Chin Chun Ooi

Molecular profiling of single circulating tumor cells from lung cancer patients

Significance There exists an urgent need for minimally invasive molecular analysis tools for cancer assessment and management, particularly in advanced-stage lung cancer, when tissue procurement is challenging and gene mutation profiling is crucial to identify molecularly targeted agents for treatment. High-throughput compartmentalization and multigene profiling of individual circulating tumor cells (CTCs) from whole-blood samples using modular gene panels may facilitate highly sensitive, yet minimally invasive characterization of lung cancer for therapy prediction and monitoring. We envision this nanoplatform as a compelling research tool to investigate the dynamics of cancer disease processes, as well as a viable clinical platform for minimally invasive yet comprehensive cancer assessment. Circulating tumor cells (CTCs) are established cancer biomarkers for the “liquid biopsy” of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non–small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.

High performance wash-free magnetic bioassays through microfluidically enhanced particle specificity

Magnetic biosensors have emerged as a sensitive and versatile platform for high performance medical diagnostics. These magnetic biosensors require well-tailored magnetic particles as detection probes, which need to give rise to a large and specific biological signal while showing very low nonspecific binding. This is especially important in wash-free bioassay protocols, which do not require removal of particles before measurement, often a necessity in point of care diagnostics. Here we show that magnetic interactions between magnetic particles and magnetized sensors dramatically impact particle transport and magnetic adhesion to the sensor surfaces. We investigate the dynamics of magnetic particles’ biomolecular binding and magnetic adhesion to the sensor surface using microfluidic experiments. We elucidate how flow forces can inhibit magnetic adhesion, greatly diminishing or even eliminating nonspecific signals in wash-free magnetic bioassays, and enhancing signal to noise ratios by several orders of magnitude. Our method is useful for selecting and optimizing magnetic particles for a wide range of magnetic sensor platforms.

Magneto-nanosensor platform for probing low-affinity protein-protein interactions and identification of a low-affinity PD-L1/PD-L2 interaction.

Substantial efforts have been made to understand the interactions between immune checkpoint receptors and their ligands targeted in immunotherapies against cancer. To carefully characterize the complete network of interactions involved and the binding affinities between their extracellular domains, an improved kinetic assay is needed to overcome limitations with surface plasmon resonance (SPR). Here, we present a magneto-nanosensor platform integrated with a microfluidic chip that allows measurement of dissociation constants in the micromolar-range. High-density conjugation of magnetic nanoparticles with prey proteins allows multivalent receptor interactions with sensor-immobilized bait proteins, more closely mimicking natural-receptor clustering on cells. The platform has advantages over traditional SPR in terms of insensitivity of signal responses to pH and salinity, less consumption of proteins and better sensitivities. Using this platform, we characterized the binding affinities of the PD-1—PD-L1/PD-L2 co-inhibitory receptor system, and discovered an unexpected interaction between the two known PD-1 ligands, PD-L1 and PD-L2.

Magnetic Nanoparticle‐Based Upregulation of B‐Cell Lymphoma 2 Enhances Bone Regeneration

Clinical translation of cell‐based strategies for tissue regeneration remains challenging because survival of implanted cells within hostile, hypoxic wound environments is uncertain. Overexpression of B‐cell lymphoma 2 (Bcl‐2) has been shown to inhibit apoptosis in implanted cells. The present study describes an “off the shelf” prefabricated scaffold integrated with magnetic nanoparticles (MNPs) used to upregulate Bcl‐2 expression in implanted adipose‐derived stromal cells for bone regeneration. Iron oxide cores were sequentially coated with branched polyethyleneimine, minicircle plasmid encoding green fluorescent protein and Bcl‐2, and poly‐β‐amino ester. Through in vitro assays, increased osteogenic potential and biological resilience were demonstrated in the magnetofected group over control and nucleofected groups. Similarly, our in vivo calvarial defect study showed that magnetofection had an efficiency rate of 30%, which in turn resulted in significantly more healing compared with control group and nucleofected group. Our novel, prefabricated MNP‐integrated scaffold allows for in situ postimplant temporospatial control of cell transfection to augment bone regeneration. Stem Cells Translational Medicine 2017;6:151–160

An intravascular magnetic wire for the high-throughput retrieval of circulating tumour cells in vivo

The detection and analysis of rare blood biomarkers is necessary for early diagnosis of cancer and to facilitate the development of tailored therapies. However, current methods for the isolation of circulating tumour cells (CTCs) or nucleic acids present in a standard clinical sample of only 5–10 ml of blood provide inadequate yields for early cancer detection and comprehensive molecular profiling. Here, we report the development of a flexible magnetic wire that can retrieve rare biomarkers from the subject’s blood in vivo at a much higher yield. The wire is inserted and removed through a standard intravenous catheter and captures biomarkers that have been previously labelled with injected magnetic particles. In a proof-of-concept experiment in a live porcine model, we demonstrate the in vivo labelling and single-pass capture of viable model CTCs in less than 10 s. The wire achieves capture efficiencies that correspond to enrichments of 10–80 times the amount of CTCs in a 5-ml blood draw, and 500–5,000 times the enrichments achieved using the commercially available Gilupi CellCollector.A magnetic wire for the intravascular recovery of labelled circulating tumour cells improves cell capture in anaesthetized pigs by up to two orders of magnitude with respect to a standard blood draw.

Multigene profiling of single circulating tumor cells

ABSTRACT Numerous techniques for isolating circulating tumor cells (CTCs) have been developed. Concurrently, single-cell techniques that can reveal molecular components of CTCs have become widely available. We discuss how the combination of isolation and multigene profiling of single CTCs in our platform can facilitate eventual translation to the clinic.

Exchange-Biased Anisotropic Magnetoresistive Field Sensor

IrMn exchange-biased anisotropic magnetoresistive sensors have been optimized for increased outputs and reduced noise. An ~ 56% increase in peak-to-peak outputs has been achieved in IrMn exchange-biased NiFe thin-film sensors, relative to sensors without IrMn. Critically, these sensors’ transfer functions show a linear and symmetric magnetic field response with small hysteresis. The tradeoff between detection range and sensitivity has also been explored, and an inverse proportional relationship is observed. In addition, sensor noise performance can be improved with an IrMn exchange bias layer and further enhanced by the addition of a thin MgO layer between the NiFe and the Ta capping layer. The decrease in magnetic 1/

High-throughput full-length single-cell mRNA-seq of rare cells

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Abstract 3796: An intravascular magnetic wire for high-throughputin vivoenrichment of rare circulating cancer biomarkers

noise can be attributed to magnetization stabilization by IrMn and a reduction in pinning sites by MgO. This improved performance from IrMn exchange-biased anisotropic magnetoresistive sensors renders them promising for many different applications.

Abstract LB-280: Gene expression profiling of individual circulating tumor cells from non-small cell lung cancer (NSCLC) patients via integrated nanotechnologies

Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 107 per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial cells, with demonstrably high-quality transcriptomic data.