Microfluidics and cancer: are we there yet?

Zhuo Zhang & Sunitha Nagrath

# Springer Science+Business Media New York 2013

Abstract More than two decades ago, microfluidics beganto show its impact in biological research. Since then, thefield of microfluidics has evolving rapidly. Cancer is one ofthe leading causes of death worldwide. Microfluidics holdsgreat promise in cancer diagnosis and also serves as anemerging tool for understanding cancer biology.Microfluidics can be valuable for cancer investigation dueto its high sensitivity, high throughput, less material-consumption, low cost, and enhanced spatio-temporal con-trol. The physical laws on microscale offer an advantageenabling the control of physics, biology, chemistry andphysiology at cellular level. Furthermore, microfluidicbased platforms are portable and can be easily designedfor point-of-care diagnostics. Developing and applying thestate of the art microfluidic technologies to address theunmet challenges in cancer can expand the horizons of notonly fundamental biology but also the management of dis-ease and patient care. Despite the various microfluidic tech-nologies available in the field, few have been testedclinically, which can be attributed to the various challengesexisting in bridging the gap between the emerging technol-ogy and real world applications. We present a review of roleof microlfuidcs in cancer research, including the history,recent advances and future directions to explore where thefield stand currently in addressing complex clinical chal-lenges and future of it. This review identifies four criticalareas in cancer research, in which microfluidics can changethe current paradigm. These include cancer cell isolation,molecular diagnostics, tumor biology and high-throughputscreening for therapeutics. In addition, some of our lab’scurrent research is presented in the corresponding sections.

Keywords Microfluidics . Cancer . Cancer diagnostics .

Circulating tumor cells . Tumor biology . BioMEMS .

Cancer therapeutics

1 Background

Microfluidics handles microliter volumes in microchannelsof 1 μm to 1000 μm size. In such regime, fluid flow isstrictly laminar, hence concentrations of molecules can bewell controlled (Whitesides 2006). Microfluidic technologywas introduced as a biological tool in the early 1990s (Hongand Quake 2003). Since then, this interdisciplinary technol-ogy, which is well known for manipulating reagents withinminiaturized platforms, has been developing rapidly (Reyeset al. 2002; Auroux et al. 2002). The material used forpreparing microfluidic devices has evolved from traditionalsilicon and glass, to elastomers rendering the device morebiocompatible and lower cost (Whitesides 2006). There areseveral inherent advantages of microfluidics, including re-duced sample size and reagent consumption, short process-ing times, enhanced sensitivity, real-time analysis andautomation (Manz et al. 1992). One of the motivations forapplying microfluidic techniques in life science is to auto-mate the labor-intensive experimental processes similar tothat accomplished in electronic circuits (Hong and Quake2003). Polymerase chain reaction, electrophoresis on chipand DNA microarrays are among the earliest (Hong andQuake 2003) microfluidic ventures. With a decade of devel-opment, microfluidic integrated systems were extended tomanipulating RNA, proteins and mammalian cells usingbiosensors, single cell assays for disease diagnosis andprognosis, among several other applications. Biologicmicrofluidic devices can now be used to explore and re-search cancer in new and unconventional ways (Tables 1, 2,3 and 4).

Z. Zhang : S. Nagrath (*)Department of Chemical Engineering, University of Michigan,2300 Hayward Street,Ann Arbor, MI 48109, USAe-mail: snagrath@umich.edu

Biomed MicrodevicesDOI 10.1007/s10544-012-9734-8

Cancer research has long been at the forefront of medicaland scientific research. Its seemingly incurable nature andlarge prevalence in society have made cancer a popular andwell-funded area of research for decades. Cancer is a

chronic disease involving changes or mutations in multiplegenes. It was estimated that in 2008, 12.7 million cancercases and 7.6 million cancer-related deaths occurred global-ly (Jemal et al. 2011). In 2011 in the United States alone, 1.6

Table 1 Microfluidic technologies for isolation of CTCs

List of technology Principle Application Clinical study

CTC-chip (Nagrath et al. 2007) Immunoaffinity Isolation of CTCs (1 ml/h flowrate, 60–65 % efficiency,50 %purity from patient samples)

68 patients with metastaticlung, prostate, pancreatic,breast and colon cancer

CTC-chip (Maheswaran et al.2008)

Immunoaffinity Identify EGFR mutations 27 patients with metastaticnon-small-cell lung cancer

CTC-chip (Stott et al. 2010a) Immunoaffinity Automated imaging of captured CTCs 62 patients with prostate cancer

CEE microchannel(Nora Dickson et al. 2011)

Immunoaffinity Isolate of cancer cells from blood cells N/A

Herringbone-chip (Stott et al.2010b)

Immunoaffinity High-throughput mixing and isolationof CTCs (1.5–2.5 ml/h flow rate,90 % efficiency, 14 % purity fromspiking cells in blood)

15 patients with metastaticprostate cancer

Self-assembled magneticarrays (Saliba et al. 2010)

Immunomagnetic Isolation of B-lymphocytes(9 μl/h flow rate, 94 % yield)

7 patients with B-cellhematological malignanttumors (leukemia andlymphoma)

Aptamer selection chip(Dharmasiri et al. 2009)

Immunoaffinity throughaptamers

Isolation of prostate cancer cells fromblood (2 ml/h flow rate, 90 % recovery,100 % purity—cell line test)

N/A

Geometrically enhanceddifferential immunocapture(GEDI) chip (Gleghorn et al.2010)

Immunoaffinity Isolation of prostate cancer circulatingtumor cells (1 ml/h flow rate, 85 %efficiency, 68 % purity from spikingcells in blood)

Blood samples of castrate-resistantprostate cancer patients

E-selectin biomimetic chip(Myung et al. 2010)

Immunoaffinity & Biomimic Isolation of cancer cells from mixtureof leukocytes (1.2 ml/h flow rate,35 % efficiency)

N/A

Integrated CTC selectionchip (Dharmasiri et al. 2011)

Immunoaffinity &electrokinetics

Isolation, enumeration, enrichment ofCTCs (1.5 ml/h flow rate, 96 % efficiency)PCR/LDR detection of KRAS colorectalcancer cell mutations

N/A

Immunomagnetic chip(Hoshino et al. 2011)

Immunomagneticnanoparticles

Capture cancer cells spiked in blood N/A

Micromagnetic chip(Kang et al. 2012b)

Immunomagnetic Isolation CTCs and release for culturing(90 % efficiency)

N/A

Membrane microfilter(Zheng et al. 2007, 2011)

Size Separation of cancer cells from blood(89 % recovery)

N/A

Filtration chip (Kuo et al. 2010) Size & Deformability Separation cancer cells from blood cells(0.72–0.96 ml/h flow rate 50–90 %recovery)

N/A

Deformability-based chip(Hur et al. 2011)

Size & Deformability High-throughput separation and enrichmentof CTCs from diluted blood (1.5–27 ml/hflow rate, 96 % yield, 3.2–5.4 foldenrichment)

N/A

Particle focusing chip(Lim et al. 2012)

Size Use particle trajectory analysis to studycancer cell focusing in whole blood

N/A

MagSweeper(Talasaz et al. 2009)

Immunomagnetic Isolation and enrichment of breast cancercells from whole blood (process 9 mlbloodper hour, 62 % efficiency, 51 % purity)

Blood samples from 17 femalepatients with metastaticbreast cancers

MOFF and DEP chip(Moon et al. 2011)

Size and Dielecrophreticproperties

Isolation of breast cancer cells from blood(126 μl/min flow rate, 99 % efficiency)

N/A

Negative selection disk(Chen et al. 2011)

Immunomagnetic Isolation of breast cancer cells frommononuclear cells mixture (60 % yield)

N/A

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million people were newly diagnosed with cancers and571,950 cancer-related deaths were projected. Prostate,breast, lung and colorectal cancers are the leading cause ofcancer deaths in the US (Siegel et al. 2011). Since 2004,considerable funding has been allocated for technologyadvancement in search of more effective anti-cancer strate-gies (Alexis et al. 2008). Cancer prevention strategies, earlycancer diagnosis and effective drug treatment need to be

more affordable and easily accessible to improve overallsurvival.

