liquid biopsy on chip: a paradigm shift towards the understanding of cancer...

22 | Integr. Biol., 2017, 9, 22--49 This journal is©The Royal Society of Chemistry 2017

Cite this: Integr. Biol., 2017,9, 22

Liquid biopsy on chip: a paradigm shift towardsthe understanding of cancer metastasis

Amogha Tadimety,†a Abeer Syed,†a Yuan Nie,a Christina R. Long,a Kasia M. Kreadya

and John X. J. Zhang*ab

This comprehensive review serves as a guide for developing scalable and robust liquid biopsies on chip

for capture, detection, and analysis of circulating tumor cells (CTCs). Liquid biopsy, the detection of

biomarkers from body fluids, has proven challenging because of CTC rarity and the heterogeneity of

CTCs shed from tumors. The review starts with the underlying biological mechanisms that make liquid

biopsy a challenge before moving into an evaluation of current technological progress. Then, a

framework for evaluation of the technologies is presented with special attention to throughput, capture

rate, and cell viability for analysis. Technologies for CTC capture, detection, and analysis will be

evaluated based on these criteria, with a focus on current approaches, limitations and future directions.

The paper provides a critical review for microchip developers as well as clinical investigators to

build upon the existing progress towards the goal of designing CTC capture, detection, and analysis

platforms.

Insight, innovation, integrationDetection of rare biomarkers, such as circulating tumor cells (CTCs), has proven challenging because of the rarity and heterogeneity of targets in samples.Quantitative detection and downstream analyses of biomarkers have become increasingly feasible with advances in micro/nanoscale engineered systems.Emerging microchip based technologies enable high throughput screening with single cell resolution, which helps understand tumor heterogeneity and themetastatic process by performing ‘‘omics’’ (genomics, proteomics) studies and by characterizing cell phenotypes based on surface protein expression anddeformability. In this review, the significance of throughput, accuracy, and efficiency is detailed for capture, detection, and analysis of cancer cells. This reviewprovides bioengineers and clinical investigators a framework for developing simple, sample-to-answer diagnostic platforms for initial diagnosis and continuedmonitoring.

1. Introduction

Although researchers, medical caregivers, and informed citizenshave curbed cancer’s death rate by 22% from 1991 to 2011 in theUSA, cancer remains the greatest cause of non-accidental deathsglobally.1 The Cancer Moonshot Initiative encourages scientists tobuild upon these advances through better predictive screening,diagnostics, prognostics, targeted therapies, and monitoring.Cancer diagnostic tools sustainably contribute to this success,especially excisional biopsies.

Once the presence of abnormal tissue or fluid is discerned,medical professionals have invasive and minimally invasiveprocedures to remove, or ‘biopsy’, a portion of it in order to

perform diagnostic tests.2 These procedures prevail as the solediagnostic means of determining whether a suspicious area iscancerous. Unfortunately in the case of breast biopsies, manywomen must undergo these procedures only to reveal the newsthat these suspicious regions are benign.3,4 In certain cases, thesuspicious tissues are located in regions of the body that areinaccessible for biopsies. Additionally, excisional biopsies onlyprovide a time dependent snapshot of the dynamic geneticfluctuations of a tumor. An invasive or even minimally invasivebiopsy is risky to spatially and temporally perform on a patientto monitor disease progression. The aforementioned informationpresents an outstanding opportunity to improve upon the state ofthe art methods of biopsies, especially when rates of survivalcorrelate with earlier detection.

The burgeoning field of microfluidics is producing innova-tive liquid biopsies on chip as a non-invasive cancer diagnostictool. Liquid biopsies on chip consist of this general scheme:liquid from a patient (i.e. blood or serum) is loaded into a

a Thayer School of Engineering at Dartmouth College, Hanover NH, 03755, USA.

E-mail: [email protected] Dartmouth-Hitchcock Norris Cotton Cancer Center, Lebanon NH, 03766, USA

† These authors contributed equally.

Received 27th September 2016,Accepted 30th November 2016

DOI: 10.1039/c6ib00202a

www.rsc.org/ibiology

Integrative Biology

REVIEW ARTICLE

Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42

.

View Article OnlineView Journal | View Issue

http://crossmark.crossref.org/dialog/?doi=10.1039/c6ib00202a&domain=pdf&date_stamp=2016-12-08http://dx.doi.org/10.1039/c6ib00202ahttp://pubs.rsc.org/en/journals/journal/IBhttp://pubs.rsc.org/en/journals/journal/IB?issueid=IB009001

This journal is©The Royal Society of Chemistry 2017 Integr. Biol., 2017, 9, 22--49 | 23

microfluidic device that performs a combination of capturing,detecting, and or analyzing tumor derived material, such ascirculating tumor cells (CTCs), circulating tumor DNA (ctDNA),or exosomes, that are shed by a tumor into the circulatorysystem (Fig. 1). In addition to being a novel non-invasive cancerbiopsy, liquid biopsies on chip are low cost, less exhaustiveof resources, superior at reproducing results, conducive tosequential sampling, and allow automation. However, micro-chip developers encounter numerous challenges in developingfunctional devices. One of the greatest challenges is that thetactics employed by cancer to evade the immune system createsophisticated hurdles that researchers must overcome to integratecancer biology into chips to capture, detect, and analyze cancerbiomarkers. Even within the same type of cancer, there arestratifications consisting of different biological signatures,or ‘biomarkers’ to capture the CTCs, ctDNA, or tumor-derived

exosomes. A major design challenge is integrating the biomarkersof cancer into microchip design.

CTCs remain the biomarkers with the greatest wealth ofresearch, analysis, and validation, although there have beenmany recent strides in ctDNA5 and extracellular vesicle analysis.6–9

There are over 3500 papers from the last ten years on PubMedrelating to CTC analysis, including papers relating to their impacton therapeutics,10–12 capture,13–15 and underlying biology.16–18

CTCs are larger than the other biomarkers and their molecularcontents are protected inside intact cells by a bilayer. Furthermore,validation of capture is easier using CTCs than it is for ctDNA andvesicles due to their larger size and well-characterized surfacemarkers. CTCs provide rich data about the current tumor statusof the patient, and provide a large enough sample for a variety ofmolecular analyses. The integrated findings from CTC capture(count, molecular contents, surface expression) provide information

Amogha Tadimety

Amogha Tadimety completed herBSE at Princeton University in2014, with a major in Chemicaland Biological Engineering andcertificates in Engineering Biologyand Values & Public Life. Duringthat time she completed researchinternships at the WeatherallInstitute of Molecular Medicine atOxford University and the WyssInstitute at Harvard. Amogha iscurrently pursuing her PhD atDartmouth College’s ThayerSchool of Engineering in the

Zhang Research Group. Her research focuses on the developmentof nanoplasmonic sensors for capture, enrichment, and detection ofcirculating biomarkers, with a focus on low-cost nanostructurefabrication.

Abeer Syed

Abeer Syed received her PhD inBiomedical Engineering from theUniversity of Glasgow, UK in2013 followed by postdoctoralresearch at New York UniversityAbu Dhabi, UAE and theDartmouth College, USA. Herresearch focuses on design,fabrication and use of micro-fluidic systems for low cost,point-of-care diagnostics.

Yuan Nie

Yuan Nie received her BS degreein Mechanical Engineering fromHuazhong University of Scienceand Technology, Wuhan, China,in 2011, and her MS degree inMechanical Engineering fromHong Kong University of Scienceand Technology, Hong Kong, in2014. She is currently a PhDstudent in Thayer School ofEngineering, Dartmouth College,NH, USA. Her main research isfocused on the development of theimmunomagnetic microfluidic

platform for the capture and analysis of rare cancer biomarkers.She is also interested in the finite volume based simulation offluidic dynamics at micro/nano-scale.

Christina R. Long

Christina Long is a Dartmouthundergraduate studying engineeringmodified with computer science.She has been working as anassistant to the Zhang lab groupsince summer of 2015. Her workfocuses on improving methods forthe immunomagnetic capture ofcirculating tumor cells.

Review Article Integrative Biology

Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42

. View Article Online

http://dx.doi.org/10.1039/c6ib00202a


for patient diagnosis, monitoring, drug response, and prognosis.Clinically relevant analysis of CTCs majorly factors into the design offuture liquid biopsies on chip.

Beyond molecular characterization of a population of cells,there have also been a number of recent studies conducted atthe single cell level.19–22 Transcriptional profiling, surfacemarker analysis, and deformability all vary greatly betweenCTCs within the same patient. This heterogeneity is charac-terized in our current gold standard biopsy through mutationanalysis within tumor tissue; similarly, a recent phenomenonhas been shown with CTCs.23 This type of analysis can provideclues as to the status of the full tumor because CTCs may shedfrom different sites within the tumor. Single cell molecularanalysis also has the potential to prevent leukocyte contaminationthrough an additional check on molecular contents. Thus, the

capture and analysis of single CTCs on chip is valuable to thedetermination of prognosis and therapy from liquid biopsies.Because of this, the review will outline technologies with specialattention to platforms that are compatible with a single-cellanalysis.

