looking back, to the future of circulating tumor cells

Pharmacology & Therapeutics xxx (2014) xxx–xxx

JPT-06643; No of Pages 10

Contents lists available at ScienceDirect

Pharmacology & Therapeutics

j ourna l homepage: www.e lsev ie r .com/ locate /pharmthera

Associate editor: B. Teicher

Looking back, to the future of circulating tumor cells

Terence W. Friedlander a,⁎, Gayatri Premasekharan b, Pamela L. Paris b

a Division of Hematology & Medical Oncology, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, United Statesb Department of Urology, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, United States

Abbreviations: ALDH, Aldehyde dehydrogenase; ACollagen adhesion matrix; CK, Cytokeratin; CTC(s), CircDielectrophoretic field-flow fractionation; ELISPOT, Enzymassay; EMT, Epithelial to mesenchymal transition; EpCAMcule; ER, Estrogen receptor; FAST, Fibre-optic arrayImmunomagentic cell separation; MCC, MicrofluidMesenchymal to epithelial transition; mCRPC, Metastatcancer; PR, Progesterone receptor; PSA, Prostate-specific amembrane antigen; VEGF, Vascular endothelial growth fa⁎ Corresponding author at: University of California, S

Street, Box 1711, San Francisco, CA 94143, San FranciTel.: 415 514 6380; fax: 415 353 7779.

E-mail address: [email protected] (T.W. Fr

0163-7258/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.pharmthera.2013.12.011

Please cite this article as: Friedlander, T.W.,http://dx.doi.org/10.1016/j.pharmthera.2013

a b s t r a c t
a r t i c l e i n f o
Keywords:
Circulating tumor cellsEpithelial to mesenchymal transitionEpCAMStem-cellsCellSearch
Detection and analysis of circulating tumor cells (CTCs) frompatientswithmetastaticmalignancies have becomeactive areas of research in recent years. CTC enumeration has already proven useful in establishing prognosis forpatientswithmetastatic breast, colon, and prostate cancer.More recently, studies are going beyond enumeration,exploring the CTCs as a means to better understand the mechanisms of tumorigenesis, invasion, and metastasisand the value of CTC characterization for prognosis and tailoring of treatment. Analysis of CTC subpopulations, forexample, is highlighting the importance of the epithelial to mesenchymal transition (EMT), a process whichmaybe crucial for allowing tumors to invade into and grow at sites distant from the original tumor site. Similarly, thedetection of CTCs expressingmarkers of stemnessmay also have important implications for treatment resistance.Genomic analysis of CTC and CTC subpopulations may allow for selection of novel therapeutic targets to combattreatment resistance. CTCs become a particularly valuable biospecimen resource when tissue biopsies are un-available or not feasible and liquid biopsies allow for serial monitoring. Lastly, cultures of patient-derived CTCsmay allow for an evaluation of therapeutic strategies performed ex vivo and in real time. This review articlewill focus on these developments, starting with the CTC pathogenesis, going on to discuss the different platformsavailable for CTC isolation and their use to date in these arenas, then will explore multiple topics including theexisting data concerning CTC subpopulations and their clinical relevance, genomic characterization, and lastly,avenues for future research.

© 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Pathogenesis of circulating tumor cells and the role of the epithelial to mesenchymal transition . . . . . . . . . 03. Methods of circulating tumor cell isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. Clinical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05. Circulating tumor cell subpopulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06. Circulating tumor cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 07. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Authors' disclosures of potential conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
R, Androgen receptor; CAM,ulating tumor cell(s); depFFF,e-linked immunosorbent spot, Epithelial cell adhesion mole-scanning technology; MACS,ic cell concentrator; MET,ic castration-resistant prostatentigen; PSMA, Prostate specificctor.an Francisco, 1600 Divisaderosco, California, United States.

iedlander).

ghts reserved.

et al., Looking back, to the fu.12.011

1. Introduction

Isolation and analysis of circulating tumor cells (CTCs) from the blood-streamof patientswithmetastaticmalignancies have emerged asways tobetter understand tumorigenesis as well as the processes of invasion andmetastasis. Although epithelial CTCs were first described well over100 years ago (Ashworth, 1869), only recently has CTC enumerationbeen shown to be clinically useful as a prognostic biomarker in epithelialmalignancies including breast (Cristofanilli et al., 2004), colon (Cohenet al., 2008), and prostate cancer (De Bono et al., 2008). In comparisonto traditional metastatic tissue biopsy, isolation of CTCs offers numerous

ture of circulating tumor cells, Pharmacology & Therapeutics (2014),

http://dx.doi.org/10.1016/j.pharmthera.2013.12.011

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2 T.W. Friedlander et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

advantages: collection of peripheral blood is easy toperform, relatively in-expensive, presents significantly less risk to patients, is lessmorbid, and iseasily repeatable over time. The idea of ‘real-time’ serial monitoring ofCTCs is attractive as it allows for an estimate of circulating disease burdenover time, can permit researchers to track and discover novel changes incancer cells (Murtaza et al., 2013), and can shed light on some of the fun-damental processes that lead to an aggressive phenotype, metastasis, andresistance to current therapies.

In order to best understand how CTCs are currently being deployedin the clinical arena, and their utility in understanding cancer cell biolo-gy, we will first review the fundamental processes that lead to therelease of tumor cells into the bloodstream, then focus on currentlyavailable platforms for CTC detection/isolation and clinical uses ofCTCs, then discusswhat is known about CTC heterogeneity and subpop-ulations including CTCs expressing mesenchymal markers and/orstemness, and finally discuss the potential for ex vivo CTC expansionand the possibility of using CTCs to select targets for therapy.