Traditionally, cancer diagnosis is highly dependent uponsampling of tumor tissues or indirect quantification of pro-teins (Seigneuric et al. 2010). Often, these conventional sam-pling approaches are invasive which lead to tissue damage,limited access and ability to get reliable samples and causehigh levels of patient discomfort. Although, proteomic and

Table 2 Emerging microfluidics based approaches for molecular diagnosis of cancers

List of technology Principle Application Clinical study

Microfluidic digital PCR(Yung et al. 2009)

BioMark System (Fluidigm) Detect EGFR mutations Plasma and tissue samplesfrom patients with non-smallcell lung cancer

Microfluidic single-cellRT-PCR (White et al. 2011)

300 parallel chambers Single nucleotide variant inmetastatic breast cancer cells

N/A

Droplet-based quantitativePCR microfluidics(Pekin et al. 2011)

Compartmentalization ofDNA in droplets

KRAS mutations in sixcancer cell lines

N/A

Microarray-based miRNAprofiling (Hoheiselet al. 2012)

Geniom Biochip Identify miRNA in whole blood 48 early stage breast cancerpatients

Microfluidic screening ofmiRNAs (Mitchellet al. 2008)

Microfluidic TaqMan miRNAqRT-PCR array

Detect tumor-derived miRNAsfrom plasma and serum

25 patients with metastaticprostate cancer

Digital microfluidic devicefor estrogen detection(Mousa et al. 2009)

Droplet-based electrokinetic assay Detect estrogen level in tissuesamples, blood, and serum

Breast tissue samples from2 breast cancer patients

Microvesicle-isolation chip(Chen et al. 2010)

Immunoaffinity Isolation of serum microvesiclesand RT-PCR analysis ofpoint mutations

Brain tumor specimens frompatients with glioblastomamultiforme

Table 3 Microfluidic platforms to explore the fundamental biology of cancer

List of technology Application Clinical significance

Microfluidics for tumor cellmigration (Chaw et al.2006, 2007)

Studying of tumor cell migration anddeformation through microgaps

Cell membrane as a novel drug targeting

Cancer cell migration assay(Irimia and Toner 2009)

Cancer cell migration in one directionwithin confined microchannels, quantifythe cancer cell motility

Drug targets for cancer invasion, motility screening

Compartmentalizing cellmigration microfluidics(Huang et al. 2011)

Brain cancer stem cell migration andmorphology characterization

Study brain cancer stem cell infiltration ofbrain parenchyma

Microfluidic vasculature system(Song et al. 2009)

Mimicking circulating cancer cell targetingon endothelium

Drug targeting, organ specific targeting

Microfluidic co-culture(Hsiao et al. 2009)

Formation of 3D spheroids of prostate cancercells, osteoblasts and endothelial cells

Drug testing platform

Microfluidic co-culture(Huang et al. 2009)

3D culture of metastatic breast cancer cells withmacrophages in patterned hydrogels

Drug testing and targeting

Microfluidic co-culture andangiogenesis(Chung et al. 2009)

Study endothelial cell migration with cancercell co-culture

Understand capillary morphogenesis

Microfluidic co-culture(Sung et al. 2011)

Understand fibroblasts associated cancercell progression

Test for cancer progression inhibitors

Microfluidic co-culture(Domenech et al. 2009, 2012)

No-flow microfluidic cell culture systemenabling hedgehog signaling between prostatecancer cells and fibroblasts

Study the paracrine signaling pathways betweentumor and stromal cells

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genomic research has identified a list of candidate cancerbiomarkers in body fluids such as blood and saliva (Fabianet al. 2008), still there is a lack of point of care devices forthese assays for rapid, non-invasive diagnosis. Inherently,microfluidics is suitable for analyzing complex fluids in vitroand thus offers a non-invasive alternative for cancer diagnosisand disease management. Possible biomarkers available in theblood include DNA, miRNA, proteins and circulating tumorcells (CTCs). However, these specific cancer biomarkers areoften present at low levels against massive background sig-nals. For example, there are only 1–10 CTCs in 1 ml of wholeblood containing 106–107 blood cells (den Toonder 2011;Krivacic et al. 2004); the ratio between targeted proteinsversus the background is approximately 1:105 (Heath andDavis 2008). Given this challenge of detecting the actualsignal from vast noise, researchers turned to MEMS(Microelectromechanical systems) based approaches, asmicrofluidics is capable of performing separations with highsensitivity thus becoming a highly useful tool for this appli-cation (Whitesides 2006). Also because of the ease of manu-facturing and low cost of microfluidic devices, biomarkerscan be assessed fairly routinely with the necessary sensitivityand is evolving as one of the promising avenues to developpersonalized medicine (Sorger 2008).

During the past decade, significant progress has beenmade in gaining fundamental understanding of cancer biol-ogy through advances in gene profiling (Ferrari 2005). Toeffectively target cancer and examine therapeutic response,it is vital to understand the aberrant expression profilesrelated to mutated genes (Weinberg 2006). In addition todirect DNA sequencing, mRNAs and proteins associatedwith specific pathways are often used to examine therapeu-tic response. Microfluidics offers efficient and sensitivetools to perform PCR, electrophoresis and hybridizationarrays on chip to make multiplexed analyses possible (Palet al. 2005). In addition, Lab-on-a-Chip(LOC) technologiescan enable high-throughput drug screening by spatio-

temporal delivery of drugs or parallel drug stimulation withminimal cross-contamination because diffusion dominatesthe local solute transport (Takayama et al. 2001, 2003;Wlodkowic et al. 2009a; Sia and Whitesides 2003).Furthermore, LOC systems provide a powerful alternativenot only to traditional cell culture, but also cell sorting andlive cell arrays (Chan et al. 2003; Huh et al. 2005;Wlodkowic et al. 2010). Additionally, single-cell analysison chip can reveal cell-to-cell variability in terms of phar-macokinetic response toward different stimuli (Wlodkowicand Darzynkiewicz 2010). Compared to conventional cellculture techniques, microfluidics presents a better approxi-mation to cellular environment by precisely controlling con-centration gradients, extracellular matrix components andcell-cell interactions (El-Ali et al. 2006).

This review will outline the latest advances in microflui-dics technology that have impacted cancer research and havechanged the current paradigm of strategies for cancer diag-nosis, monitoring and therapeutics. Particularly, approachesfor isolating circulating tumor cells (CTCs), molecular diag-nosis, understanding tumor biology and high-throughputmultiplex screening systems will be described (Fig. 1).

Four distinct areas have been defined to show the influ-ence of microfluidics technology on cancer research. Figure 1summarizes the four areas: one, isolation of CTCs has beenapproached by immunoaffinity-based, size-based andmagnetic-based separation methods (Nagrath et al. 2007;Kang et al. 2012a; Hur et al. 2011); two, detection or char-acterization of tumor cells through molecular diagnostics canbe expanded from single-cell RT-qPCR, droplet-based DNAmutation arrays to protein detection assays (White et al. 2011;Pekin et al. 2011; Mousa et al. 2009); three, tumor biologycan be focused on understanding tumor cell migration andcell culturing in microchannels such as multi-cellular spher-oid formation (Hsiao et al. 2009; Chung et al. 2009; Irimiaand Toner 2009); four, high-throughput screening can beachieved including blood protein measurement, single-cell

Table 4 High throughput MEMS based strategies for biomarker discovery and drug screening

List of technology Principle Application Clinical study

Integrated barcode chip(Fan et al. 2008)

DNA-encoded antibodylibrary technique

Detect multiplex tumor-associated proteinsfrom blood serum in short time

22 patients with breast andprostate cancer

Nano-Bio-Chip (Jokerstet al. 2009)

Quantum dot immunoassay Detect colon, breast and ovariancancer biomarkers

N/A

Label-free detection chip(Stern et al. 2010)

Immunoassay Detect PSA and CA15.3 markers forprostate and breast cancer

N/A

Single-cell microassay(Wlodkowic et al. 2009b)

Mechanically trapping Real-time monitoring apoptosis of cancer cells N/A

Cell culture microarray(Kim et al. 2012)

Concentration gradients Drug testing N/A

Droplet-based drug screeningmicrofluidics (Milleret al. 2012)

Taylor-Aris dispersion,droplet generation

Screen a library of potential inhibitortowards protein tyrosine phosphatase 1B

N/A

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arrays for studying drug-induced apoptosis and a drug testingplatform (Fan et al. 2008; Kim et al. 2012; Wlodkowic et al.2009b). Next, Fig. 2 demonstrates a time-evolution of micro-fluidic technologies for cancer across the past 20 years.Recently many technologies have been emerging to betterserve cancer diagnosis and treatment (Wlodkowic et al.2009a, b, Nagrath et al. 2007; Fan et al. 2008; Cheng et al.1998; Gasperis et al. 1999; DeRisi et al. 1996; Chiu et al.2000; Fang et al. 1999; Mohamed et al. 2004, 2005; Wang etal. 2004, 2005; Tian 2000; Hung et al. 2005; Chaw et al.2006; Zheng et al. 2005; Brouzes et al. 2009; Di Carlo et al.