In order to facilitate the design process of liquid biopsies onchip, this review functions as a guide for microchip develop-ment. The review is structured to explain and evaluate existingtechnologies in the context of cancer biology, throughput,and ease of analysis. We begin this review with the relevantbackground knowledge of invasive and minimally invasivebiopsies, and then delve into the perspectives and limitationsof liquid CTC biopsies on chips. We then present a frameworkwith key parameters for liquid CTC biopsies on chip basedupon state of the art technology and clinically significantdata. We then quickly overview the mechanisms of cancerand the theorized journey CTCs, so that we can critically analyzemethods and existing technologies integrating biomarkers ofCTCs into microchip designs for capture, detection, and analysis.Where appropriate, this review focuses on breast cancer, not onlybecause of its impact on healthcare, but because of power anddepth of existing knowledge to illustrate the opportunities forliquid biopsies on chip.

2. Current biopsy gold standardsand liquid biopsy2.1 Surgical biopsy

Early in the 10th century, Al Zahrawi, an Arab surgeon, inventeda hollow needle to examine abnormal tissues in thyroidglands.24 Since Al Zahrawi’s first recorded biopsy, major foun-dations of biopsies have not changed. Today, the existingcategories of biopsies, surgical, core-needle, and fine-needle,

Fig. 1 Liquid biopsy as a diagnostic and prognostic tool. The informationderived from liquid biopsy can be used for continuous monitoring of thepatients, from initial screening to personalized treatment.

Kasia M. Kready

Kasia Kready, currently an under-graduate at Dartmouth College,studies engineering with a focusin biotechnology at the Thayerschool of Engineering. Broadly, sheis interested in ‘gaps’ in women’shealthcare, nanotechnology, andbioengineering. Kasia has been anundergraduate research assistant inJohn Zhang’s lab since 2014. Herwork involves fabricating nano-plasmonic arrays for biosensingapplications. Additionally, shestudies Arabic, French, and Greek.She is expected to graduate inJune 2017.

John X. J. Zhang

John X. J. Zhang is a Professorat Thayer School of Engineering,Dartmouth College, and anInvestigator of Dartmouth-Hitchcock Medical Center. Hereceived his PhD from StanfordUniversity, and was a ResearchScientist at Massachusetts Instituteof Technology. Dr Zhang is a Fellowof American Institute for Medicaland Biological Engineering, andrecipient of NIH Director’sTransformative Research Award,NSF CAREER Award, DARPA Young

Faculty Award and many other recognitions. Zhang’s research focuseson exploring bio-inspired nanomaterials, scale-dependent biophysics,and nanofabrication technology, towards developing new diagnosticdevices and methods on probing complex cellular processes andbiological networks critical to development and diseases.

Integrative Biology Review Article

Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




still require the excision of suspicious tissues by way of aninvasive or minimally invasive procedure (Table 1).

Surgical biopsies are the most invasive; the patient receivesa local anesthetic, allowing the surgeon to remove all or part ofthe abnormal tissue, nearby normal tissue, and implant a smallpiece of metal to mark the location of the tumor in case furthersurgery is required.25 The removal of part or all of the abnormaltissue conveniently decreases the amount of treatment necessaryfor remission.26 Once excised, the biopsied material is sent topathologists, who then produce a report with clinically relevantinformation indicating the presence of cancerous cells.If cancerous cells are discovered, the type, the grade, and thereceptor status of the tumor are reported along with thelocation of the tumor, the distance of the tumor from normaltissue, and the margins.27 Margins are a measure of how wellthe tumor was removed, and they are broken down into threedifferent types. A positive margin signifies that cancer cells areat the boundary of the tumor and suggest that the tumorhas advanced beyond the immediate area.26 A negative marginindicates that cancer cells are not found at the boundary of thetumor, and that the cancer is localized to that specific region.A close margin means that the distance between canceroustissue and normal tissue is less than 3 millimeters. Surgicalbiopsies grant the most data to be gleaned from the tumor andthe most of the tumor removed.26 However, this data is only asnapshot of the dynamic genetic fluctuations of the tumor, andrepeated surgical biopsies cannot be performed to monitorpatients in real-time because of surgical risks and patientdiscomfort.

2.2 Minimally invasive biopsy

The focus of minimally invasive techniques is obtaining thediagnosis, not complete excision or treatment of cancer. In addition

to reducing morbidity and mortality, minimally invasive techniquesoffer unparalleled cosmetic results and a higher degree of patientcomfort.2 These techniques have allowed for cost-effective andholistic medical care by focusing surgery on negative margins.28–31

Core-needle biopsy (CNB),32,33 fine-needle aspiration cytology(FNAC),34,35 large core biopsy,33,36 and vacuum-assisted corebiopsy35,37 are the cutting-edge procedures.

During a CNB, a hollow needle is inserted into a locallyanesthetized region above the abnormal tissue and a ‘core’ ofthe abnormal tissue is excised. Large core biopsies remove theentirety of small abnormal lesions through the hollow core ofa large needle.38 Fine-needle aspirations are performed whenan abnormal tissue is expected to be fluid filled or when anabnormal tissue is superficial. Fine-needle aspirations are usedto drain the fluid and remove cells for testing from suspectedfluid filled abnormal tissues.38 FNACs excise abnormaltissue from superficial regions through minimally invasivefine-needles.34 Vacuum-assisted core biopsies apply a vacuumto a hollow needle, pulling the tissue into the center of theneedle where it is cut and retrieved after the surgery. All ofthese procedures are guided by imaging techniques such asmammograms, magnetic resonance imaging (MRI), or ultrasounds.These image guidance techniques allow the visualization of thelocation of the abnormal tissue without physically viewing it.

Similar to surgical biopsies, minimally invasive biopsiesdo not support real-time monitoring of the patient’s responseto therapies. Although minimally invasive surgeries are morecosmetically appealing and diminish discomfort in comparisonto surgical biopsies, they require local anesthetics, imagingguidance, and only remove small abnormal lesions. Invasiveand minimally invasive biopsies suffer from a reliance onappreciable symptoms of tumors to signal that the operation isrequired.39 They also require a highly skilled pathologist whocannot robotically reproduce results, and surgeons who couldpotentially perturb the CTCs into proliferation. In some cases,regardless of the surgeon’s and pathologist’s expertise, thetumor could be anatomically challenging to biopsy with sub-stantial risks.39 Even with these results, they do not represent atumor’s heterogeneity or its evolving resistance mechanisms.Without these, cancers cannot be precisely stratified, which isimperative for personalized treatments.

Innovative, non-invasive biopsies must be developed to revampoutdated procedures that rely less upon perceivable symptoms andimage guidance. By the nature of biopsy, symptoms need to bepresent before a biopsy can be performed and definitive diagnosisformed. There is some debate over whether improved life expec-tancy of cancer patients is due to earlier detection or bettertreatment methods. A study of life expectancy by Chie et al. foundthat for breast, cervical, colorectal, gastric, and liver cancer, earlydetection was responsible for a larger share of improved lifeexpectancy than improved treatment methods.40 This result showsgreat promise for the impact of early detection on patient prognosis.

2.3 Liquid biopsy

Liquid biopsies are a non-invasive means of testing the patient’sblood to identify CTCs and circulating tumor DNA (ctDNA) cast

Table 1 Advantages of biopsy techniques. A comparison of surgicalbiopsy,24–26 minimally invasive biopsy,27–38 and liquid biopsy,40–45 includ-ing advantages and disadvantages

Advantages Disadvantages

Surgical| Most data retrieved (type,

grade, receptor status, margins)– Requires local anesthetic

| Removes part or all of tumor – Requires image guidance– Invasive and uncomfortable

for patient– No real-time monitoring

Minimally invasive| Greater patient comfort – Requires local anesthetic| Better cosmetic results – Requires image guidance| Can fully excise small lesions – No real-time monitoring

Liquid biopsy| Does not require appreciable

symptoms for diagnosis– Collecting rare biomarkers

is challenging| Far less invasive – Does not provide a treatment| Enables real-time monitoring – Very few FDA approved

technologies| Does not require pathologist – Location of tumor unknown

– Size of tumor unknown– Margins unknown


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




out by the primary tumor and metastatic sites.41 As such, liquidbiopsies are appealing because they offer an up-to-date insightinto the patient’s condition and allow for situation-specificresponses and informed treatment choices.42 A number ofsamples, including blood, plasma, urine, CSF, and breast milkare being studied for liquid biopsy platform development.41

Apart from providing accurate information regarding the cancer’sadvancement and intensity, liquid biopsies can effectively gaugethe body’s responses to the chosen method of treatment, and theycan detect early recurrence and identify possible causes behindresistance to treatment.43 Liquid biopsy analysis can combine withongoing treatment to map molecular disease changes, allowingclinicians to chart the growth of secondary resistance and isolateheterogeneous subclonal populations of tumor cells that continueto grow.44

Liquid biopsy enables real-time monitoring of ongoingchanges in the tumor at the molecular level, allowing for moreextensive insights into disease state and progression.44 The useof liquid biopsies holds significant potential and it is likely toreplace extensive imaging and invasive tissue biopsies. Likely,liquid biopsies will frequently monitor and inform cancertreatment choices. With technological advances, liquid biopsymay conceivably detect early tumors yet invisible on conven-tional imaging.

