Clinical Validation of an Ultra High-Throughput Spiral Microfluidics for the Detection and Enrichment of Viable Circulating Tumor Cells

Background: Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation. Methodology/Principal Findings: Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12–1275 CTCs/ml; Lung cancer samples: 10–1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples. Conclusions/Significance: We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.Singapore-MIT Alliance for Research and Technolog

Concurrent Single-Cell RNA and Targeted DNA Sequencing on an Automated Platform for Comeasurement of Genomic and Transcriptomic Signatures

BACKGROUND: The comeasurement of both genomic and transcriptomic signatures in single cells is of fundamental importance to accurately assess how the genetic information correlates with the transcriptomic phenotype. However, existing technologies have low throughput and laborious work flows. METHODS: We developed a new method for concurrent sequencing of the transcriptome and targeted genomic regions (CORTAD-seq) within the same single cell on an automated microfluidic platform. The method was compatible with the downstream library preparation, allowing easy integration into existing next-generation sequencing work flows. We incorporated a single-cell bioinformatics pipeline for transcriptome and mutation analysis. RESULTS: As proof of principle, we applied CORTAD-seq to lung cancer cell lines to dissect the cellular consequences of mutations that result in resistance to targeted therapy. We obtained a mean detection of 6000 expressed genes and an exonic rate of 50%. The targeted DNA-sequencing data achieved a 97.8% detection rate for mutations and allowed for the identification of copy number variations and haplotype construction. We detected expression signatures of tyrosine kinase inhibitor (TKI) resistance, epidermal growth factor receptor (EGFR) amplification, and expansion of the T790M mutation among resistant cells. We also identified characteristics for TKI resistance that were independent of EGFR T790M, indicating that other alterations are required for resistance in this context. CONCLUSIONS: CORTAD-seq allows assessment of the interconnection between genetic and transcriptomic changes in single cells. It is operated on an automated, commercially available single-cell isolation platform, making its implementation straightforward. (C) 2018 American Association for Clinical Chemistr

Enumeration of CTC from cancer patients.

<p>(A) Immunofluorescence staining of isolated CTCs. CTCs (marked by white arrow) were identified by the following criteria: Hoechst+, pan-CK+ and CD45-. Scale bar: 20 µm (B) Box plot summary indicating the range of CK+cells/ml recovered from the sample outlet for blood samples extracted from healthy volunteers, as wells as breast and lung cancer patients. The box plot presents the median, lower and upper quartiles (25^th,75^th percentiles). Data points that lie outside the 10^th and 90^th percentiles are shown as outliers (Anova, p<0.001). Encapsulated image of PAP stained isolated cells shows a large CTC with high nucleus to cytoplasmic (N/C) ratio (labeled with white arrow). (C) Staining of CTC for pan-CK and CD45. Upper panel depicts a representative image of cells which were double positive (CK+/CD45+); while lower panel shows a double negative (CK-/CD45-) cell. Scale bar: 20 µm (D) Staining of CTC for pan-cytokeratin and EpCAM. Scale bar: 20 µm.</p

Overview of sample preparation and processing procedures.

<p>(A) Sample processing workflow showing different steps of enrichment and identification. (i) The blood sample is collected; (ii) Plasma is separated using standard centrifugation (1500×g for 10 min) and stored at −80 degree Celsius for DNA analysis. (iii) RBCs are lysed using ammonium chloride and (iv) sample is processed through multiplexed spiral chip within 10 min. (v) The isolated CTCs are available for immunostaining using standard markers or FISH (fluorescence in situ hybridization). DNA or RNA can be extracted from the CTCs and subjected to next-generation sequencing and q-PCR. Viable cells can be released and propagated in cell culture for various applications including cancer stem cell (CSC) study or drug discovery. (B) Illustration of the design of a multiplexed device (left) and optical image of an actual multiplexed spiral microfluidic device (middle) for capturing CTCs with two inlets and two outlets. Blood sample and sheath fluid are pumped through the device using two separate syringe pumps. Under the influence of inertial lift and Dean drag forces in the fluid flow, CTCs focus near microchannel inner wall (Region A-A) while WBCs and Platelets goes through one Dean cycle and migrate back towards the outer wall (Region B-B), thus achieving separation.</p