Emerging role of nanomaterials in circulating tumor cell isolation and analysis

Abstract

Circulating tumor cells (CTCs) are low frequency cells found in the bloodstream after having been shed from a primary tumor. These cells are research targets because of the information they may potentially provide about both an individual cancer as well as the mechanisms through which cancer spreads in the process of metastasis. Established technologies exist for CTC isolation, but the recent progress and future of this field lie in nanomaterials. In this review, we provide perspective into historical CTC capture as well as current research being conducted, emphasizing the significance of the materials being used to fabricate these devices. The modern investigation into CTCs initially featured techniques that have since been commercialized. A major innovation in the field was the development of a microfluidic capture device, first fabricated in silicon and followed up with glass and thermopolymer devices. We then specifically highlight the technologies incorporating magnetic nanoparticles, carbon nanotubes, nanowires, nanopillars, nanofibers, and nanoroughened surfaces, graphene oxide and their fabrication methods. The nanoscale provides a new set of tools that has the potential to overcome current limitations associated with CTC capture and analysis. We believe the current trajectory of the field is in the direction of nanomaterials, allowing the improvements necessary to further CTC research.

Figure 1

Schematic view of the metastatic process showing CTC transit: the CTCs exit the primary tumor, intravasate into the bloodstream, circulate, and extravasate into a secondary site where they may ultimately achieve different fates including dormancy and full-blown metastasis. Adapted with permission from ref (3). Copyright 2002. Nature Publishing Group.

Figure 2

Recently developed CTC technologies. Isolation by Size of Epithelial Tumor Cells (ISET). Adapted with permission from ref (33). Copyright 2000 Elsevier. CellSearch. Adapted with permission. Copyright 2014. Janssen Diagnostics LLC. CTC-chip. Adapted with permission from ref (46). Copyright 2007 Nature Publishing Group. High-throughput microsampling unit (HTMSU). Adapted with permission from ref (66). Copyright 2008 American Chemical Society. Herringbone-chip. Nanopillars. Adapted with permission from ref (90). Copyright 2009 John Wiley & Sons, Inc. Carbon nanotubes. Adapted with permission from ref (86). Copyright 2011 John Wiley & Sons, Inc. Nanodots. Adapted with permission from ref (104). Copyright 2011 John Wiley & Sons, Inc. Nanofibers. Adapted with permission from ref (98). Copyright 2012 John Wiley & Sons, Inc. Graphene oxide. Adapted with permission from ref (124). Copyright 2013 Nature Publishing Group.

Figure 3

Size-based filtration techniques. (a) Melanoma cells preincubated with immunobeads captured on a nylon monofilament filter. Adapted with permission from ref (32). Copyright 1997 Elsevier. (b) Culture of captured melanoma cells on nylon monofilament filter. Adapted with permission from ref (32). Copyright 1997 Elsevier. (c) Stained cells as separated using the polycarbonate filter found in ISET (Isolation by Size of Epithelial Tumor Cells) technology [1, spiked tumor cells; 2, membrane pores; 3, leukocytes]. Adapted with permission from ref (33). Copyright 2000 Elsevier. (d) Parylene C microfilter released from silicon mold. Adapted with permission from ref (36). Copyright 2007 Elsevier. (e) Two-layer microfilter to protect cells from damaging forces. Adapted with permission from ref (38). Copyright 2010 Springer Science+Business Media, LLC. (f) CTC clusters isolated by RIA (reversible bead attachment for cell isolation and analysis) and different levels of HER2 expression in CTCs isolated from metastatic breast cancer patients. Adapted with permission from ref (39). Copyright 2007 John Wiley & Sons, Inc.

Figure 4

Silicon-based microfluidic CTC separation technologies. (a and b) Setup of post-based CTC-chip developed by Nagrath et al. Captured NCI-H1650 cell imaged with scanning electron microscopy (color added) with high magnification inset. Adapted with permission from ref (46). Copyright 2007 Nature Publishing Group. (c and d) Computer simulated particle paths for larger cells (blue) compared with smaller cells (yellow) for geometrically enhanced differential immunocapture (GEDI). Prostate cancer tumor cell captured on octagonal post of GEDI microdevice. Adapted with permission from ref (49). Copyright 2010 The Royal Society of Chemistry. (e) Filtration unit with two different filter gaps for the capture of CTCs that have undergone size amplification with magnetic particles. Adapted with permission from ref (52). Copyright 2012 The Royal Society of Chemistry. (f) Hybrid nanoparticles consisting of antibodies, quantum dots, and specific DNA sequences for the labeling, capture, and release of CTCs in a capture and recovery chip. Adapted with permission from ref (53). Copyright 2013 Elsevier.

Figure 5

Glass/PDMS-based microfluidic CTC separation technologies. (a) Multiple flow channels shown with blood flowing through the device. The Herringbone-chip was able to isolate clusters of CTCs in addition to single cells (shown in lower inset). (b) Microfluidic device consisting of over 59 000 micropillars functionalized with DNA aptamers. Adapted with permission from ref (58). Copyright 2012 American Chemical Society. (c) PDMS post structure device fabricated using a 2-step AGEpp-PEI inking process. Adapted with permission from ref (61). Copyright 2011 The Royal Society of Chemistry. (d) Alginate hydrogel containing PEG conjugated to capture antibodies (inset). Adapted with permission from ref (62). Copyright 2011 American Chemical Society. (e) Deterministic lateral displacement is used to sort cells that are ultimately captured on an antibody-functionalized substrate. Adapted with permission from ref (64). Copyright 2013 Elsevier.

