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Keywords:

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

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledegments
  8. References

During development, renal stem cells reside in the nephrogenic blastema. Wilms’ tumour (WT), a common childhood malignancy, is suggested to arise from the nephrogenic blastema that undergoes partial differentiation and as such is an attractive model to study renal stem cells leading to cancer initiation and maintenance. Previously we have made use of blastema-enriched WT stem-like xenografts propagated in 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 putative renal, cadherin 11), are expressed in WT cell sub-populations in varying levels. Of all markers, NCAM, CD133 and FZD7 were constantly detected in low-to-moderate portions likely to contain the stem cell fraction. Sorting according to FZD7 resulted in extensive cell death, while sorted NCAM and CD133 cell fractions were subjected to clonogenicity assays and quantitative RT-PCR analysis, exclusively demonstrating the NCAM+ fraction as highly clonogenic, overexpressing the WT ‘stemness’ genes and topoisomerase2A (TOP2A), a bad prognostic marker for WT. Moreover, treatment of WT cells with the topoisomerase inhibitors, Etoposide and Irinotecan resulted in down-regulation of TOP2A along with NCAM and WT1. Thus, we suggest NCAM as a marker for the WT progenitor cell population. These findings provide novel insights into the cellular hierarchy of WT, having possible implications for future therapeutic options.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledegments
  8. References

Wilms’ tumour (WT; nephroblastoma) is the most frequent tumour of the genitourinary tract in children and rated fourth in overall incidence among childhood cancers [1]. It is viewed as a prototype of differentiation failure in human neoplasia as it recapitulates the histology of the nephrogenic zone of the growing foetal kidney and contains a stem cell compartment (termed ‘blastema’) along with more differentiated structures such as tubular epithelia, stromal elements and also other mesoderm elements (rhabdomyoblasts, cartilage, osteoid tissue and fat) [2]. Recent molecular data demonstrate that WTs systematically overexpress nephric-progenitor genes corresponding to the earliest stages of normal metanephric kidney development [3, 4], connecting tumourigenesis and organogenesis in the kidney [1]. With improved multimodality therapy, WT survival rates have risen over the last 40 years to 85–90%; however, for those whose disease relapses or metastizes, even intensive salvage regimens result in subsequent survival closer to 50%[5]. Moreover, survivors are at increased risk for a broad spectrum of adverse outcomes caused by chemotherapy and radiation therapy, such as late mortality and secondary cancers [6, 7].

It is becoming clear that many, if not most, malignancies arise from a population of cells that exclusively maintain the ability to self-renew and sustain the tumour via the expression of tumour-progenitor genes [8, 9]. These ‘cancer stem cells’ are often biologically distinct from the differentiated cancer cells that comprise most of the tumour bulk. Because cancer stem cells are believed to be primarily responsible for tumour initiation as well as resistance to chemo- and radiotherapy, their persistence may account for relapsing disease in WT [10–13]. The presence of such 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 the WT stem cells. Serial passages of WT xenografts in immunodeficient mice resulted in in vivo selection of the stem cell compartment (WT blastema) whereas the more differentiated structures that 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 development, regeneration and tumourigenesis [19], revealed overexpressed 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 have recently emerged as critical regulators of self-renewal signals of stem and cancer cells [20, 21].

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

In the present study, we sought to implement our previous findings in a collection of primary WTs. Unsorted, heterogeneous populations of WT cells were examined for the protein expression of the microarray up-regulated antigenic markers as well as for other stem cell markers that have potential to define the WT stem cell population (haematopoietic stem cell markers: CD34, c-Kit [14, 23]; mesenchymal stem cell markers: CD105, CD90, CD44 [24–26] and markers of normal 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 characterized in WT. This phenotypical characterization revealed that most WTs contain various portions of cells expressing stem cell markers. Sorting experiments of primary WT cells according to several candidate proteins (FZD7, CD133 and NCAM), suggested NCAM and not CD133, a common cancer stem cell marker [15, 17, 18], as a putative marker for the renal malignant stem/progenitor population.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledegments
  8. References

