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Table 1: List of CTC isolation methods and techniques with their advantages and
limitations.

Name Basic Properties Advantages Limitations

Acoustophoresis Cells are suspended The strength of the With direct pressure
(Deshmukh et al., 2014; in fluid and are acoustic radiation source, any instability in
Gossett et al., 2010; Li, exposed to force is dependent the pressure source can
P. et al., 2015). ultrasound waves upon the volume of cause deviation in the

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and pressure the particle, the intended flow lines

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amplitude density of both the
This results in an particle and the fluid

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acoustic radiation Isolation factors
force include the size of
The radiation force the particle and its
will then push compressibility

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particles towards the
pressure anode or
pressure anti-anode,
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ultimately
separating the
particles
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AdnaTest Separation by way The variety of Possible false-positive

(Andreopoulou et al., of anti-EpCAM and selection markers finding due to expression
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2012; Müller et al., anti-MUC1 (antibodies) allows of a selection marker being

2012; Zieglschmid et al., antibody-targeting for the possibility of present in other cells other
2005) immunomagnetic characterizing cells than CTCs, such as with
beads for multiple markers, nucleic acid contamination.
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all simultaneously
CTCs are then CTCs that undergo EMT
detected via RT- are EpCAM negative
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Antibody cocktails
PCR assay for are specific to a
tumor-associated certain cancer type Cells are not viable after
transcripts detection
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CAM assay (Lu et al., Used as a functional High sensitivity and Isolation step requires
2010; Monteiro-Riviere cell separation specificity, leading to more than 12 hours
et al., 2009) method based on effective enrichment Biomarker dependent
CTC invasiveness and identification
compared to other based on CTC
cells. invasiveness.
CTCs can be
identified using the Downstream analysis
CAM uptake is possible.
criteria.
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Name Basic Properties Advantages Limitations

CellSearch (Attard et al., The only FDA- CTCs expressing low
2009; Beije et al., 2015;Separation by anti- approved blood test levels of EpCAM are
Harouaka et al., 2014; EpCAM targeting for the use in patient unlikely to be captured
Hong and Zu, 2013; Kim immunomagnetic care diagnostics,
et al., 2012; beads used for metastatic Cells captured appear to be
Swennenhuis et al., breast, prostate, are more apoptotic
2009) colorectal cancer

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CTC-Chip (McKeown Unique microfluidic High sensitivity and Long blood processing

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and Sarosi, 2013; approach specificity time (1.67mL/hr)
Nagrath et al., 2007) Use of antibody 99% success rate in Cells no longer viable

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(anti-EpCAM) identifying CTCs in
microposts the peripheral blood
Controlled laminar of metastatic
flow conditions pancreatic, prostate,

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colon cancer, lung,
and breast patients
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CTC Cluster-Chip Utilizes bifurcating CTC clusters were
(Sarioglu et al., 2015) traps to capture CTC clusters can be identified in only 30-40%
CTCs, release via isolated of patients suffering from
flow reversal Sensitive enough to metastatic breast, prostate,
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capture CTC clusters and melanoma cancers

as small as 2 cells Identification of CTCs is
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Does not require the lower versus the CTC-Chip

use of tumor specific
markers for isolation
Low flow rates
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preserve cell viability

CTC-iChip (Karabacak Size-based High throughput Limited to single or small
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et al., 2014; Ozkumur et enrichment with Fast processing time 2-4 cell clusters, not tumor
al., 2013) either EpCAM- More sensitive in microemboli
(Bioengineering, 2014) based positive detecting low levels
enrichment or CD45 of CTCs, versus
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negative depletion commercial

Microfluidic design technology
for rapid sorting Capacity to isolate
Magnetically CTCs by methods
labeled target cells either dependent or
are sorted out independent of tumor
Inertial based membrane epitopes,
capture platform making this method
applicable to all
cancers
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Name Basic Properties Advantages Limitations

Dielectrophoresis (DEP) Separation can be Can be used for
(Gascoyne et al., 2009; positive or negative, selective isolation of Requires specific
Gossett et al., 2010; which will affect the cells parameters such as cell
Zieglschmid et al., 2005) position of the cell Either AC or DC type and electric field
in the field currents may be used frequency.
Cells are placed in a
non-uniform electric DC current can result in a

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field, the field then electrochemical reaction on
the electrode surface,

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imparts a net force
on the cell due to an causing a reduction in
convective flow. This

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induced or
permanent dipole reduction can cause cell
Isolation based on isolation to be inhibited, as
polarizability and well as free radical

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size generation that results in
cellular damage.
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DynaBeads Magnetic separation Can be pre-coupled Only 3 types of DynaBeads
(Hardingham et al., and isolation based with biomolecules are available for human
1993) on binding to with an affinity for a tumor cell isolation
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desired target and desired target, such

beads responding to as: antibodies,
magnetic field proteins, antigens, or
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DNA/RNA probes

