Developmental tumourigenesis: NCAM as a putative marker for the malignant renal stem/progenitor cell...

Introduction

Wilms’ tumour (WT; nephroblastoma) is the most frequent tumourof the genitourinary tract in children and rated fourth in overallincidence among childhood cancers [1]. It is viewed as a proto-

type of differentiation failure in human neoplasia as it recapitulatesthe histology of the nephrogenic zone of the growing foetal kidneyand contains a stem cell compartment (termed ‘blastema’) alongwith more differentiated structures such as tubular epithelia, stromal elements and also other mesoderm elements (rhabdomy-oblasts, cartilage, osteoid tissue and fat) [2]. Recent moleculardata demonstrate that WTs systematically overexpress nephric-progenitor genes corresponding to the earliest stages of normalmetanephric kidney development [3, 4], connecting tumourigene-sis and organogenesis in the kidney [1]. With improved multi-modality therapy, WT survival rates have risen over the last 40 years to 85–90%; however, for those whose disease relapses

Developmental tumourigenesis: NCAM as a putative marker

for the malignant renal stem/progenitor cell population

Naomi Pode-Shakked a, f, Sally Metsuyanim a, Eithan Rom-Gross b, Yoram Mor c, f, Eduard Fridman e, f, Itamar Goldstein d, Ninette Amariglio d, f, Gideon Rechavi d, f,

Gilmor Keshet d, #, Benjamin Dekel a, f, #, *

a Department of Pediatrics and Pediatric Stem Cell Research Institute, Sheba Medical Center, Israelb Department of Pediatric Surgery, Hadassah Medical Center, Hebrew University, Israel

c Department of Urology, Sheba Medical Center, Israeld Department of Pediatric Hemato-Oncology and Sheba Cancer Research Center, Sheba Medical Center, Israel

e Department of Pathology, Sheba Medical Center, Israelf Sackler School of Medicine, Tel Aviv University, Israel

Received: April 28, 2008; Accepted: October 27, 2008

Abstract

During development, renal stem cells reside in the nephrogenic blastema. Wilms’ tumour (WT), a common childhood malignancy, is sug-gested to arise from the nephrogenic blastema that undergoes partial differentiation and as such is an attractive model to study renal stemcells leading to cancer initiation and maintenance. Previously we have made use of blastema-enriched WT stem-like xenografts propagatedin vivo to define a ‘WT-stem’ signature set, which includes cell surface markers convenient for cell isolation (frizzled homolog 2 [Drosophila] –FZD2, FZD7, G-protein coupled receptor 39, activin receptor type 2B, neural cell adhesion molecule – NCAM). We show by fluorescence-activated cell sorting analysis of sphere-forming heterogeneous primary WT cultures that most of these markers and other stem cell surface antigens (haematopoietic, CD133, CD34, c-Kit; mesenchymal, CD105, CD90, CD44; cancer, CD133, MDR1; hESC, CD24 and puta-tive renal, cadherin 11), are expressed in WT cell sub-populations in varying levels. Of all markers, NCAM, CD133 and FZD7 were constantlydetected in low-to-moderate portions likely to contain the stem cell fraction. Sorting according to FZD7 resulted in extensive cell death, whilesorted NCAM and CD133 cell fractions were subjected to clonogenicity assays and quantitative RT-PCR analysis, exclusively demonstrat-ing the NCAM� fraction as highly clonogenic, overexpressing the WT ‘stemness’ genes and topoisomerase2A (TOP2A), a bad prognosticmarker for WT. Moreover, treatment of WT cells with the topoisomerase inhibitors, Etoposide and Irinotecan resulted in down-regulation ofTOP2A along with NCAM and WT1. Thus, we suggest NCAM as a marker for the WT progenitor cell population. These findings providenovel insights into the cellular hierarchy of WT, having possible implications for future therapeutic options.

Keywords: stem/progenitor cells • Wilms’ tumour • renal cancer • renal development • stem cell markers

J. Cell. Mol. Med. Vol 13, No 8B, 2009 pp. 1792-1808

#These authors contributed equally to this manuscript.*Correspondence to: Benjamin DEKEL M.D., Ph.D., Pediatric Stem Cell Research Institute, Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, 52621, Israel.Tel.: �972-3-5302517Fax: �972-3-5305787E-mail: benjamin.dekel@gmail.com

doi:10.1111/j.1582-4934.2008.00607.x

J. Cell. Mol. Med. Vol 13, No 8B, 2009

or metastizes, even intensive salvage regimens result in subse-quent survival closer to 50% [5]. Moreover, survivors are atincreased risk for a broad spectrum of adverse outcomes causedby chemotherapy and radiation therapy, such as late mortality andsecondary cancers [6, 7].

It is becoming clear that many, if not most, malignancies arisefrom a population of cells that exclusively maintain the ability toself-renew and sustain the tumour via the expression of tumour-progenitor genes [8, 9]. These ‘cancer stem cells’ areoften biologically distinct from the differentiated cancer cells thatcomprise most of the tumour bulk. Because cancer stem cells arebelieved to be primarily responsible for tumour initiation as well asresistance to chemo- and radiotherapy, their persistence mayaccount for relapsing disease in WT [10–13]. The presence ofsuch cells, which have been identified in a number of cancers [11, 14–18], have yet to be proven in WT.

We have recently made progress towards identification of theWT stem cells. Serial passages of WT xenografts in immunodefi-cient mice resulted in in vivo selection of the stem cell compart-ment (WT blastema) whereas the more differentiated structuresthat were present in the primary tumour, disappeared [3].Microarray analysis of these stem-like WT xenografts and later on,closer examination of target genes in models of renal develop-ment, regeneration and tumourigenesis [19], revealed overex-pressed genes which are likely to represent the WT-’stem’ signature set. These included a combination of nephric-patterning,Wnt pathway and polycomb group genes, some of which haverecently emerged as critical regulators of self-renewal signals ofstem and cancer cells [20, 21].