2006; Dharmasiri et al. 2011; Liu et al. 2010; Domenech et al.2012). Below, specific microfluidic approaches for each areawill be discussed beginning with circulating tumor cellisolation.

2 Microfluidic technologies for Isolation CirculatingTumor Cells (CTCs)

Recently, several hypotheses have been developed regard-ing cancer metastasis and progression. CTCs, tumor cells

Fig. 1 Role of microfluidic technologies in cancer research. Isolationof CTCs using immunoaffinity-based (Nagrath et al. 2007), immuno-magnetic-based (Kang et al. 2012a; Reproduced by permission of TheRoyal Society of Chemistry) and size-based (Hur et al. 2011; Repro-duced by permission of The Royal Society of Chemistry) methods.Molecular Diagnosis: on-chip single-cell RT-qPCR carried out in eachof the reaction chambers (White et al. 2011), droplet-based PCR fordetecting rare mutations (Pekin et al. 2011; Reproduced by permissionof The Royal Society of Chemistry), droplet-scale estrogen assay formeasuring small amounts of tissue (Mousa et al. 2009). Tumor Biol-ogy: formation of 3D co-culture spheroids for studying the metastatic

microenvironment of prostate cancer (Hsiao et al. 2009), cell migrationplatform to study the effect of co-culture environments (Chung et al.2009; Reproduced by permission of The Royal Society of Chemistry),cancer cell migration in microcapillary array in conditions of mechan-ical confinement (Irimia and Toner 2009; Reproduced by permission ofThe Royal Society of Chemistry). High-throughput Screening: inte-grated blood barcode chip to detect plasma proteins (Fan et al. 2008),programmable cell culture array for drug screening (Kim et al. 2012;Reproduced by permission of TheRoyal Society of Chemistry), single-cell array composed of micromechanical traps to screen anti-cancerdrugs that induce apoptosis (Wlodkowic et al. 2009b)

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shed by primary tumors into circulation, have been identi-fied as the lethal drivers in the metastatic cascade. They canultimately lodge, invade and proliferate in distant secondarysites initiating metastatic lesions. Metastasis is the leadingcause of cancer related deaths (Hanahan and Weinberg2000). In some cancers, metastasis may occur in the earlystage of tumor progression (Maheswaran and Haber 2010).In addition, metastatic cells acquire mutations beyond thoseinitiated within primary tumors (Nguyen and Massagué2007). Therefore detecting CTCs can be extremely valuableto cancer diagnosis in early stages and help with treatmentdecisions. The emerging microfluidic technologies can iso-late CTCs, based on their biochemical or physical proper-ties, using a variety of methods.

2.1 Immunoaffinity-based isolation

One of the major CTC isolation method is based onantibody-antigen interactions. The most commonly usedsurface antigen is Epithelial Cell Adhesion Molecule orEpCAM, first identified in the late 70’s (Baeuerle andGires 2007). EpCAM is overexpressed in breast, colon,lung, prostate, gastric, ovarian and renal carcinomas(Baeuerle and Gires 2007; Went et al. 2004, 2006) andhence widely employed as the target antibody in almost allimmunoaffinity based CTC isolation strategies.

Early in 2004 one of the first CTC detection technologies,CellSearch, demonstrated CTC-based diagnostic potential by

separating CTCs using EpCAM-coated magnetic beads andcorrelating the number of isolated CTCs to prognosis in breastcancer patients. It is the only device approved by the U.S.Food and Drug Administration for isolation of CTCs in meta-static breast, colon and prostate cancers (Kaiser 2010). In2007, a microfluidic-based CTC capture device, which con-sisted of 78,000 EpCAM-coated microposts embedded on asilicon chip, was first published in Nature (Nagrath et al.2007). The chip can capture cancer cells from milliliters ofunprocessed whole blood with high sensitivity and purity. Thecaptured cancer cells were maintained in an appropriate con-dition for molecular analysis through immunostaining, orDNA/RNA extraction. The chip successfully detected CTCin all but one of 116 blood samples from 68 patients withmetastatic lung, prostate, pancreatic, breast and colon cancer.

Later in 2008, it was shown that the CTC-chip technol-ogy was successfully applied to monitor the epidermalgrowth factor receptor gene (EGFR) mutations in patientswith non-small-cell lung cancer. Sufficient DNAwas isolat-ed from the captured CTCs to allow allele specific assaytesting and in few instances direct sequencing. Not onlywere rare somatic genetic mutations detected in 19 out of20 EGFR positive patient samples but the device alsodetected secondary resistant mutation from 11 out of 12patients, who developed resistance to tyrosine kinase inhib-itors (Maheswaran et al. 2008). This type of information thatis vital for diagnosis, prognosis, and therapeutics was earlierprovided only by invasive tissue biopsies. Non-invasive

Fig. 2 Timeline of development of microfluidics based technologiesfor cancer (Wlodkowic et al. 2009a, b; Nagrath et al. 2007; Fan et al.2008; Cheng et al. 1998; Gasperis et al. 1999; DeRisi et al. 1996; Chiuet al. 2000; Fang et al. 1999; Mohamed et al. 2004, 2005; Wang et al.2004, 2005; Tian 2000; Hung et al. 2005; Chaw et al. 2006; Zheng et

al. 2005; Brouzes et al. 2009; Di Carlo et al. 2006 (Reproduced bypermission of The Royal Society of Chemistry); Dharmasiri et al.2011; Liu et al. 2010; Domenech et al. 2012 (Reproduced by permis-sion of The Royal Society of Chemistry)

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genotyping or so called “blood biopsies” in patients wasmade possible with the CTC-chip.

In 2010, another step forward was taken in the clinicaldevelopment of the CTC-chip by developing an automatedstrategy to characterize CTCs of prostate cancer (Stott et al.2010a). CTCs were fixed and stained with the prostate-specific antigen (PSA) and DNA content. The chip wasimaged in a semiautomated fashion and CTCs were charac-terized by an image processing algorithm in terms of fluo-rescence intensity, cell shape and other morphological traits.Applying this method enabled easier monitoring of CTClevels for different patients during the course of therapy.

The CTC-chips discussed so far immobilized antibodieson microposts through surface chemistry. Antibodies linkedto magnetic arrays can self-assemble in plain microchannels(Saliba et al. 2010). This system can sort B-lymphocytesfrom patient with leukemia and lymphoma. Also cancercells were separated from a mixture of cancer and endothe-lial cells with an efficiency of 80 % using the same device.Another immobilization technique was demonstrated byDharmasiri et al. using aptamers to target membrane pro-teins expressed on prostate cancer cells and captured cancercells with high efficiency and purity (Dharmasiri et al.2009). Dickson et al. reported a streptavidin coated micro-fluidic device can isolate cancer cells from blood cellsincubated with biotin-tagged anti-EpCAM (Nora Dicksonet al. 2011).

Despite the ability to specifically target CTCs, anotherchallenge for CTC isolation remains. CTCs are very rare (1–10 per mL of whole blood) compared with billons of whiteblood cells and red blood cells. The rarity poses a significantengineering problem in designing a device to capture CTCswith high specificity and purity and simultaneously keep thecells viable for subsequent molecular analysis (den Toonder2011). Researchers have been developing sophisticatedmicrofluidic devices to address these issues.

Stott et al. made a high-throughput polydimethylsiloxane(PDMS) based CTC-chip with enhanced capture efficiencyand optical properties (Stott et al. 2010b). The microchannelwas fabricated into a herringbone shape which generatedpassive mixing via microvortices. It can process larger vol-ume of blood as compared to the micropost-based CTC-chipwhile maintaining the same capture efficiency. For example,the herringbone chip can maintain >40 % capture efficiencyat flow rate up to 4.8 mL/h but efficiency of CTC-chipdropped significantly above 2–3 mL/h. After being cap-tured, cancer cells were viable and intact for molecularcharacterization and imaging.