Primary Wilms’ tumours cell cultures

Primary WT samples were retrieved from patients with WT within an hour after surgery, from both Sheba Medical Center and Hadassah-Ein Kerem Hospital. All studies were approved by the local ethical committee and informed consents were provided by the legal guardians of the patients involved 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 Modified Dulbecco’s Medium (IMDM) medium supplemented with 10% Fetal Bovine Serum (FBS) and growth factors: 50 ng/ml of basic Fibro blast Growth Factor (bFGF), 50 ng/ml of Epidermal Growth Factor (EGF) and 5 ng/ml of Stem Cell Factor (SCF) (R&D Systems). Sphere formation was tested by plating the cells in ultra-low attachment plates (Corning Life Sciences, Wilkes Barre, PA, USA), with medium generally used to culture embryoid bodies from human embryonic stem cells [30], which consists of knockout Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco-Invitrogen, Paisley, Scotland UK), 20% FBS defined (HiClone, Logan, UT, USA), L-glutamine, 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-human CD133/1-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, rat anti-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, San Antonio, TX, USA); goat anti-human Cadherin-11 (Novus biologicals, Littleton, CO, USA).

In order to visualize the primary unconjugated antibodies, appropriate secondary antibodies were used conjugated to either Alexafluor-488 or Alexafluor-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 solution (Sigma-Aldrich, St. Louis, MO, USA). Surface antigens were labelled by incubation with either fluorochrome conjugated or unconjugated primary antibodies described above, for 45 min. in the dark at 4°C to prevent internalization of antibodies. When unconjugated primary antibodies were 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 cell gating. All washing steps were performed in FACS buffer consisting of 0.5% bovine serum albumin (Sigma-Aldrich) and 0.02% sodium azide in Dulbecco’s Phosphate Buffered Saline (PBS). Quantitative measurements were made from the cross point of the IgG isotype graph with the specific antibody graph.

FACS sorting

Cells were harvested as described above, filtered through a 30-μm nylon mesh before final centrifugation, then resuspended in flow cytometry buffer consisting of 0.5% bovine serum albumin (Sigma-Aldrich) and 0.02% 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 in order to enrich for cells expressing these markers. A 100-μm nozzle (BD Biosciences), sheath pressure of 20–25 pounds per square inch, and an acquisition rate of 1000–3000 events per second were used as conditions optimized for WT cell sorting. Single viable cells were gated on the basis of 7AAD (eBioscience), and then physically sorted into collection tubes for limiting dilution plating and RNA extraction. Data were additionally analysed and presented using FlowJo software (Tree Star, Ashland, OR, USA). Purity of sorted fractions was tested by FACS analysis. Prior to aseptic sorting, the nozzle, sheath and sample lines were washed with bleach or 70% ethanol for 15 min., followed by washes with sterile water to remove remaining decontaminant.

Cell viability

Cell-survival quantification with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (XTT) assay (Biological Industries, Beit Haemek, Israel) was performed according to the manufacturer’s protocol. This method determines the ability of metabolically active cells to reduce the yellow salt XTT to an orange formazan dye. Therefore, the conversion only occurs in living cells, and the amount of orange formazan 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 to either 2 μg of anti-FZD7 antibody or to 0.25 μg of sFRP-1 in respect to untreated controls for 72 hrs. After the indicated time, the cells were washed with PBS and incubated with the XTT solution according to the kit specifications for 3 hrs. After this incubation period, quantification of the formazan dye formed was determined using a spectrophotometer at a wavelength of 450 nm minus the absorbance at 620 nm. Each experiment was performed in triplicates, and each series was repeated at 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 apoptotic cells can be stained by annexin V, which binds to phosphotidyl-serines normally found in the inner aspect of the cell membrane but can be found on the outer aspect of the cell membrane in apoptotic cells. On the other hand, during early apoptosis, 7AAD stains DNA and is excluded from the nucleus, so staining does not occur. During necrosis and late apoptosis, membrane integrity is compromised, and cells are stained by both annexin V and 7AAD; cells were treated with 5 μg of anti-FZD7 antibody with respect to untreated control. Both samples were kept in 4 degrees for 12 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-μl samples and appropriate controls. Subsequently, 5 μl of either FITC or APC-conjugated annexin V and 5 μl of 7AAD were added to the samples and/or controls followed by incubation for 15 min. at room temperature in the dark. Annexin V binding and 7AAD staining were evaluated by using FACSort (Becton Dickinson) with CELLQUEST software (BD Biosciences).