2.3.1 Challenges of liquid biopsy. There are a number ofchallenges associated with liquid biopsy, including the fact that

the margins, grade, tumor size, and exact location cannot bedetermined from liquid biopsy alone. Because of this thereare a number of recent studies validating liquid biopsy andbenchmarking it to gold standard. There are also biologicalchallenges associated with CTC capture as a diagnostic. Adulthumans have 5 L of total blood; considering the rarity of theCTCs, a small aliquot of the patient blood (7.5 mL) might not beenough to detect the cells. Of patients with progressive breastcancer, only 1.43% reported more than 500 CTCs per 7.5 mL ofblood.45 CTCs are difficult to detect because they may undergofiltration in smaller capillaries or become cloaked by plateletsand coagulation factors, thereby escaping the immune system(Fig. 2).46 Any CTC detection system has to be sensitive enoughto detect that small number of cells assuming that CTCs are notlost during preprocessing and multistep batch purification.

3. CTC liquid biopsy framework

Information from CTC capture can aid in predictive screening,diagnostics, prognostics, targeted therapies, and monitoring.For predictive screening and diagnostics, just the presence andenumeration of CTCs is valuable, and the analysis may includevalidation that the captured cells are CTCs and whether thecell is metastatic. A more advanced diagnostic may measurethe surface markers, secreted proteins, or mutations inherentwithin the CTCs genome. For determining patient prognosis,enumeration is very important, as number of captured CTCs hasbeen shown to correlate with overall survival rate. A mutational orepigenetic analysis may also be done because certain mutationsresult in a more aggressive cancer presentation. Similarly formonitoring, the number of captured CTCs and whether theydecrease as a patient is being treated can give the clinicianvaluable information about patient status and recurrence ofdisease. In terms of targeted therapies, a more in-depth levelof genomic analysis may be done or drug response can beevaluated by CTC culture. Through this method clinicians canlearn which treatment the CTCs respond to most readily, whichcould help them prescribe the correct therapy from the start.Some advantages of liquid biopsy include the possibility forserial sampling, automation, and reproducible results. For eachof the applications discussed, including diagnostics, monitoring,and prognosis, the ability to serially sample and automate thetesting allows for validation and confirmation of the result(Table 2).

When designing liquid biopsy chips, it is ideal to integratecapture, detection, and analysis into simple platforms. Captureis the first step, and involves separating out CTCs from thestarting sample, most commonly whole blood. A number oftechniques exist for CTC capture and involve separating CTCsbased on distinctions in physical or chemical characteristicsfrom other cells. This can involve binding the antigens, separatingthem by size or optical properties, or by mechanical properties.The ideal capture technology would have a high capture rate(above 90% effective), high throughput (milliliters of sample ranin a few hours), low contamination rate, and do minimal damage

Fig. 2 A schematic representation of challenges in CTC research. CTCsare difficult to detect because they may (A) undergo filtration in smallercapillaries, (B) form clusters likely to lodge in capillaries, (C) becomecloaked by platelets and coagulation factors, thereby escaping theimmune response, (D) may not provide useful information, (E) are hetero-geneous, (F) show stemness, (G) show partial EMT, and (H) unclearmetastatic seeding potential. Reprinted from Science, 341, Plaks, Circulatingtumor cells, 1186–1188, Copyright (2013), with permission from AAAS.


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




to the cells (above 90% cell viability). This would mean that thetechnology quickly picks out the most CTCs while leaving behindother types of cells, and does this quickly and leaves the cellsviable. The capture technologies discussed later in the paper willbe evaluated based on these criteria.

A very similar set of criteria can be used to evaluate thequality of a method of detection, or confirming the presence ofthe captured CTCs. Among available methods include immuno-fluorescent staining, light microscope imaging, nanoparticlebased methods, and label-free detection. A clinically translatabledetection method would allow for high throughput (milliliters ofsample ran in a few hours), enumeration and high detectionefficiency (above 90% effective), and significant contrastbetween the CTC and both the background and other cells.Ideally, an advanced detection method would maintain cellviability for analysis (above 90% cell viability).

Finally, the analysis step may be as simple as enumerationbut often involves a molecular analysis of the captured cells.First, the effectiveness of the enumeration through analysisshould be confirmed because of the importance of CTC count.This analysis step is key to benchmarking the liquid biopsytechnology against current gold standard. Because of this, theaccuracy of the analysis, repeatability, and comparison tocurrent clinical standard are some of the criteria for evaluationof a method of CTC analysis. Based on the kind of molecularanalysis that is typically done on a tumor biopsy, the analysis ofthe CTCs should be able to provide similar data. The best wayto do this kind of validation is to run, in parallel and on thesame patients, the liquid biopsy assay and a tissue biopsyanalysis. An analytic validation of the diagnostic capabilitiesrequired for FDA approval of a liquid biopsy chip.47 Theanalysis methods reviewed in this paper will be described interms of their accuracy, repeatability, and in the context of thecurrent gold standards.

Oftentimes it makes the most sense to work backwards fromthe clinical need to determine the best capture and detectiontechnology. For example, immunofluorescence staining and fixa-tion can damage the cells, so it would not be an apt technology touse if you wanted to culture or do a genomic analysis on the cellsafterwards. In this case, you may want to use a label free methodof capture and detection, such as optical detection or mechanicalmethod, in order to minimize the potential harm to the cells.On the other hand, if the goal is simply to detect whether CTCsare present and to count them, antibody–antigen bindingfollowed by immunofluorescence staining may be the mostsuccessful method because of its demonstrated capture effi-ciency and ease of detection. Where appropriate, this paper will

describe these design decisions and the possible technologiesdepending on desired analysis.

4. Biological background:mechanisms, EMT, & biomarkers

Integrating biology on chip for CTC capture, detection, andanalysis requires an understanding of the underlying biologicalworkings of cancer. In this section we discuss the mechanismsof cancer, the epithelial mesenchymal transition (EMT), bio-markers, and corresponding molecular analyses. The mecha-nism of cancer explains how six features of cancer are necessaryfor malignant growth and how tumor cells evade the hostimmune system, and many of these features underlie thechallenges associated with CTC capture, detection, and analysis.Presumably, EMT boosts the challenges posed to CTC capturebecause the process involves additional heterogeneity during theprocess of cancer metastasis. Finally, some of the molecularstudies discussed show this variability among CTC populationsare the basis for single cell capture and analysis technologies. Thisbiology section serves as a background for understanding relevantbiomarkers and optimizing liquid biopsy chip design.

4.1 Mechanisms of cancer

The unified efforts of researchers to understand the mechanismsof cancer have assembled an abounding body of knowledge thathas elicited a well-accepted theory regarding the genesis of cancer.Hanahan and Weinberg listed six hallmarks of cancer (Fig. 3), andlater other critical features have been used as the starting pointfor cancer research.48,49 Any combination of three of these sixfeatures are necessarily characteristic of any malignant growth:sustaining proliferative signaling, evading growth suppressors,activating invasion and metastasis, enabling replicative immor-tality, inducing angiogenesis, and resisting cell death.

4.1.1 Self-sufficiency in growth signals. For normal cells totransition from a quiescent state to an active proliferative state,they need mitogenic growth signals transmitted into the cellthrough transmembrane receptors. These membrane receptorsbind typical classes of signaling molecules, and Hanahan andWeinberg stated that no normal cells could proliferate in theabsence of such signals. This signaling requirement is evidentwhen propagating normal cells in culture because diffusiblemitogenic factors and a proper substratum are necessary tofacilitate proliferation. Many oncogenes in the cancer catalogare capable of mimicking normal growth signaling, so cancer

Table 2 Technology analysis metrics. Methods of evaluation of capture, detection, and analysis techniques to be covered in this review

Capture Detection Analysis

| High throughput | High throughput | Effective enumeration| High capture efficiency | High detection efficiency | Accuracy| Low contamination | Minimal damage to cells | Repeatability| Minimal damage to cells | Contrast with background | Comparison to clinical Gold standard

| Contrast with contaminant


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




cells are less dependent on stimulation from normal tissuemicroenvironments.48,50

4.1.2 Insensitivity to antigrowth signals. In order to maintaincellular quiescence and tissue homeostasis, a number of anti-proliferative or growth-inhibitory signals are transmitted bytransmembrane cell surface receptors. Such signals may blockproliferation by forcing the cells out of proliferative cycles into thequiescent (G0) state. Alternatively, the signals may stimulatecells into permanently surrendering their proliferative ability byinducing them to enter post-mitotic states. For developing cancercells to function and thrive, they must evade such signals.48,51

4.1.3 Evading apoptosis. Apoptosis, or programmed celldeath, in addition to other causes of attrition, is an importantfactor in determining the growth of a cell population. Resistancetowards programmed deaths is characteristic of cancer. Tumorcells acquire resistance to apoptosis through a number ofstrategies, most often through a mechanism involving the p53tumor suppressor gene.48,52

4.1.4 Limitless replicative potential. All types of mammaliancells have an autonomous function that limits their uninhibitedpropagation, acquiring self-sufficiency in growth signals, insen-sitivity to antigrowth signals and resistance to apoptosis. Normalcells in culture have a limited replicative potential and eventuallyachieve senescence, a state where the cells stop replicating aftera number of duplications. For any group of tumor cells tocomplete the progression into a life-threatening, macroscopictumor, they have to become immortal and achieve limitlessreplicative potential at some stage of the multistep tumordevelopment.48,53

4.1.5 Sustained angiogenesis. When a tissue is formed,angiogenesis, the formation of new blood vessels is carefullyregulated. Cells within aberrant proliferative lesions lack thisability in the initial phase, which in turn curtails their expandingcapability. In order to grow significantly, incipient neoplasiasneeds to develop sustained angiogenic ability.48,54,55

4.1.6 Tissue invasion and metastasis. Metastasis, thecolonization of distant parts of the body by pioneering tumorcells, and the invasion of neighboring tissues enables cells tomove out of the primary tumor and propagate to new partswithin the body with better access to space and nutrients.The process of metastasis and the associate genetic and bio-chemical determinants are not completely understood.48,56

Since it is believed that at least three of these hallmarks aremandatory for malignancy, different categories of cancers arise;each one possibly shedding off a variety of heterogeneousstrata of CTCs with contrasting phenotypic and functionalcharacteristics.