Gleghorn et al. reported a geometrically enhanced differ-ential immunocapture (GEDI) chip to capture prostate CTCswith high-efficiency and high-purity (Gleghorn et al. 2010).The researchers optimized the displacement, size and shapeof posts to maximize the interactions between large CTCs

(15–25 μm) and the antibody coated surface while smallblood cells (4–18 μm) can escape capture. Testing clinicalblood samples exhibited increased capture efficiency andpurity on the GEDI device.

Myung et al. demonstrated immobilization of E-selectinand anti-EpCAM on the microfluidic channels enhancedcapture of CTCs. E-selectin induced rolling of leukocytesand cancer cells at different velocities resulting in increasedantibody accessibility to cancer cells (Myung et al. 2010).The cancer cells tended to roll faster as the shear stressincreased while the rolling velocity of leukocyte remainedstable. Therefore, this approach achieved separation of leu-kocytes and CTCs with increased cell capture efficiency.

Dharmasiri et al. have recently reported an integratedmicrofluidic system which incorporates immunoaffinity-based capture, enzymatic release, conductivity enumerationand electrokinetic enrichment of colorectal CTCs(Dharmasiri et al. 2011). This method allows consequentmanipulation and molecular profiling of CTCs using PCRcoupled with a ligase detection reaction (LDR) assay. Sincethis system contains an electrokinetic enrichment compo-nent, it can concentrate the mass-limited DNA samplesextracted from CTCs. They were able to detect the KRASoncogene mutation in the SW620 cell line but not in theHT29 cell line, which was consistent with the knowngenotypes.

Hoshino et al. utilized immunomagnetic separationmechanism to isolate cancer cells in a microchip. Bloodsamples containing spiked cancer cells were incubated withmagnetic nanoparticles conjugated to anti-EpCAM priorpassing through the device. This device can isolate as fewas 5 cells per mL of blood and can be operated at 10 ml/h flow rate without a significant reduction in capture rate(Hoshino et al. 2011).

Most recently, Kang et al. presented a novel CTC isola-tion approach which incorporated magnetic separation withmicrofluidic devices that permitted removal of capturedCTC and culturing in vitro (Kang et al. 2012b). The deviceconsisted of a main channel flanked by two rows of dead-end side chambers for magnetically labeled CTC collection.The mouse blood sample was first treated with EpCAMcoated magnetic beads followed by flow through the isola-tion channel. After that, CTCs can be released by movingthe magnet to the opposite side of the device. CTCs werethen cultured and checked for viability.

In our lab, poly-dimethylsiloxane (PDMS) is used toconstruct microfluidic devices that can capture low numbersof cancer cells from whole blood using the EpCAM basedimmunoaffinity capturing principle. For example, a non-small cell lung cancer (NSCLC) cell line, H1650 cells werespiked into whole blood and run through a microdevicecontaining thousands of EpCAM coated microposts. Thecapture efficiency was around 97 %. The chip was then

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immunostainedwith cytokeratin 7/8, CD45 and correspondingsecondary antibodies followed by 4′,6-Diamidino-2-Phenylindole, Dilactate (DAPI) for nuclear counterstaining(Fig. 3). Compared with the previous reported results(Nagrath et al. 2007), this PDMS based CTC capture chipexhibited higher capture efficiency and purity, more than 90%as compared to previous 60–70 %. Also this device can beeasily fabricated with low cost. And because of the transparentmaterials used, the quality of imaging is improved.

The major drawback of immunoaffinity-based isolation isthat the expression level of EpCAM on CTCs varies amongcancer types and for a given cancer. Some of the CTCsmight not express EpCAM, particularly cells that undergothe epithelial-mesenchymal transition (EMT) (Santos et al.2010). Therefore, immunoaffinity CTC-chips might misssome subpopulations of CTCs which may carry importantgenetic information about primary tumors. Utilizing size-based separation and filtration in addition to immunoaffinitymethods may increase capture of CTCs with minimal loss oflow EpCAM expressing cells.

2.2 Size-based separation

CTCs are often larger in size and may have a differentspecific gravity than blood cells therefore they can be sep-arated from blood cells either by physical filtration or byhydrodynamic forces (Hou et al. 2011). One advantage ofthe size-based separation is that cells can be enriched with-out using a specific biomarker. With hydrodynamic separa-tion, there is an added advantage that the system can beoperated at relatively high flow rate which is valuable toenrich rare CTCs. Furthermore, isolated CTCs can be col-lected without compromising cell viability or gene expres-sion profile thereby enabling off-chip cellular and molecularcharacterizations. Microfluidic devices enable sorting ofCTCs based on size followed by single cell analysis or cellculture on chip as well.

Zheng et al. presented an efficient membrane microfilterdevice made of parylene-C for isolation of prostate cancercells from whole blood (Zheng et al. 2007). The membranefilter contained 16,000 evenly distributed pores of 10 umdiameter and 20 um space in between. The membrane wasintegrated with electrodes for direct electrolysis of theretained cancer cells and then polymerase chain reaction(PCR) was carried out on the cell lysate. In successiveapproaches, researchers used two-layer membranes to filterviable prostate and breast cancer cells (Zheng et al. 2011).The captured cells were cultured on device for 2 weeks. Twoissues arose with increasing volumes of blood processed;the membrane was easily clogged and whole blood neededto be diluted before filtering.

Kuo et al. demonstrated a microfluidic filtration systemwhich can separate breast cancer cell spiked into whole

Fig. 3 Capture efficiency and purity of lung cancer cells H1650 spikedinto whole blood (left), Immunofluorescence images of H1650 cellswith blood cells (right) (green: cytokeratin 7/8, red: CD45, blue:DAPI)

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blood with 50–90 % recovery rate (Kuo et al. 2010). Thedevice consisted of a serpentine channel interconnected withtwo outer filtrate channels with rectangular apertures. Theforce experienced by cells during the filtration process wascarefully assessed and the dimensions of the apertures wereadjusted accordingly to minimize cell damage.

Hur et al. presented a device to enrich cancer cells indiluted blood by a factor of 5.4 (Hur et al. 2011). Theyutilized the principle that distinct focusing positions of de-formable particles can be created by a balance betweeninertial lift forces and viscoelastic forces in microchannelswith high aspect ratio. Despite high throughput and ease ofoperation, the blood samples needed to be diluted to avoiddefocusing caused by cancer and blood cell interactions.

Lim et al. utilized a particle tracking analysis (PTA)method to study the particle focusing in microchannels(Lim et al. 2012). Polystyrene beads, white blood cellsand prostate cancer cells (PC-3) were tested in both di-luted and whole blood generating two-dimensional focus-ing profiles as guidelines for isolating cells from wholeblood.

Despite some key advantages of size-based separation,the performance of this technique is still limited due to theheterogeneity of CTCs in size and morphology (Allard2004). To overcome this challenge, additional downstreamprocesses might be needed to increase detection accuracyand sensitivity.

2.3 Other MEMS based methods

Talasaz et al. demonstrated a magnetic sweeper utilizing animmunomagnetic separation mechanism with enhanced pu-rity and recovery rate (Talasaz et al. 2009). The device wascomposed of magnetic rods sweeping wells of a six-wellplate to capture magnetically labeled breast cancer cellsfrom blood. The cells were released and underwent genomicsequencing and other molecular analyses. Moon et al. com-bined both hydrodynamic focusing and dielectrophoresis toisolate high purity cancer cells from blood at high flow rates(Moon et al. 2011). Diluted blood was passed through amulti-orifice micochannel for separating blood cells fromcancer cells by different equilibrium positions of cells.Cancer cells, now pooled with fewer blood cells were thenflowed into a non-uniform electric field for further separa-tion. The combined modules achieved efficient enrichmentof cancer cells in a reduced time period. Chen et al. pre-sented a microfluidic disk to negatively deplete non-tumorcells via immunomagnetic principles to achieve isolation ofrare cancer cells (Chen et al. 2011). Non-target cells werelabeled with magnetic beads and as samples passed througha multistage magnetic field, those cells got trapped.Compared to positive immunoaffinity selection, negativedepletion accommodates the need to capture CTCs that

don’t express the typical surface markers, such as cellsundergoing the epithelial-mesenchymal transition (EMT)(Sieuwerts et al. 2009).

