Polymer Microsystems for the Enrichment of Circulating Tumor Cells and their Clinical Demonstration

Cancer research is centered on the discovery of new biomarkers that could unlock the obscurities behind the mechanisms that cause cancer or those associated with its spread (i.e., metastatic disease). Circulating tumor cells (CTCs) have emerged as attractive biomarkers for the management of many cancer-related diseases due primarily to the ease of securing them from a simple blood draw. However, their rarity (~1 CTC per mL of whole blood) makes enrichment analytically challenging. Microfluidic systems are viewed as exquisite platforms for the clinical analysis of CTCs due to their ability to be used in an automated fashion, minimizing sample loss and contamination. This has formed the basis of the reported research, which focused on the development of microfluidic systems for CTC analysis. The system reported herein consisted of a modular design and targeted the analysis of CTCs using pancreatic ductal adenocarcinoma (PDAC) as the model disease for determining the utility of the system. The system was composed of 3 functional modules; (i) a thermoplastic CTC selection module consisting of high aspect ratio (30 µm x 150 µm) channels; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for phenotype identification of selected CTCs. The system could exhaustively process 7.5 mL of blood in \u3c45 min with CTC recoveries \u3e90% directly from whole blood. In addition, significantly reduced assay turnaround times (8 h to 1.5 h) was demonstrated. We also show the ability to detect KRAS gene mutations from CTCs enriched by the microfluidic system. As a proof-of-concept, the ability to identify KRAS point mutations using a PCR/LDR/CE assay from as low as 10 CTCs enriched by the integrated microfluidic system was demonstrated. Finally, the clinical utility of the polymer-based microfluidic device for the analysis of circulating multiple myeloma cells (CMMCs) was demonstrated as well. Parameters such as translational velocity and recovery of CMMCs were optimized and found to be 1.1 mm/s and 71%, respectively. Also demonstrated was on-chip immunophenotyping and clonal testing of CMMCs, which has been reported to be prognostically significant. Further, a pilot study involving 26 patients was performed using the polymer microfluidic device with the aim of correlating the number of CMMCs with disease activity. An average of 347 CMMCs/mL of whole blood was recovered from blood volumes of approximately 0.5 mL

Messenger RNAs localized to distal projections of human stem cell derived neurons

The identification of mRNAs in distal projections of model organisms has led to the discovery of multiple proteins that are locally synthesized for functional roles such as axon guidance, injury signaling and regeneration. The extent to which local protein synthesis is conserved in human neurons is unknown. Here we used compartmentalized microfluidic chambers to characterize the transcriptome of distal projections of human embryonic stem cells differentiated using a protocol which enriched for glutamatergic neurons (hESC-neurons). Using gene expression analysis, we identified mRNAs proportionally enriched in these projections, representing a functionally unique local transcriptome as compared to the human neuronal transcriptome inclusive of somata. Further, we found that the most abundant mRNAs within these hESC-neuron projections were functionally similar to the axonal transcriptome of rat cortical neurons. We confirmed the presence of two well characterized axonal mRNAs in model organisms, β-actin and GAP43, within hESC-neuron projections using multiplexed single molecule RNA-FISH. Additionally, we report the novel finding that oxytocin mRNA localized to these human projections and confirmed its localization using RNA-FISH. This new evaluation of mRNA within human projections provides an important resource for studying local mRNA translation and has the potential to reveal both conserved and unique translation dependent mechanisms

Multi-compartment Microfluidic Device Geometry and Covalently Bound Poly-D-Lysine Influence Neuronal Maturation