4.2 Epithelial mesenchymal transition

In addition to the heterogeneity of cancer developed during itsgenesis, a second tier of heterogeneity is layered on through thehypothesized, and not well understood, process of epithelialmesenchymal transition (EMT). EMT is believed to simulate aspatial and temporal flux of CTCs between different diseasestates (epithelial, stemness, and mesenchymal) during primarytumor metastasis.57 First off, EMT enables an apical-basalpolarized epithelial cell to transition through a number ofbiochemical changes to become a mesenchymal cell, leadingto improved migratory capacity, invasiveness, improved resis-tance to apoptosis, and highly elevated extracellular matrix(ECM) component production (Fig. 4).58 Numerous molecularprocesses such as specific cell-surface protein expression,cytoskeletal protein rearrangement, synthesis of ECM-degrading enzymes, and changes in specific microRNAexpression are involved in initiating the EMT process andfacilitating its completion.59,60 Finally, the completion of thisbiological process is marked by the destruction of the under-lying basement membrane, giving the cell, now classified asmesenchymal, the ability to escape the epithelial layer inwhich it was formed.

Fig. 3 Schematic representation of six hallmarks of cancer. The cancerous cell produces sufficient growth signals while blocking the antigrowth signalsto proliferate uncontrollably. Cells evade apoptosis, angiogenesis and activate metastasis. Reprinted from Cell, 14(5), Hanahan and Weinberg, Hallmarksof cancer: the next generation, 646–674, Copyright (2011), with permission from Elsevier.


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




Another type of cancer cell that supposedly emerges duringEMT is a tumorigenic circulating cancer stem cell (CSC). CSCs areoften seen as the most dangerous because they mimic physio-logical cells, maintain multipotency, sustained self-renewalabilities and capabilities to initiate tumors.61 Researchers offertwo hypotheses on the origin of CSCs. The first hypothesisproposes that during EMT cancerous somatic stem cells movefrom the primary tumor into the blood and become mesenchymalCSCs. The second hypothesis claims that circulating CSCs mayoriginate from fully differentiated cancer cells and then adoptmigratory properties during EMT as new pathways are developed.A number of studies have indicated that CSCs develop fromdifferentiated progenitor cells or somatic stem cells. Sun andQui (2013) claimed that only this particular subpopulation isresponsible for relapse or metastasis in cancer patients.62 If it isthe case that CSCs are the major cause of metastasis, presentand future CTC research should focus on recognizing thisspecific circulating-CSC population.

There is a notable difference between normal and pathologicalEMT. EMT is a process to disperse cells in embryos and causemesenchymal cells in injured tissues to induce invasive/metastatic behavior of epithelial cancers.59 All the stepsinvolved in EMT during the embryological process are notcrucial for establishing invasive phenotypes, and partial epithelialmesenchymal transition is sufficient to initiate tumor cells.63

Researchers must improve upon the theory of EMT to fully

understand how pathological EMT differs from normal EMTand the essential signals necessary for transitioning betweenthe stages of EMT.

4.3 Molecular heterogeneity

Research on CTCs is in the process of transitioning fromcapture and enumeration to molecular characterization ofthe cell’s surface and contents.64 Significant variability hasbeen found from transcriptional profiling and surface markeranalysis, leading to an understanding that CTCs are highlyheterogeneous.65 The heterogeneity of cells in primary breastcancer is visible in distinct mutations within the same slice oftumor tissue, but this principle was only recently shown inCTCs.65 In 2012, it was shown that CTC variability is highlyconsistent with tumor tissue variability, meaning that pheno-typing of CTCs will bring to light many of the characteristicspresent in the tumor itself.65

Recently, Ni et al. developed a method for whole genomeamplification from single lung cancer CTCs to capture informa-tion for individual therapy and drug resistance.23 After capturingCTCs, a method of multiple annealing and looping was usedto determine variations and deletions in the CTC exomes.It was found that individual patients had reproducible patternsof copy number variation, and that these variations differedbetween metastatic and non-metastatic tumors. Additionally,single nucleotide variations on a specific genome can strongly

Fig. 4 Schematic of EMT. During EMT, epithelial cells undergo cytoskeletal rearrangements, synthesize ECM, lose cell–cell junctions and showincreased motility. The increased motility enables them to migrate to other organs through the blood vessels where they proliferate to initiate metastases.‘‘Reprinted from Nature Medicine, 19, Tam and Weinberg, The epigenetics of epithelial-mesenchymal plasticity in cancer, 1438–1449, Copyright (2013),with permission from NPG.


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




indicate metastasis. This variability shows the additional layerof heterogeneity due to specific genomic mutations that can befound in CTCs.23

The two major classes of single cell molecular profilinginclude mutational profiling studies and gene expression profilingstudies.39 Both of these can elucidate the regulatory status of thetumor cells because they will shed light on the mutations or theover- and under-regulation of specific tumor-related genes. Amongthe molecular techniques used for mutational profiling are Sangersequencing21,66 and comparative genomic hybridization (CGH).20,67

These studies show a high level of copy number variation andheterogeneity in expression-related mutations in a variety of cancersincluding breast, prostate, and colorectal cancer. For the expression-related studies, RNA analysis is typically performed and can be donethrough qRT-PCR microarray or RNA sequencing panels.65,68 Theseexperiments can show profiles distinct from white blood cells, butalso unique to specific cancer types. These studies also show a widerange of regulation of tumor and growth factor related genes.

These types of molecular analyses performed on populationsof CTCs demonstrate the heterogeneity of CTCs. An under-standing of the molecular heterogeneity of CTCs is particularlyuseful because it will inform development of targeted thera-peutics for personalized medicine. Thus, the capture and analysisof single CTCs on chip is hugely valuable to the determination ofprognosis and therapy from liquid biopsy. As it stands, there arevery few technologies that employ novel, highly efficient capturetechnologies with single-cell level analysis. Some examples ofhybrid devices to be developed include a monitoring device thatalso measures drug response at the single cell level or a diagnosticdevice that measures surface marker expression.

4.4 Biomarkers

Biomarkers are commonly used in research, both clinical anddiscovery-based; therefore, many disease-state specific bio-markers have been defined and characterized.69 Biomarkers can

include clinical signs such as pulse or blood pressure, or bio-chemical signs such as surface markers and metabolites.69 Aworking definition according to the World Health Organizationand the UN is ‘‘any substance, structure, or process that can bemeasured in the body or its products and influence or predict theincidence of outcome or disease’’.69

In the case of liquid biopsy of CTCs, there are two mainclasses of biomarkers that this review covers in detail. The firstis surface protein biomarkers, mostly antigens that can beexploited for the capture and detection of CTCs. There are anumber of heterogeneous sub-populations of CTCs, and thesurface antigens of CTCs vary widely with function and statethroughout EMT.70 The epithelial, mesenchymal and stemnessmarkers are the most recognized families of antigens found onthe circulating tumor cells and will be discussed in more detailin this section.70

There are also some studies that infer the presence of CTCsthrough expression-level analysis by capture of DNA ormRNA.39 These studies either analyze specific genes (suchas cytokeratin-19 and cytokeratin-20)71 or detect epigeneticpatterns such as methylation.72 Such analysis determines thespecific mechanisms of aberrant behavior and similar geneticanalysis provides clues as to the best method of treatment(Table 3).

4.4.1 Protein biomarkers for CTC capture & detectionEpithelial CTC markers. Post CTC enrichment, if evidence of

two or more of the markers – EpCAM, E-cadherin, cytokeratins– is found at single cell level, a particular CTC can be markedas a differentiated epithelial cancer cell; however, it shouldbe confirmed that stem and mesenchymal markers are notobserved.