Tumour-initiating stem cells have been identified through anexperimental strategy that combines sorting of tumour cell sub-populations, identified on the basis of the different expression ofsurface markers, with functional analyses of the sorted cells [11, 16, 22]. Therefore, we were particularly interested in signi -ficantly overexpressed genes that encode for cell surface mark-ers (neural cell adhesion molecule – NCAM, frizzled homolog 7[Drosophila] – FZD7, FZD2, G-protein coupled receptor 39 –GPR39, activin receptor type 2B – ACVRIIB) and can thereforeset the basis for sorting of WT cell sub-populations with cancerstem/progenitor potential.

In the present study, we sought to implement our previous find-ings in a collection of primary WTs. Unsorted, heterogeneous popu-lations of WT cells were examined for the protein expression of themicroarray up-regulated antigenic markers as well as for other stemcell markers that have potential to define the WT stem cell population(haematopoietic stem cell markers: CD34, c-Kit [14, 23]; mesenchy-mal stem cell markers: CD105, CD90, CD44 [24–26] and markers ofnormal and cancer stem cells in other tissues, CD133 [15, 17, 18, 27],MDR1 [28] and CD24 [27, 29]), most of which have yet to be char-acterized in WT. This phenotypical characterization revealed that mostWTs contain various portions of cells expressing stem cell markers.Sorting experiments of primary WT cells according to several candi-date proteins (FZD7, CD133 and NCAM), suggested NCAM and notCD133, a common cancer stem cell marker [15, 17, 18], as a putativemarker for the renal malignant stem/progenitor population.

Material and methods

Primary Wilms’ tumours cell cultures

Primary WT samples were retrieved from patients with WT within an hourafter surgery, from both Sheba Medical Center and Hadassah-Ein KeremHospital. All studies were approved by the local ethical committee andinformed consents were provided by the legal guardians of the patientsinvolved in this research according to the declaration of Helsinki.

The samples were minced in Hank’s Buffered Salt Solution (HBSS),soaked in collagenase overnight and then cultured in Iscove’s ModifiedDulbecco’s Medium (IMDM) medium supplemented with 10% Fetal BovineSerum (FBS) and growth factors: 50 ng/ml of basic Fibro blast GrowthFactor (bFGF), 50 ng/ml of Epidermal Growth Factor (EGF) and 5 ng/ml ofStem Cell Factor (SCF) (R&D Systems). Sphere formation was tested byplating the cells in ultra-low attachment plates (Corning Life Sciences,Wilkes Barre, PA, USA), with medium generally used to culture embryoidbodies from human embryonic stem cells [30], which consists of knock-out Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco-Invitrogen,Paisley, Scotland UK), 20% FBS defined (HiClone, Logan, UT, USA), L-glut-amine, pen-strep, 10% non-essential amino acids (Gibco-Invitrogen),supplemented with 100 ng/ml bFGF, 100 ng/ml EGF and 10 ng/lm SCF(R&D Systems, Minneapolis, MN, USA).

Antibodies for fluorescence-activated cell sorting(FACS) analysis and sorting

Primary fluorochrome conjugated antibodies: mouse anti-humanCD133/1-PE/allophecoaritin (APC) (Miltenyi Biotech, Bergisch Gladbach,Germany); mouse anti-human NCAM-APC (Biolegend, San Diego,California, USA); anti-human CD34-fluoroscein isothiocyanate (FITC)(R&D Systems); anti-human C-Kit-PE, anti-human CD24:PE/FITC, anti-human CD44-APC (eBioscience, San Diego, CA, USA); anti-human CD105-FITC (Serotec, Oxford, UK); anti-human CD45-APC, anti-human CD90-APC(BD Biosciences, San Jose, CA, USA).

Primary unconjugated antibodies: mouse anti-human ACVRIIB, ratanti-human FZD7 (R&D Systems, Minneapolis, MN, USA); mouse anti-human MDR1 (Chemicon, Temecula, CA, USA); rabbit anti-human FZD2(Acris, Herford, Germany); rabbit anti-human GPR39 (Genetex, SanAntonio, TX, USA); goat anti-human Cadherin-11 (Novus biologicals,Littleton, CO, USA).

In order to visualize the primary unconjugated antibodies, appropriatesecondary antibodies were used conjugated to either Alexafluor-488 orAlexafluor-647 (Molecular Probes, Inc., Invitrogen, Eugene, OR, USA).

FACS analysis

Cells were harvested using 0.05% trypsin/ethylenediaminetetraacetic acid(Gibco, Grand Island, NY, USA) or non-enzymatic cell dissociation solu-tion (Sigma-Aldrich, St. Louis, MO, USA). Surface antigens were labelledby incubation with either fluorochrome conjugated or unconjugated pri-mary antibodies described above, for 45 min. in the dark at 4�C to pre-vent internalization of antibodies. When unconjugated primary antibodieswere used, after a washing step, the cells were incubated for 20–30 min.with the appropriate fluorochrome conjugated secondary antibodies in

addition to 7-amino-actinomycin-D (7AAD; eBioscience) for viable cellgating. All washing steps were performed in FACS buffer consisting of0.5% bovine serum albumin (Sigma-Aldrich) and 0.02% sodium azide inDulbecco’s Phosphate Buffered Saline (PBS). Quantitative measurementswere made from the cross point of the IgG isotype graph with the specificantibody graph.

FACS sorting

Cells were harvested as described above, filtered through a 30-�m nylonmesh before final centrifugation, then resuspended in flow cytometrybuffer consisting of 0.5% bovine serum albumin (Sigma-Aldrich) and0.02% sodium azide in PBS. Cells were labelled with either anti-CD133-APC (Miltenyi Biotech, Germany) or anti-NCAM-APC (Biolegend).Fluorescence-activated cell sorter FACSAria (BD Biosciences) was used inorder to enrich for cells expressing these markers. A 100-�m nozzle (BDBiosciences), sheath pressure of 20–25 pounds per square inch, and anacquisition rate of 1000–3000 events per second were used as conditionsoptimized for WT cell sorting. Single viable cells were gated on the basisof 7AAD (eBioscience), and then physically sorted into collection tubes forlimiting dilution plating and RNA extraction. Data were additionallyanalysed and presented using FlowJo software (Tree Star, Ashland, OR,USA). Purity of sorted fractions was tested by FACS analysis. Prior to asep-tic sorting, the nozzle, sheath and sample lines were washed with bleachor 70% ethanol for 15 min., followed by washes with sterile water toremove remaining decontaminant.

