Biochimica et Biophysica Acta 1796 (2009) 27–32

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Biochimica et Biophysica Acta

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Review

The multiple personality disorder phenotype(s) of circulating endothelialcells in cancer

Francesco Bertolini a,⁎, Patrizia Mancuso a, Paola Braidotti b, Yuval Shaked c, Robert S. Kerbel d

a European Institute of Oncology, Division of Hematology-Oncology, Department of Medicine, European Institute of Oncology, via Ripamonti 435, 20141 Milan, Italyb University of Milan, Department of Medicine, Surgery, Odontology, San Paolo Hospital and IRCCS Fondazione Policlinico, Mangiagalli, Regina Elena, Milan, Italyc Technion-Israel Institute of Technology, Rappaport Faculty of Medicine, Department of Molecular Pharmacology, Haifa, Israeld Sunnybrook Health Sciences Centre, Molecular and Cellular Biology, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M4N 3M5

⁎ Corresponding author. Tel.: +39 02 57489535; fax:E-mail address: francesco.bertolini@ieo.it (F. Bertolin

0304-419X/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.bbcan.2009.04.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 November 2008Received in revised form 20 March 2009Accepted 20 April 2009Available online 3 May 2009

Keywords:AngiogenesisEndothelial cellsEndothelial progenitors

Circulating endothelial cells (CECs) and circulating endothelial progenitors (CEPs) are currently beinginvestigated in a variety of diseases as markers of vascular turnover or damage and, also in the case of CEPs,vasculogenesis. CEPs appear to have a “catalytic” role in different steps of cancer progression and recurrenceafter therapy, and there are preclinical and clinical data suggesting that CEC enumeration might be useful toselect and stratify patients who are candidates for anti-angiogenic treatments. In some types of cancer, CECsand CEPs might be one of the possible hidden identities of cancer stem cells. The definition of CEC and CEPphenotype and the standardization of CEC and CEP enumeration strategies are highly desirable goals in orderto exploit these cells as reliable biomarkers in oncology clinical trials.

© 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272. Molecular, ultrastructural and phenotypic definition of CECs and CEPs: a work in progress, but there is light at the end of the tunnel . . . . . . 283. CEC number and viability is modulated by cancer and treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294. CEPs: role in development of macro-metastases and resistance to chemotherapy, VDAs and anti-angiogenic drugs . . . . . . . . . . . . . . . 295. Cancer-specific CEC/CEPs: possible biomarkers of occult cancer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296. Genetically aberrant CEC/CEPs: the hidden identity of cancer stem cells, or just another site to look for minimal residual disease? . . . . . . . 307. CECs/CEPs in the clinic: catalytic versus biomarker roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

1. Introduction

Cells with an endothelial morphology were found to circulate inthe blood more than 35 years ago [1]. Twenty years later, theendothelial nature of these cells was confirmed by immunohisto-chemistry (IHC) studies, and their enumeration by means of positiveenrichment, IHC or flow cytometry (FC) indicated that levels ofcirculating endothelial cells (CECs) are increased in a very widespectrum of disorders encompassing vascular, autoimmune, infectiousand ischemic diseases [2–8]. Over the past ten years, increased CEC

+39 02 57489537.i).

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counts were observed in some cancer patients [9–21], and these cellswere studied as surrogate biomarkers of angiogenesis and anti-angiogenic drug activity in preclinical models and medical oncology[22–26]. These studies also indicated that the endothelial phenotypewas expressed by cells displaying a wide variety of different features[6–8]. Some CECs had a phenotype compatible with terminallydifferentiated endothelial cells (EC), in some cases being apoptoticor necrotic and thus most likely derived from the turnover of vesselwalls. Some other cells expressed progenitor-associated antigens inaddition to endothelial antigens, and were considered candidates as“circulating endothelial progenitors” (CEPs). This concise review willfocus on some of the most recent findings about CECs and CEPs incancer. Some novel (and possibly provocative) hypotheses emergingfrom these studies are discussed.