Ephesia CTC Chip Isolation by way of Capture efficiency Currently has not been
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(Alix-Panabières et al., immunomagnetic range of 90-94% formatted to accommodate

2012; Karabacak et al., sorting coupled with Ephesia technology large volumes of blood
2014; Saliba et al., 2010) microfluidics can allow for a more
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Sample flows across flexible platform to

immobilized perform advanced
magnetic beads cell biology testing
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on cancer cells
Required sample
volumes are one-
tenth those of flow
cytometry
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Name Basic Properties Advantages Limitations

EPISPOT (Alix- Removes leukocytes This assay creates a Requires efficient antigen
Panabières, 2012; via CD45 depletion new way to detect binding and specific
Kalyuzhny, 2005; viable CTCs and epitope presentation
Millner et al., 2013) and during short- DTCs (disseminated Demands high antigen
term cell cultures, tumor cells) levels
detects specific With an extension of Transition into in vitro
marker proteins a multi-parameter cultures may decrease cell

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shed/ secreted/ analysis, this could viability and reduce
released from single reveal the CTC/DTC

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detection rates
epithelial cancer protein fingerprint
cells

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An adaptation of the
enzyme- linked
immunospot

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(ELISPOT)
technology
GILUPI CellCollector Overcomes sample Only used for extraction of
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Nanodetector (Alix- Ex vivo (FSMW) blood volume CTCs directly from
Panabières and Pantel, Functionalized limitations patient’s bloodstream, not
2013; Krebs et al., 2014; Structured Medical Increases diagnostic for use of extraction in
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Luecke et al., 2015; Wire is antibody sensitivity of CTC blood sample

Maheswaran et al., 2008; (anti-EpCAM) isolation More studies need to be
Nagrath et al., 2007; coated and applied done to verify clinical
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Saucedo-Zeni et al., into peripheral arm No evident side applicability

2012) vein effects
Isolation occurs in
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vivo
Herringbone CTC-Chip An advanced CTC- Future goal is to be It does not consider the
(Sarioglu et al., 2015; chip utilizing a used for large scale inherent particulate
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Stott et al., 2010; Wang strategy of including clinical applications properties of cells.
et al., 2016) herringbones or Successfully isolated Cells are no longer viable
surface ridges in the 93% CTCs from
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walls of the device patients with prostate

to disrupt cancer
streamlines and Captures CTC
encourage collisions clusters
between antibody-
coated walls and
CTCs
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Name Basic Properties Advantages Limitations

ISET (De Giorgi et al., Utilizes a filter- CTCs can be damaged or
2010; Joosse et al., 2015; based, size High throughput fragmented due to multi-
Vona et al., 2004; Zheng exclusion approach Downstream step cell processes
et al., 2011) to isolate epithelial morphological CTC heterogeneity
cells studies can be regarding morphology and
performed size
IsoFlux Rare Cell Uses next generation High sensitivity of Maximum daily analysis is

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Access System (Harb et sequencing (NGS), CTCs from many 12 samples

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al., 2013) with quantitative tumor types
polymerase chain CTC may have biomarker

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reactions (qPCR), The variety of heterogeneity Those that
fluorescence in situ selection markers undergo EMT are EpCAM
hybridization (antibodies) allows negative
(FISH), and for the possibility of

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immunofluorescence characterizing cells
Uses flow control for multiple markers,
and all simultaneously
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immunomagnetic Multiple kits for lab
capture to increase usage are available,
CTC isolation both for cell
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enrichment, and
downstream analysis
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MagSweeper (Ao et al., Isolates CTCs The CTCs possessing low

2016; Powell et al., Separation by way without or no EpCAM expression
2012; Talasaz et al., of anti-EpCAM contaminating will be over-looked (CTCs
2009) antibody-targeting leukocytes that undergo EMT are
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immunomagnetic The variety of EpCAM negative)

beads selection markers
(antibodies) allows
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for the possibility of

characterizing cells
for multiple markers,
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all simultaneously
MagDense (Durmus et Platform enables Further studies are required
al., 2015) Magnetic levitation single-cell density for capture efficiency and
based on specific measurements and specificity for CTCs
densities of CTCs imaging
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Name Basic Properties Advantages Limitations

Metacell Filtration Size based Filtration techniques Filters have a larger pore
(Dolfus et al., 2015) separation technique are sensitive enough size (8μm) versus
driven by capillary- to allow ScreenCellCyto (6.5μm)
action cytomorphological
Commercial device and
used for isolation of immunocytochemical
CTCs, allowing for analysis of CTCs

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cytological

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identification
Microfluidic Silicon Antibody based Antibody coated Further CTC separation