Cell viability

Cell-survival quantification with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (XTT) assay (Biological Industries, BeitHaemek, Israel) was performed according to the manufacturer’s proto-col. This method determines the ability of metabolically active cells toreduce the yellow salt XTT to an orange formazan dye. Therefore, theconversion only occurs in living cells, and the amount of orange for-mazan formed directly correlates to the number of living cells.Nephroblastoma cells (from four different donors) were plated in 96-well plates at 5 � 103 cells/well in culture medium and exposed toeither 2 �g of anti-FZD7 antibody or to 0.25 �g of sFRP-1 in respect tountreated controls for 72 hrs. After the indicated time, the cells werewashed with PBS and incubated with the XTT solution according to thekit specifications for 3 hrs. After this incubation period, quantificationof the formazan dye formed was determined using a spectrophotome-ter at a wavelength of 450 nm minus the absorbance at 620 nm. Eachexperiment was performed in triplicates, and each series was repeatedat least twice.

Assessment of apoptosis

In order to evaluate the effect of anti-FZD7 antibody on WT cells’ survival,an annexin-V staining kit (Roche Applied Science, Indianapolis, IN, USA)was used according to the manufacturer specifications. Early apoptoticcells can be stained by annexin V, which binds to phosphotidyl-serinesnormally found in the inner aspect of the cell membrane but can be foundon the outer aspect of the cell membrane in apoptotic cells. On the other

hand, during early apoptosis, 7AAD stains DNA and is excluded from thenucleus, so staining does not occur. During necrosis and late apoptosis,membrane integrity is compromised, and cells are stained by bothannexin V and 7AAD; cells were treated with 5 �g of anti-FZD7 antibodywith respect to untreated control. Both samples were kept in 4 degrees for12 hrs. After the indicated incubation time cells, (1 � 106) were collected,washed and re-suspended in annexin V binding buffer (Gibco-Invitrogen)for preparation of 100-�l samples and appropriate controls.Subsequently, 5 �l of either FITC or APC-conjugated annexin V and 5 �lof 7AAD were added to the samples and/or controls followed by incuba-tion for 15 min. at room temperature in the dark. Annexin V binding and7AAD staining were evaluated by using FACSort (Becton Dickinson) withCELLQUEST software (BD Biosciences).

Immunohistochemical staining of human foetalkidney

Sections, 4-�m thick, were cut from whole blocks of human foetal kidneyfrom 20 weeks of gestation, human adult kidney and WT for immunohis-tochemistry. Immunostainings were performed as previously described[31]. In brief, the sections were processed within 1 week to avoid oxida-tion of antigens. Before immunostaining, sections were treated with 10 mM citrate buffer, pH 6.0 for 10 min. at 97�C in a microwave oven forantigen retrieval, followed by treatment of 3% H2O2 for 10 min. The slideswere subsequently stained by the labelled strepavidin-biotin method usinga Histostain plus kit (Zymed, San Francisco, CA, USA). Anti-human NCAMantibody (LifeSpan Biosciences, Inc., Seattle, WA, USA), anti-human FZD7antibody (R&D Systems) and anti-human CD133 antibody (MiltenyiBiotech, Germany), at a dilution of 1:50 were used. Controls were preparedby omitting the primary antibodies or by substituting the primary antibod-ies with goat IgG isotype. The immunoreaction was visualized by an HRP-based chromogen/substrate system, including Diaminobenzidine(DAB) (brown) chromogen (liquid DAB substrate kit – Zymed).

Single-cell cloning by limiting dilution

Limiting dilution assay was performed as previously described [32]. WTtumour cells were sorted according to either NCAM or CD133 expressionand the sorted cell fractions were plated in 96-well micro well plates(Greiner Bio-One, Mediscan, Kremsmunster, Austria) in 150 �l of culturemedia, at 0.3 or 1 cells per well dilution. The low cell concentration wasachieved by serial dilutions reaching 1000 cells per ml. The number of col-onized wells was recorded after 4 weeks.

Real time PCR analysis

Quantitative real time reverse transcription PCR (qPCR) reactions wascarried out as previously described [33], to determine fold changes inexpression of a selection of genes for stemness and diffrentiationbetween WT cells cultured either in adherence promoting conditions oras spheres and in sorted WT cells. RNA was extracted using themicroRNeasy kit (Qiagen, Hilden, Germany) according to the manufac-turer’s instructions. cDNA synthesis was carried out using the Highcapacity cDNA RT kit (Applied Biosystems, Foster City, CA, USA). Eachanalysis reaction was performed in duplicates. �-actin or Glyceraldehyde-3

phosphate dehydrogenase (GAPDH) were used as endogenous controlthroughout all experimental analyses.

Gene expression analysis was performed with TaqMan GeneExpression Assays on an ABI Prism 7900HT sequence detection system(Applied Biosystems).

In vitro effects of chemotherapies on WT cellgene expression

In order to determine the lethal dose for 50% of WT cells (LD50) with each of the studied drugs, WT cells were seeded in 96-well plates at104 cells/well for 24 hrs. After the indicated time the medium wasreplaced with medium containing a range of concentrations for each ofthe drugs evaluated: For Etoposide and Irinotecan �1�M–250�M weretested, for Cisplatin �1�M–100�M were tested. After 48 hours, a XTTproliferation assay was performed as described above and the lethal dosefor 50% of cells (LD50) was determined. For Etoposide and Irinotecan anLD50 of 40�M and for Cisplatin an LD50 of 10�M were observed.