2. Pathogenesis of circulating tumor cells andthe role of the epithelial to mesenchymal transition

Despite the understanding that tumor cells must travel through thebloodstream or lymphatics in order to establish metastases, a singular,precise definition of a CTC remains elusive. It is widely hypothesizedthat CTCs originate from cells within the primary or metastatic tumorthat acquire the ability to detach from the basement membrane, invadethrough the tissue stroma, and enter into the blood vessels (Nowell,1976). The acquisition of this invasive phenotype is hypothesized tooccur in response to the increasing tissue hypoxia that develops astumors grow and compete for resources (Semenza, 2012), which inturn leads to neovascularization and lymphangiogenesis (Cao et al.,2013). A critical concept that has emerged to be relevant to CTCs isthe epithelial tomesenchymal transition (EMT), a process first observedin embryonic development (Shook & Keller, 2003) (Fig. 1). EMT enablesepithelial cells to lose their apical–basal polarity, detach from neigh-boring cells, acquire a fibroblast-like morphology, invade throughthe surrounding stroma, and become more resistant to apoptosis(Vincent-Salomon & Thiery, 2003). During this process, tumor cellslose expression of specific epithelial markers including E-cadherin(Comijn et al., 2001), EpCAM (Went et al., 2004), and cytokeratin(Savagner, 2010), gain expression of mesenchymal cytoskeletal andadhesion proteins such as vimentin and N-cadherin (Kokkinos

Fig. 1. CTCs, EMT, and cancer progression: A schematic of the reversible EMTmodel for cancer pfrom the basement membrane through EMT activation. Metastasizing cells then directly enter tOnce they extravasate they may proliferate and reverse back to an epithelial phenotype to form

Please cite this article as: Friedlander, T.W., et al., Looking back, to the fuhttp://dx.doi.org/10.1016/j.pharmthera.2013.12.011

et al., 2007), and upregulate kinases and growth factors includingc-MET (Giordano et al., 2000), TGF-β (Oft et al., 1996), Wnt (Chenet al., 2012), and FOXC1 (Xia et al., 2013). EMT is thought to be con-trolled by a family of genes including, among others, Snail, Twist,Zeb, and E47 (Berx et al., 2007).

Whether nascent CTCs undergo EMT in order to escape the primaryor metastatic tumor and enter the bloodstream, or whether epithelial-like CTCs are simply ‘shed’ from a tumor that has eroded into a bloodvessel and undergo EMT while in transition, is still a matter of debate.CTCs derived from epithelial tumors can be detected using multipledifferent epithelial markers (Table 1). While the presence of epithelialCTCs in circulationwould seem to support the notion that CTCs are sim-ply shed by large tumors, it is also possible that primary tumor cells thathave undergoneEMT in order to enter into and survive in the circulationcan subsequently undergo a reverse process termed mesenchymal toepithelial transition (MET), whereby cells revert back to their epithelialstate. This concept is supported by the finding of CTCs expressing mes-enchymalmarkers including vimentin (Kallergi et al., 2011), N-cadherin(Balasubramanian et al., 2012), CD44 (Theodoropoulos et al., 2010), aswell as reports of CTCs expressing markers of both epithelial andmesenchymal lineage (Armstrong et al., 2011), implying the presenceof a transition state between epithelial and mesenchymal. Similar sup-port comes from the clinical observation that epithelial tumors withmesenchymal differentiation (for example, sarcomatoid renal cell can-cers) are more aggressive and prone to early dissemination (Cangianoet al., 1999). The concept of a dynamic flux between epithelial andmes-enchymal states may also better explain the process of metastasis,which allows for individual or small groups of mesenchymal-liketumor cells to invade and colonize distant sites, then revert back to anepithelial state once a suitable niche is found. It should be stressedthat to date no one singlemodel of metastasis encompasses the entiretyof all observational findings and a number of other factors, including thesite of origin of the tumor cell and the degree of cell heterogeneity with-in a tumor, may come into play. Nevertheless, the EMT/MET model ofdissemination has advantages in explaining the wide variety andplasticity of CTCs observed to date, and has important implications forthe direction of future research.

3. Methods of circulating tumor cell isolation

CTCs occur at very low frequency in the bloodstream, generallyestimated at fewer than 1 CTC per million leukocytes. Nucleated non-

rogression. Invasion involves epithelial cells losing their polarity and detaching themselveshe circulation. Circulating tumor cells then travel to distant sites and exit the bloodstream.macrometastases.


image of Fig.�1


3T.W. Friedlander et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

hematopoietic cells expressing EpCAM and cytokeratin are detectableacross a wide range of metastatic malignancies, with counts rangingfrom b1/ml to greater than 3000/ml (Allard et al., 2004). The challengein making these estimates is that there are differences in: both intra-and inter-individual variation in CTC counts over time (Camara et al.,2006), average CTC detection frequency across specific malignancies(N50% in prostate cancer compared to b25% in pancreas cancer (Allardet al., 2004)), and most importantly, in the methods used to isolateand define a CTC.

As reflected in Table 1, multiple enrichment techniques are availableto isolate CTCs. Each enrichment platform isolates and identifies CTCsbased on a certain set of characteristics. The majority of available plat-forms utilize immunocytochemical enrichment, relying on the expres-sion of cell surface markers, generally epithelial ones. A minority ofplatforms enrich for CTCs based not on marker expression, but ratherbased on physical characteristics that distinguish cancer cells from nor-mal ones, including cell size, deformability, or bioelectric properties.Functional assays exist which enrich CTCs based on their capacity toinvade into a collagenous matrix, or their ability to produce tumor-specific proteins.

