Microfluidics and Circulating Tumor Cells
2012; Elsevier BV; Volume: 15; Issue: 2 Linguagem: Inglês
10.1016/j.jmoldx.2012.09.004
ISSN1943-7811
AutoresYi Dong, Alison M. Skelley, Keith D. Merdek, Kam Sprott, Chunsheng Jiang, William E. Pierceall, Jessie Lin, Michael Stocum, Walter P. Carney, Denis A. Smirnov,
Tópico(s)Microfluidic and Capillary Electrophoresis Applications
ResumoCirculating tumor cells (CTCs) are shed from cancerous tumors, enter the circulatory system, and migrate to distant organs to form metastases that ultimately lead to the death of most patients with cancer. Identification and characterization of CTCs provides a means to study, monitor, and potentially interfere with the metastatic process. Isolation of CTCs from blood is challenging because CTCs are rare and possess characteristics that reflect the heterogeneity of cancers. Various methods have been developed to enrich CTCs from many millions of normal blood cells. Microfluidics offers an opportunity to create a next generation of superior CTC enrichment devices. This review focuses on various microfluidic approaches that have been applied to date to capture CTCs from the blood of patients with cancer. Circulating tumor cells (CTCs) are shed from cancerous tumors, enter the circulatory system, and migrate to distant organs to form metastases that ultimately lead to the death of most patients with cancer. Identification and characterization of CTCs provides a means to study, monitor, and potentially interfere with the metastatic process. Isolation of CTCs from blood is challenging because CTCs are rare and possess characteristics that reflect the heterogeneity of cancers. Various methods have been developed to enrich CTCs from many millions of normal blood cells. Microfluidics offers an opportunity to create a next generation of superior CTC enrichment devices. This review focuses on various microfluidic approaches that have been applied to date to capture CTCs from the blood of patients with cancer. Circulating tumor cells (CTCs) are defined as tumor cells in the circulation. The dissemination of cancer cells from the primary tumor is a first step of the metastatic process in distant organs and is the leading cause of death in patients with cancer. Approximately 90% of patients with epithelial cancers, accounting for approximately 80% of all cancers throughout the world, die of metastatic disease rather than of the direct effects of the primary tumor. An average tumor may release an estimated million cells per day into the bloodstream1Butler J.E. Ni L. Nessler R. Joshi K.S. Suter M. Rosenberg B. Chang J. Brown W.R. Cantarero L.A. The physical and functional behavior of capture antibodies adsorbed on polystyrene.J Immunol Methods. 1992; 150: 77-90Crossref PubMed Scopus (272) Google Scholar; however, most dispersed cancer cells do not survive. Nevertheless, the cells that do survive clearly pose an existential threat to the host organism. From the moment that CTCs were first identified in 1869, an underlying hypothesis has emerged that these cells originate from primary tumors and perhaps from metastatic sites. For solid tumors, the presence of these cells is evident late in disease and is most apparent when metastatic disease sites are already established. Identification and characterization of CTCs offers an opportunity to study, monitor, and, ultimately, alter the metastatic process. CTCs are exceedingly rare cells. Ordinary blood cell types are present in tremendous numbers by comparison, and patients with cancer may have altered levels of other blood cell types to consider. These other cell types may comprise leukocytes (approximately 7 million/mL of blood) and red blood cells (approximately 5 billion/mL of blood). Assuming that all CTCs shed from the primary tumor remain in the circulatory system, an estimated yield of <200 CTCs/mL of blood will be derived from an average male patient (5 L of total blood volume). Therefore, CTCs should, theoretically, be present at 4 to <30 μm, even in cells from a single patient.10Allard W.J. Matera J. Miller M.C. Repollet M. Connelly M.C. Rao C. Tibbe A.G.J. Uhr J.W. Terstappen L.W.M.M. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.Clin Cancer Res. 2004; 10: 6897-6904Crossref PubMed Scopus (2000) Google Scholar Therefore, finely tuned filters can be manufactured that retain only tumor cells and let most of the blood cells go through. Although filter-based approaches are not limited by specific biological properties of cells, they are less likely to capture cells similar in size to blood cells. Filter-based approaches will also be unable to capture clinically informative tumor cell fragments. After capture, identification of tumor cells is mostly accomplished by staining cancer cells with specific markers that distinguish these cells from blood cells that are falsely co-captured along with CTCs, a challenge encountered by all existing CTC-capturing technologies. Microfluidic methods are an effective means to interrogate the constituents of biological fluids for diagnostic purposes, just as they are useful for precise measurements and assays for other analytical processes, such as drug screening, nucleic acid amplification, and enzymatic reactions. Microfluidic methods naturally lend themselves to solving the problem of capturing rare cells because device performance can be tailored to exploit physical and/or biological differences between CTCs and the background cells, enabling isolation. In addition, microfluidic approaches allow for gentle capturing of live rare cells so that further analysis can be performed using cellular, microscopic, or molecular techniques. Furthermore, microfluidics presents an opportunity to combine isolation and detection methods in a single device, opening the door for the development of true point-of-care diagnostic CTC devices. A tradeoff of microfluidics is the challenge of analyzing large sample volumes to access key information about rare cells in the circulation. The small dimensional features of chip design and complex fluid dynamics can interfere with efficient, large-scale capture of specific, rare cells unless its format and microfluidics are styled to meet those specific requirements. In the following section, we review the microfluidic approaches to CTC isolation that have been described to date (summarized in Table 1).Table 1Summary of Microfluidics-Based Approaches for the Capture of CTCsCTC properties usedStrategyCommercializationSize filtrationMicromachined device of four successively narrower channels11Mohamed H. Murray M. Turner J.N. Caggana M. Isolation of tumor cells using size and deformation.J Chromatogr A. 2009; 1216: 8289-8295Crossref PubMed Scopus (180) Google ScholarClearbridge Biomedics (Singapore)Arrays of pillars forming crescent-shaped isolation wells12Tan S. Yobas L. Lee G. Ong C. Lim C. Microdevice for the isolation and enumeration of cancer cells from blood.Biomed Microdevices. 2009; 11: 883-892Crossref PubMed Scopus (320) Google ScholarMicrofilter with embedded microelectrodes, capable of on-membrane electrolysis13Zheng S. Lin H. Liu J.-Q. Balic M. Datar R. Cote R.J. Tai Y.-C. Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells.J Chromatogr A. 2007; 1162: 154-161Crossref PubMed Scopus (514) Google ScholarSize streamline sortMicrodevice with multiple asymmetrical channels for pinch flow fractionation and hydrodynamic filtration of cells14Yamada M. Nakashima M. Seki M. Pinched flow fractionation: continuous size separation of particles utilizing a laminar flow profile in a pinched microchannel.Ann Chem. 2004; 76: 5465-5471Crossref PubMed Scopus (608) Google ScholarMicrochannels using changing channel dimension to sort cells into individual laminar flow streamlines15Carlo D.D. Inertial microfluidics.Lab Chip. 2009; 9: 3038-3046Crossref PubMed Scopus (1188) Google ScholarMicrodevice with three sequential regions to focus, pinch, and collect cells by contraction and expansion of channel dimensions16Bhagat A.A.S. Hou H.W. Li L.D. Lim C.T. Han J. Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation.Lab Chip. 2011; 11: 1870-1878Crossref PubMed Scopus (289) Google ScholarSize and particle polarizabilityDEP for retention of cells through differential flow17Becker F.F. Wang X.B. Huang Y. Pethig R. Vykoukal J. Gascoyne P.R. Separation of human breast cancer cells from blood by differential dielectric affinity.Proc Natl Acad Sci U S A. 1995; 92: 860-864Crossref PubMed Scopus (615) Google ScholarApoCell (Houston, TX)Thin DEP microchamber for retention of cells through differential flow18Gascoyne P.R.C. Noshari J. Anderson T.J. Becker F.F. Isolation of rare cells from cell mixtures by dielectrophoresis.Electrophoresis. 