To study the in vitro effect of topoisomerase inhibitors – the topoiso-merase II inhibitor, Etoposide and the topoisomerase I inhibitor,Irinotecan – on WTs gene expression in comparison to Cisplatin treatedor untreated cells, WT cells from three different WTs were distributedinto 4 � 25 T flasks and cultured for 24 hrs at 37�C/5% CO2. After theindicated time, medium was replaced with either a medium containingone of the above mentioned chemotherapies or with normal growthmedium as control. After additional 48 hrs in culture, cells weretrypsinized and RNA was extracted for quantative real time PCR analysisof topoisomerase2A (TOP2A), NCAM and WT1. This procedure wasrepeated twice with each tumour.

Statistical analysis

Results are expressed as the mean values � S.E.M of the mean, unless otherwise indicated. Statistical differences between WT cellpopulations were evaluated using the non-parametric, one sided signtest. Statistical differences of two group data were compared byStudent’s t-test. For all statistical analysis, the level of significance wasset as P 0.05.

Results

Primary WT cultures

We have analysed eight WTs of different histological subtypes(Table 1). Primary WT cultures were established in growth pro-moting medium as well as in anchorage independence promot-ing conditions. Cell cultures from six tumours displayed mostlyspindle-shaped morphology (Fig. 1A, a–f). Nevertheless, phe-notypic plasticity was demonstrated as cells originating from aspecific tumour, cultured under identical conditions andobserved at a similar passage number, showed both cobble-stone (a characteristic feature of epithelial cells) and spindle-like cell morphology (Fig. 1B, a and b). In addition, when grownin low-attachment promoting conditions, WT cells originatingfrom different tumours were able to form floating sphere-likeclusters (Fig. 1C, a–d) and created secondary spheres after dis-sociation and replating. Thus, primary WT cultures show mor-phological plasticity and sphere formation capability. Sphereformation has been shown to maintain stem-cell potential invarious primary culture systems, especially neural [15, 22]. Inorder to examine whether this form of WT propagation is advan-tageous for maintaining primary WT cells in an un-differentiatedstate, we compared WT cells grown in adherence promotingconditions with WT spheres for the expression of a selection ofWT-‘stem’ signature and stemness genes [19, 29, 34] (Fig. 1D).Investigation of ‘stemness’ genes has not been previously per-formed in WT. Analysis of four different WTs showed that whileall stemness genes displayed a coordinated expression patternin a particular tumour, this was not associated with a certainculturing method; Two of the tumours (WOO4, WOO5) showedhigher expression in the WT spheres while WOO2 overex-pressed the stemness genes in the adherent culture and WO1Oshowed mostly comparable expression levels (Fig. 1D). Thislack of clear advantage for the WT spheres led us to furtherexamine WT cells in adherent cultures.

Patient Code Gender Age Pattern Histology Remarks

W002 Female 4 years Triphasic Favourable histology Lung metastasis

W003 Male 10 years Blastemal Unfavourable histology Recurrent with diffused anaplasia

W004 Female 6 years Triphasic Favourable histology Bilateral

W005 Male 2 years Triphasic Favourable histology –

W006 Male 2 years Triphasic Favourable histology Focal anaplasia

W007 Female 3 years Triphasic Favourable histology Recurrent WT with focal anaplasia

W009 Male 2 years Triphasic Unfavourable histology Recurrent with diffused anaplasia

W010 Female 1 years Triphasic Favourable histology –

Table 1 Patient and tumour characteristics

Fig. 1 Primary Wilms’ tumour (WT) cell cul-tures. (A) Pictures by confocal microscopeof six primary WTs in culture, showingmostly mesenchymal cell morphology.(magnification �10); (B) Morphologic plas-ticity of cells from the same primary WT(WOO2) after an equal number of passages,showing different morphologic cell struc-tures in culture – (a) – cobblestone shapedcells, (b) – spindle shaped cells. (magnifica-tion �10); (C) Primary WTs of different his-tologic subtypes form sphere-like clustersin low attachment conditions. Photo -micrographs of cultured WT cells (magnifi-cation �20) at 5 days after plating the cellsin ultra-low attachment plates with mediumsuitable for growing embryoid bodies fromhESC [30]; (i) WOO2, (ii) WOO3, (iii), WOO4and (iv) WOO5; (magnification �20). (D)Quantitative RT-PCR analysis for the expres-sion of WT-stem signature (nephric-progen-itor- WT1, SIX2, Sall1, polycomb group-EZH2, BMI1, Wnt pathway-FZD7, �-cateninand ESC – nanog, Oct4) genes in WT cells grown in adherence promoting condi-tions (open bars) in comparison to WTspheres (grey bars). Shown are experimentsin cells derived from four different WTs(WOO2, WOO4, WOO5 and WO1O). Thevalue for the spheres was used as the cali-brator (therefore 1) and all other valueswere calculated with respect to it. Resultsare presented as the mean � S.E.M of atleast three separated experiments.Quantitative transcript levels were normal-ized to expression of �-actin or GAPDH.

Phenotypical analysis of low-passage cultures of WT

We initially examined unsorted populations of low-passage WTcells by flow cytometry in order to identify sub-populationsexpressing putative stem cell markers. Table 2 summarizesimmunophenotyping data of primary WT cells. Each surfacemarker was analysed in at least five different WTs. Figure 2presents the results of FACS analysis grouped into earlyblastemal markers, haematopoietic and mesenchymal stem cellmarkers, markers that were overexpressed in the stem-like WTxenografts and other non-specific stem cell markers (MDR1-cancer, CD24-pluripotent embryonic). A representative FACSanalysis for each marker of the various groups and detailedanalysis of NCAM, FZD7 and CD133 in the remaining fivetumours with the corresponding isotype controls are shown inFig. 2, a–f and Fig. 3, a–c, respectively. Four main immunophe-notypes with regard to the various cell markers were identifiedin the primary heterogeneous cultures: constant low expres-sion, 10% (ACVRIIB, GPR39, cadherin 11), variable low-to-moderate expression, 11–50% (FZD7, FZD2, NCAM, CD133,CD34, CD24, MDR1), variable moderate-to-high expression,�50% (CD90, CD44) and absent expression (c-Kit, CD105).Thus, sub-populations of cells in primary WTs express all

surface markers predicted by microarray analysis, as well asother known stem cell markers.