Despite the large number and wide variety of CTC enrichment plat-forms, a major challenge for many immunologic platforms, especiallythose based on expression of cell surface markers, is that one limitsthe detectable CTC pool. While epithelial antigens like EpCAM andcytokeratins are widely expressed on most epithelial malignancies,many of the platforms that rely on these markers for CTC enrichmentwill by definition not be able to enrich for CTCs which have downregu-lated or lost epithelialmarkers, as we discussed previously for EMT. Thisholds true for non-epithelial malignancies, such as melanoma, in whichEpCAM expression is low or absent. Even if CTCs are not actively under-going EMT, platforms that rely principally on epithelial marker expres-sion will miss CTCs that have lost expression of key epithelial markersthrough mutation, genomic deletion, clonal selection, or reversion to amore mesenchymal state. That being said, the enumeration of CTCs ex-pressing epithelial antigens has beenwidely studied in clinical trials andas we will discuss in the next section, has been useful in assessingpatient prognosis.

4. Clinical applications

4.1. Baseline circulating tumor cell enumeration

The largest clinical experience to date with CTCs has been with theCellSearch platform (Veridex). CellSearch immunomagnetically en-riches for CTCs, and enumeration is defined by the expression ofEpCAM, cytokeratins 8, 18, and 19, the absence of CD45 expression,and positive nuclear staining with DAPI. In a landmark study, 102women with metastatic breast cancer starting a new line of treatmentwere included in a training set to define a CTC threshold that distin-guished patients with rapid progression of disease from those withslow progression (Cristofanilli et al., 2004). This threshold, determinedto be 5 CTCs/7.5 ml of blood, was then shown to be prognostic forboth progression-free and overall survival in a validation set of 75 addi-tional women with breast cancer. Similar studies using the CellSearchplatform have shown that the baseline epithelial CTC counts are prog-nostic for survival in both metastatic colon cancer (Cohen et al., 2008)and metastatic castration-resistant prostate cancer (CRPC) (De Bonoet al., 2008). A recent study confirmed the prognostic utility ofCellSearch inmetastatic breast cancer, butwas unable to showprognos-tic validity for the AdnaTest Breast platform, which incorporates rtPCRof breast cancer specific genes after enrichment via expression of epi-thelial markers (Muller et al., 2012). Taken together these studies ofenumeration suggest that a high epithelial CTC count portends aworse prognosis in a number of epithelial malignancies, and should beuseful for counseling and risk-stratifying patients at the outset of thera-py.While knowing that a particular patient has a poor prognosis can be


useful in some circumstances, the clinical relevance of a baseline prog-nostic test such as CTC enumeration is still however a subject of debate.

4.2. Treatment-related changes in circulating tumor cell counts

The change in CTC counts in response to therapy, as opposed tomea-surement of baseline CTC counts, has also been investigated for its clin-ical relevance. In a study of 276 men starting a new line ofchemotherapy for metastatic CRPC, it was found that the prognosis forthe 45 patients whose CTC counts (as measured by CellSearch) de-creased from ≥5 CTCs/7.5 ml at the start of therapy to b5 CTCs/7.5 mlhad a significantly improved survival (21.3 vs. 6.8 months) comparedto men whose counts did not decrease below the 5 CTC threshold. Itwas similarly observed that the 26 men whose counts started belowthe 5 CTCs/7.5 ml threshold and increased above ≥5 after treatmenthad a significantly worse overall survival (9.3 vs. 26 months) comparedto those whose counts remained b5 throughout treatment. This effectappeared independent of known prognostic variables including changein PSA (H.I. Scher et al., 2009). This suggests that serial monitoring ofCTC counts has a role in better understanding prognosis compared toone-timebaseline assessment, and also raises the possibility of selectionpressure due to treatment-induced resistance. To explore this better,CTC enumeration and molecular characterization are being performedin two ongoing clinical trials in prostate cancer sponsored by Stand Upto Cancer and the Prostate Cancer Foundation.

4.3. Circulating tumor cells as a surrogate endpoint for clinical trials

CTC counts have also been explored as interim or potential surrogatemarkers for clinical endpoints, with the hypothesis that changes in CTCcounts may be an early sign of therapeutic efficacy. This use builds onthe association of CTC counts with prognosis in a muchmore statistical-ly strict sense. In order for a biomarker (CTCs) to qualify as a true surro-gate for an outcome such as survival, the biomarker must fulfill criteriafor surrogacy, which states that for all treatments under considerationthere is a single pathway from treatment to true endpoint that goesthrough the surrogate biomarker, such that once the surrogate endpointis known, no other information is needed to determine the distributionof the true endpoint (Prentice, 1989; Schwab, 2009).

One study demonstrated that persistently high CellSearch CTCcounts are a poor prognostic marker for survival in metastatic breastcancer patients evenwhen radiologic evaluation shows disease stability,suggesting that CTC enumerationmay detect disease progression earlierthan radiologic assessment (Budd et al., 2006). More work will need tobe done to see if these CTCs meet the strict criteria for surrogacy, how-ever. Inmen taking abiraterone acetate, a novel, recently approved hor-monal therapy in advanced prostate cancer, the change in CTCs countscoupled with changes in the level of serum lactate dehydrogenase ap-pears to correlate with survival better than changes in PSA (H.I. Scheret al., 2011; H. Scher et al., 2013). This exciting finding, if validated asa true surrogate for survival in the context described above, could po-tentially streamline the Phase III clinical trials process, allowing for anassessment of benefit at a much earlier time point and potentially re-ducing the need for long-term clinical follow up prior to drug-approval.

4.4. Circulating tumor cells in clinically localized (non-metastatic) disease

In non-metastatic disease, the greatest clinical application exploredto date has been CTC enumeration as a prognostic biomarker for diseaserecurrence after definitive therapy. The SUCCESS trial analyzedCellSearch CTCs from almost 1500 women with node-positive andhigh-risk node-negative breast tumors undergoing adjuvant chemo-therapy and followed for a median of 32 months. More than 1 CTCwas detectable in 9.4% of subjects prior to chemotherapy, which corre-lated with the number of lymph nodes involved. In a multivariate anal-ysis, the presence of N1 post-treatment CTC was an independent



Table 1CTC enrichment and detection platforms.