2009; 30: 1388-1398Crossref PubMed Scopus (359) Google ScholarBinding cell surface markersMicrochip with staggered microposts coated with anti-EpCAM19Davies J. Dawkes A.C. Haymes A.G. Roberts C.J. Sunderland R.F. Wilkins M.J. Davies M.C. Tendler S.J.B. Jackson D.E. Edwards J.C. A scanning tunneling microscopy comparison of passive antibody adsorption and biotinylated antibody linkage to streptavidin on microtiter wells.J Immunolog Meth. 1994; 167: 263-269Crossref PubMed Scopus (47) Google ScholarCytoScale Diagnostics (Los Angeles, CA)Microtube coated internally with halloysite nanotubes presenting selectin20Spitznagel T.M. Clark D.S. Surface-density and orientation effects on immobilized antibodies and antibody fragments.Biotechnology (NY). 1993; 11: 825-829Crossref PubMed Scopus (69) Google ScholarSilicon nanopillar arrays coated with anti-EpCAM21Hughes A.D. King M.R. Use of naturally occurring halloysite nanotubes for enhanced capture of flowing cells.Langmuir. 2010; 26: 12155-12164Crossref PubMed Scopus (122) Google ScholarMicrochip with channels in a herringbone design generating microvortexes to enhance contact with anti-EpCAM–coated walls22Wang S. Wang H. Jiao J. Chen K. Owens G.E. Kamei K. Sun J. Sherman D.J. Behrenbruch C.P. Wu H. Tseng H. Three-dimensional nanostructured substrates toward efficient capture of circulating tumor cells.Angew Chem Int Ed Engl. 2009; 48: 8970-8973Crossref PubMed Scopus (419) Google ScholarAnti-EpCAM–coated fluid-permeable membrane sandwiched between two polydimethylsiloxane flow chambers to promote cell rolling and adhesion23Wang S. Liu K. Liu J. Yu Z.T.-F. Xu X. Zhao L. Lee T. Lee E.K. Reiss J. Lee Y. Chung L.W.K. Huang J. Rettig M. Seligson D. Duraiswamy K.N. Shen C.K. Tseung H.R. Highly efficient capture of circulating tumor cells by using nanostructured silicon substrates with integrated chaotic micromixers.Angew Chem Int Ed Engl. 2011; 50: 3084-3088Crossref PubMed Scopus (542) Google ScholarMicrochip with a combination of solid- and porous-coated micropost arrays to enhance antibody capture at material boundaries24Mittal S. Wong I.Y. Deen W.M. Toner M. Antibody-functionalized fluid-permeable surfaces for rolling cell capture at high flow rates.Biophys J. 2012; 102: 721-730Abstract Full Text Full Text PDF PubMed Scopus (30) Google ScholarMicrochip with magnetic beads coated with anti-EpCAM that form self assembling pillars (Ephesia)25Chen G.D. Fachin F. Fernandez-Suarez M. Wardle B.L. Toner M. Nanoporous elements in microfluidics for multiscale manipulation of bioparticles.Small. 2011; 7: 1061-1067Crossref PubMed Scopus (59) Google ScholarBinding cell surface markers and electrokinetic manipulationDEP for migration of cells bound to antibody-coated magnetic nanoparticles (MIRACLE)26Liu C. Stakenborg T. Henry O. O'Sullivan C. Borgen E. Schirmer C. Laddach N. Roeser T. Latta D. Ritzi-Lehnert M. Fermer C. van de Flierdt J. Hauch S. Lagae L. Lab-on-a-Chip for the magnetic isolation and analysis of circulating tumor cells. 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS 2011). 2011 October 2-6, Seattle.WA. Chemical and Biological Microsystems Society. 2011; 1: 344-346Google ScholarBioFluidica (Baton Rouge, LA)High-throughput microsampling unit combining sinusoidal channels coated with anti-EpCAM for capture and electromigration for concentration27Saliba A.-E. Saias L. Psychari E. Minc N. Simon D. Bidard F.-C. Mathiot C. Pierga J.-Y. Fraisier V. Salamero J. Saada V. Farace F. Vielh P. Malaquin L. Viovy J.-L. Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.Proc Natl Acad Sci U S A. 2010; 107: 14524-14529Crossref PubMed Scopus (274) Google ScholarSize and binding cell surface markersMicrochip with microposts coated with anti-EpCAM arranged in a gradient design for affinity capture and size filtration28Dharmasiri U. Njoroge S.K. Witek M.A. Adebiyi M.G. Kamande J.W. Hupert M.L. Barany F. Soper S.A. High-throughput selection, enumeration, electrokinetic manipulation, and molecular profiling of low-abundance circulating tumor cells using a microfluidic system.Anal Chem. 2011; 83: 2301-2309Crossref PubMed Scopus (154) Google ScholarOn-Q-ity Inc. Open table in a new tab Because tumor cell diameter tends to be larger than that of normal blood cells,29Seal S.H. A sieve for the isolation of cancer cells and other large cells from the blood.Cancer. 