Immunostaining of human foetal kidney

We chose to further analyse the NCAM and FZD7 markers, whichwere overexpressed in the stem-like WT xenografts (at the genelevel) and were now found in primary WTs to be constantlyexpressed in low-to-moderate levels likely to contain the stem cellfraction. CD133, a widespread cancer stem cell marker [15, 17, 18]which showed a similar expression pattern in the primary WT cul-tures, was also tested. In human foetal kidney, the nephrogenicmesenchyme is the region containing the normal kidney stem cells.Taking into account both the presence of these normal renal stemcells within this region and the close relations of WT with normalnephrogenesis [1], we determined the in situ localization of thesemarkers in mid-gestation human foetal kidneys (Fig. 4). BothNCAM and FZD7 were intensely expressed by cells of the nephro-genic mesenchyme (Fig. 4A, B), suggestive of cell origin within thistissue. We could not achieve CD133 staining in the human foetalkidney. Nevertheless, CD133 was abundantly expressed in tubularepithelial cells of the adult human kidney, whereas in WT it local-ized predominantly to the tumour vasculature (Fig. 4C), staining inboth cases differentiated rather than un-differentiated cell types.

Table 2 Immunophenotype of primary WTs

Primary Wilms’ Tumours

Marker groups Surface markers WOO2 WOO3 WOO4 WOO5 WOO6 WOO7

Early blastemal marker Cadherin-11 � � � � � �

Haematopoietic stem/progenitor cell markers CD133 ��

C-KIT � � � � � �

CD34 � � � � � �

Mesenchymal stem cell marker CD105 � � � ND � �

CD90 �� ND ��

Markers that were high in the WT stem like Xn NCAM ��

ACVRIIB � � � � � ND

GPR39 � � � � � ND

FZD7 ��

FZD2 �� ?

Additional normal and cancer stem cell markers CD24 ��

MDR1 �� ND

Pan haematopoietic marker CD45 � � � � � �

�0.5–10% of the cells express the marker; �� 11%-50% of the cells express the marker; �� 50% of the cells express the marker; – noneof the cells express the marker; ND not determined.

Fig. 2 Flow cytometric analyses of primary Wilms’ tumours. A representative FACS analysis for each marker of the various groups: (A) Blastemal mark-ers, (B) Mesenchymal stem cell markers, (C) Pan haematopoietic marker, (D) Markers that were overexpressed in stem-like WT xenografts (E)haematopoietic stem cell markers, (F) Markers of cancer and normal stem cells in other tissues, demonstrates the expression of Cadherin-11, CD133,NCAM, GPR39, ACVRIIB, FZD7, FZD2, CD34, CD90, CD44 in WT cell sub-populations and the absence of CD105, C-kit, and CD45 expression. Data arerepresentative of no less than two separate experiments for each marker in at least five tumours.

Fig. 3 Flow cytometric analyses of NCAM, FZD7 and CD133 expression in primary Wilms’ tumours. Detailed analysis of (A) NCAM, (B) FZD7 and (C)CD133 in five additional tumours (WOO3, WOO4, WOO5, WOO6, WOO7) and corresponding isotype controls showing low-moderate expression in alltumours examined. Upper panels represent isotype control antibody analysis of the corresponding stained cells in bottom panels.

Exclusion of FZD7 and CD133 as markers for theisolation of WT stem/progenitor cells

We next sorted WT cell sub-populations according to FZD7, CD133or NCAM expression, in order to test whether these cell fractionspossess stem cell potential. Cell sorting with an anti-FZD7 antibodyresulted in extensive cell death (as was shown by trypan blue stain-ing of the sorted cells) and precluded us from obtaining a positivecell fraction. In order to elucidate the reason for this cell loss, we performed cell proliferation assays (XTT) on four different WTs in

the absence or presence of either the Wnt signalling antagonist atthe frizzled receptor level – secreted frizzled-related protein 1(sFRP1) or the anti-FZD7 purified antibody used for sorting.Decreased survival of WT cells was detected in all preparations byeither compound (Fig. 5A, a and b, P 0.05). In addition, weanalysed apoptosis and cell death in WT cells treated with anti-FZD7by FACS (Fig. 5B). We found an increase in apoptotic cells(annexin�) after anti-FZD7 treatment compared to untreated cells(cells were assayed after overnight incubation with/without anti-FZD7 antibody) (Fig. 5Ba). Double staining revealed increase in both

Fig. 4 In situ localization of FZD7, NCAM and CD133. (A, B) Immunostaining of mid-gestation human foetal kidney demonstrates expression of (A) Frizzled7(original magnification: upper left panel �4, lower left panel �20) and (B) NCAM (original magnification: upper right panel �4, lower right panel �10) inthe nephrogenic mesenchyme (MM). (C) Staining of human adult kidney (a) and WT (b) for CD133 demonstrates expression in renal tubular epithelia (C,a – asterix) and in tumour vasculature (C, b – arrows) – (original magnification: �40); Cells were counterstained with haematoxylin and eosin.

annexin� 7AAD� (early apoptotic cells) and annexin�7AAD� (lateapoptotic/necrotic) cells and decrease in annexin�7AAD� viablecells (Fig. 5Bb). Thus, application of anti-FZD7 reduces WT cell sur-vival and increases WT cell death and apoptosis, limiting the use ofFZD7 for functional cell sorting.