Approach CTC enrichment platforms CTC enrichmentmarkers Post-enrichment CTCdetection techniques

CTC detection markers Subpopulationsidentifiedduring enrichment

Subpopulationsidentifiedduring detection

Cancers studied References

Immunological CTC-iChip CD15,CD45 +/− EpCAM

Immunofluorescence,cytopathologic, FISH

Multimarker Epithelial, mesenchymal Epithelial, mesenchymal Prostate, lung, breast Ozkumur et al., 2013

Immunological Magnetic Cell Sorting(MACS)

CD45 Immunofluorescence EpCAM, CK(7,8), CD45 Epithelial Epithelial Breast Tkaczuk et al., 2008

Immunological Microfluidic cellconcentrator (MCC)

CD45 MCC imaging EpCAM Epithelial Epithelial Prostate, breast Casavant et al., 2013

Immunological MACS CK, human epithelialantigen (HEA-125)

Immunocytochemistry CK 5,6,8,17 and 19 Epithelial Epithelial Renal Bilkenroth et al., 2001

EpCAM Chip-based nanostructuredsensors

PSA, TMPRSS/ERG Epithelial Epithelial Prostate Ivanov et al., 2012

Immunological Immunomagentic cellseparation unit (autoMACS)

CD45 Immunocytochemistry CK Epithelial Epithelial Prostate, bladder,renal cell carcinoma

Meye et al., 2002

Immunological MACS plus nested mutantallele-specific amplification(nested MASA)

CD45 RT-PCR p53 and/or K-ras – – Colorectal Iinuma et al., 2011

Immunological PowerMag* CD45, Dextran coatedmagnetic nanoparticles

Immunofluorescence EpCAM, CD45 Epithelial Epithelial Head and neck Lin et al., 2013

Immunological Immunomagentic labeling* CD45 Immunocytochemistry,RT-PCR

CK, CD45, EGFR mRNA Epithelial Epithelial Head and neck Yang et al., 2009

Immunological Cell Search Veridex EpCAM Immunocytochemistry CK(8,18,19), CD45 Epithelial Epithelial Prostate, breast, colon Hayes et al., 2006;M. Cristofanilli et al., 2004;G. T. Budd et al., 2006;Allard et al., 2004;Thalgott et al., 2013

Immunological Cell Search Veridex plusNanoVelcro Chip

EpCAM Immunocytochemistry CK(8,18,19), CD45 Epithelial Epithelial Breast Lu et al., 2013

Immunological Magnetic cell sorting system EpCAM, CD45 Immunocytochemistry Plastin3 (PLS3),Vimentin, CK

Epithelial Epithelial, mesenchymal,EMT

Colorectal Yokobori et al., 2013

Immunological Adna Test (Immunomagneticenrichment)

EpCAM, MUC-1,Her2 Transcripts

– Twist, Akt2, PI3Kα Epithelial Epithelial, mesenchymal,EMT, stem cells

Breast B. Aktas et al., 2009

Immunological Immunomagentic Dynabeads CD45 Enzyme-linked immunosor-bentspot assay (ELISPOT)

Cath-D, MUC1, CK Epithelial Epithelial Breast Alix-Panabières et al., 2005

Epithelial antibody(Ber-EP4)

Immunocytochemistry, RT-PCR CK Epithelial Epithelial Head and neck,breast, colon

Zigeuner et al., 2000;Partridge et al., 1999;Sieben et al., 2001;Park et al., 2001

Immunological Immunomagnetic beads EpCAM Immunocytochemistry,Flow cytometry

CK, CD45 Epithelial Epithelial Breast, prostate Liberti et al., 2001;Moreno et al., 2001

Immunological CTC microseparator EpCAM Immunocytochemistry CK, CD45 Epithelial Epithelial Lung, breast Kim et al., 2013Immunological Magsweeper EpCAM, CD45 qPCR PSA, KLK3, TMPRSS2,CD45 Epithelial Epithelial Prostate Cann et al., 2012Immunological Cynvenio Biosystems EpCAM Next generation sequencing

and/or quantitativeallele-specific PCR

EpCAM Epithelial Epithelial Lung, breast Parker et al., 2012

Immunological Fluxion Biosystems EpCAM CK,CD45 Epithelial Epithelial Breast, pancreatic,colon

Stresemann et al., 2012

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Mutation detection,next-generation sequencing,FISH, and gene expression

Immunological GEDI microfluidic device PSMA Confocal microscopy TMPRSS/ERG – – Prostate Kirby et al., 2012Immunological +

Immuno-depletion

CD45, EpCAM Flow cytometry EpCAM, CK(7,8), CD45 Epithelial Epithelial Skin Liu et al., 2011

Immunological +Immuno-depletion

Cell Search Veridex andRosetteSep

EpCAM (Veridex),CD45 (RosetteSep)

Flow cytometry CD44, CD47, EpCAM,MET, CD45

Epithelial (Veridex) Epithelial, mesenchymal,EMT, stem cells

Breast Baccelli et al., 2013

Size 2D,3D microfilter size Marker independent Scanning electron microscopy,confocal microscopy,immunofluorescence

CK Epithelial Epithelial MCF-7 and LnCAPcell lines

Zheng et al., 2011;Lin et al., 2010

Size Isolation by size of epithelialtumor cells (ISET)

Marker independent Immunocytochemistry CK Epithelial Epithelial Hepatocellular (liver) Vona et al., 2000;Vona et al., 2004

Size Screencell Marker independent Immunocytochemistry,RT PCR

EpCAM, CK Epithelial Epithelial Breast, colorectal, skin Desitter et al., 2011;Attard & de Bono, 2011

Size/Deformity/Inertia

ClearCell FX Marker independent – – Epithelial Epithelial Bladder and lungcancer cell line

Warkiani et al., 2013

Density Oncoquick density gradientseparation

Marker independent Immonocytochemistry,RT PCR

CK(8,18,19), EpCAM Epithelial Epithelial Breast, colon, rectal,gastric, ovarian, prostate

