Materials and microfluidics: enabling the efficient isolation and analysis of circulating tumour cells

Abstract

We present a critical review of microfluidic technologies and material effects on the analyses of circulating tumour cells (CTCs) selected from the peripheral blood of cancer patients. CTCs are a minimally invasive source of clinical information that can be used to prognose patient outcome, monitor minimal residual disease, assess tumour resistance to therapeutic agents, and potentially screen individuals for the early diagnosis of cancer. The performance of CTC isolation technologies depends on microfluidic architectures, the underlying principles of isolation, and the choice of materials. We present a critical review of the fundamental principles used in these technologies and discuss their performance. We also give context to how CTC isolation technologies enable downstream analysis of selected CTCs in terms of detecting genetic mutations and gene expression that could be used to gain information that may affect patient outcome.

Figures

Figure 1

A Scopus survey of articles published from 2004 to 2016 that reference CTCs in general or specifically the subject of CTCs and CellSearch™ or microfluidics. Scopus results were restricted to articles only and used the fields specified in the legend.

Figure 2

Applications of CTC analyses including enumeration, genomic mutation screening (FISH, Sanger sequencing, aCGH, and NGS), RNA expression profiling (RNA-ISH, qRT-PCR, expression microarrays, and single cell RNA-seq), protein analysis (EPISPOT), and ex vivo culturing (CTC expansion, xenograft models, and drug susceptibility). Adapted from Pantel and Speicher. Abbreviations: FISH – fluorescence in situ hybridization; WGA – whole genome amplification; aCGH – array comparative genomic hybridization; NGS – Next Generation Sequencing; RNA-ISH – fluorescence RNA in situhybridization; qRT-PCR – quantitative reverse transcription polymerase chain reaction; EPISPOT – epithelial immunospot. Figure panels reproduced from reference with permission from Wiley, copyright 2015; reference with permission from Elseveir, copyright 2009; reference with permission from Nature Publishing Group, copyright 2014; reference with permission from The American Association for the Advancement of Science, copyright 2013; reference with permission from Nature Publishing Group, copyright 2014; and reference with permission from The American Association for the Advancement of Science, copyright 2014.

Figure 3

Magnetic CTC isolation technologies. (A) Workflow of the CellSearch™ CTC Test versus the CellSearch™ Profile Kit.(B) Workflow and diagram of the iChip, here shown in positive selection mode. The blood is debulked, the remaining cells are focused, and magnetically labelled cells (CTCs in positive selection mode, WBCs in negative selection) are preferentially forced into a separate outlet.(C) A diagram of the Ephesia microfluidic technology, which aligns anti-EpCAM magnetic microbeads into solid supports for CTC isolation that can be released by removing the magnetic field.,(D) Velocity valley, and magnetic ranking technologies for isolating magnetically labelled CTCs in zones of varying velocity or magnetic field strength, respectively, which provides phenotypic ranking of CTC antigen (e.g., EpCAM) expression in addition to enumeration. X-shaped microstructures reduce fluid velocity so magnetic forces can provide efficient CTC recovery. (E) The μHall device detects CTCs labelled with magnetic nanoparticles passing over a μHall sensor, which induces a voltage proportional to antigen expression. The sample stream (pink) is focused over 8 staggered μHall sensors that compensate for variable CTC position. Figure panels reproduced from reference with permission from The American Association for the Advancement of Science, copyright 2013; reference with permission from Wiley, copyright 2015; reference with permission from Nature Publishing Group, copyright 2017; and reference with permission from The American Association for the Advancement of Science, copyright 2012.

Figure 4

Direct comparisons to the CellSearch™ CTC Test by (A) the CellSearch™ Profile Kit,(B) Apostream™,(C) the posiChip,(D) the magnetic ranking microfluidic device,(E) the GEDI micropillar device, and (F) the Ephesia microfluidic device. Note that magnetic ranking and Ephesia technologies collected blood in CellSave™ tubes in comparisons,, and the GEDI device selected PSMA(+) CTCs, whereas the CTC Test targeted EpCAM(+) CTCs. In this study, Kirby et al. noted that 60% (median) of CTCs were PSMA(+)/EpCAM(+), indicating the GEDI yields were roughly 10-fold greater than by the CellSearch™ CTC Test. Figure panels reproduced from reference with permission from Nature Publishing Group, copyright 2010; reference with permission from The American Association for the Advancement of Science, copyright 2013; and reference with permission from Nature Publishing Group, copyright 2017.