3 Microfluidic approaches for molecular diagnosisof cancer

In addition to cellular approaches, other biomolecules aremonitored for cancer diagnostics such as circulating tumorDNA, microRNAs, proteins and serum microvesicles (Diehlet al. 2008; Mitchell et al. 2008; Roessler 2005; Valenti2006). Microfluidics exhibits high sensitivity and accuracyfor detecting cancer specific biomarkers present at low con-centrations. Additionally, microfluidics can be developedinto point-of-care devices with reduced cost which willlikely lead to routine minimally invasive-clinical testing.

Yung et al. demonstrated a microfluidic digital PCRplatform for detecting rare EGFR mutations from tumortissue and plasma in non-small cell lung cancer patients(Yung et al. 2009). White et al. presented an integratedmicrofluidic device to perform RT-qPCR with high through-put and precision (White et al. 2011). The device consistedof 300 parallel assay chambers processing 20 μl samples. Itwas used to detect single-cell mutations from metastaticbreast cancer cells. Pekin et al. recently showed a droplet-based microfluidic device to quantitatively and sensitivelydetect rare KRAS mutations of tumor DNA encapsulated indroplets (Pekin et al. 2011). The mutant DNA and wild-typeDNA were readily identified by reading the fluorescencefrom the array of droplets. This technology reduced the costand simplified the procedure of the digital PCR process.

MicroRNAs (miRNAs) are small, non-coding RNA mol-ecules that regulate gene expression and can play an impor-tant role in cancer development (Esquela-Kerscher andSlack 2006). Circulating miRNAs in blood are stable andcan be regarded as a potential biomarker for early cancerdetection (Tsujiura et al. 2010). Schrauder et al. utilized acommercial microfluidic-based array, Geniom Biochip, toperform miRNA-profiling from the blood of 48 patientswith early stage breast cancers (Hoheisel et al. 2012).They were able to detect 46 down-regulated and 13 up-regulated miRNAs in the patients compared to healthy con-trols. They found miR-202 was significantly up-regulatedand may be involved in carcinogenesis.

In another study, Mitchell et al. extracted miRNAs fromthe blood serum of prostate cancer patients followed byquantitative RT-qPCR measurement using TaqMan micro-fluidic arrays (Mitchell et al. 2008). They found that miR-141 is expressed specifically in prostate tumors and canserve as a cancer-specific biomarker. Despite the manyadvantages miRNA detection may offer, challenges are thatthe sensitivity, specificity and accuracy of the system is still

Biomed Microdevices

limited for processing the small amount of miRNAsextracted from whole blood.

Digital microfluidic devices, which incorporate electro-des and use electrical forces to move liquids, can be ap-plied to sensitively detect rare amount of hormones forcancer diagnosis. For example, the level of estrogen is animportant risk indicator of breast cancer. Mousa et al.demonstrated a highly sensitive droplet-based estrogendetection assay to process tissue samples, blood, and serumwithin a short period of time (Mousa et al. 2009). Thedevice used electrical forces to drive sample manipulationexploiting the conductive nature of most biologically rele-vant liquids.

The search for blood biomarkers for cancer diagnosis hasbeen extended to microvesicles that are derived from cellsand circulate in blood. Microvesicles (exosomes) are smallmembrane-bound particles that are abundant in plasma. Thesize of exosomes is 60–100 nm (Taylor et al. 2005).miRNAs, proteins and lipids are packaged in exosomesmaking them potential biomarkers for cancer detection(Ratajczak et al. 2006). Chen et al. demonstrated a micro-fluidic device that can isolate microvesicles (exosomes)from blood serum of glioblastoma multiforme (GBM)patients via anti-CD63 functionalized surface. RNA wasextracted from the captured exosomes followed by RT-PCR analysis (Chen et al. 2010). This microfluidic isolationapproach provided quick exosome identification and en-abled the extraction of high quality RNA. One major chal-lenge of isolating exosomes is the ability to discriminateexosomes specifically from other similarly sized biologicalstructures.

Overall, microfluidic devices are able to detect smallmolecules present in complex bodily fluids with accuracyand specificity. This alternative approach may lead toadvances in monitoring cancer progression, personalizedtreatments based on tumor make up, and insight into thehow cancer genetics alter through therapeutics.

4 Microfluidic systems to explore and understand cancerbiology

Microfluidics can mimic the physiological cues in the cel-lular environment through spatial and temporal control overgradients of soluble factors and cell-cell contacts in extra-cellular matrix (El-Ali et al. 2006). Additionally, compo-nents for downstream analysis such as imaging or molecularcharacterization can be connected with cell culture moduleto make an integrated system. All of these factors makemicrofluidics an excellent tool for studying cell biology.When it comes to exploring cancer cell biology, variousmodels have been developed to investigate cancer cell mi-gration, angiogenesis and tumor microenvironment.

Chaw et al. performed a quantitative study on tumor cellmigration through microgaps with or without Matrigel-endothelial cells lining (Chaw et al. 2006, 2007). Cancercells were cultured in side channels with serum deficientmedium and individual cells migrated towards the centralchannel which contained complete medium. They were ableto calculate the cell migration rate and observe cell defor-mation which provided novel drug targets for metastasis.Another cell migration study in microfluidic channels waspresented by Irima et al. in which individual cancer cellswere mechanically confined within channels then, withinseveral hours, cell movement was observed as fast andpersistently in one direction (Irimia and Toner 2009).This device can be viewed as a cancer-cell invasionassay mimicking cancer cell migration from primarytumor sites at the onset of metastasis. Most recently,Huang et al. reported a compartmentalized microfluidicdevice to study brain tumor stem cell migration and toisolate the stem-like cancer cells for subsequent cultur-ing and analysis (Huang et al. 2011).

To gain insight into the effects of tumor microenviron-ment on cancer progression, a microfluidic system thatcaptured the transient interactions between endothelial cellsand cancer cells during metastasis was developed (Song etal. 2009). The microfluidic device consisted of two PDMSlayers sandwiching a layer of porous polyester membrane.In particular, the effects of chemokine CXCL12 acting onendothelium through CXCR4 receptors was investigatedand shown to regulate the organ specific homing of meta-static cancer cells. Using the two-layer design principle, thesame group reported the formation of co-cultured 3D sphe-roids of prostate cancer cells, osteoblasts and endothelialcells in microfluidic channels as a model resembling themetastatic prostate cancer bone microenvironment (Hsiaoet al. 2009). The researchers were also able to identify thecancer stem cell subpopulations inside the spheroids.Compared with 2D co-culture environment, the 3D sphe-roids can better mimic the physiologic microenvironment interms of cell proliferation rate, intercellular interactions andpreserving stem-cell like subpopulations.

Huang et al. presented another microfluidic design to co-culture cells in 3D (Huang et al. 2009). Different cell typeswere loaded in distinct parallel channels connected by aseries of juxtaposed channels partitioned by regularlyspaced posts acting as a partition to the cell loading chan-nels. It was observed that macrophages invaded toward thebreast cancer cells after one week of co-culturing. Thepurpose of this co-culture device is to mimic the interactionsbetween tumor cells and their surrounding stromal environ-ment, and by varying environmental cues, epigeneticchanges can be revealed.

Chung et al. demonstrated another cellular environmentinduced migration assay using 3D collagen gel separated

Biomed Microdevices

flow channels (Chung et al. 2009). They were able to cultureendothelial cells in the middle channel and stimulate theirmigration through the gel region by either a gradient ofgrowth factor or by culturing cancer cells (MTLn3 orU87MG) or muscle cells in the side channels. Quantitativeassessment of the effect of applied growth factor gradients,the stiffness of the gel and the co-culture environment wasperformed.

Sung et al. constructed a microfluidic co-culture systemthat enabled patterning of cancer cells (MCF-DCIS) andfibroblasts in adjacent laminar flow regimes (Sung et al.2011). It was shown that fibroblasts promoted invasivetransition of the cancer cells. Domenech et al. demonstratedthe ability to use a microfluidic co-culture system to under-stand the paracrine signaling between cancer cells and stro-mal cells (Domenech et al. 2009, 2012). In particular, thehedgehog signaling between prostate cancer cells and myo-fibroblasts was captured for the first time in vitro.

One of the challenges of culturing cells in microfluidicscomes from controlling the tiny environment surroundingthe cells. Parameters like medium composition, shear stress,chemical gradients and temperature are of important consid-eration when designing the system (Walker et al. 2004; Sungand Shuler 2012). However, once the appropriate conditionsare obtained microfluidic devices provide a tailored, con-trolled environment for cellular studies.