Balic et al., 2005;Baker et al., 2003

Density Ficoll-Paque Marker independent RT PCR CK, PSA Epithelial Epithelial Colon, breast, prostate Paterlini-Brechot & Benali,2007

Density RosetteSep Marker independent Flow cytometry Folate – – Ovarian, prostate He et al., 2008Biological Vitatex Cell adhesion

Matrix (CAM) platformMarker independent Immunocytochemistry,

flow cytometry, aCGHEpCAM, CK, CD44, CD34,CD45, Vimentin

Epithelial, mesenchymal,EMT, stem cells


Ovarian, prostate, breast Fan et al., 2009; Lu et al., 2010;Paris et al., 2009

Enzymatic EPISPOT CD45, CXCR4 Enzyme-linked immunosor-bentspot assay (ELISPOT)

PSA, MUC-1, Cath-D,others

– – Breast, prostate, colon Alix-Panabières et al., 2005

Electrophoresis Dielectrophoreticfield-flow fractionation(depFFF) chamber

Marker independent Flow cytometry CK – Epithelial Colon, melanoma,colorectal

Gascoyne et al., 2009;Shim et al., 2013;Sabuncu et al., 2010

Electrophoresis ApoStream Marker independent Immunocytochemistry EpCAM, CK Epithelial/EMT Epithalial/EMT Ovarian, Breast cancercell lines

Gupta et al., 2012

Nucleic acid Real time polymerasechain reaction (RT-PCR)

PSA, Prostate specificmembrane antigen(PSMA)

– – – – Prostate Yates et al., 2012;Morgan et al., 2007;Olsson et al., 1996

Nucleic acid Single and multiplexRT-PCR

CK19, MAGE-A3, HER-2,TWIST1, Htert α+β+

– – – Epithelial, mesenchymal,EMT, stem cells

Breast Strati et al., 2011

Microscopy Fibre-optic arrayscanning technology(FAST)

CK – – – Epithelial Breast Hsieh et al., 2006;Krivacic et al., 2004

Microscopy TelomeScan OBP-401(GFP-expressingattenuated adenovirus)

Immunocytochemistry,Flow cytometry, RT-PCR

CK Epithelial, mesenchymal,EMT, stem cells


Non-small lung, gastric,colorectal, prostate, breastand cervical cancer cell lines

Kojima et al.., 2009

Microscopy Epic Sciences CK FISH PTEN – Epithelial Prostate Punnoose et al., 2013

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predictor of worse disease-free and overall survival. Taken in context,this study suggests that CTC enumeration before and after adjuvant che-motherapy, coupled with clinical predictors of recurrence includingtumor size, lymph node status and HER2 receptor amplification, couldaid in the assessment of disease recurrence risk in women with breastcancer.

A second study performed in the Netherlands explored the po-tential for CTCs to predict recurrence in non-metastatic breast can-cer. In this study, CellSearch CTC counts were evaluated in 404women undergoing surgery for stage I–III breast cancer (Frankenet al., 2012). The authors found that women with ≥1 CTC/30 ml atthe time of surgery were more likely to have disease recurrencethan women who had no CTCs detected (4 year relapse-free survival78.9% vs. 88.4% p = 0.0038). While statistically significant, the factthat CTCs were detected in 15% of women with benign tumors and19% of women with DCIS, that recurrences occurred in CTC-negative subjects, and the somewhat small absolute difference inrelapse rates between women with detectable and undetectableCTCs raises questions about how to best incorporate CellSearch CTCenumeration for disease recurrence prediction going forward. In asimilar trial, the GEPARQuattro study, changes in HER2+ CTC countswere evaluated in response to neoadjuvant chemotherapy fornon-metastatic breast cancer (Riethdorf et al., 2010). The CTC chang-es were not correlated with primary tumor response to therapy;however long term results evaluating disease recurrence areawaited.

4.5. Circulating tumor cells aspredictive biomarkers to guide treatment selection

While it is encouraging that CTC enumeration has been developed asa clinical tool to establish prognosis for many metastatic epithelial ma-lignancies, and may be useful in predicting recurrence, a potentiallymore useful clinical application would be to use either baseline CTCcounts, or the change in CTCs in response to a specific therapy, as a bio-marker predictive of response to a specific therapy. The reason a predic-tive biomarker is generally more useful clinically than a prognosticbiomarker is that a predictive biomarker (e.g. decline in CTC counts)can tell clinicians when it is appropriate to continue or discontinue aspecific therapy, and can therefore lead to a more personalized ap-proach to treatment. CTC enumeration for predictive purposes is attrac-tive because it is less costly and relatively less invasive than radiologicimaging, andmay provide amore rapid assessment of treatment relatedchanges than radiographs, which typically require a longer periodof treatment (generally 2–3 cycles of systemic therapy) before tumorregression becomes readily apparent.

The ongoing SWOG S0500 study (NCT00382018) in non-metastaticbreast cancer is one of the largest studies currently evaluating the use-fulness of CTC enumeration in guiding choice of therapy. This study ran-domizes women with N5 CTCs/7.5 ml both at baseline and after 1 cycleof adjuvant therapy to receive eithermore of the same adjuvant chemo-therapy or to switch to a different regimen, with the hypothesis thatpersistent elevation of CTCs after 1 cycle of treatment indicates thatthe residual tumor cells may harbor therapeutic resistance and that achange to an alternative treatment will be beneficial. The DETECT IIIstudy (NCT01619111) is another randomized Phase III trial evaluatingthe role of lapatinib, a multi-target tyrosine kinase with affinityfor HER2, in HER2-negative metastatic breast cancer patients withdetectable HER2-positive CTCs. Similarly, in the TREAT-CTC study(NCT01548677) women with HER2-negative primary breast cancerwith detectable CTCs after the completion of neoadjuvant chemothera-py are randomized to receive trastuzumab or placebo, with the suppo-sition that more therapy will reduce the chance of disease relapse inwomen at high risk of recurrence. Lastly, the COMETI P2 study(NCT01701050), while not a therapeutic trial, is evaluating the valueof an algorithm combining the level of CTC expression of the estrogen


receptor (ER), Bcl-2, HER2, and Ki-67 to predict sensitivity to hormonaltherapy in ER-positive, HER2-negative metastatic cancer. If successfuland validated prospectively, this could have the potential to helpguide selection of endocrine vs. chemotherapy for these women.