Multi-compartment microfluidic devices have become valuable tools for experimental neuroscientists, improving the organization of neurons and access to their distinct subcellular microenvironments for measurements and manipulations. While murine neurons are extensively used within these devices, there is a growing need to culture and maintain human neurons differentiated from stem cells within multi-compartment devices. Human neuron cultures have different metabolic demands and require longer culture times to achieve synaptic maturation. We tested different channel heights (100 μm, 400 μm, and open) to determine whether greater exposure to media for nutrient exchange might improve long-term growth of NIH-approved H9 embryonic stem cells differentiated into glutamatergic neurons. Our data showed an opposite result with both closed channel configurations having greater synaptic maturation compared to the open compartment configuration. These data suggest that restricted microenvironments surrounding neurons improve growth and maturation of neurons. We next tested whether covalently bound poly-D-lysine (PDL) might improve growth and maturation of these neurons as somata tend to cluster together on PDL adsorbed surfaces after long culture periods (>30 days). We found that covalently bound PDL greatly improved the differentiation and maturation of stem cell-derived neurons within the devices. Lastly, experimental paradigms using the multi-compartment platform show that axons of human stem cell derived neurons intrinsically regenerate in the absence of inhibitory cues and that isolated axons form presynaptic terminals when presented with synaptic targets

Circulating Tumor Cells as a Biomarker of Response to Treatment in Patient-Derived Xenograft Mouse Models of Pancreatic Adenocarcinoma

Circulating tumor cells (CTCs) are cells shed from solid tumors into circulation and have been shown to be prognostic in the setting of metastatic disease. These cells are obtained through a routine blood draw and may serve as an easily accessible marker for monitoring treatment effectiveness. Because of the rapid progression of pancreatic ductal adenocarcinoma (PDAC), early insight into treatment effectiveness may allow for necessary and timely changes in treatment regimens. The objective of this study was to evaluate CTC burden as a biomarker of response to treatment with a oral phosphatidylinositol-3-kinase inhibitor, BKM120, in patient-derived xenograft (PDX) mouse models of PDAC. PDX mice were randomized to receive vehicle or BKM120 treatment for 28 days and CTCs were enumerated from whole blood before and after treatment using a microfluidic chip that selected for EpCAM (epithelial cell adhesion molecule) positive cells. This microfluidic device allowed for the release of captured CTCs and enumeration of these cells via their electrical impedance signatures. Median CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (26.61 to 2.21 CTCs/250 µL, p = 0.0207) while no significant change was observed in the vehicle group (23.26 to 11.89 CTCs/250 µL, p = 0.8081). This reduction in CTC burden in the treatment group correlated with tumor growth inhibition indicating CTC burden is a promising biomarker of response to treatment in preclinical models. Mutant enriched sequencing of isolated CTCs confirmed that they harbored KRAS G12V mutations, identical to the matched tumors. In the long-term, PDX mice are a useful preclinical model for furthering our understanding of CTCs. Clinically, mutational analysis of CTCs and serial monitoring of CTC burden may be used as a minimally invasive approach to predict and monitor treatment response to guide therapeutic regimens

Discrete microfluidics for the isolation of circulating tumor cell subpopulations targeting fibroblast activation protein alpha and epithelial cell adhesion molecule

Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens. Clinical sensitivities of 100% showed substantial improvement compared to epithelial cell adhesion molecule selection alone. Owing to high purity (>80%) of the selected circulating tumor cells, molecular analysis of both circulating tumor cell subpopulations was carried out in bulk, including next generation sequencing, mutation analysis, and gene expression. Results suggested fibroblast activation protein alpha and epithelial cell adhesion molecule circulating tumor cells are distinct subpopulations and the use of these in concert can provide information needed to navigate through cancer disease management challenges

Isolation of circulating plasma cells from blood of patients diagnosed with clonal plasma cell disorders using cell selection microfluidics

Blood samples from patients with plasma cell disorders were analysed for the presence of circulating plasma cells (CPCs) using a microfluidic device modified with monoclonal anti-CD138 antibodies. CPCs were immuno-phenotyped using a CD38/CD56/CD45 panel and identified in 78% of patients with monoclonal gammopathy of undetermined significance (MGUS), all patients with smouldering and symptomatic multiple myeloma (MM), and none in the controls. The burden of CPCs was higher in patients with symptomatic MM compared with MGUS and smouldering MM (p < 0.05). FISH analysis revealed the presence of chromosome 13 deletions in CPCs that correlated with bone marrow results. Point mutations in KRAS were identified, including different mutations from sub-clones derived from the same patient. The microfluidic assay represents a highly sensitive method for enumerating CPCs and allows for the cytogenetic and molecular characterization of CPCs