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Chip (Sequist et al., microposts maximize from microfluidic device is
2009) CTC-antibody challenging
interactions

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NanoVelcro Microfluidic device This is a more Only EpCAM-positive
Microfluidic Device uses tiny rods coated advanced prototype CTCs are detected
(Zhe et al., 2011) with antibodies that adds a new
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After blood passes transparent substrate
through, a laser is for isolating CTCs
used to extract
CTCs
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Uses 3 color
immunofluorescence
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for detection of
CTCs
Negative Enrichment A negative selection Improved sample CTC expressing CD-45
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QMS (Ignatiadis and technique for cell yield and purity maybe inadvertently
Reinholz, 2011; Jing et enrichment followed High throughput and remove from the sample
al., 2007) by separation by cost effective Contamination with
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Quadrupole WBC’s might result in

Magnetic Flow unintentional loss of CTCs
Sorter (QMS)
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OncoQuick® Loss of sample while

(Rosenberg, R et al., Polypropylene tube High throughput, depleting mononuclear
2002; Rosenberg, R. et is inserted above the inexpensive cells
al., 2002) separation medium Increases sensitivity Detection depends upon
The medium allows and specificity for only cytokeratine-20
for elimination of tumor cells biomarker
erythrocytes, Significant reduction
granulocytes, in co-enriched
lymphocytes, and number of
mononuclear cells mononuclear cells,
with a high CTC
recovery rate
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Name Basic Properties Advantages Limitations

Parsortrix (Joosse et al., Isolation of CTCs Purity of CTCs CTC heterogeneity
2015; Xu et al., 2015) based on size and harvested is 3.1% regarding size
compressibility Ability to capture
CTC clusters
Harvests CTCs with
both epithelial and
mesenchymal

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features

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Uses cassette device
to for easy

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detachment
pluriSelect(Nadezhda Antibody based Non-magnetic cell Currently limited for use in
Frolova, 2012) CTC separation separation, can be colon carcinoma
pluriSelect uses added directly to a diagnostics

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pluriBead® carrying whole blood sample
a tumor-associated
anti-EpCAM
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antibody
Reactive Ion Etching Adhesion of the The capture Suboptimal capture purity
(RIE) (Chen et al., CTCs on the nano- efficiency is up to
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2012a; Nalepa et al., roughsurface 80% within one hour

2013) regardless of size of cell incubation
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and without using Does not require

any capture EpCAM expression
antibody. for the cell capture
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RoboSep/EasySep(Wang Magnetically label Rely on biomarkers for

et al., 2013; Wilcox et and separate cells by High throughput capturing the cells
al., 2009) positive or negative Fully automated
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selection instrument, reducing

possibilities of cross
contamination
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Design for a
customized cell
separation protocol
can be created by
stem cell
technologies
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Name Basic Properties Advantages Limitations

ScreenCellCyto (Dolfus Size-exclusion In addition to Poor specificity
et al., 2015; Wang et al., based isolation ScreenCell devices Unable to capture CTCs
2013) method being used for smaller than WBCs
Peripheral blood isolation of CTCs,
sample is mixed they are also used for
isolation of
with filtration buffer
Diluted sample is Circulating Fetal

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then filtered by Cells (CFCs) from

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maternal peripheral
aspiration created by
a vacuum tube blood for prenatal

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collector diagnostics
Disposable Filters in only 3
minutes
Not dependent upon

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EpCAM
TelomeScan (Kim et al., Detects elevated May more effectively May also detect
2011; Kojima et al., telomerase activity detect epithelial to hematopoietic stem cells
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2009) via a telomerase- mesenchymal for false-positive results
specific replication transition (EMT) or
selective adenovirus EpCAM negative
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tumor cells
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Table 2: Gene categorization/grouping on the basis of functional categories after the evaluation
of CTCs

Functional categories Associated genes

Epithelial phenotype KRT8 (Powell et al., 2012; Ting et al., 2014),
KRT18 (Powell et al., 2012; Ting et al., 2014),
KRT19 (Powell et al., 2012; Smirnov et al., 2005;
Ting et al., 2014),

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CTNNB1 (Powell et al., 2012),
Krt7 (Ting et al., 2014),

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Epcam (Lu et al., 2010; Smirnov et al., 2005; Sun
et al., 2013; Ting et al., 2014),

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EGFR (Chen, C.L. et al., 2013; Kallergi et al.,
2008; Lu et al., 2010; Ting et al., 2014),
CDH1 (Armstrong et al., 2011; Ting et al., 2014),