A challenge in evaluating CTCs as a predictive biomarker is that notall therapies work in the same way, and while some, such as most che-motherapies, lead to direct tumor necrosis or apoptosis, others, likeagents targeting VEGF or the tumor microenvironment, may have littledirect effect on CTCs. Therefore, results from any one trial exploring thepredictive value of changing from one therapy to another based on CTCcountsmay not be generalizable to other treatments or othermalignan-cies. Thus more data from large clinical trials will be needed to fullyassess the utility of CTCs in predicting response.

4.6. New clinical directions for circulating tumor cells

A more recent study explored the dynamic change between breastcancer CTCs expressing epithelial and mesenchymal markers, andsuggests that a change from epithelial to mesenchymal phenotype cor-relates with disease progression, and that subsequent ‘effective’ chemo-therapy can cause a reversion back to an epithelial phenotype (M. Yuet al., 2013). This is an exciting observation, as it suggests that dynamic,treatment-induced changes are occurring in CTCs, and raises the ques-tion of whether agents designed to inhibit EMT or to target mesenchy-mal cells will have therapeutic value in combination with agents thattarget epithelial tumor cells. The prognostic value of this will need tobe better assessed in larger, prospective studies, to ascertain whetherthe switch to amoremesenchymal phenotype can be used to predict re-sponse to subsequent therapy.

Lastly, a recent study explored whether molecular profiling of CTCscan guide treatment decisions in prostate cancer (Miyamoto et al.,2012). In this study, single cell immunofluorescence was performedon androgen-stimulated and androgen-deprived LNCaP cell lines to es-tablish a signature of active androgen receptor (AR) signaling. This sig-nature of active AR signaling (PSA+, PSMA−) was then applied to CTCsfrom men with metastatic prostate cancer captured on an EpCAMantibody-coatedmicrofluidic chip. The authors found that initial andro-gen deprivation in patientswith castration-sensitive diseasewas associ-ated with a profound downregulation in AR signaling, althoughsomewhat variable responses were detected in response to secondaryhormonal therapies when given to men with CRPC. Interestingly, inmen with CRPC the presence of a persistent “AR-on” signal despitetreatment with abiraterone acetate, an inhibitor of androgen synthesis,was associated with poor outcomes, suggesting that abiraterone wasfailing to suppress the AR and likely leading to treatment resistanceand disease progression. This work, while preliminary and requiringfurther confirmation in larger studies, suggests the possibility that serialmolecular phenotyping of CTCs could be used clinically to monitorresponse to therapy, and to select patients with adverse features to re-ceive different treatments.

The vast majority of clinical trials incorporating CTCs as biomarkershave used platforms that detect CTCs expressing epithelial markers.While the results of many of these studies are promising and newresults are eagerly awaited, future clinical trials incorporating CTC sub-populations, such as those undergoing EMT or those with stem-likecharacteristics, may be additionally informative.

5. Circulating tumor cell subpopulations

The majority of the aforementioned clinical trials have focused onthe clinical utility of CTC enumeration, andmany of these trials have fo-cused only on CTCs expressing epithelial markers. Yet, as discussed pre-viously there is emerging evidence that EMT plays a significant role inthe establishment of metastases. Similarly, there is increasing interestin characterizing CTCs with stem-like features, and in using CTCs to




explore the relationship between EMT, stemness, andmetastasis in can-cer cell biology.

5.1. Stem-like circulating tumor cells

The concept that tumor cells within a primary tumor are hetero-geneous in terms of their metastatic and renewal potential is a not arecent one. Studies done more than 50 years ago demonstrated thatonly some tumors could be grown from a single tumor cell, and sim-ilarly that the daughter cells could bemorphologically quite differentfrom the parent (Polyak & Hahn, 2006). This work laid the founda-tion for the modern cancer stem cell hypothesis, which posits thatwithin tumors there exists a subset of cells that are capable of contin-uous self-renewal and that can give rise to differentiated progeny(Polyak & Weinberg, 2009). Supporting this notion is the fact thatbenign epithelial tissues exhibit heterogeneity, with evidence ofslowly-dividing epithelial stem cells, more mitotically-active transitamplifying cells, and terminally differentiated epithelial cells thatserve various tissue-specific functions (Smalley & Ashworth, 2003).As metastatic epithelial malignancies retain a large number oftissue-specific characteristics (e.g.: PSA secretion in prostate cancer,ER/PR expression in luminal breast cancers), it is reasonable toassume that a similar process of differentiation from a morededifferentiated stem-like cancer cell to a terminally-differentiatedepithelial cancer cell may take place.

As CTCs arewidely thought to arise fromprimary tumors and to like-ly represent metastases in transition, they provide an ideal platform inwhich to explore the role that stem cells play in metastasis. One of thefirst challenges in discussing CTCs that bear stem-like qualities is defin-ing exactly what phenotypically defines these cells. The observationthat as few as 100 CD44+/CD24−/low cells can establish tumors that re-capitulate the phenotypic heterogeneity of the primary tumor, whereastens of thousands of cells with alternate phenotypes cannot, has led tothe incorporation of the CD44+/CD24−/low phenotype as a marker ofpotential cancer stem cells (Al-Hajj et al., 2003). Other markers studiedinclude Seprase, CD133, alpha-6 integrin, B1-integrin, Oct-4, expressionALDH, and dysregulation of theWnt and Hedgehog signaling pathways(Singh et al., 2004; X. Yu et al., 2011; Friedlander et al., in press).