Furthermore, microfluidics provides an excellent oppor-tunity to easily pattern cells to create the desired environ-mental cues for cell growth and proliferation. In our lab, weused laminar flow (Sung et al. 2011; Wong et al. 2008) topattern multiple cell types in microchannels and were able tostudy the cellular interactions between cancer cells and non-tumorous cells existing in the tumor microenvironment. Forexample, MCF7 breast cancer cells were co-cultured withfibroblasts in a side-by-side pattern in a 3D gel environment.After 5 days of culturing, the cells reached confluence.MCF7 cells were immunostained with cytokerain 7/8,EpCAM and DAPI. Fibroblasts were only stained withDAPI (Fig. 4).

5 High-throughput biomarker and drug screening

High throughput multiplex screening using microfluidicshas gathered momentum recently as a method in cancerresearch. The microfluidic high-throughput screening sys-tem requires fewer reagents, small sample volumes and canprocess multiple compounds with various concentrations inshorter time. Also the system can be automated to increasethe efficiency of anti-cancer drug development. Cell-basedmicroarrays enable large-scale single-cell study to identifyrare cancer subpopulations, like stem cells and progenitorcells (El-Ali et al. 2006). Single cell analysis has gained

increasing attention for elucidating the heterogeneity amongcancer cells.

Fan et al. developed a point-of-care diagnostic devicewhich detects multiple proteins from small blood serumsamples within 10 min (Fan et al. 2008). A DNA-encodedantibody library technique was used to construct the barcodearrays for immuno-detection of 12 tumor-associated bio-markers. 22 patients with breast and prostate cancers wereexamined and confirmed the reliability of the system.Jokerst et al. presented another approach for multiplex

Fig. 4 Microfluidics based co-culture systems. MCF7 cells in themiddle co-cultured with fibroblasts on the sides patterned by laminarflow (green: EpCAM, red: cytokeratin 7/8, blue: DAPI). MCF7 cellsare indicated by red cytokeratin, green EpCAM and blue DAPI nucle-us. Fibroblasts are only stained with DAPI surrounding the cancer cellson the two sides. Cancer cells tend to grow into the fibroblasts lanes

Biomed Microdevices

serum protein detection employing a quantum dot-relatedbiosensor (Jokerst et al. 2009). Stern et al. proposed a label-free biomarker detection platform which was composed of amicrofluidic purification chip for pre-concentration and ananosensor chip for detection (Stern et al. 2010). This sys-tem can handle a 10 μl of blood sample for quantitativelydetecting multiple soluble proteins present in blood in20 min. Two model biomarkers PSA and CA15.3 weretested in the device and exhibited highly sensitive andsimultaneous detection. Wlodkowic et al. demonstrated amicrofluidic single-cell assay to study tumor cell apoptosisand screen for anti-cancer drugs (Wlodkowic et al. 2009b).The device consisted of an array of 440 traps that canimmobilize leukemia cancer cells for consequent character-izations. This platform enabled real-time imaging and mon-itoring of apoptosis.

Kim et al. developed an automated microfluidic cell cul-ture array consisting of 64 chambers which can examine 64pair-wise drug combinations from two input streams (Kim etal. 2012). Prostate cancer cells PC3, were tested with twosensitizer drugs and a cell-death inducing drug, TRAIL. Thetwo-layer PDMS device was controlled by LabVIEW soft-ware enabling large-scale testing of different combinations ofdrugs which can be valuable for identifying effective therapy.Miller et al. approached high-throughput drug screeningusing a droplet-based microfluidic platform (Miller et al.2012). The system made use of the Taylor-Aris dispersionmechanism to generate different concentration of drugs en-capsulated in droplets. The concentration of drugs can beindicated by the fluorescence from enzymatic reactions.Higher fluorescence corresponds to a better inhibition effectby the drugs. Potential inhibitors towards protein tyrosinephosphatase 1B, a known diabetes and cancer target, weretested and the dose-dependent response was plotted.

Overall, microfluidics based high throughput platformsare very promising and highly attractive for rapid testing ofdrugs and biomarker discovery. This is one of the rapidlyevolving field along with cell based diagnostics.

6 Challenges and future directions

Despite the promise of emerging technologies for cancerdiagnosis and therapy, most of them haven’t been validatedwith relevant clinical samples and almost none reachedclinical trials (Schattner 2009). There exists a considerablegap between laboratory investigation and clinical applica-tion. One of the key challenges is that nearly all samples ofclinical relevance are complex in nature and handling ofthese in microfluidic environment needs special design con-sideration. It is of great importance to take into account thecomplexities involving patient samples from the conceptualstage itself. In a sense, a bridge needs to be built to cover the

gap during technology development. This will make clinicalimplementation of the technology much more feasible.Access to clinical samples while testing and optimizingdevices is very important as it can reduce development time,by avoiding total re-optimization and re-designing, if therequired tools, assays and parameter space are realized in theinitial optimization stage itself.

Cancer diagnosis can be viewed as the first stage of treat-ment and is important for designing personalized medicine.The ability to detect cancers at early stage, to target validatedcancer biomarkers, and to characterize certain mutations lead-ing to drug resistance in tumor progression has always been agreat effort in cancer research and will remain a challenge forthe future. Low-cost and point-of-care diagnosis tools andtechnologies will be desperately needed to enable cancermanagement and reduction in mortality rates.

Microfluidics holds great promise for miniaturization andautomation through handling small amount of materials andincorporating control systems. Microfluidics can detectCTCs from peripheral blood and collect genetic informationto validate stage-specific markers for diagnosis and moni-toring treatment response or relapse. The next generation ofmicrofluidic devices would possibly make use of multiplebiochemical and biophysical cues that are unique to cancerbiomarkers to achieve high detection efficiency, high cellviability, and high throughput, which would enhance theclinical relevance of microfluidic technologies for cancerdetection. A high throughput technique for immunoaffinitybased assay should be able to process approximately at10 mL/h such that meaningful volumes can be processedin a reasonable time. The assay can take anywhere up to 3 hfor completion from the sample input to enumeration. Withan emerging interest to apply microfluidic-based technolo-gies in cell biology, the devices can be made effective toolsfor understanding basic tumor biology in terms of tumormicroenvironment, gene redundancy, and tumor stem cells.All of these would foster drug and therapeutic developmentin the battle with cancer.

Acknowledgements This work was supported by the NIH Director’sNew Innovator Award 1DP2 OD006672-01, and 3M Non tenuredfaculty award to Prof. Nagrath. The work was performed in part atthe Lurie Nanofabrication Facility, a member of the National Nano-technology Infrastructure Network, which is supported by the NationalScience Foundation.

References

F. Alexis, J.W. Rhee, J.P. Richie, A.F. Radovic-Moreno, R. Langer,O.C. Farokhzad, New frontiers in nanotechnology for cancertreatment. Urol. Oncol. 26(1), 74–85 (2008)

W.J. Allard, Tumor cells circulate in the peripheral blood of all majorcarcinomas but not in healthy subjects or patients with nonmalig-nant diseases. Clin. Cancer Res. 10(20), 6897–6904 (2004)

Biomed Microdevices

P.A. Auroux et al., Micro total analysis systems. 2. Analytical standardoperations and applications. Anal. Chem. 74(12), 2637–2652(2002)

P.A. Baeuerle, O. Gires, EpCAM (CD326) finding its role in cancer.Br. J. Cancer 96(3), 417–423 (2007)

E. Brouzes et al., Droplet microfluidic technology for single-cell high-throughput screening. Proc. Natl. Acad. Sci. U. S. A. 106(34),14195–14200 (2009)

S.D. Chan et al., Cytometric analysis of protein expression and apo-ptosis in human primary cells with a novel microfluidic chip-based system. Cytom. A 55(2), 119–125 (2003)

K.C. Chaw et al., A quantitative observation and imaging of singletumor cell migration and deformation using a multi-gap micro-fluidic device representing the blood vessel. Microvasc. Res. 72(3), 153–160 (2006)

K.C. Chaw et al., Multi-step microfluidic device for studying cancermetastasis. Lab Chip 7(8), 1041–1047 (2007)

C. Chen et al., Microfluidic isolation and transcriptome analysis ofserum microvesicles. Lab Chip 10(4), 505–511 (2010)

C.L. Chen et al., Separation and detection of rare cells in amicrofluidic disk via negative selection. Lab Chip 11(3),474–483 (2011)