1964; 17: 637-642Crossref PubMed Scopus (81) Google Scholar several microfluidic systems have been developed to isolate CTCs by virtue of their increased relative size. The fabrication techniques used to design and construct microfluidic devices allow for control of device properties at the micrometer scale, so these devices naturally lend themselves to assays in which size-based filtration is used to separate cells. Microfluidic size-based CTC capture systems aim to achieve superior results compared with macrosize-based approaches, such as filter membranes. Mohamed et al11Mohamed H. Murray M. Turner J.N. Caggana M. Isolation of tumor cells using size and deformation.J Chromatogr A. 2009; 1216: 8289-8295Crossref PubMed Scopus (180) Google Scholar designed a micromachined device of arrays of four successively narrower channels to isolate cells from eight tumor cell lines. Their devices have channels ranging from 20 to 5 μm in width and depth. The 20-μm segment was used to disperse samples evenly across the chip. The subsequent segments were used to trap increasingly smaller cells. Spacing between microchannels was carefully designed to prevent clogging of the chip. Often, microfluidics is used to complement traditional size-based CTC selection methods. Tan et al12Tan S. Yobas L. Lee G. Ong C. Lim C. Microdevice for the isolation and enumeration of cancer cells from blood.Biomed Microdevices. 2009; 11: 883-892Crossref PubMed Scopus (320) Google Scholar, 30Tan S.J. Lakshmi R.L. Chen P. Lim W.-T. Yobas L. Lim C.T. Versatile label free biochip for the detection of circulating tumor cells from peripheral blood in cancer patients.Biosens Bioelectron. 2010; 26: 1701-1705Crossref PubMed Scopus (171) Google Scholar designed a device with multiple arrays of crescent-shaped isolation wells that achieved CTC isolation by size and deformability differences between CTCs and white blood cells. Each well consists of three pillars with 5-μm gaps in between that ensure efficient CTC trapping while allowing more deformable white blood cells to be removed. Using microelectromechanical system–based technology and a membrane filter, Zheng et al13Zheng S. Lin H. Liu J.-Q. Balic M. Datar R. Cote R.J. Tai Y.-C. Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells.J Chromatogr A. 2007; 1162: 154-161Crossref PubMed Scopus (514) Google Scholar integrated CTC isolation and on-chip cell lysis for PCR-based genomic analysis. A parylene membrane microfilter was used in this case for cell enrichment. An array of microelectrodes was built with microfabrication processes to address each individual pore. Once CTCs were captured, on-membrane electrolysis was performed to lyse cells and allow for subsequent PCR analysis on the CTCs. One concern for size-based capture is that its efficiency may be undermined by heterogeneous CTC size in patient blood. Selection of a size cutoff may yield reliable CTC isolation in most of the patient samples but inadequate performance across all patients. In addition, cells are deformable, and if the operating conditions are not carefully controlled, passage through small openings may cause shear stress and potential damage to or loss of the CTCs. Determining the optimum cutoff size involves considerations such as the range in viscosities of patient samples and the desired throughput. The clinical usefulness of these approaches remains to be validated. Streamline sorting is an alternative means of size-based filtration in which cells of different sizes migrate into unique streamlines based on fluid forces. After separation into streamlines has taken place, the bulk flow is segmented to collect these streamlines at different outputs. The advantage is that the cells do not pass through any physical constrictions, so shear forces are reduced. In addition, these devices typically operate at relatively high flow rates, resulting in high throughput. However, to enable sorting, the fluid forces in the device have to be precisely controlled, often requiring that the patient sample be diluted with a carrier liquid of known properties. This enables the device to function equally, regardless of the properties of the patient