In a study of womenwithmetastatic breast cancer, CTCs bearing theputative stem-like signature of CD44+/CD24−/low were detectable in80% of women, with anywhere from 8.3 to 100% of cells bearing thisphenotype (Theodoropoulos et al., 2010). In this study approximatelyhalf of these women also had CTCs that stained ALDHhigh. Another sim-ilar study showed that 69% of womenwith breast cancer bear CTCs thatexpress ALDH transcripts, and suggested that high ALDH is a marker ofpoor prognosis in advanced disease (Aktas et al., 2009). Similarly, otherinvestigators have correlated the presence of CTCs expressing the com-bination of CD133/cytokeratin/carcinoembroynic antigen with signifi-cantly worse disease-free survival (DFS) and OS in patients withresected colon cancer who might benefit from adjuvant chemotherapy(Iinuma et al., 2011). These findings have led to the proposition thatmarkers of stemness on CTCs could be used as prognostic biomarkers.Keep inmind,many of these stem-like CTCs do not have EpCAMexpres-sion, but show evidence of EMT, and thus would be missed by mostepithelial platforms (Wicha & Hayes, 2011). This work, although stillpreliminary also raises the possibility that targeting CTCs and tumorsthat bear stem-like characteristics with antibodies specific for markersof stemness could have a therapeutic effect. Indeed, a monoclonal anti-body directed against CD44 called RG7356was recently shown to resultin complete clearance of engrafted chronic lymphocytic leukemia cellsin a mouse model (S. Zhang et al., 2013). Whether this will have thera-peutic effect in humans, and particularly in epithelial malignancies isunknown. Anti-CD44 antibody targeted therapy is under early investi-gation in hepatocellular carcinoma and in pancreatic cancer as well(Tang et al., 2011; Wang et al., 2012).


5.2. Mesenchymal circulating tumor cells

In addition tomarkers of stemness, multiple studies have focused onbetter defining the EMT subpopulation, and in establishing its relevancyto cancer progression, invasion, and metastasis. Multiple studies haveshown that CTCs bear EMT markers including N-cadherin, O-cadherin,vimentin, TWIST1, Snail, Slug, Zeb1, and FOXC2 (Armstrong et al.,2011; Kasimir-Bauer et al., 2012; M. Mego et al., 2012) Specifically,one study quantified the presence of CTCs undergoing EMT in womenwith primary or metastatic breast cancer, including some women withinflammatory breast cancer (M. Mego et al., 2009). After depletion ofCD45-positive cells, the authors extracted RNA from CTCs in order to as-sess the level of expression of TWIST1, SNAIL1, SLUG, ZEB1, FOXC2 andEpCAM gene transcripts. At least one of these transcripts wasoverexpressed compared to a healthy control in 21% of patients, andthere was a higher frequency of detection of these transcripts inwomen with triple-negative breast cancer (30.8% vs. 13.3%). Taken to-gether, this highlights the heterogeneity present in the CTC pool. Thepresence of CTCs bearing a mesenchymal phenotype has also been cor-related with worse outcomes in breast cancer (Gradilone et al., 2011).

Delving deeper into the biology of EMT, a particularly intriguingstudy was able to demonstrate that a dynamic flux in breast cancerCTCs between epithelial and mesenchymal states is detectable and ap-pears to correlate with response to therapy (M. Yu et al., 2013). In thisstudy, the authors analyzed CTCs captured on a herringbone chip con-taining antibodies directed against EpCAM, EGFR, and HER2, and thenperformed mRNA in-situ hybridization to detect the presence of tran-scripts associated with either an epithelial (cytokeratins 5, 7, 8, 18,and 19, EpCAM and CDH1) or mesenchymal (fibronectin 1, CDH2, andSERPINE/PAI1) state. The authors were then able to quantitate the per-centage of epithelial vs. mesenchymal CTCs at any time point and wereable to show that the mesenchymal fraction increased with resistanceto targeted therapy, suggesting a possible mechanism by which tumorsovercome effective therapies. Using RNA-sequencing and Gene Set En-richment Analysis in a CTC sample containing a high fraction of mesen-chymal cells, the authors were also able to show upregulation ofmultiple genes associated with EMT including genes associated withthe extracellular matrix, TGF-B, FOXC1, and WNT signaling.

5.3. Circulating tumor cell clusters

CTC clusters have been observed in a number of studies, and their re-lationship to stemness, EMT, and tumor progression is under investiga-tion (Molnar et al., 2001; Paris et al., 2009; Friedlander et al., in press). Amajor challenge in the study of CTC clusters is the fact that many CTCisolation platforms disrupt cell–cell contact and presumably lead tothe breakdown of these clusters into individual cells. Similarly, there isdispute over whether clusters are an artifact of the CTC isolation proce-dure. Therefore, comparatively less is known currently about thephenomenon of CTC clusters.

The study of CTC clusters is warranted though, as their observationraises the possibility that conglomerates of tumor emboli either shedby the primary tumor or formed/aggregated in circulation becomelodged in distant sites and are able to survive and proliferate, in contrastto the hypothesis that solitary EMT or stem-like cells are responsible fordistal spread of tumors. The notion that tumor cell clusters play a role inmetastasis is supported by the observation that tumor emboli are capa-ble of forming metastases in multiple preclinical models (Watanabe,1954; Fidler, 1973) and by the fact that there may be less autolysis inclusters as evidenced by relative hematoxylin and eosin stainingintensities (Zheng et al., 2007; Z. Zhang et al., 2008), suggesting thatcluster formationmay serve to protect CTCs from the stresses of circula-tion. Recent work has shown that E-cadherin is overexpressed inlymphovascular emboli and that E-cadherin cleavage may directly me-diate the formation of these clusters and promote tumor cell survival(Ye et al., 2012). Interestingly, in a recent study (M. Yu et al., 2013)




CTC clusters were observed more frequently in patients with primarilymesenchymal CTCs andwere absent from specimenswithmore epithe-lial CTCs.Whether thismeans that epithelial clusters are able to undergoEMT, that solitary mesenchymal cells are able to form clusters (possiblydue to TGF-β-mediated interaction with platelets), or a totally separatephenomenon remains to be seen. Further study will need to betterexplore the correlation of the presence of CTC clusters with risk ofmetastasis and overall prognosis in patients.