Epithelial cellular adhesion molecule. Epithelial cellularadhesion molecule (EpCAM) is a cell surface glycoprotein foundin epithelial cells. Its expression in epithelial cancer cells ishigher than in normal epithelial cells, so it differentiates cancer

Table 3 Biomarkers for CTC capture, detection, and analysis. Surface protein biomarkers based upon EMT stage, detection biomarkers, and commongenes for analysis along with references

Biomarkers for CTC capture, detection, and analysis

Description Application Ref.

EpithelialEpCAM Surface glycoprotein, downregulated in EMT Capture, detection, enumeration 70, 73 and 74E-cadherin Component of adherens junctions, involved in cell architecture Capture, detection, enumeration 75 and 76Cytokeratins Cytoskeletal filaments, downregulated in EMT Capture, detection, enumeration, analysis 71, 77 and 78

MesenchymalN-cadherin Adherens junctions, upregulated in EMT Capture, detection, enumeration 79–81Vimentin Mesenchymal phenotype, enhances mobility Capture, detection, enumeration 80Plastin-3 Induces EMT, activates mesenchymal genes Capture, detection, enumeration 82 and 83

StemnessALDH-1 Correlates to stemness characteristics and patient outcome Capture, detection, enumeration 84CD44 Cell surface glycoprotein, cell migration and metastasis Capture, detection, enumeration 89

GenesKRAS Commonly mutated in many cancers, especially colorectal Analysis 39VIM EMT associated gene, often used for qPCR Analysis 65BRMS1 Breast cancer regulator, epigenetic analysis Analysis 72


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




cells from healthy epithelial cells.73 The development ofEpCAM as a biomarker was mainly carried out by CellSearchsystem, and was used for the enumeration and detection of theCTCs (Veridex, Warren, New Jersey, USA).74 However, theexpression of EpCAM depends on the stage of EMT, whichwhen regulated downwards, is expressed in combination withN-cadherin and Vimentin and can eventually disappear fromcell surfaces.70 This means that EpCAM alone as a surfacebiomarker will not capture all CTCs at various stages of EMT.

E-cadherin. As a component of adherens junctions (whichmaintain cell–cell interactions), E-cadherin is a hallmark ofepithelial cells and functions closely with the actin cytoskeletonto provide resistance against cell detachment. Like some otherproteins, E-cadherin is also involved in supporting the epithelialtissue architecture and its regulation plays an important role inthe progression of epithelial mesenchymal transition: the expres-sion decreases as the cell approaches mesenchymal state.75

E-cadherin may be inactivated or down-regulated by initiationof processes such as hypermethylation, histone deacetylation,and transcriptional repression once the cancer cells manage toget out of the primary tumor.76

Cytokeratins. Cytokeratins (CKs) are the largest class ofintermediate filaments of the cytoskeleton and are the bio-markers of regular epithelial differentiation. CKs are used as adiagnostic tool for the detection of circulating carcinoma cells.To enable the detection, antibodies have to recognize mostepitopes displayed by cancer cells. Epithelial cancer cells,besides expressing CKs, also express K8, K18 and K19. Thesespecific markers are expressed by cells with epithelial phenotype;however, their expression is down-regulated during epithelialmesenchymal transition.77 Enabling the use of anti-CK anti-bodies in combination with others helps in tracking cells thatlose the partial or whole epithelial phenotype.78

Mesenchymal-like CTC markers. Varying degrees of EMT takeplace in individual cells, making it hard to define a mesenchymalCTC subpopulation in pure state as they evolve from epithelialstatus to a mesenchymal state. The identification of mesenchymalbiomarkers only shows the active status of an EMT process. WhenEMT is over and before the transition to the next stage, mesenchymalepithelial transition (MET), these markers can aid in distinguishinga pure population of cells with mixed phenotype (both epithelialand mesenchymal features). A cell can be defined to have amesenchymal character when two mesenchymal markers areconnected to epithelial or stem markers.70

N-cadherin. E-cadherin and N-cadherin subfamilies constitutethe proteins of adherens junctions and N-cadherin is expressed innumerous cells, including the mesenchymal cells, making it apotential biomarker of EMT. While EMT is occurring, N-cadherinis active while E-cadherin is inactive. Upregulation of N-cadherin isconsidered a hallmark of mesenchymal characterized CTCs.79

Vimentin. Vimentin is a protein expressed in mesenchymalcells that induces a mesenchymal phenotype and enhancesmobility.80 Vimentin has also been found in normal blood cells,

however, Lustberg et al., who conducted this research, neverfound a cell that was positive for both CD45 and Vimentin. Thisfinding indicates that Vimentin cannot mark hematopoieticcells. Vimentin expression alone cannot differentiate a mesenchymalphenotype from an epithelial phenotype, and evidence supportingthe efficiency of this biomarker in identifying mesenchymal cellsneeds to be demonstrated.81

Plastin-3. It has been shown that Plastin-3 (PLS3) has theability to induce EMT in colorectal cancer cells.82 While thisstudy focuses on the colorectal cancer cells, one can designatethis factor as a biomarker of mesenchymal CTCs because itactivates a high level of mesenchymal genes.67 Plastin-3 canalso be considered a putative clue to differentiate CTCs withboth epithelial and mesenchymal traits.83

Stemness-like CTCs eiomarkers. This strata of CTCs, CTCswith stemness phenotypes and CSCs, are thought to be respon-sible for relapse or metastasis in cancer patients.30 Differentiatingand isolating CSCs among CTCs will allow a fuller understandingof prognosis and treatment plans. The following markers may beused to identify CSCs in blood.

Aldehyde dehydrogenase-1 (ALDH1). ALDH1 is expressed in1–2% of breast epithelial cells and was first described in a studyby Ginestier et al.84 The group was able to demonstrate ALDH1as a biomarker for increased risk of breast cancer usingimmunostaining. Although a number of cells have this char-acteristic, only a few are able to grow into differentiated solidtumor in NOD/SCID mice.84 Barriere et al. conducted a clinicaltrial with the aim of detecting CTCs in early breast cancerdiagnosis and found that of the 130 patients in question,49 were CTC positive and 17 of those were ALDH1 positive.These results show the presence of stemness CTCs in bloodsamples of cancer patients without metastasis.85 This level ofbiological understanding can inform specific personalizedtreatments.

CD44. CD44, a cell surface glycoprotein, has been implicatedin cell migration and metastasis.86 Breast CSCs have the ability tointravasate into the blood; their characteristics are defined by theexpression of CD44+/CD24low/�/Lin – cell surface markers.The involvement of CSCs in tumorigenesis was shown byAl-Hajj et al. who demonstrated that the transplantation of asfew as a hundred CD44 CD24-breast carcinoma cells was enoughto initiate differentiated solid tumor growth in NOD/SCID mice.On the other hand, even 105 CD44�/CD24+ tumor cells did notinitiate the growth of tumors in animal models. It is worthremarking that cells populations with CD44+/CD24low/� maynot necessarily coincide with cell populations consisting ofALDH1 positive cells.87

This evidence suggests that these proteins may be helpful indifferentiating between epithelial and mesenchymal charac-teristics of stem cells.88 Alternative splicing of CD44 can resultin a number of isoforms. A few of these isoforms can act ashighly specific markers of stemness and need to be extensivelystudied. Similarly, CD44 is encoded by 20 exons, and 10 ofthem can be regulated by alternative splicing; these are named


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




variants of v exons. The isoforms obtained are expressedexclusively in proliferating cells, cancer cells in particular. CSCsexpress CD44v6 in colorectal cancer and this expression givesthe CSCs metastatic ability. When it comes to the detection ofCSCs, CD44v6 appears to be one of the best markers.89

4.4.2 Expression biomarkers for analysis. A number ofstudies have used quantitative PCR (q-PCR) or quantitativereal-time PCR (qRT-PCR) to measure the gene expression ofcaptured CTCs.65 The specific panel for analysis is dependenton the type of cancer, and can include several genes andmutations of interest. Powell et al. studied the transcriptionof 87 cancer-related and control genes among a population ofbreast cancer CTCs and cell lines to determine the heterogeneityassociated with different subgroups.65 Among the genes studiedwere metastasis-associated genes NPTN, S100A4, S100A9, andepithelial mesenchymal transition associated genes VIM,TGFß1, ZEB2, FOXC1, and CXCR4. These provide a backgroundof the types of genetic biomarkers that are used for analysis ofcaptured CTCs. Another application of qRT-PCR was inmeasurement of mRNA markers for determination of patientprognosis.71 This group used carcinoembryonic antigen (CEA),cytokeratin (CK) 19, CK20, and CD133 (CEA/CK/CD133) mRNAmarkers.71

Another group used qPCR on whole genome amplified DNAto determine specific mutations in the KRAS gene in patientswith colorectal cancer.90 They studied a number of clinicallyrelevant mutations, including c.34G 4 T, c.34G 4 A, c.34G 4C, c.35G 4 T, c.35G 4 A, c.35G 4 C, and c.38G 4 A in genomicDNA samples in order to quantify the heterogeneity of muta-tions among the CTC genome.90 Different primers were usedfor each of these point mutations in order to ensure singlenucleotide specificity.