Fig. 2. Overview of cellular events with an EC phenotype enumerated by flowcytometry. The majority of CD45−,CD31+CD146+events are apoptotic or necroticcellular fragments (1–10/μL in the blood). The use of Syto16 allows the enumeration ofDNA-containing CD45−CD31+CD146+CECs (0.01–1/μL). Along with CECs, TEMshowed the presence of some small, viable and lymphoid-like cells that are compatiblewith a progenitor cell morphology, lack CD45 expression andmay express CD133 and/orCD34 [62].

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2. Molecular, ultrastructural and phenotypic definition of CECsand CEPs: a work in progress, but there is light at the end of thetunnel

Although a comprehensive description of the different enumera-tion strategies of CECs and CEPs is well beyond the scope of thisconcise review, it should be mentioned that the two most frequentlyused CEC enumeration strategies, IHC and FC, have provided in manycases different CEC frequencies in health and disease [6–8]. Accordingto IHC enumeration, CECs are large cells present in a frequency of 10–100/mL in healthy subjects [6–7]. According to FC, events with an ECphenotype show inmost cases small dimensions and are countedwitha frequency of 100–10,000/mL [8–11,16–18]. Antigenic promiscuitybetween CECs and platelets has prompted the development andvalidation of new FC enumeration procedures where DNA stainingreagents have allowed the count and the sorting of platelet-depleted,DNA-containing cells with an EC phenotype (CD45−,CD31+CD146+;[62]). Studies which have used transmission electron microscopy(TEM), confirmed that these sorted CECs are of endothelial nature byvirtue of the presence of EC-specific Weibel-Palade bodies (Fig. 1) andof RNA transcripts for the EC-specific gene VE-cadherin. Transmissionelectron microscopy (TEM) studies also offered an explanation of thecontroversies about CEC frequency in the blood. Themajority of sortedCECs, in fact, were found to be apoptotic or necrotic cellular fragments,most likely lost at count after the cell processing involved in IHCenumeration. Along with apoptotic CECs, however, TEM showed thepresence of small, viable and lymphoid-like cells that are compatiblewith a progenitor cell morphology (Fig. 2).

TEMwill most likely be of help for the next crucial steps in CEC andCEP studies, namely, to dissect the functions of candidate CEC and CEP

Fig.1. Transmission electronmicroscopy images of sorted DNA+,CD45−CD31+CD146+CECapoptotic/necrotic cells most likely derived from vessel wall turnover along with lymphocycentriole is present). The arrow points to a Weibel-Palade body seen more detailed in theapoptotic cell.

subpopulations. Both these cell families, in fact, encompass subpopu-lations with different roles. Multiparametric FC has shown that amongDNA+,CD45−,CD31+,CD146+CECs there are some expressing otherEC-related antigens such as CD143, CD144, VEGFR1, VEGFR2, VEGFR3,along with activation antigens such as CD105 (endoglin), amongothers [8]. The need for a detailed phenotypic profile is particularlyurgent for CEPs, because CD34 and VEGFR2 antigens, used by manyinvestigators for CEP enumeration by FC [5,8,27–28], are expressedalso by mature CECs, and the use of CD133 antigen for CEPidentification [29–30] has led to the sorting of cells that not all

s. (a) Low-magnification overview of the sorted cell population, showing the presence ofte-like cells (arrows). (b) Endothelial (precursor?) cell undergoing cellular division (ainset. (c) A mature endothelial cell with a Weibel-Palade body (arrow). (d) An overtly

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laboratories were able to differentiate in vitro and in vivo along theendothelial lineage [31–32]. Even more controversies exist for theenumeration of CEPs in mice, because the expression and function ofCD34 and CD133 antigens are not well characterized in micecompared to humans. Thus a candidate phenotype for CEPs in miceis CD45−, VEGFR2(Flk)+, CD117+ [22–24], and antibodies reactingwith a particular configuration of CD144 are also used [33–34].

3. CEC number and viability is modulated by cancer and treatment

FC and IHC studies have indicated that in some types of cancerpatient CEC numbers and viability are increased when compared tohealthy controls [9–21]. Possible explanations for these findingsinvolve the angiogenic switch associated with cancer growth and therobust production of angiogenic growth factors such as VEGF, bFGF,HGF and many others by cancer cells and/or various host cells [8]. Therecent and unexpected finding of an autocrine loop in ECs [35] is ofparticular interest, because it might be that the increase of viable CECsin the blood of cancer patients mirrors an aberrant vascular turnover/remodeling associated with high local levels of VEGF produced bycancer cells.