In contrast to FZD7, an enriched viable fraction of CD133expressing cells could be readily obtained and further subjected toclonogenicity assays from single cells (i.e. limiting dilution) andqRT-PCR analysis. No difference in clonogenicity was observedbetween the sorted CD133� and CD133� WT cell fractions of threedifferent tumours examined (Fig. 5B). We further compared the cellfractions for the expression of ‘tumour-progenitor’ genes (Fig. 5C,a). These genes were previously found to be overexpressed in theprogressive WT stem-like xenografts [3] and comprise of Wnt path-way (FZD7, �-catenin), polycomb group (BMI-1, EZH2) andnephric-progenitor (SIX2, WT1) genes. In addition, both Wnt path-way and polycomb group of gene have been implicated in self-renewal of normal and cancer stem cells [12, 20, 21, 34–37] andOCT4 in pluripotentiality [38]. These WT-stem signature and stem-ness genes were inconsistent in their expression pattern betweenthe CD133� and CD133� cell fractions showing either similarexpression levels or elevated expression in the CD133� cell fraction.Thus, both clonogenicity assays and real-time RT-PCR suggest thatthe CD133� sub-population is not a putative WT stem cell fraction.

Functional analysis of sorted NCAM� and NCAM�

WT sub-populations

Sorting experiments for NCAM resulted in a highly enriched positivecell fraction (�92%) and a highly purified negative cell fraction(2%) (Fig. 6A, a–c). Clonogenicity assays from single cells(shown are experiments originating from three different tumours)demonstrated the NCAM� cell fraction to be highly enriched with

clonogenic cells compared to the NCAM� fraction (Fig. 6B, a and b).In addition, real-time RT-PCR analysis showed the NCAM� cellfraction to overexpress the ‘tumour-progenitor’ gene set comparedto the negative fraction, i.e. nephric-progenitor (WT1, SIX2), poly-comb group (EZH2, BMI-1), Wnt pathway (FZD7, �-catenin) and theembryonic stem cell pluripotency and self-renewal (nanog) genes.Thus, as opposed to CD133, NCAM expressing cells are highlyclonogenic and overexpress a set of WT ‘stemness’ genes.

Analysis of the epithelial elements of WTs suggests that theserepresent regions of metanephric blastema that have attempted toundergo a normal process of mesenchyme (Vimentin�)-to-epithe-lial (E-cadherin�) transition (MET) but failed to proceed tonephron formation [39]. We therefore examined the differentiationstatus of the cell fractions by analysing expression of Vimentinand E-cadherin. High Vimentin and low E-cadherin expressionindicated that the NCAM� fraction is likely to originate from earlystages of mesenchymal to epithelial transition, characteristic ofearly kidney development (Fig. 6C, a and b).

Finally, we examined the NCAM gene expression in theunsorted WT cells grown as spheres in comparison to the adher-ent cultures, previously analysed for the expression of stemnessgenes (Fig. 1D). We found that independent of the culture method(adherent versus spheres), NCAM followed the expression patternof the stemness genes; elevated in WOO4 and WOO5 in the WTspheres, elevated in WOO2 in the adherent cultures and compara-ble in WO1O (Fig. 6D). This intimate relation between NCAM andthe WT-stem signature genes in the unsorted cells support thefindings obtained for the NCAM sorted population.

TOP2A is overexpressed in the NCAM� cell fraction

To begin investigating the clinical relevance of the NCAM� cells wesought to determine whether TOP2A, a molecular marker known

Fig. 5 Exclusion of FZD7 and CD133 as markers for the isolation of WT stem/progenitor cells. (A) Cell viability was determined by XTT cell proliferationassay (black bars, WT cells treated with either sFRP1 or anti-FZD7 antibody; white bars, untreated� WT cells), which measures mitochondrial respira-tory function as described in materials and methods, on four WT cultures derived from four different tumours in the absence or presence of either (A)the Wnt signalling antagonist at the frizzled receptor level – secreted frizzled-related protein 1 (sFRP1) or (B) anti-FZD7 antibody showing decreasedsurvival rate in all preparations by either compound in comparison to untreated controls. Data are means � S.E.M derived from two independent exper-iments with triplicate wells per condition, P 0.05; (B) Enhanced WT Cell death after exposure to 5 �g of anti FZD7 antibody for 12 hrs. (a) Flow cyto-metric analysis of WT cells incubated overnight with (treated) or without (untreated) anti-FZD7 antibody. Cell number is plotted as a function of theintensity of staining for annexin V; cells stained positive with annexin V antibodies are apoptotic. The percentages of apoptotic cells are indicated. (b)Flow cytometry profile represents annexin-V-APC staining in x axis and 7AAD in y axis. Shown is a marked elevation in the early apoptotic (annexin V�

7AAD�) and a substantial reduction in the surviving (annexin V� 7AAD�) WT cells after treatment with anti-FZD7 antibody, in comparison to untreatedcontrol. Data presented are representative of three independent experiments. (C) Clonogenicity assays of the sorted CD133� and CD133� WT sub-popu-lations performed on three different WTs. Columns represent the mean number of colonized wells. Limiting dilutions followed by plating of a single cell/wellin 96-well plates were performed in order to compare the clonogenic capabilities between WT CD133� and CD133� cell fractions. No significant differencein clonogenic capacity was observed between the two cell fractions; (D) Quantitative RT-PCR analysis of the WT-stem signature genes (nephric-progenitor-WT1, SIX2, polycomb group- EZH2, BMI1, Wnt pathway-FZD7, �-catenin and self-renewal/multipoteniality- OCT4) in CD133� and CD133� WT sorted cellsfrom at least three different tumours, demonstrates no difference between the cell fractions or higher expression in the CD133� in comparison to theCD133� sub-population. The value for the CD133� was used as the calibrator (therefore 1) and all other values were calculated with respect to it. Resultsare the mean � S.E.M of four separate experiments, * P 0.05. Quantitative transcript levels were normalized to expression of �-actin.

