Accordingly, we demonstrate that white blood cells recovered from this Lab-on-a-chip device are suitable for PCR-based molecular diagnosis procedures employing DNA sequencing or biospecific interaction analysis using surface plasmon resonance and biosensor technology.

Applications to cancer research of "lab-on-a-chip" devices based on dielectrophoresis (DEP).

The recent development of advanced analytical and bioseparation methodologies based on microarrays and biosensors is one of the strategic objectives of the so-called post-genomic. In this field, the development of microfabricated devices could bring new opportunities in several application fields, such as predictive oncology, diagnostics and anti-tumor drug research. The so called "Laboratory-on-a-chip technology", involving miniaturisation of analytical procedures, is expected to enable highly complex laboratory testing to move from the central laboratory into non-laboratory settings. The main advantages of Lab-on-a-chip devices are integration of multiple steps of different analytical procedures, large variety of applications, sub-microliter consumption of reagents and samples, and portability. One of the requirement for new generation Lab-on-a-chip devices is the possibility to be independent from additional preparative/analytical instruments. Ideally, Lab-on-a-chip devices should be able to perform with high efficiency and reproducibility both actuating and sensing procedures. In this review, we discuss applications of dielectrophoretic(DEP)-based Lab-on-a-chip devices to cancer research. The theory of dielectrophoresis as well as the description of several devices, based on spiral-shaped, parallel and arrayed electrodes are here presented. In addition, in this review we describe manipulation of cancer cells using advanced DEP-based Lab-on-a-chip devices in the absence of fluid flow and with the integration of both actuating and sensing procedures.