Following the preclinical evidence that CEC count can be used as asurrogate biomarker for angiogenesis and anti-angiogenic drugactivity by means of determining the optimal biological dosage ofanti-angiogenic drugs [24–26], their numbers and viability have alsobeen measured in different clinical trials involving cancer patientstreated with various anti-angiogenic therapies [10–21]. An increase inthe number of apoptotic CECs after 60 days of therapy was associatedwith prolonged progression-free survival and overall survival inmetastatic breast cancer patients treated with a doublet low-dosemetronomic (anti-angiogenic) chemotherapy regimen [16]. When thehumanized anti-VEGF antibody, bevacizumab, was added to themetronomic chemotherapy for the treatment of metastatic breastcancer, patients who showed a clinical response in a phase II clinicaltrial (as well as a larger population of patients who had a clinicalbenefit from the treatment) had significantly greater baseline levels ofviable CECs than did patients who failed to respond; furthermore, thenumber of apoptotic CECs before therapy initiatedwas associatedwithprolonged progression-free survival [36]. In patients with renal cancertreated with the small molecule anti-angiogenic agent, sunitinib,changes in CECs differed between the patients with clinical benefitand those with progressive disease [18]. Taken together, these datasuggest that the investigation of CEC number and viability by FC haspotential for the stratification of cancer patients who are more likelyto benefit from anti-angiogenic treatments [8,25,37]. This possibilityawaits confirmation in prospective randomized clinical trials.

4. CEPs: role in development of macro-metastases and resistanceto chemotherapy, VDAs and anti-angiogenic drugs

The concept of CEP-dependent new blood vessel formation in adultlife was first proposed by Asahara et al. in 1997 [27] and Shi et al. in1998 [28]. These two seminal studies identified endothelial progeni-tors as cells that are capable of generating mature ECs in vitro and invivo. A study from Lyden et al. [38] was the first to identify a role forCEPs in cancer growth using Id (Id1+/−Id3−/−) deficient mice. Thesemice have a severe CEP defect leading to impaired angiogenesis andtumor growth. More recently it was found that CEP generationdepends on the ability of Id to restrain the expression of its target genep21 [39]. Genetic ablation of p21 rescued CEP generation in Id1deficient mice, re-establishing normal tumor growth [39]. Under-standing whether all types of tumor rely, at least in part, on CEP-dependent vessel generation has been elusive, primarily because therelative contribution of CEP-derived vessels was found to be extremelyvariable, and in most cases low, in different preclinical models ofcancer [38–43]. Clinical studies in patients who received a gender-

mismatched bone marrow transplant before cancer recurrence [44]indicated that CEP-derived vessels were indeed present, albeit at a lowfrequency (on average, 5% of all vessels).

Benezra's group reconciled these apparently conflicting data bydemonstrating that the recruitment of CEPs into tumor vasculaturedepends on the tumor grade [43] and also by showing that CEPs arekey contributors in the first steps of tumor vascularization in smalltumors. However, following the establishment of cancer vessels, theirrelative contribution to neoplastic angiogenesis is quantitatively lessrelevant as these cells become progressively diluted with the divisionof differentiated endothelial cells [33].