J. Cheng et al., Isolation of cultured cervical carcinoma cells mixedwith peripheral blood cells on a bioelectronic chip. Anal. Chem.70(11), 2321–2326 (1998)

D.T. Chiu et al., Patterned deposition of cells and proteins onto surfa-ces by using three-dimensional microfluidic systems. Proc. Natl.Acad. Sci. U. S. A. 97(6), 2408–2413 (2000)

S. Chung et al., Cell migration into scaffolds under co-culture con-ditions in a microfluidic platform. Lab Chip 9(2), 269–275(2009)

J. den Toonder, Circulating tumor cells: the grand challenge. Lab Chip11(3), 375–377 (2011)

J. DeRisi, P.S. Meltzer, L. Penland, P.O. Brown (Group 1); M.L.Bittner, M. Ray, Y. Chen, Y.A. Su, J.M. Trent (Group 2). Use ofa cDNA microarray to analyse gene expression patterns in humancancer. Nat. Genet. 14, 457–460 (1996)

U. Dharmasiri et al., Highly efficient capture and enumeration of lowabundance prostate cancer cells using prostate-specific membraneantigen aptamers immobilized to a polymeric microfluidic device.Electrophoresis 30(18), 3289–3300 (2009)

U. Dharmasiri et al., High-throughput selection, enumeration, electro-kinetic manipulation, and molecular profiling of low-abundancecirculating tumor cells using a microfluidic system. Anal. Chem.83(6), 2301–2309 (2011)

D. Di Carlo, L.Y. Wu, L.P. Lee, Dynamic single cell culture array. LabChip 6(11), 1445–1449 (2006)

F. Diehl et al., Circulating mutant DNA to assess tumor dynamics. Nat.Med. 14(9), 985–990 (2008)

M. Domenech et al., Cellular observations enabled by microculture:paracrine signaling and population demographics. Integr. Biol.(Camb.) 1(3), 267–274 (2009)

M. Domenech et al., Hedgehog signaling in myofibroblasts directlypromotes prostate tumor cell growth. Integr. Biol. (Camb.) 4(2),142–152 (2012)

J. El-Ali, P.K. Sorger, K.F. Jensen, Cells on chips. Nature 442(7101),403–411 (2006)

A. Esquela-Kerscher, F.J. Slack, Oncomirs—microRNAs with a role incancer. Nat. Rev. Cancer 6(4), 259–269 (2006)

T.K. Fabian, P. Fejerdy, P. Csermely, Salivary genomics, transcriptom-ics and proteomics: the emerging concept of the oral ecosystemand their use in the early diagnosis of cancer and other diseases.Curr. Genomics 9(1), 11–21 (2008)

R. Fan et al., Integrated barcode chips for rapid, multiplexed analysisof proteins in microliter quantities of blood. Nat. Biotechnol. 26(12), 1373–1378 (2008)

B. Fang, M. Zborowski, L.R. Moore, Detection of rare MCF-7 breastcarcinoma cells from mixtures of human peripheral leukocytes bymagnetic deposition analysis. Cytometry 36(4), 294–302 (1999)

M. Ferrari, Cancer nanotechnology: opportunities and challenges. Nat.Rev. Cancer 5(3), 161–171 (2005)

G.D. Gasperis et al., Microfuidic cell separation by 2D dielectropho-resis. Biomed. Microdevices 2(1), 41–49 (1999)

J.P. Gleghorn et al., Capture of circulating tumor cells from wholeblood of prostate cancer patients using geometrically enhanceddifferential immunocapture (GEDI) and a prostate-specific anti-body. Lab Chip 10(1), 27 (2010)

D. Hanahan, R.A. Weinberg, The hallmarks of cancer. Cell 100(1), 57–70 (2000)

J.R. Heath, M.E. Davis, Nanotechnology and cancer. Annu. Rev. Med.59, 251–265 (2008)

J.D. Hoheisel et al., Circulating Micro-RNAs as potential blood-basedmarkers for early stage breast cancer detection. PLoS One 7(1),e29770 (2012)

J.W. Hong, S.R. Quake, Integrated nanoliter systems. Nat. Biotechnol.21(10), 1179–1183 (2003)

K. Hoshino et al., Microchip-based immunomagnetic detection ofcirculating tumor cells. Lab Chip 11(20), 3449–3457 (2011)

H.W. Hou et al., Microfluidic devices for blood fractionation.Micromachines 2(3), 319–343 (2011)

A.Y. Hsiao et al., Microfluidic system for formation of PC-3 prostatecancer co-culture spheroids. Biomaterials 30(16), 3020–3027(2009)

C.P. Huang et al., Engineering microscale cellular niches for three-dimensional multicellular co-cultures. Lab Chip 9(12), 1740–1748 (2009)

Y. Huang et al., Evaluation of cancer stem cell migration using com-partmentalizing microfluidic devices and live cell imaging. J.Visual. Exp. 58 (2011)

D. Huh et al., Microfluidics for flow cytometric analysis of cells andparticles. Physiol. Meas. 26(3), R73–R98 (2005)

P.J. Hung et al., Continuous perfusion microfluidic cell culture arrayfor high-throughput cell-based assays. Biotechnol. Bioeng. 89(1),1–8 (2005)

S.C. Hur et al., Deformability-based cell classification and enrichmentusing inertial microfluidics. Lab Chip 11(5), 912–920 (2011)

D. Irimia, M. Toner, Spontaneous migration of cancer cells underconditions of mechanical confinement. Integr. Biol. (Camb.) 1(8–9), 506–512 (2009)

A. Jemal et al., Global cancer statistics. CA Cancer J. Clin. 61(2), 69–90 (2011)

J.V. Jokerst et al., Nano-bio-chips for high performance multiplexedprotein detection: determinations of cancer biomarkers in serumand saliva using quantum dot bioconjugate labels. Biosens.Bioelectron. 24(12), 3622–3629 (2009)

J. Kaiser, Medicine. Cancer’s circulation problem. Science 327(5969),1072–1074 (2010)

J.H. Kang et al., A combined micromagnetic-microfluidic device forrapid capture and culture of rare circulating tumor cells. Lab Chip(2012a)

J.H. Kang et al., A combined micromagnetic-microfluidic device forrapid capture and culture of rare circulating tumor cells. Lab Chip12(12), 2175–2181 (2012b)

J. Kim et al., A programmable microfluidic cell array for combinatorialdrug screening. Lab Chip (2012)

R.T. Krivacic et al., A rare-cell detector for cancer. Proc. Natl. Acad.Sci. U. S. A. 101(29), 10501–10504 (2004)

J.S. Kuo et al., Deformability considerations in filtration of biologicalcells. Lab Chip 10(7), 837–842 (2010)

E.J. Lim et al., Visualization of microscale particle focusing in dilutedand whole blood using particle trajectory analysis. Lab Chip 12(12), 2199 (2012)

Biomed Microdevices

L. Liu et al., A microfluidic device for continuous cancer cell cultureand passage with hydrodynamic forces. Lab Chip 10(14), 1807–1813 (2010)

S. Maheswaran, D.A. Haber, Circulating tumor cells: a window intocancer biology and metastasis. Curr. Opin. Genet. Dev. 20(1), 96–99 (2010)

S. Maheswaran et al., Detection of mutations in EGFR in circu-lating lung-cancer cells. N. Engl. J. Med. 359(4), 366–377(2008)

A. Manz et al., Planar chips technology for miniaturization and inte-gration of separation techniques into monitoring systems: capil-lary electrophoresis on a chip. J. Chromatogr. 593, 253–258(1992)

O.J. Miller, A. El Harrak, T. Mangeat, J.-C. Baret, L. Frenz, B. ElDebs, E. Mayot, M.L. Samuels, E.K. Rooney, P. Dieu, M. Galvan,D.R. Link, A.D. Griffiths, High-resolution dose–response screen-ing using droplet-based microfluidics. PNAS 109(2), 378–383(2012)

P.S. Mitchell et al., Circulating microRNAs as stable blood-basedmarkers for cancer detection. Proc. Natl. Acad. Sci. 105(30),10513–10518 (2008)

H. Mohamed et al., Development of a rare cell fractionation device:application for cancer detection. IEEE Trans. Nanobiosci. 3(4),251–256 (2004)

H.Mohamed,M.Murray, J.N. Turner, M. Caggana, Circulating tumor cells:captured with a micromachined device. NSTI-Nanotech 1 (2005)