6. Circulating tumor cell culture

Although there are many different techniques available to isolateand enumerate CTCs from peripheral blood, downstream molecularand genomic characterization can be challenging due to the lownumberof cells that can be isolated at any one time point. Study of fixed CTCsprovides relatively little data about their functional capability and met-astatic potential. Culture of CTCs both in vitro and in animal models cantherefore allow for a better understanding of the functional capacity andmetastatic potential of CTCs. Similarly, CTC culture could aid in the eval-uation of drug sensitivity and allow for the generation of a larger pool ofcells from which genomic and correlative work can be performed.

It has been observed that tumor cells injected into themurine blood-stream can be recovered, and that these recovered cells have increasedcolony formation compared to the parental cultured cells. Interestingly,when these recovered cells are reinjected into new murine hosts theydisplay an increased metastatic capacity, hinting at the possible pres-ence of stem-like precursors. Recently, it has been shown that prostatecancer cells are recoverable in murine models after spiking and thatcell clusters are observable directly on a CTC-chip after multiday culture(Helzer et al., 2009).

In humans, CTCs isolated on different platforms are expandable inculture. In our work with the VitaAssay (Vitatex) platform, we haveshown that isolated CTCs staining positively for pan-cytokeratins canbe expanded in vitro for up to 14 days (Paris et al., 2009). Similarwork has been performed with CTCs isolated from breast cancer andlung cancer patients (Hughes et al., 2012).

Xenotransplantation of CTCs into immunodeficient mice is an activearea of exploration. In small cell lung cancer, CTCs captured using theRosetteSep platform and transplanted subcutaneously into immunode-ficient mice will grow into tumors that histologically resemble the pri-mary malignancy. These tumors are reimplantable after growth in themouse and retain their metastatic capacity (Hodgkinson et al., 2013).In this small study, tumor take rate was approximately 40%, with mosttumors forming within 3–4 months on transplantation. CTCs havebeen successfully passaged in orthotopic models for a number of othermalignancies, including breast cancer and melanoma (Benali-Furetet al., 2013; Mihalcioiu, 2013).

Determination of sensitivity to chemotherapy and other new agentsis another potential application for CTCs grown in culture. In particular,in one study CRPC CTCs were captured on the GEDI microfluidic deviceand exposed to different types of taxane chemotherapy (Kirby et al.,2012). The authors observed that the microtubule response to chemo-therapy in CTCs trendedwith clinical progression, raising the possibilitythat ex vivo drug testing could be performed on CTCs, thereby guidingtherapy selection and sparing patients ineffective treatment. Whethertreatment of CTCs with novel therapeutics either in vitro or in animalmodels will fully recapitulate the responses observed clinically will re-quire more study; the payoff to patients and clinicians however, is po-tentially very large, and more studies of this nature are eagerlyanticipated.

7. Conclusion

Taken together the aforementioned studies suggest that there is afundamental shift in the CTCfield frombench to bedside,with a numberof studies focusing on translating CTC characteristics into clinical utility.


Still there is much more to learn about CTCs, their subpopulations, andthe potential downstream uses including molecular and genomiccharacterization, and ex-vivo expansion and xenotransplantation.

CTC enumeration has demonstrated utility in establishing prognosisin both localized and metastatic cancers, and current investigations inmultiple tumor types are focused on their use as a biomarker predictiveof response in order to help guide therapy selection. Change in CTCcounts in response to a specific therapy also has an important potentialto serve as a surrogate biomarker in clinical trials, allowing patients andclinical investigators to knowwhether a specific therapy will affect sur-vival potentially years ahead of time.

In recent years, the field has expanded beyond enumeration and alarge proportion of current work is focused on better characterizingCTC subpopulations including CTC clusters and mesenchymal andstem-like CTCs in order to investigate tumor biology and mechanismsof metastasis. At the same time, culture and xenotransplantation ofCTCs have the potential to inform individual patients and clinicians,essentially in real time, about the functional biology of a given tumor.

Genomic study of CTCs and cell free DNA in serum, while notdiscussed in depth here, holds potential to identify genomic targets fortherapy, and currently a large number of researchers are exploringthis possibility (Dawson et al., 2013; Diaz et al., 2012). Because singlecell analysis is challenging both targeted sequencing of cancer-relatedgenes, as well as whole genome approaches, are being explored, andmay allow for a more personalized approach to therapy selection.

One of the remaining significant challenges to this work is the ques-tion of tumor and CTC heterogeneity.While not included in the scope ofthis review, novel techniques such as the DEPArray system that allowfor the isolation and genomic amplification of single CTCs (Peeterset al., 2013) can potentially shed more light on the prevalence oftumor heterogeneity within an individual's tumor as well as potentiallyallow for the identification of rare targets that may have important clin-ical implications. Taken together, CTCs are an easily accessible resourcethat can potentially provide awealth of important clinical, genomic, andbiologic data, and are rapidly becoming an important tool in the fightagainst cancer.

Authors' disclosures of potential conflicts of interest

The authors declare no potential conflict of interest. This work hasnot been published previously and is not under consideration for publi-cation elsewhere. This publication is approved by all authors.

The authors received no financial support or external funding forthis publication.

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looking back, to the future of circulating tumor cells

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