Three previously unrecognized and crucial roles for CEPs in tumorprogression have been recently suggested. Angiogenesis-mediatedprogression from micro- to lethal macro-metastasis is a leading causeof death in cancer patients. Using preclinical models of pulmonarymetastasis, the Mittal laboratory reported that tumors induce Id1expression in CEPs. Id1 suppression after metastatic colonizationblocked CEP mobilization, caused angiogenesis inhibition, impairedpulmonary macro-metastases, and increased survival of tumor-bearing animals [34]. In addition, a new perspective has recentlyemerged regarding what could be a critical role for CEPs in tumorangiogenesis, namely, following acute types of cytotoxic therapy. Forexample, Shaked et al. [45] found that treatment of tumor-bearingmice with vascular disrupting agents (VDAs), i.e., drugs which targetthe established but abnormal tumor vasculature, causing a rapidshutdown of blood flow followed by extensive tumor hypoxia andnecrosis, leads to an acute mobilization of CEPs, which subsequentlyhome to the viable tumor rim that usually remains after such therapy,and drives ‘rebound revascularization’ and tumor regrowth/recoveryfollowing VDA therapy. In another study, we [46] found that certainchemotherapy drugs (taxanes in particular) administered at max-imum tolerated doses (MTDs) can also induce a rapid CEP mobiliza-tion, most likely generated – at least in part – by the modulation ofcirculating SDF-1 levels. Prevention of the CEP spike by concurrenttreatment with targeted anti-angiogenic drugs, e.g. treatment withanti-VEGFR-2 or anti-VEGF monoclonal antibodies, or by geneticmanipulation strategies (e.g., undertaking treatment of tumors in Idmutant mice), or by the use of anti-SDF-1 neutralizing antibodies,resulted in enhanced antitumor activity of the administered cytotoxicchemotherapeutic drug. These findings raise the possibility thattherapeutic strategies which aim to reduce CEP mobilization, mightenhance the efficacy of certain cytotoxic anti-cancer therapies and – atthe same time – reduce the risk of cancer metastases.

These results point to a sudden and “catalytic” function for CEPs,which may be a consequence of the aforementioned cytotoxic drugsnot only being able to induce rapidmobilization of CEPs from the bonemarrow, but also because the agents can damage the tumorvasculature, thereby creating the need and favorable circumstancesfor their physical incorporation into damaged vessels as part of a rapidhost repair response. Indeed, cardiovascular researchers have for anumber of years been studying the hypotheses that rapidmobilizationof CEPs following damage to blood vessels caused by such pathologicevents as stroke or infarcts represents such an adaptive (reactive) hostrepair process. Interestingly, when certain chemotherapy drugs areadministered at much lower doses in a frequent repetitive fashion(i.e., “metronomic” chemotherapy), the acute CEP mobilizationresponse seen with MTDs is not only avoided, but that such cells areactually targeted [23,26]. This may be one of the mechanisms bywhich low-dose metronomic chemotherapy can cause an anti-angiogenic effect [47].

5. Cancer-specific CEC/CEPs: possible biomarkers of occult cancer?

A number of investigators have suggested that vessel-lining ECs intumors might express a specific antigenic profile. The St Croix groupcompared gene expression patterns of ECs obtained from normal

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resting tissues, tumors, and regenerating liver [48]. They identified 25transcripts overexpressed in tumor versus normal ECs, including 13that were not found in the angiogenic endothelium of regeneratingliver. Those EC tumor-specific antigens were primarily cell surfacemolecules of uncertain function. Should the tumor-specific expressionof these antigens be confirmed in CECs, it would be possible –

following the intuition of the late Folkman [49] – to monitor tumor-specific CECs in subjects at high risk for cancer development orrecurrence after therapy, in order to treat them with low-toxic anti-angiogenic (or other) targeted drugs before overt cancer symptoms orrecurrence. In other words, it might be possible to monitor a nascentcancer before it becomes malignant, and try to keep it dormant andavoid development of metastases. Anti-angiogenic drugs, given in thisway, may play a role similar to that of statins for the prevention ofvascular diseases.

6. Genetically aberrant CEC/CEPs: the hidden identity of cancerstem cells, or just another site to look for minimal residualdisease?

In contrast to cancer cells, tumor-lining ECs have long beenconsidered for many years a genetically stable host cell drug target[50]. Preclinical and clinical evidence has recently challenged thisdogma, and in hematological malignancies such as non-Hodgkin'slymphoma, myeloma, chronic and acute leukemias and myelodys-plastic syndromes there is evidence that ECs might share the samegenetic abnormalities found in cancer cells [51–55]. Although it is stillnot clear whether these unexpected findings were due to a commoncancer/EC progenitor, to cancer-to-EC trans-differentiation, or tofusion between cancer and ECs [8], there are at least two relatedquestions that deserve investigation.