High-Throughput Selection, Enumeration, Electrokinetic Manipulation, and Molecular Profiling of Low-Abundance Circulating Tumor Cells Using a Microfluidic System

A circulating tumor cell (CTC) selection microfluidic device was integrated to an electrokinetic enrichment device for preconcentrating CTCs directly from whole blood to allow for the detection of mutations contained within the genomic DNA of the CTCs. Molecular profiling of CTCs can provide important clinical information that cannot be garnered simply by enumerating the selected CTCs. We evaluated our approach using SW620 and HT29 cells (colorectal cancer cell lines) seeded into whole blood as a model system. Because SW620 and HT29 cells overexpress the integral membrane protein EpCAM, they could be immunospecifically selected using a microfluidic device containing anti-EpCAM antibodies immobilized to the walls of a selection bed. The microfluidic device was operated at an optimized flow rate of 2 mm s(-1), which allowed for the ability to process 1 mL of whole blood in < 40 min. The selected CTCs were then enzymatically released from the antibody selection surface and hydrodynamically transported through a pair of Pt electrodes for conductivity-based enumeration. The efficiency of CTC selection was found to be 96% +/- 4%. Following enumeration, the CTCs were hydrodynamically transported at a flow rate of 1 mu L min(-1) to an on-chip electromanipulation unit, where they were electrophoretically withdrawn from the bulk hydrodynamic flow and directed into a receiving reservoir. Using an electric field of 100 V cm(-1), the negatively charged CTCs were enriched into an anodic receiving reservoir to a final volume of 2 mu L, providing an enrichment factor of 500. The collected CTCs could then be searched for point mutations using a PCR/LDR/capillary electrophoresis assay. The DNA extracted from the CTCs was subjected to a primary polymerase chain reaction (PCR) with the amplicons used for a ligase detection reaction (LDR) to probe for KRAS oncogenic point mutations. Point mutations in codon 12 of the KRAS gene were successfully detected in the SW620 CTCs for samples containing < 10 CTCs in 1 mL of whole blood. However, the HT29 cells did not contain these mutations, consistent with their known genotype.close6

CTC isolation using a microfluidic chip.

<p>(A) Design of the CTC microfluidic chip with sinusoidally shaped capture channels and brightfield images of (B) the capillary tube inserted into the on-chip entry channel where whole blood enters the microfluidic chip, (C) sinusoidally shaped capture channels where anti-human EpCAM antibodies are immobilized for CTC capture, (D) exit channel, and (E) impedance sensor with two Pt electrodes located adjacent to the exit channel to detect released CTCs. (F) Cells captured from whole blood of PDX-tumor bearing mice visualized directly on the microfluidic chip following immunostaining with DAPI (blue), human cytokeratin 8/19 (CK, red), and mouse CD45 (green). The staining pattern of DAPI-positive, human CK-positive, and mouse CD45-negative is characteristic of human CTCs. (G) A rare contaminating mouse leukocyte bound non-specifically to the microfluidic chip stained DAPI-positive, human CK-negative, and mouse CD45-positive. Contaminating leukocytes were excluded from enumeration due to the high specificity of the electrical impedance detector for cancer cells.</p

Response of CTC burden to BKM120 treatment.

<p>PDAC PDX mice were treated with vehicle or BKM120 for 28 days. CTCs were enumerated from whole blood on day 0 prior to the first treatment and on day 28 after the last treatment. CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (pre-treatment, pre: median  = 26.61 CTCs/250 µL, range  = 7–63 CTCs/250 µL, n = 8; post-treatment, post: median  = 2.21 CTCs/250 µL, range  = 0–79 CTCs/250 µL, n = 8; p = 0.0207, Wilcoxon) while no significant change was observed in the vehicle group (pre: median  = 23.26 CTCs/250 µL, range  = 4–43 CTCs/250 µL, n = 4; post: median  = 11.89 CTCs/250 µL, range  = 6–146 CTCs/250 µL, n = 8; p = 0.8081, Wilcoxon) One post BKM120 treatment sample had no detectable CTCs and is not plotted on scale.</p