Another method of analysis determines epigenetic and regu-latory changes to the genome by measuring sequence-specificmethylation.72 They specifically studied the methylation of thebreast cancer metastasis suppressor-1 (BRMS1) promoter. Theyfound that methylation was not detected in noncancerous tissue,but in CTCs and breast cancer tissue more than one third ofsamples had methylated promoters.72 This study shows anotherbiomarker, the BRMS1 promoter, which can be used for analysisof CTC heterogeneity and expression.

Methods of CTC analysis attempting to understand geneexpression and regulation use a number of molecular biologytechniques including amplification and epigenetic monitoring.Some of the relevant genes include VIM, CK, the KRAS gene,and, specific to breast cancer, the BRMS1 promoter. Any ofthese genes can be targeted and studied after CTC separationand detection in order to monitor regulatory changes in thetumor environment.

5. Macroscale technologiesand challenges

As a precursor to liquid biopsy on chip, macroscale liquidbiopsies such as ISET,91,92 Magnetic Activated Cell Sorting

systems (MACs),93 CellSearch,93 MagSweeper,93 and GILUPINanomedizin,94 strive to make the detriments of surgical biopsiesand minimally invasive biopsies obsolete. These devices strengthis the capture of CTCs in the pursuit of a snapshot of the globalgenomic fluctuations of a tumor. Some devices, such as ISET, arebetter adapted for downstream analysis of captured CTCs. Withthe exception of GILUPI Nanomedizin, these devices require asimple blood draw, afflicting the patient less than minimallyinvasive biopsies. The scrutiny of liquid biopsy on chip againstthese technologies at each iteration of the design process enablesmicrochip developers to hone in on an elegant platform.

Sized-based filtration is a rudimentary macroscale methodof CTC separation. One of the most commonly used techniquesincludes a physical filtration based ISET array developed byVona et al.91 Using as little as 1 mL of blood, ISET can detecta single micropipetted CTC; additionally, ISET enumerates,characterizes immunomorphologically, and lends itself wellto molecular analysis, such as Fluorescence in situ Hybridization(FISH) and real-time PCR (RT-PCR) of CTCs.91 ISET essentiallyfilters CTCs that are larger than leukocytes; however, leukocytesrange in sizes comparable to CTCs, which commonly contami-nates and impedes the effectiveness of size-based enrichment(Fig. 5a). Furthermore, smaller CTCs are not captured in thefiltration system.

More predominant and mainstream approaches buildupon Magnet Activated Cell Sorting Systems, MACS (Fig. 5b).Typically, MACS systems magnetically force CTCs labeled withmagnetic particles to the side of conical tubes in whichthe samples are stored. Unlabeled cells are flushed, and themagnetically labeled CTCs are removed from the tube afterwithdrawing the external magnetic field.93 Manual MACS systems,such as those developed by Miltenyi Biotech, offer low throughputs,prolonged separation times, and weak external fields generated bybulky magnets for the enrichment of rare cells.93

The only FDA approved and commercial system for theseparation of breast, colorectal, and lung CTCs is CellSearch(Fig. 5c). CellSearch is a two-part system for the capture andenumeration of EpCAM and Cytokeratin positive CTCs labeledwith ferrofluids. CellSearch is limited to capturing CTCswith epithelial phenotypes, and possibly neglects CTCs withmesenchymal and stemness phenotypes. Furthermore, afterfluorescence imaging for enumeration, captured CTCs arere-suspended in a solution. The troublesome recovery of theseCTCs for further analyses hinders the effectiveness of CellSearch.93

Another device-based macroscopic system for the capture ofCTCs is MagSweeper (Fig. 5d). A magnetic rod whirls above thebottom of a well containing a sample of CTCs labeled withmagnetic particles. Weakly bound cells are flushed from therod, and the strongly bound CTCs remain. This system canaccommodate multiplex assays and screen multiple samplessimultaneously. However, the magnetic rods are covered in asheath for easy separation of the CTCs from the magnetic rodthat consequently weakens the magnetic force and thwartssome of its capture efficiency.93

GILUPI Nanomedizin (Germany) developed a minimallyinvasive liquid biopsy, in which a biocompatible structured


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




medical Seldinger guidewire (FSMW) functionalized with anti-EpCAM antibodies is inserted into the cubital vein for half anhour (Fig. 5e). After removal, the wire is rinsed, and theattached CTCs counted by immunocytochemically stainingwith EpCAM and/or cytokeratins.94 This device can samplelarge volumes of blood with minimal distress to the patient.Unfortunately, the hydrodynamic forces acting on the targetCTCs cannot be controlled, which could interfere the devicescapturing capabilities.

The popularity and success of immunomagnetic separationtechnologies is a sound takeaway from this brief reviewof macroscale liquid biopsy technologies. However, there iscompelling evidence to suggest that it is advantageous toincorporate multiple separation methods into a simple platform.Additionally, it is imperative to juxtapose future microscaletechnologies in the pipeline towards clinical adoption; not onlywith industrial gold standards, but also with macroscale equiva-lents to fully assure that what is being developed is of clinicalimportance and practicality.

6. Microscale technologies

There are many advantages of using microfluidics in cancer cellenrichment and isolation. Microfluidics can minimize the totalsteps involved in cell pre-treatment and reduce lost or damagedcells.95 Additionally, microscale technologies consume smallsample and reagent volumes, are low cost, and support higherthroughput, sensitivity, resolution, portability, and automation.96

At the microscale, physical forces like laminar flow, diffusion,fluidic resistance, and surface tension become dominant.97 Shiftingto a microscale also leads to the increase in relative amount of

surface area exposure that a volume of fluid experiences. Whenthe surface area contact increases, it leads to quicker diffusionand elevated heat conduction. Laminar flow enables free-flowingadjacent perfusion of various reagents that are useful in thelocalized treatment of cells, and when used in combination withcontrolled diffusion, it can aid the construction of chemicalgradients with subcellular resolution.98

The main strategies at the microscale include positiveenrichment, negative enrichment, and label-free techniques.Positive enrichment typically refers to a process that selectsfor the CTCs while leaving the other particles behind.99 Thistypically has a very high accuracy, and one of the most commonmethods involves antibody tagging surface antigens on theCTC. Another strategy is negative enrichment, which involvestargeting and removing other types of cells, in this case whiteand red blood cells.95 This generally leads to lower purity butdoes not have the challenge of removal of binding probes fromthe surface of the CTC, and is often able to bypass the challengeof CTC heterogeneity.99 A label-free technique is one thatavoids biochemically tagging the desired molecule.100 Thismeans that rather than using immune affinity for capture orsample cleaning, another method that does not involve labelingcells is used such as size-based, mechanical property based,acoustic, or optical. The following are methods employed in thefield of microfluidics to capture and separate CTCs (Fig. 6).

6.1 Hydrodynamic

The hydrodynamic method of CTC separation, deterministiclateral displacement (DLD), operates by causing the smallparticles to follow the laminar flow streams whereas largeparticles are continuously forced to change their laminar flow

Fig. 5 (a) Physical separation based ISET. CTCs are captured by a filter that allows red blood cells and leukocytes to pass through. The first two panelsshow individual isolated CTCs, the third shows a cluster. Reprinted from British Journal of Cancer, 105, Farace, A direct comparison of CellSearch andISET for circulating tumour-cell detection in patients with metastatic carcinomas, 847–853, Copyright (2011), with permission from NPG. (b) Schematicof magnetic activated cell sorting systems (MACS). CTCS are captured by immunomagnetic particles that cling to the side of a tube in the presence of amagnetic field, allowing red blood cells and leukocytes to elute away. Reprinted from Lab on a Chip, 14(3), Chen, Multiscale immunomagneticenrichment of circulating tumor cells: from tubes to microchips, Copyright (2013), with permission from RSC. (c) CellSearch. An FDA two part separationand enumeration platform that captures CTCs with epithelial phenotypes. Reprinted from Lab on a Chip, 14(3), Chen, Multiscale immunomagneticenrichment of circulating tumor cells: from tubes to microchips, Copyright (2013), with permission from RSC. (d) MagSweeper. A magnetic rodfunctionalized with EpCAM antibodies swirls above the bottom of a dish containing whole blood, capturing CTCs. Reprinted from Lab on a Chip, 14(3),Chen, Multiscale immunomagnetic enrichment of circulating tumor cells: from tubes to microchips, Copyright (2013), with permission from RSC.(e) GILUPI Nanomedizin. An antibody conjugated medical wire is inserted into the vein for 30 min, capturing CTCs circulating in the blood of the patient.Reprinted with permission from authors.


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




streams causing them to be displaced laterally.101 This methodis well suited for label-free and negative enrichment of CTCswithout significantly harming them. However, since leukocytesand CTCs are comparable sizes, this method suffers fromsignificant leukocyte contamination and low throughput.

6.2 Dielectrophoretic

When subjected to an inhomogeneous electric field, a cellbecomes polarized, but the electrostatic forces on the two endsof the dipole are not equal inducing dielectrophoretic (DEP)based cell sorting.102–104 This method is label-free, has moderateto high capture efficiencies, and high throughput, but moreneeds to be understood about how a CTC charges changethroughout the metastatic journey.