First, are oncogene-bearing CEC/CEPs potentially able to causecancer recurrence, and if so, should they be investigated as a new ‘site’for minimal residual disease after therapy?

The second question stems from the recent findings of Shen et al.[56]. These authors have found that in some preclinical tumormodels, most blood vessels were derived from precancerous cancerstem cells (pCSCs). These pCSCs expressed VEGFR-2, and were muchmore potent in tumor vasculogenesis than the differentiated tumormonocytic cells from the same tumor. They also observed tumor-cellrelated vasculogenesis in human cervical cancer and breast cancers.These findings suggest that oncogene-bearing CEC/CEPs might beone of the possible hidden identities of cancer stem cells, andsuggest a possible mechanism for resistance to anti-angiogenic drugtherapy of cancer, currently a subject of considerable interest andimportance [57].

7. CECs/CEPs in the clinic: catalytic versus biomarker roles

Viewed in the light of the results summarized above, we foreseetwo separate fields of clinical investigation for CEPs and CECs. SomeCEPs (alongwith other hematopoietic cells) appear to have a transient“catalytic” but critical role [58] in promoting angiogenesis duringtumor growth, in stimulating growth of micro- to macro-metastases,and in ‘rebound’ revascularization after certain therapies are stopped[30,33–34,38–39,45,58–59]. These cells are potentially promisingtargets for anti-cancer therapies and for adjuvant therapeuticstrategies in patients at risk for cancer relapse. Also, one can try toexploit the tumor tropism of these cells for delivering anti-cancerdrugs specifically at tumor site (reviewed in [8]). On the other side ofthe coin, these catalytic functions are in most cases associated with apulsating presence of these cells in the blood and in the tissues, andthus it might be difficult to exploit measurement of these cells asbiomarkers for selection and stratification of patients. Regarding CEPs,two recent reviews are a step towards a better definition of thenomenclature and putative roles of different CEP types and related

bone marrow-derived cells that have been described in the literature[60–61].

CECs in most cases are apoptotic or necrotic cells, being releasedinto the circulation as a consequence of vascular turnover, and thusthey would not represent a “druggable” target for anti-cancertherapies. On the other hand, CEC presence in the blood seems lesspulsating (i.e., more stable) than CEPs and there is increasing evidenceof their potential as surrogate biomarkers of cancer angiogenesis andof anti-angiogenic drug activity [8–21].

8. Conclusions

In the 1983 movie Zelig, the title character, Leonard Zelig (playedby Woody Allen), is a man who has the ability to change hisappearance to that of the people he is surrounded by. For example, ifhe is among doctors, he transforms into a doctor; if around overweightpeople, he quickly becomes heavy himself. Zelig is called the “humanchameleon”. In cancer, the CEC/CEP field currently seems to suffer asimilar multiple personality disorder. There are hints that CECs andCEPs might serve as biomarkers for cancer or resurgence (or both),and there are preclinical and clinical data suggesting that theenumeration of these cells might be useful to select and stratifypatients who are candidates for anti-angiogenic treatments; further-more, they might be crucial for the process of cancer metastases. Insome types of cancer, CEC/CEPs might be new clothes for cancer(stem?) cells. Considering the current plethora of medical acronyms(CEP, for instance, is used for “care evaluation program”, “chroniceosinophilic pneumonia”, and “cortical evoked potentials”, in additionto circulating endothelial progenitors), one might consider referringto the broad CEC/CEP family as “Zelig cells”.

Acknowledgments

We apologize to the numerous investigators whose papers couldnot be cited because of space limitations.

Supported in part by AIRC (Associazione Italiana per la Ricerca sulCancro), ISS (Istituto Superiore di Sanità), Ministero della Salute grantRF-IMI-2006-411189 and the sixth EU Framework Programme(Integrated Project ‘Angiotargeting’; contract no 504743) in the areaof ‘Life sciences, genomics and biotechnology for health’. FB is ascholar of the US National Blood Foundation. RSK is a recipient of aTier I Canada Research Chair and is supported by grants from theNational Institutes of Health, USA (CA-41233), the Ontario Institutefor Cancer Research (OICR), and the National Cancer Institute ofCanada (NCIC).

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