Figure 6

Thermopolymer-based microfluidic CTC separation technologies. (a) Surface modification of PMMA by UV exposure. Adapted with permission from ref (65). Copyright 2005 American Chemical Society. (b) HTMSU facilitates capture using serpentine channels and integrates enumeration using a conductivity sensor near the outlet. Adapted with permission from ref (66). Copyright 2008 American Chemical Society. (c) Overall schematic of CTC immunocapture and release for downstream analysis. Adapted with permission from ref (67). Copyright 2011 American Chemical Society. (d) Picture of the assembled microfluidic modules for CTC isolation and analysis: (I) HT-CTC selection module; (II) impedance sensing module; (III) staining and imaging module. Adapted with permission from ref (70). Copyright 2013 American Chemical Society.

Figure 7

Magnetic nanoparticles (MNPs) in CTC capture and analysis. (a) Anti-EpCAM functionalized MNPs bind to CTCs and remove them from solution through the use of block magnets. Adapted with permission from ref (71). Copyright 2011 The Royal Society of Chemistry. (b) Schematic of an antibody modified using a heterofunctional linker and an immunomagnetic nanocarrier. Adapted with permission from ref (73). Copyright 2013 American Chemical Society. (c) DSPE-PEG-NH₂ and C18-PMH-mPEG functionalized graphite-coated magnetic nanocrystals. Adapted with permission from ref (74). Copyright 2013 American Chemical Society. (d) The magnetic moment of a MNP conjugated with a specific antibody is sensed as the MNP-covered cell flows over a series of micro-Hall sensors. Adapted with permission from ref (75). Copyright 2012 American Association for the Advancement of Science.

Figure 8

Vertically aligned carbon nanotubes (VACNTs). (a) Scanning electron microscopy images of VACNTs grown by chemical vapor deposition and enclosed in a PDMS chamber. Adapted with permission from ref (84). Copyright 2010 IEEE. (b) Increased capture via porous structures is a result of streamline manipulation and hydrodynamic resistance reduction, allowing for more collisions occurring at closer distances. Adapted with permission from ref (85). Copyright 2012 The Royal Society of Chemistry. (c) Porous elements of varying diameters improve cell capture and allow more of the post to be active in capture when compared with a comparably size solid control pillar. Adapted with permission from ref (86). Copyright 2011 John Wiley & Sons, Inc.

Figure 9

Nanopillar, nanowire, and nanofiber structures. (a) Chaotic micromixer induces increased contact between flowing cells and anti-EpCAM functionalized silicon nanopillars (SiNPs) substrates. Adapted with permission from ref (91). Copyright 2011 John Wiley & Sons, Inc. (b) T lymphocyte cell capture on DNA-silicon nanowire arrays (SiNWAs) and cell release using exonuclease I to break down aptamers. Adapted with permission from ref (93). Copyright 2011 John Wiley & Sons, Inc. (c) Aptamer-coated NanoVelcro Chip for capturing and releasing NSCLC CTCs Adapted with permission from ref (94). Copyright 2013 John Wiley & Sons, Inc. (d) CD4+ T lymphocytes (shown by SEM images) may be selectively captured by quartz nanowires (QNWs) functionalized with anti-EpCAM.^, Adapted with permission from refs (95) and (96). Copyright 2010, 2012, American Chemical Society. (e) Titanium nanofibers are fabricated through electrospinning and calcination prior to functionalization for ultimate use in cell capture. Adapted with permission from ref (98). Copyright 2012 John Wiley & Sons, Inc.

Figure 10

Nanotextured surfaces. (a) Nanostructured microelectrodes with two different redox-active probes, Ru(NH₃)₆³⁺, which accumulates on the sensor based on the amount of target mRNA, and Fe(CN)₆^3-, which can regenerate the Ru(II) species. Adapted with permission from ref (101). Copyright 2012 American Chemical Society. (b) PDMS surfaces characterized by increased roughness as shown by atomic force microscopy show increased cell spreading and attachment (inset: scanning electron microscope images). Adapted with permission from ref (102). Copyright 2011 American Cancer Society. (c) Enhanced cell capture using gold nanoparticles with multiple aptamers for multivalent interaction. Adapted with permission from ref (103). Copyright 2013 American Chemical Society. (d) Poly(3, 4-ethylenedioxy)thiophene (PEDOT) dots functionalized with anti-EpCAM antibodies can capture EpCAM-positive cells. Adapted with permission from ref (104). Copyright 2011 John Wiley & Sons, Inc. (e) Cancer cells preferentially adhere to reactive ion etched glass surfaces, shown as a schematic and with scanning electron microscopy. Adapted with permission from ref (100). Copyright 2012 American Chemical Society.

Figure 11

Graphene oxide. (a) Functionalized reduced graphene oxide nanoparticles for use in HER2 detection. Adapted with permission from ref (121). Copyright 2011 John Wiley & Sons, Inc. (b) Ultrasensitive graphene-enhanced fluorescent and electrochemical CTC detection procedures. Adapted with permission from ref (122). Copyright 2012 American Chemical Society. (c) Schematic of the micropillar device with modified GO-Fe₃O₄ MNPs (GO-F) and images of a single nickel micropillar capturing a cancer cell. Adapted with permission from ref (123). Copyright 2011 John Wiley & Sons, Inc. (d) Functionalization chemistry stemming from the self-assembly of PEG-functionalized graphene oxide dispersed with tetrabutylammonium, resulting in anti-EpCAM end groups that capture target cells; SEM images and fluorescence microscope image of captured and cultured MCF-7 cells. Adapted with permission from ref (124). Copyright 2013 Nature Publishing Group.