sample; however, only a relatively small sample volume can be handled at a time, and enrichment efficiency is limited.15Carlo D.D. Inertial microfluidics.Lab Chip. 2009; 9: 3038-3046Crossref PubMed Scopus (1188) Google Scholar Yamada and colleagues14Yamada M. Nakashima M. Seki M. Pinched flow fractionation: continuous size separation of particles utilizing a laminar flow profile in a pinched microchannel.Ann Chem. 2004; 76: 5465-5471Crossref PubMed Scopus (608) Google Scholar, 31Takagi J. Yamada M. Yasuda M. Seki M. Continuous particle separation in a microchannel having asymmetrically arranged multiple branches.Lab Chip. 2005; 5: 778-784Crossref PubMed Scopus (283) Google Scholar, 32Yamada M. Seki M. Hydrodynamic filtration for on-chip particle concentration and classification utilizing microfluidics.Lab Chip. 2005; 5: 1233-1239Crossref PubMed Scopus (409) Google Scholar proposed a series of size- and shape-based cell-sorting microdevices, including pinch flow fractionation and hydrodynamic filtration. The central idea of these technologies is based on the fact that at a low Reynolds number (a measure of the ratio of inertial forces to viscous forces), the center of a particle will follow streamlines. By controlling flow rates through one or more inlets and optimizing asymmetrical channel geometry, cells of different sizes and shapes will move along different streamlines in the flow. With carefully positioned or configured outlets, cells of different sizes and shapes move into corresponding outlets and are separated. Di Carlo and coworkers33Mach A.J. Kim J.H. Arshi A. Hur S.C. Di Carlo D. Automated cellular sample preparation using a Centrifuge-on-a-Chip.Lab Chip. 2011; 11: 2827-2834Crossref PubMed Scopus (234) Google Scholar, 34Hur S.C. Mach A.J. Di Carlo D. High-throughput size-based rare cell enrichment using microscale vortices.Biomicrofluidics. 2011; 5: 22206Crossref PubMed Scopus (248) Google Scholar demonstrated that flowing particles also migrate across streamlines in microchannels experiencing laminar flow, consistent with observations made some 100 years ago by Hele-Shaw. The shear gradient lift force tends to push particles toward the wall, and the wall effect lift force pushes particles toward the center of the channel. When the particle size is comparable with the channel dimension, these lift forces are significant and lead to lateral migration across flow streamlines. By biasing the balance of the two lift forces, cells migrate to distinct vertical and lateral equilibrium positions in the channel. Thus, different cells are focused at different locations. In addition, expanding the microchannel quickly in width can generate microvortices. In the expansion region, the wall effect lift is not significant as the neighboring channel wall is no longer within its vicinity. The shear gradient lift force dominates and drives cells across streamlines toward the center of the vortices. Cells above a critical size enter the vortex and maintain a stable position in the vortex. With this system, they were able to concentrate 10 mL of blood with 500 spiked cancer cells to 200 μL within 3 minutes. They recovered a mean ± SD of 102 ± 21 cancer cells with 221 ± 155 blood cells. Although the ∼20% recovery rate is low, the 40% purity achieved is higher than that in most reported processes. Bhagat et al16Bhagat A.A.S. Hou H.W. Li L.D. Lim C.T. Han J. Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation.Lab Chip. 2011; 11: 1870-1878Crossref PubMed Scopus (289) Google Scholar used lift force–induced cell separation combined with size constriction to isolate spiked cancer cells in blood. The microchannel design of this device consists of three regions in sequential order: focused volumetric flow, size-based constriction, and cell-isolating regions. The focusing and constriction regions are high aspect ratio rectangular channels patterned with contraction-expansion arrays. The focusing region consists of 70 contraction-expansion subunits and has a 20-μm width at the contraction parts of the channel. All cells migrating through this region equilibrate efficiently along the channel sidewalls under the influence of shear-modulated inertial lift force. The constriction region consists of five contraction-expansio
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