6.3 Acoustic

An external transducer is used to generate a standing ultrasonicwave across a microchannel. When the cells experience this force,they either move towards the pressure node or the anti-node.105

This method is label-free, offers high throughput, and has demon-strated detection of pigmented cancer but still suffers from leukocytecontamination and limited number of detectable cancers.

6.4 Size-based filtration

Size based filtration involves fabricating pores of appropriatesize so that the RBCs can pass through whereas the WBCs andCTCs can be collected from the filter.106,107 This method hasthe potential to be used for label-free, positive, or negativeenrichment of CTCs with high capture efficiencies while mini-mally damaging them. However, leukocyte contamination andmoderate throughput are hindrances. Sized based filtration tendsto allow more deformable CTCs with a greater metastatic potentialto pass through the filter.

6.5 Immunomagnetic

Scientists widely study immunomagnetic separation methods,and they have developed numerous capture platforms. Simply,this method involves conjugating an antibody to a magneticparticle, incubating the functionalized magnetic particle with asolution containing cells to capture them, and applying amagnetic field to separate them from the bulk solution.67,108

Positive immunomagnetic capture permits integration ofcancer biology into the capture mechanism, and generallydemonstrates high throughput, high capture efficiency, andlow leukocyte contamination. Negative immunomagneticenrichment has been employed to improve the diversity andheterogeneity of the enriched CTC population.

Each of these methods is widely used at the microscale tocapture CTCs from either an enriched or unprocessed sample.When considering these techniques for liquid biopsy, it isimportant to consider ease of detection, confirmation, andfurther analysis. Because immunomagnetic capture involvesimmunoaffinity, the success of the technology is highly dependenton the choice of biomarker. Another important aspect given ourunderstanding of the biological mechanisms governing cancer isthe ability to perform single cell tests, which will be discussed laterin more detail. Thus, this review will focus on some immuno-magnetic and microfluidic technologies that enable single cellmolecular analysis (Table 4).

7. Capture technologies

According to the presented framework, optimal chips for thecapture of CTCs should feature: a high capture rate, highthroughput, low contamination rate, and high cell viability.The following immunomagnetic, capture and release, and single

Fig. 6 Microscale technologies for CTC capture. Overview of microscale CTC capture mechanisms.


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




cell capture technologies are critically reviewed in order to illus-trate the current optimization of these key parameters. A focuswill be given to immunomagnetic technologies because of thenumerous advantages for rare CTC capture, and the recent trendtowards immunomagnetic microchip development.93 Some of theadvantages of immunomagnetic chips include their sensitivityand specificity due to their use of antigen–antibody capture, andtheir high throughput and tunability due to their power over alarger area than some other capture methods which require directinput.93 For these reasons, immunomagnetic chips do well withour framework based on capture rate, throughput, contamination,and viability. For completeness, we also review other methods asdiscussed, including capture and release and studies at the singlecell level.

7.1 Immunomagnetic

Immunomagnetic microchips provide a versatile platform forCTC enrichment and isolation. In the future, a combination ofboth negative and positive enrichment, or highly integrateddevices that take advantage of other techniques should bedeveloped to increase capture efficiency and purity and betterdistribution of captured CTCs. Combining more than onetechnique to increase the capture efficiency of CTCs hasrecently been gaining considerable popularity.109 Apart from thedesign, other important feature to consider for next generationtechniques is the diversity of surface biomarkers and how toexploit them to achieve higher sensitivity and specificity. Thissection will detail a number of immunomagnetic microscaletechnologies.

Kang et al., designed a PDMS based device with one mainchannel and two dead-end side chambers for capture andculture of CTCs (Fig. 7a).110 The breast cancer cells were labeledwith 2.8 mm magnetic beads conjugated to EpCAM. The CTCs

were collected in the side chambers that had an NdFeB N52permanent magnet beneath it. The microchannel at the inletwas oriented at 60 degrees to main channel to ensure cellswould be closer to the magnetic field. The flow rate wasoptimized to be 1.2 mL h�1 based on the residence time ofthe bead-bound cells and time required to attract cells into thedead-end chambers. A capture efficiency of 90% was reportedfor breast cancer cells spiked in 1 mL of blood, and the capturedcells were cultured and harvested for further molecularanalysis.110 This device has great potential to impact the field oftargeted therapies because it cultures CTCs from the patient, andshowcases a moderate throughput and capture efficiency gearedtowards early detection, and monitoring. However, the biomarkerof choice, EpCAM, is known to not capture the full heterogeneouspopulation of CTCs.

Another platform that uses positive selection of CTCs wasdeveloped by Earhart et al.111 The magnetic sifter biochip(Fig. 7b) had a dense array of magnetic pores with highmagnetic field gradients existing along the pore edges. WhenCTCs bound to magnetic beads labeled with anti-EpCAMantibodies are injected through the pores, they are capturedat the pore edges under the influence of the external magneticfield whereas the unlabeled cells pass through the poresunhindered. The capture efficiencies for high EpCAM expressingCTCs was B90% compared to the low expressing CTCs whichranged from 17.7% to 48%. In addition to a high throughput andcapture efficiency of only high EpCAM expressing CTCs, thesimple and quick release of the captured CTCs from the sifterallows for enumeration and advanced genomic analysis appro-priate for early detection, monitoring, prognosis, diagnosis, andtargeted therapy.

The CTC–iChip combines immunomagnetic capture withDLD and inertial focusing (Fig. 7c).109 In the first stage, DLD was

Table 4 Evaluation of CTC capture technologies. Summary of included CTC capture technologies and evaluation metrics. For capture efficiency, high isdefined as greater than 90%, and moderate is defined as 60–90%. For throughput, high is defined as greater than 5 mL h�1, and moderate is defined as1–5 mL h�1

Evaluation of CTC capture technologies

Technology BiomarkerCaptureefficiency Throughput

Normal cellcontamination

Cellviability Ref.

ImmunomagneticPermanent magnet EpCAM Moderate Moderate Low High 110Magnetic sifter EpCAM High High Moderate High 111CTC-iChip Biomarker for WBCs High High Moderate to high Moderate 109Array of micromagnets EpCAM High High Moderate High 15, 112 and

113

Capture & releaseLasers & microfilms EpCAM High Moderate Low High 113Thermoresponsive EpCAM High Moderate — High 115pH & glucose — Moderate Moderate — High 116Aptamers Protein tyrosine kinase 7

(PTK7)Moderate Moderate Low Moderate 117

Single cell captureMicrowells — Moderate High — High 118Programmable valves &channels

— — High — High 119

Capture & culture — — High — High 120


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




used to achieve hydrodynamic size based sorting by removingnon-nucleated cells such as RBCs and platelets from the wholeblood. The next stages used inertial microfluidics to focus theCTCs and WBCs for magnetophoretic sorting. Once the cells enterthe magnetic field, the magnetically labeled CTCs are deflectedand separated from the WBCs.109 The apt combination of DLDand immunomagnetic capture, in addition to negative enrich-ment of CTCs, grasps the most diverse population of CTCs. Theplatform primarily enumerates CTCs through fluorescence-baseddetection methods, which can damage the cells, preventing furthergenomic downstream analysis. One disadvantage is that thisdevice has been demonstrated at spiking concentrations muchhigher than typical CTC concentrations, at about 200–1000 CTCsper mL but has also shown lower detection concentrations. Thisdevice does not satisfactorily meet the requirements for discoveringtargeted therapies, but it has demonstrated its early detection,prognosis, diagnosis, and monitoring capabilities.

Similarly, Zhang et al., developed immunomagneticmicrochips for capture and release of CTCs at single celllevel.15,112,113 Fig. 8a and b show the original design of themicrochip. The first generation device was PDMS based hexagonalmicrochannel with permanent magnets placed below the PDMSdevice. Magnetic nanoparticles conjugated to anti-EpCAM wereadded to the whole blood sample spiked with cancer cells and

then pumped into the microchip. The CTCs were captured on theglass slides and the waste blood cells were eluted. This simpledesign enabled a higher flow rate of 10 mL h�1 compared withthe commercially available CellSearch system and demonstratedconsiderably high capture rates of 90% and 86% for COLO205 andSKBR3 cells, respectively, with a very low tumor to blood cell ratios(about 1 : 10, including red blood cells).15 The next generationmicrochip designs from Chen et al., had an array of ultra-thinmicromagnets of sub-micron height with a spacing of 100 mmfabricated on the glass slides (Fig. 8c), to increase the magneticattraction force by creating areas where the magnetic field inten-sity is locally increased. The micromagnet array both uniformlydistributes the intensity throughout the glass slide and efficientlycaptures CTCs labeled with magnetic nanoparticles.112 In compar-ison to the CTC-iChip, this one has a higher throughput, demon-strated low normal cell contamination, and analogous captureefficiency. The positive selection of EpCAM phenotypic CTCscaptures a uniform population of CTCs, but has the same dis-advantages that we mentioned earlier due to the heterogeneity ofthe CTC population. This device allows for the quick enumerationof CTCs, ideal for early detection, prognosis, and basic diagnosis,but it is not suitable for an advanced analysis for target therapies.