to be associated with poor prognosis of WT [40–43] is elevated inthis cell fraction. Real-time PCR demonstrated TOP2A to be signif-icantly elevated (P 0.05) in NCAM� compared to NCAM� cellsobtained from five independent WTs (Fig 7A). These data comple-ment our results showing overexpression of WT1 and EZH2 in theNCAM� cells (Fig. 6C), as both of these markers are consideredpoor prognostic markers in WT [40, 44]. Having established theelevation of TOP2A in the NCAM cell fraction, we next determinedwhether in vitro treatment of heterogeneous WT cells with thetopoisomerase inhibitors, Etoposide and Irinotecan influencesexpression of both TOP2A and NCAM. This was compared totreatment with Cisplatin, used in refractory childhood solidtumours [45, 46] and to untreated WT cells. Etoposide, a topoiso-merase II inhibitor had a dramatic effect on TOP2A expressionwith concomitant reduction in NCAM expression in two of thethree tumours evaluated (WOO2 and WOO4, but not in WO1O) inrespect to the untreated control. Interestingly, Irinotecan, a topoi-somerase I inhibitor, also reduced both TOP2A and NCAM expres-sion in the three tumours evaluated (Fig. 7B). In contrast, applica-tion of Cisplatin did not show a similar decrease and in someinstances, up-regulation of marker levels was observed (Fig. 7B).Moreover, application of either one of the topoisomeraseinhibitors (Etoposide or Irinotecan), but not of Cisplatin, on theWT cells, resulted in a reduction in WT1 expression in both of thetumours examined, suggesting additional coupling of TOP2A withWT1 (Fig. 7C).

Discussion

Herein we wanted to investigate for the first time whether humanWT could contain tumour cell subsets that have potential as can-cer stem/progenitor or tumour initiating cells. This investigation isbased on our previous findings on the intimate link betweentumourigenesis and organ development in the kidney [3, 19]. Wechose to examine unsorted, heterogeneous populations of WT

cells for expression of the cell surface markers based on our previous studies, in which such markers were overexpressed in aDNA microarray screen of progressive stem-like WT xenograftsand human foetal kidneys [3]. To identify additional potential WTprogenitor sub-populations for further investigation, we also stud-ied the presence of cellular fractions within WTs that expressmarkers consistent with stem cells of human haematopoietic,mesenchymal, neuronal and pluripotent embryonic origin. Thistour de force of phenotypic characterization is critical becausespecific markers of human multi-potent renal embryonic progeni-tors that lead to formation of WT are unknown. Accordingly, wehave detected sub-populations of WT cells that are immunoreac-tive for various stem cell markers and may contribute to ourunderstanding of the WT cell of origin. For instance, lack ofexpression of markers such as c-Kit (CD117), CD45 or CD105,which strongly characterize haematopoietic and mesenchymalstem cells, respectively, indicates the specificity of WT cells inregard to their lineage. Of all protein markers analysed, thosefound to be expressed in low-to-moderate portions in all primaryWT samples (see Table 2) are likely to contain the stem/progenitorcell fraction and could serve as initial markers for sorting of puta-tive cell sub-populations, an approach which has not been previ-ously carried out in WT. Based on that criteria, we chose to moreclosely examine NCAM, FZD7 and CD133. NCAM and FZD7 wereamong the most up-regulated genes in the WT stem-likexenografts, while CD133 has been found to be expressed by manyother tumour initiating cells [8, 13, 15, 17, 18]. The demonstrationof the presence of NCAM and FZD7 in the human nephrogenicmesenchyme, which contains the normal renal stem cells, maycontribute to our understanding of the WT cell of origin.

Sorting experiments and functional assays revealed the diffi-culties in sorting WT cells according to FZD7 expression whichresulted in loss of cell viability. FZD7 is one of the Wnt pathwayreceptors associated with stem cell self-renewal and proliferation[47]. The Wnt pathway plays critical roles in multiple cellularevents that occur during the development of the mammalian kid-ney [48], including regulation of renal epithelial progenitors [49].

Fig. 6 Functional analysis of sorted NCAM� and NCAM� WT sub-populations. (A) Sorting experiments of WTs according to NCAM expression. Seen are(a) Primary WT cells prior to sorting, and after sorting (b) a highly enriched positive cell fraction (�92%) and (c) a highly pure negative fraction (2%).(B) Clonogenic capacity of the NCAM� versus the NCAM� WT cells. Shown are representative experiments performed on three WTs obtained from threedifferent donors, demonstrating the NCAM� to be highly clonogenic compared to the NCAM� WT cells. (C) Quantitative RT-PCR analysis of the WT-stemsignature genes (WT1, SIX2, EZH2, BMI1, FZD7, �-catenin and nanog) and renal differentiation associated genes (Vimentin and E-cadherin) in NCAM�

and NCAM� WT sub-populations demonstrates (C, a) elevated mRNA levels of the ‘tumour-progenitor’ genes in the NCAM� cell fraction compared to thenegative one; (C, b) elevated Vimentin mRNA levels and low E-cadherin. Experiments were performed on four WTs derived from four different donors.The value for the NCAM� was used as the calibrator (therefore 1) and all other values were calculated with respect to it. Results are presented as themean � S.E.M of at least three separate experiments. P 0.05 for elevation of all the WT-stem signature genes in the NCAM� relative to the NCAM�

WT cells. (D) Quantitative RT-PCR analysis of NCAM mRNA levels along with that of the WT-stem signature and stemness genes in WT spheres and adher-ent cultures. NCAM expression in WT cells follows the expression pattern of the stenmess genes, shown in Fig. 1D, regardless of the culturing methodemployed. Experiments were performed on four WTs derived from four different donors. For each tumour experiments were repeated at least three times.The value for the spheres was used as the calibrator (therefore1) and all other values were calculated with respect to it. Results are presented as themean � S.E.M of three separate experiments. Quantitative transcript levels were normalized to expression of �-actin or GAPDH.

Indeed, blocking the Wnt pathway (specifically with the FZD7 anti-body or with a general antagonist at its level) was shown to bedetrimental for these cells. Thus, FZD7 is a functional receptor forWT cells and therefore is not optimal for cell sorting. Nevertheless,since 15% of WTs have activating mutations in �-catenin [50] thatwould render them unresponsive to Wnt ligands and are likely toremove the need for signalling through the �-catenin pathway,there might be cases in which FZD7� cell isolation and character-ization can be accomplished. Accordingly, future studies aimed atdetecting such mutations and correlating their presence to FZD7�

cell viability and function are warranted.