H.-S. Moon et al., Continuous separation of breast cancer cells fromblood samples using multi-orifice flow fractionation (MOFF) anddielectrophoresis (DEP). Lab Chip 11(6), 1118 (2011)

N.A. Mousa et al., Droplet-scale estrogen assays in breast tissue, blood,and serum. Sci. Transl. Med. 1(1), 1ra2 (2009)

J.H. Myung et al., Enhanced tumor cell isolation by a biomimeticcombination of E-selectin and anti-EpCAM: implications for theeffective separation of circulating tumor cells (CTCs). Langmuir26(11), 8589–8596 (2010)

S. Nagrath et al., Isolation of rare circulating tumour cells in cancer patientsby microchip technology. Nature 450(7173), 1235–1239 (2007)

D.X. Nguyen, J. Massagué, Genetic determinants of cancer metastasis.Nat. Rev. Genet. 8(5), 341–352 (2007)

M. Nora Dickson et al., Efficient capture of circulating tumor cells with anovel immunocytochemical microfluidic device. Biomicrofluidics 5(3), 34119–3411915 (2011)

R. Pal et al., An integrated microfluidic device for influenza and othergenetic analyses. Lab Chip 5(10), 1024–1032 (2005)

D. Pekin et al., Quantitative and sensitive detection of rare mutations usingdroplet-based microfluidics. Lab Chip 11(13), 2156–2166 (2011)

J. Ratajczak et al., Membrane-derived microvesicles: important andunderappreciated mediators of cell-to-cell communication.Leukemia 20(9), 1487–1495 (2006)

D.R. Reyes et al., Micro total analysis systems. 1. Introduction, theory,and technology. Anal. Chem. 74(12), 2623–2636 (2002)

M. Roessler, Identification of nicotinamide N-methyltransferase as anovel serum tumor marker for colorectal cancer. Clin. Cancer Res.11(18), 6550–6557 (2005)

A.E. Saliba et al., Microfluidic sorting and multimodal typing of cancercells in self-assembled magnetic arrays. Proc. Natl. Acad. Sci. U.S. A. 107(33), 14524–14529 (2010)

J. Santos et al., Molecular biomarker analyses using circulating tumorcells. PLoS One 5(9), e12517 (2010)

E. Schattner, A chip against cancer. Sci. Am. 300(4), 21–22 (2009)R. Seigneuric et al., From nanotechnology to nanomedicine: applica-

tions to cancer research. Curr. Mol. Med. 10(7), 640–652 (2010)S.K. Sia, G.M. Whitesides, Microfluidic devices fabricated in poly

(dimethylsiloxane) for biological studies. Electrophoresis 24(21), 3563–3576 (2003)

R. Siegel et al., Cancer statistics, 2011: the impact of eliminatingsocioeconomic and racial disparities on premature cancer deaths.CA Cancer J. Clin. 61(4), 212–236 (2011)

A.M. Sieuwerts et al., Anti-epithelial cell adhesion molecule antibodiesand the detection of circulating normal-like breast tumor cells. J.Natl. Cancer Inst. 101(1), 61–66 (2009)

J.W. Song et al., Microfluidic endothelium for studying the intravas-cular adhesion of metastatic breast cancer cells. PLoS One 4(6),e5756 (2009)

P.K. Sorger, Microfluidics closes in on point-of-care assays. Nat.Biotechnol. 26(12), 1345–1346 (2008)

E. Stern et al., Label-free biomarker detection from whole blood. Nat.Nanotechnol. 5(2), 138–142 (2010)

S.L. Stott et al., Isolation and characterization of circulating tumor cellsfrom patients with localized and metastatic prostate cancer. Sci.Transl. Med. 2(25), 25ra23 (2010a)

S.L. Stott, C.-H. Hsu, D.I. Tsukrov, M. Yud, D.T. Miyamoto, B.A.Waltman et al., Isolation of circulating tumor cells using amicrovortex-generating herringbone-chip. PNAS 107(43),18392–18397 (2010b)

J.H. Sung, M.L. Shuler, Microtechnology for mimicking in vivo tissueenvironment. Ann. Biomed. Eng. 40(6), 1289–1300 (2012)

K.E. Sung et al., Transition to invasion in breast cancer: a microfluidicin vitro model enables examination of spatial and temporaleffects. Integr. Biol. (Camb.) 3(4), 439–450 (2011)

S. Takayama et al., Subcellular positioning of small molecules. Nature411(6841), 1016 (2001)

S. Takayama et al., Selective chemical treatment of cellular micro-domains using multiple laminar streams. Chem. Biol. 10(2), 123–130 (2003)

A.H. Talasaz et al., Isolating highly enriched populations of circulatingepithelial cells and other rare cells from blood using a magneticsweeper device. Proc. Natl. Acad. Sci. 106(10), 3970–3975 (2009)

D.D. Taylor, C. Gerçel-Taylor, Tumour-derived exosomes and theirrole in cancer-associated T-cell signalling defects. Br. J. Cancer92(2), 305–311 (2005)

H. Tian, Rapid detection of deletion, insertion, and substitution muta-tions via heteroduplex analysis using capillary- and microchip-based electrophoresis. Genome Res. 10(9), 1403–1413 (2000)

M. Tsujiura et al., Circulating microRNAs in plasma of patients withgastric cancers. Br. J. Cancer 102(7), 1174–1179 (2010)

R. Valenti, Human tumor-released microvesicles promote the differen-tiation of myeloid cells with transforming growth factor—medi-ated suppressive activity on T lymphocytes. Cancer Res. 66(18),9290–9298 (2006)

G.M. Walker, H.C. Zeringue, D.J. Beebe, Microenvironment design con-siderations for cellular scale studies. Lab Chip 4(2), 91–97 (2004)

S.J. Wang et al., Differential effects of EGF gradient profiles on MDA-MB-231 breast cancer cell chemotaxis. Exp Cell Res 300(1), 180–189 (2004)

M.M. Wang et al., Microfluidic sorting of mammalian cells by opticalforce switching. Nat. Biotechnol. 23(1), 83–87 (2005)

R. Weinberg, The Biology of Cancer (Garland Science, New York,2006)

P.T. Went et al., Frequent EpCam protein expression in human carci-nomas. Hum. Pathol. 35(1), 122–128 (2004)

P. Went et al., Frequent high-level expression of the immunotherapeu-tic target Ep-CAM in colon, stomach, prostate and lung cancers.Br. J. Cancer 94(1), 128–135 (2006)

A.K. White et al., High-throughput microfluidic single-cell RT-qPCR.Proc. Natl. Acad. Sci. U. S. A. 108(34), 13999–14004 (2011)

G.M. Whitesides, The origins and the future of microfluidics. Nature442(7101), 368–373 (2006)

D. Wlodkowic, Z. Darzynkiewicz, Microfluidics: emerging prospects foranti-cancer drug screening. World J Clin Oncol 1(1), 18–23 (2010)

Biomed Microdevices

D. Wlodkowic et al., Biological implications of polymeric micro-devices for live cell assays. Anal. Chem. 81(23), 9828–9833(2009a)

D. Wlodkowic et al., Microfluidic single-cell array cytometry for theanalysis of tumor apoptosis. Anal. Chem. 81(13), 5517–5523(2009b)

D. Wlodkowic, J. Skommer, Z. Darzynkiewicz, Cytometry in cellnecrobiology revisited. Recent advances and new vistas. Cytom.A 77(7), 591–606 (2010)

A.P. Wong et al., Partitioning microfluidic channels with hydrogel toconstruct tunable 3-D cellular microenvironments. Biomaterials29(12), 1853–1861 (2008)

T.K.F. Yung et al., Single-molecule detection of epidermal growthfactor receptor mutations in plasma by microfluidics digital PCRin non-small cell lung cancer patients. Clin. Cancer Res. 15(6),2076–2084 (2009)

G. Zheng et al., Multiplexed electrical detection of cancer markers withnanowire sensor arrays. Nat. Biotechnol. 23(10), 1294–1301 (2005)

S. Zheng et al., Membrane microfilter device for selective capture,electrolysis and genomic analysis of human circulating tumorcells. J. Chromatogr. A 1162(2), 154–161 (2007)

S. Zheng et al., 3D microfilter device for viable circulating tumor cell(CTC) enrichment from blood. Biomed. Microdevices 13(1),203–213 (2011)

Biomed Microdevices