All of the immunomagnetic techniques described abovehave all included a step that employs positive enrichment by

Fig. 7 (a) Microchannels with dead-end side chambers. Side chambers were designed for immunomagnetic capture of CTCs Reprinted from Lab on aChip, 12, Kang et al., A combined micromagnetic-microfluidic device for rapid capture and culture of rare circulating tumor cells, 2175–2181, Copyright(2012), with permission from RSC. (b) Micro sifter Biochip. CTCs were conjugated with magnetic tags and under the influence of magnetic forces, CTCsstay on the substrate while all the other blood cells are flushed away through the holes. Reprinted from Lab on a Chip, 14, Earhart et al., Isolation andmutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips, 78–88, Copyright (2014), with permissionfrom RSC. (c) CTC-iChip. A positive isolation method is shown here: CTCs are labeled with magnetic beads, and by hydrodynamic cell sorting, onlynucleated cells like WBCs and CTCs are retained in the sample for the next stage of isolation. Then inertial focusing is applied to order the rest cells so thata better separation of CTCs and WBCs into different outlets is obtained. Reprinted from Science Translational Medicine, 5(179), Ozkumur et al., Inertial focusingfor tumor antigen-dependent and -independent sorting of rare circulating tumor cells, 179ra47, Copyright (2013), with permission from AAAS.


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




selectively targeting markers on the surface of CTCs. Usingnegative enrichment alone provides a number of advantagesbecause of its label-free nature. Karabacak et al. investigated amodification of the iChip to improve the removal of red bloodcells for higher sensitivity negative enrichment of CTCs.114 Thistechnology first separated nucleated cells and then used anti-bodies to deplete leukocytes and achieved a 97% yield of CTCs.This negative enrichment improvement is highly promising,but has not yet been demonstrated at a physiological concen-tration of CTCs.

7.2 Capture and release

As discussed previously, the capture and release of CTCs isoptimal for single cell analysis. By allowing selective release,cells can be analyzed on an individual basis at favorable temporaland spatial separations. Lasers and microfilms, thermoresponsivepolymers, pH and glucose sensitive polymers, and aptamers aremethods of capture and release reviewed below.

7.2.1 Lasers and microfilms. Hoshino et al. also developedan efficient method for releasing captured CTCs from substrateusing polyethylene naphthalate (PEN) film for downstreamanalysis. The cancer cells bound to the multiplexing nano-carriers were collected on glass slides by patterned micromagnets

as the blood flows through the microchannels. On top of thepatterned micromagnets is a 3 mm thick PEN film that has beentreated with a 0.01% (w/v) solution of poly-L-lysine to promote celladhesion. After blood screening, a buffer solution is introduced toflush unwanted leukocytes. Next the captured cells are stained andtheir captured locations recorded for the cell profiling analysis.Once a cancer cell is located, an area (B500 mm � 500 mm) of thefilm containing the target cell is cut using a focused laser beam(Fig. 8d). With the high purity of the screening microchip and theresolution of the laser tool, most leukocytes can be avoided orremoved from the cut portion, eliminating possible contaminationfrom leukocytes non-specifically remaining on the slides. A singlecell quantitative reverse transcription polymerase chain reaction(qRT-PCR) was performed on the captured CTCs to test 10 differentgenes. The results from single cell analyses was comparable to theresults obtained for thousands of cells.113 This device’s captureand release method is precise, but less portable than the afore-mentioned devices.

7.2.2 Thermoresponsive surfaces. Liu et al. used a thermo-responsive nanostructured surface to capture and release can-cer cells in a reversible manner by employing the hydrophobiceffect.115 The nanostructured surface is made up of the thermo-responsive material poly(N-isopropylacrylamide) (PNIPAAm)

Fig. 8 (a) and (b) An immunomagnetic microchip and its schematic. Whole blood sample with spiked cancer cells is flowed into the microchip andtargeted cancer cells are captured under magnetic field because of antibody–antigen reaction with antibody conjugated micro magnetic beads.Reprinted from Lab on a Chip, 11, Hoshino et al. Microchip-based immunomagnetic detection of circulating tumor cells, 3449–3457, Copyright (2011),with permission from RSC. (c) Local micro magnets for better distribution of captured CTCs. Patterned thin-film micromagnets were fabricated on thesubstrate to increase local magnetic field gradients for better distribution of captured CTCs. Reprinted from Scientific Reports, 5, Chen et al. Microscalemagnetic field modulation for enhanced capture and distribution of rare circulating tumor cells, 8745, Copyright (2015), with permission from NPG.(d) Single-cell-level capture and analysis of CTCs. PEN film was formed on top of glass slide so that after capture of CTCs, single CTC could be isolatedand qPCR could be conducted. Reprinted from Journal of Circulating Biomarkers, Hoshino et al. An Immunofluorescence-assisted Microfluidic SingleCell Quantitative Reverse Transcription Polymerase Chain Reaction Analysis of Tumor Cells Separated from Blood, ISSN 1849-4544, Copyright (2015),under a Creative Commons License.


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




atop silicon nanopillars, and this reversibly interacts withbiotinylated BSA through the hydrophobic effects. To capturethe cells, an antibody is connected to this biotin-BSA thattargets the cells. Through manipulating surface wettabilityand alternating temperature between 20 1C and 37 1C thecapture of cells was controlled (Fig. 9). This procedure capturedand released viable MCF-7 breast cancer cells and was onlyperformed on large cultures of cells, not on the single cell level.But the controllable capture and release of the cells could beapplied successfully to the capture of single cells for analysis.

7.2.3 pH and glucose. The same group developed anothertype of responsive surface for reversible capture and release ofcancer cells.116 This surface is responsive to both pH andglucose concentration through a combination of poly(acrylamido-phenylboronic acid) (polyAAPBA) on a silicon nanowire array. Thesurface can capture and release cells through variation of pH (from6.8 to 7.8) and glucose concentration (0 to 70 mM glucose). Thesurface is functional for at least 5 cycles, and cells remain viablemore than 95% of the time.116 The experiment was performed onthe MCF-7 breast cancer cell line and demonstrates the valueof the technology for capture and release of cells for furthermolecular analysis, but did not demonstrate the platform at thesingle-cell level.116

7.2.4 Aptamers. Another network that has been used forcell capture and release uses a 3D DNA network of repeatedaptamers to enhance capture.117 Zhao et al. found that usinglong rolling circle amplification for multiple aptamers cantarget cells better than single aptamers, and that these productscan be controlled for density, sequence, and length. This bio-inspired network is based off of natural sea creatures that haveevolved long tentacles to capture prey, and uses the aptamers toincrease avidity for more effective binding. A huge advantage ofthis approach is that restriction enzymes can be used to cleavethe single cells after capture for further analysis. This approachwas found to successfully separate target cells from mixtures ofmany types of cells, and then DNase I was used to releasethe cells from the device. The use of aptamers rather than

antibodies enables the cells to be released without muchdamage done, leading to most of the cells maintainingviability.117 This 3D DNA network technique holds great promisefor single CTC capture and release for downstream analysis;however, creating DNase I free environments requires a particularlevel of lab sterility, diminishing its opportunities for integratinginto portable platforms.

7.3 Single cell and cluster capture

On the premise that a small population of cancer cells drivesdrug resistance, Lin et al. recently investigated a platform formicrowell cell loading.118 The technology involves a dual-welldesign and has 77% single-cell loading. The goal of thisresearch is to trap single cells through a small microwell andinto a large microwell to allow for cell culture. The device tookadvantage of gravitational force and low flow rates to allow cellsto settle into the capture wells before washing away uncapturedcells. The cells then drop from these capture wells into largerwells that cultures the cells over the course of several days.A number of flow rates were iterated through until a ‘‘cellsweeping’’ procedure was developed to maintain a high captureefficiency, and a flow rate of 3 mL min�1 was found to be best forallowing cells to move through the device while also settlingwithin capture channels.118 While this sort of platform is lessapplicable to CTC separation, it is a feasible last step after CTCshave been separated to isolate individual cells for single-celldownstream analysis. It is also relatively low throughput,resulting in a very long timespan required for analysis.

Over ten years ago Wheeler et al. developed microfluidicdevices for single cell analysis for a number of applications,including testing cell viability and intracellular ion concentration.119

The technology builds upon the development of multilayer softlithography with a series of valves at channel intersections (Fig. 10).These digitally controlled valves can close and open channels in aprogrammable method to separate out single cells from a solution.The device is designed to both separate an individual cell andprovide it with small volumes of reagents, a function that relies on

Fig. 9 Thermoresponsive cell capture and release. Targeted cancer cells are reversibly captured and released using biotin-BSA and PNIPAAm-modifiedSINP through temperature changes between 37 1C and 20 1C. Reprinted from Advanced Materials, 25, Liu, Hydrophobic interaction-mediated captureand release of cancer cells on thermoresponsive nanostructured surfaces, 922–927, Copyright (2013), with permission from Wiley-VCH.


Publ

ishe

d on

01

Dec

embe

r 20

16. D

ownl

oade

d on

25/

01/2

017

16:2

2:42




laminar flow. The use of this system to measure intracellularcalcium ion concentration was investigated, and showed that thisdevice can be used for a number of single cell assays

liquid biopsy on chip: a paradigm shift towards the understanding of cancer...

Documents