Figure 5

Positive-affinity microfluidic selection. (A) Assembly of the silicon CTC chip, SEM of a pseudo-coloured cell isolated on the Ab-coated micropillars, and simulated fluid velocity field in the device.(B) The GEDI device arranges micropillars to hydrodynamically induce a strong bias towards recovering cells >15–18 μm (blue) and minimizing smaller WBC (yellow) interactions.(C) The herringbone chip uses convective mixing to encourage CTCs to interact with Ab-coated surfaces.(D) A schematic of the silicon nanopillar chip, where a convective mixing chamber is attached to a nano-textured, Ab-coated Si substrate.(E) Polyurethane tubing is nano-textured with naturally occurring halloysite nanotubes and coated with Abs and selectins.(F) The thermoplastic-based sinusoidal chip uses narrow, Ab-coated microchannels to isolate CTCs. CTC release, enables off-chip enumeration and viability testing by an impedance sensor and a microfluidic imaging module, which are integrated to a fluidic motherboard. Figure panels reproduced from reference with permission from Nature Publishing Group, copyright 2007; reference with permission from Wiley, copyright 2011; and reference with permission from American Chemical Society, copyright 2013.

Figure 6

Strategies to release CTCs after microfluidic affinity-selection.(A) Proteolytic digestion of Ab-antigen complex.(B) Exonuclease digestion of DNA aptamers.(C) Uracil-specific enzymatic digestion of oligonucleotide linkers that anchor Abs to surfaces.(D) Thermally responsive polymer that internalizes the attached Abs when cooled.(E) Electrostatic assembly of nano-films containing biotinylated-alginate that can be enzymatically digested.(F) Gelatin nano-films assembled by avidin cross-linking that can be thermally melted or locally dissociated by mechanically tapping with a microtip. Figure panels reproduced from reference with permission from Wiley, copyright 2013; reference with permission from Wiley, copyright 2013; reference with permission from Elsevier, copyright 2015; and reference with permission from Wiley, copyright 2015.

Figure 7

(A) Cell abundance versus cell diameter of blood cells and CTCs, and common size ranges for CTC discrimination., Note that WBC sizes can be smaller in free solution than when plated for microscopy.,(B) (i) A CK(+)/DAPI(+) CTC (red and blue) amongst CD45(+)/DAPI(+) WBCs (green and blue) on a Si filter membrane. (ii) SEM of a fixed CTC on a 2D parylene-C membrane. (iii) Picture of a clogged filter after processing 7.5 mL of blood. (iv) Schematic of a 3D parylene-C membrane. (v) Brightfield and fluorescence images of MCF-7 cells filtered after size enlargement with anti-EpCAM microbeads. (vi) Images of CTCs trapped in a micropillar-based filtration device.(C) The Cluster-Chip collects CTC clusters specifically due to their size.(D) The Vortex Technology hydrodynamically traps large CTCs in side channels at high flow rates.(E) Dean Flow Fractionation is a hydrodynamic centrifugation method for size-dependent separation of CTCs.(F) Dielectrophoretic crossover frequencies for cancer cell lines, leukemia cell lines, and WBCs.(inset) Working principle of DEP showing field lines for positive and negative DEP experienced by CTCs and WBCs at 65 kHz, respectively.(G) Schematic of the ApoStream™ technology for DEP-flow field fractionation of CTCs. Figure panels reproduced from reference with permission from Elsevier, copyright 2007; reference with permission from Nature Publishing Group, copyright 2014; reference with permission from Springer, copyright 2011; reference with permission from Elsevier, copyright 2010; reference with permission from Nature Publishing Group, copyright 2015; reference with permission from Nature Publishing Group, copyright 2013; reference with permission from AIP Publishing, copyright 2013; and reference with permission from AIP Publishing, copyright 2012.