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MUC1(Lu et al., 2010; Pierga et al., 2007; Zhu et
al., 2014),
HER2 (Kolostova et al., 2015b),
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ERBB2 (Kolostova et al., 2015b; Lu et al., 2010)
Epithelial mesenchymal transition (EMT) TGFß1(Powell et al., 2012),
FOXC1 (Powell et al., 2012),
CXCR4, (Powell et al., 2012),
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NFKB1(Powell et al., 2012),

VIM (Armstrong et al., 2011; Lu et al., 2010;
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Powell et al., 2012; Ting et al., 2014),

ZEB2 (Powell et al., 2012),
CDH11 (Ting et al., 2014),
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PTPRN2 (Chen, C.L. et al., 2013),

ALDH1 (Chen, C.L. et al., 2013),
ESR2 (Chen, C.L. et al., 2013),
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WNT5A (Chen, C.L. et al., 2013),

Twist1 (Aktas et al., 2009; Giordano et al., 2012;
Lu et al., 2010),
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Akt2 (Giordano et al., 2012; Kolostova et al.,

2015b),
PI3Kα (Aktas et al., 2009; Kolostova et al.,
2015b),
SNAIL1 (Giordano et al., 2012),
ZEB1 (Giordano et al., 2012),
TG2 (Giordano et al., 2012)
Metastasis S100A4 (Powell et al., 2012),
S100A9 (Powell et al., 2012),
S100A14 (Smirnov et al., 2005),
S100A16 (Smirnov et al., 2005),
NPTN (Powell et al., 2012),
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KLK3 (Smirnov et al., 2005),

CEACAM5 (Smirnov et al., 2005),
KRT 20 (Smirnov et al., 2005),
FABP1 (Smirnov et al., 2005)
PI3K/AKT/mTOR pathway AKT1,
AKT2,
PIK3R1,
PTEN (Powell et al., 2012)
Apoptosis BAX,
CASP3,

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CD53,

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CD59 (Powell et al., 2012)
Stem cell phenotype CD24 (Giordano et al., 2012; Lu et al., 2010;

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Powell et al., 2012),
CD44 (Giordano et al., 2012; Lu et al., 2010;
Powell et al., 2012),
CD133 (Armstrong et al., 2011; Giordano et al.,

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2012; Sun et al., 2013),
ALDH1 (Aktas et al., 2009; Giordano et al., 2012;
Kolostova et al., 2015b)
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DNA repair PARP1 (Powell et al., 2012)
Cell metabolism SLC2A1,
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TFRC (Powell et al., 2012)

Cell proliferation RRM1,
MAPK14 (Powell et al., 2012)
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List of Figures

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Figure 1: Outline of existing isolation, detection and characterization techniques and promising
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future clinical utilities.

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(A) (i) (B)

(ii)

(iii)

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(C)

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(ii)

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Figure 2: (A) (i) Insertion of the functionalized structured medical Seldinger guidewire (FSMW)
into the vein through a conventional cylindrical (instrument is also known as GILUPI GmbH
CellCollector). (ii) Wire (anti-EpCAM-antibody-functionalized) is slowly inserted inside the
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cannula till surface is in contact with the blood flow in vein lumen. (B) Seldinger guidewire-
Gold-plated with a size of 200 nm- coated with polycarboxylate hydrogel (functionalized with
anti-EpCAM-antibodies) which capture circulating cells expressing EpCAM antigen on surface.
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(C) Image illustrating breast cancer cells (SK-BR-3) captured by functionalized guidewire.
Reprinted with permissions from (Saucedo-Zeni et al., 2012).
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(A) (B)
(i)

(ii)

(iii)

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Figure 3: (A) Aptamer-mediated CTC capture and release via complementary oligonucleotide
sequences (CS= complementary sequences) (Zhang et al., 2012). (i) explain about the basic
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structure of the of the hydrogel immobilized on the glass slide (ii) illustration of the aptamer
sequence hybridized with complementary sequences (CSs) (iii) represents the process of cell
release. The stable aptamer hybridized with triggering CSs, forming a new hybridized state that
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leads to rapid dissociation of the aptamer from the hydrogel hence releasing the cells from the
hydrogel. (B) Illustrating the cell binding and aptamer formed by SAPE of anti-EGFR aptamer –
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DNA and biotin, and addition of RELease particles. After washing, the fluorescence cells
(caused by selective binding with aptamer) completely hybridizes with anti-EGFR aptamer
because of the presences of RELease RNA. This leads to opening up its herpin sturucture which
releases 76% of anti-EGFR aptamer from the cellular surface. (Wan et al., 2012)
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Figure 4: Ex-vivo CTC (breast cancer) culture (A) Nonadherent CTC cultures (B) mouse
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xenografts derived from cultured CTCs implanted in mammary fat pad. (C) Orignal breast
tumors and corresponding CTCs culture and two different CTC lines derived from mouse
xenografts. (Yu et al., 2014)
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