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

Abstract

BackgroundCirculating 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 FindingsHere, 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/SignificanceWe 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.

Circulating tumor cell technologies

“…No RBC lysis required, captures viable cells, easy to manufacture, clusters observed (Sollier et al, 2014) ClearCell Ò FX RBC lysis required, 1e1.5 mL/min, captures viable cells, easy to manufacture (Khoo et al, 2015(Khoo et al, , 2014Warkiani et al, 2014) Biophysical e Electrophoresis Separates cells based on their electrical signature using an applied electric field. (Carpenter et al, 2014;Fabbri et al, 2013;Fernandez et al, 2014;Manaresi et al, 2003;Peeters et al, 2013;Polzer et al, 2014) Biophysical e Acoustophoresis Separates cells based on acoustophoretic mobility, which is size dependent, by exposing them to acoustic waves.…”

Evaluation and consequences of heterogeneity in the circulating tumor cell compartment

“…Percentages of ALK -rearranged CTCs were confirmed by two other studies. In a first report, one-fourth of the total 177 CTCs of one patient harbored ALK rearrangements [90] and in the other, 100% of the CTCs of 5 patients were ALK -rearranged [91], whereas in the primary tumor tissue this was around 50% in both studies. Furthermore, ROS1 rearrangements were found in CTCs of four patients with lung cancer [95].…”

The incorporation of microfluidics into circulating tumor cell isolation for clinical applications

“…Generally, near the outlet of spiral devices, larger CTCs focus near the inner wall due to the combination of the inertial lift force and the Dean drag force. To this end, Hou et al designed a multiplexed spiral device that detected CTCs in clinical samples from breast and lung cancer patients [34]. …”

“…Figures used or adapted with permission from (clockwise from top right): [29] Copyright 2014 The Royal Society of Chemistry; [31] Copyright 2014 AIP Publishing LLC; [34]; [26]. …”