In contrast, highly enriched populations of WT NCAM orCD133 expressing cells could be isolated. Their analysis revealeda fundamental difference: While NCAM� cells are highly clono-genic and overexpress the WT ‘stemness genes’ (nephric-progen-itor, Wnt pathway and polycomb group genes), previouslyobserved in the ‘WT-stem’ signature set of the progressive WTxenografts [3] and in independent real-time PCR verifications ofthese stem-like tumours [19], CD133� do not possess such char-acteristics. CD133� cells previously isolated from adult kidneycancer were shown to contribute to tumour vascularization, ratherthan being tumour-initiating cells [19, 51]. Similarly, WT CD133�

Fig. 7 TOP2A is overexpresed by NCAM� WT cells. (A) Quantitative RT-PCR analysis for the expression of TOP2A in the NCAM� in comparison to theNCAM� WT cells. The NCAM� cells consistently overexpressed the WT poor prognostic factor, TOP2A. Shown are experiments performed on five dif-ferent WTs. The value for the NCAM� was used as the calibrator (therefore 1) and all other values were calculated with respect to it. Results are pre-sented as the mean � S.E.M of at least three separate experiments. P 0.05. (B) Reduction in TOP2A expression as a result of treatment with thetopoisomerase inhibitors, either Etoposide or Irinotecan, mostly correlates with reduction of NCAM expressions in WT cells in comparison to untreatedcontrol. Shown are experiments performed on three different WTs. (C) Treatment of WT cells with either of the topoisomerase inhibitors reduces theexpression of the WT1 gene in comparison to untreated control. Shown are experiments performed on two different WTs. The value for the untreatedcontrol was used as the calibrator (therefore 1) and all other values were calculated with respect to it. Results are presented as the mean � S.E.M ofat least three separate experiments for each of the tumours, * P 0.05. Quantitative transcript levels were normalized to expression of either �-actinor GAPDH.

cells are not likely to represent the epithelial malignant renal pro-genitor that drives WT initiation.

Our new functional data of the sorted WT NCAM� populationintegrates with previous data which showed by immunostainingthat NCAM is predominantly localized to WT blastema as well asin the de-differentiated cells of WTs [52]. In addition, it has beendemonstrated that following senescence and crisis of primary WTcultures, surviving cells were all shown to be NCAM�, supportingit as a marker of self-renewing cells in WT [53]. Collectively, thesedata suggest NCAM as a marker for the malignant renal progeni-tor population. We emphasize that future in vivo xenotransplanta-tion studies of the NCAM� cell population are required to deter-mine its tumour-initiating capabilities. Nevertheless, while WTxenografts are readily formed via implantation of fresh surgicalsamples into immunodeficient hosts [3], WT are notorious fortheir inability to establish xenografts after tumours have beenprocessed into single cell suspension and especially from primaryWT cultures [54]. Taking into account these inherent limitations ofWT and the fact that cell sorting and heterotransplantation inimmunodeficient host animals require large numbers of the ratherrare clinical WTs for calibration of both implantation site and celldosage, makes these experiments beyond the scope of this paper.

Others and we have very recently demonstrated a similar phe-nomenon in which progressive xenografts of human tumoursadopting a highly infiltrative and stem-like phenotype, down-regu-late angiogenesis genes and expand independent of angiogenesis[19]. From a practical point of view, cancer treatment strategies ofWT recurrence aimed at angiogenic targets might therefore notsuffice and there is a need to pursue the invasive stem-like cancercells. Since WT stem cells are likely to be defined by a combina-tion of markers including NCAM as the initial marker, we propose,at this stage, that WT patients may benefit from targeting theNCAM molecule. Even in the case of WT being a model for tumourarising from self-renewing progenitor cells that divide rapidly andare sensitive to the conventional chemotherapies along with thetumour bulk, targeting NCAM could in turn reduce the late adverse

effects resulting from current therapeutics. NCAM has been impli-cated with both adverse prognosis and metastatic behaviours ofseveral tumours [55, 56] Furthermore, WT recurrence and metas-tasis is associated the appearance of a blastemal phenotype in thetumour [57, 58]. In this regard, our demonstration of high levelsof the poor prognostic marker TOP2A in the NCAM� cell fractionand the down-regulation of both TOP2A and NCAM followingEtoposide or Irinotecan but not Cisplatin treatment of WT cells isespecially important. These preliminary data suggest that theNCAM cell population might be especially susceptible to topoiso-merase inhibitors. Nevertheless, un-coupling of TOP2A and NCAMreduction after applying Etoposide in one of the three tumoursexamined (WO1O) and their strong connection in the sametumour following Irinotecan treatment, suggests a more compli-cated mechanism for the operation of the topoisomeraseinhibitors. Future in vitro and in vivo studies that combine the twotopoisomerase inhibitors (already in trials for small cell lung can-cer showing high affectivity and relatively low toxicity [59, 60])with anti-NCAM immunotherapy for targeting the cancer initiatingcells and possibly preventing WT recurrence with limited drugcomplications are warranted. Immunotherapy directed againstNCAM in adult tumours is under intensive investigation and sev-eral newly developed reagents are readily available [61].

Acknowledegments

We thank the Kahn Family Foundation for supporting our research. Thiswork was supported by ‘Talpiot’ Medical Leadership Award, the IsraelScience Foundation (ISF) Project Grant, the Israel Cancer ResearchFoundation Clinical Career Development Award (ICRF), The Schreiber andBreteler Foundations Sackler School of Medicine, Tel Aviv University(B.D.). This work is part of the requirements towards a PhD degree,Sackler School of Medicine, Tel Aviv University (N.P.S). G.R. holds theDjerassi Chair in Oncology at the Tel Aviv University.

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Developmental tumourigenesis: NCAM as a putative marker for the malignant renal stem/progenitor cell...

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