Immunohistochemical staining of human foetal kidney

Sections, 4-μm thick, were cut from whole blocks of human foetal kidney from 20 weeks of gestation, human adult kidney and WT for immunohistochemistry. Immunostainings were performed as previously described [31]. In brief, the sections were processed within 1 week to avoid oxidation 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 for antigen retrieval, followed by treatment of 3% H2O2 for 10 min. The slides were subsequently stained by the labelled strepavidin-biotin method using a Histostain plus kit (Zymed, San Francisco, CA, USA). Anti-human NCAM antibody (LifeSpan Biosciences, Inc., Seattle, WA, USA), anti-human FZD7 antibody (R&D Systems) and anti-human CD133 antibody (Miltenyi Biotech, Germany), at a dilution of 1:50 were used. Controls were prepared by omitting the primary antibodies or by substituting the primary antibodies 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]. WT tumour cells were sorted according to either NCAM or CD133 expression and the sorted cell fractions were plated in 96-well micro well plates (Greiner Bio-One, Mediscan, Kremsmunster, Austria) in 150 μl of culture media, at 0.3 or 1 cells per well dilution. The low cell concentration was achieved by serial dilutions reaching 1000 cells per ml. The number of colonized wells was recorded after 4 weeks.

Real time PCR analysis

Quantitative real time reverse transcription PCR (qPCR) reactions was carried out as previously described [33], to determine fold changes in expression of a selection of genes for stemness and diffrentiation between WT cells cultured either in adherence promoting conditions or as spheres and in sorted WT cells. RNA was extracted using the microRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA synthesis was carried out using the High capacity cDNA RT kit (Applied Biosystems, Foster City, CA, USA). Each analysis reaction was performed in duplicates. β-actin or Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) were used as endogenous control throughout all experimental analyses.

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

In vitro effects of chemotherapies on WT cell gene 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 at 104 cells/well for 24 hrs. After the indicated time the medium was replaced with medium containing a range of concentrations for each of the drugs evaluated: For Etoposide and Irinotecan −1μM–250μM were tested, for Cisplatin −1μM–100μM were tested. After 48 hours, a XTT proliferation assay was performed as described above and the lethal dose for 50% of cells (LD50) was determined. For Etoposide and Irinotecan an LD50 of 40μM and for Cisplatin an LD50 of 10μM were observed.

To study the in vitro effect of topoisomerase inhibitors – the topoisomerase II inhibitor, Etoposide and the topoisomerase I inhibitor, Irinotecan – on WTs gene expression in comparison to Cisplatin treated or untreated cells, WT cells from three different WTs were distributed into 4 × 25 T flasks and cultured for 24 hrs at 37°C/5% CO2. After the indicated time, medium was replaced with either a medium containing one of the above mentioned chemotherapies or with normal growth medium as control. After additional 48 hrs in culture, cells were trypsinized and RNA was extracted for quantative real time PCR analysis of topoisomerase2A (TOP2A), NCAM and WT1. This procedure was repeated 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 cell populations were evaluated using the non-parametric, one sided sign test. Statistical differences of two group data were compared by Student’s t-test. For all statistical analysis, the level of significance was set as P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledegments
  8. References

Primary WT cultures

We have analysed eight WTs of different histological subtypes (Table 1). Primary WT cultures were established in growth promoting medium as well as in anchorage independence promoting conditions. Cell cultures from six tumours displayed mostly spindle-shaped morphology (Fig. 1A, a–f). Nevertheless, phenotypic plasticity was demonstrated as cells originating from a specific tumour, cultured under identical conditions and observed at a similar passage number, showed both cobblestone (a characteristic feature of epithelial cells) and spindle-like cell morphology (Fig. 1B, a and b). In addition, when grown in low-attachment promoting conditions, WT cells originating from different tumours were able to form floating sphere-like clusters (Fig. 1C, a–d) and created secondary spheres after dissociation and replating. Thus, primary WT cultures show morphological plasticity and sphere formation capability. Sphere formation has been shown to maintain stem-cell potential in various primary culture systems, especially neural [15, 22]. In order to examine whether this form of WT propagation is advantageous for maintaining primary WT cells in an un-differentiated state, we compared WT cells grown in adherence promoting conditions with WT spheres for the expression of a selection of WT-‘stem’ signature and stemness genes [19, 29, 34] (Fig. 1D). Investigation of ‘stemness’ genes has not been previously performed in WT. Analysis of four different WTs showed that while all stemness genes displayed a coordinated expression pattern in a particular tumour, this was not associated with a certain culturing method; Two of the tumours (WOO4, WOO5) showed higher expression in the WT spheres while WOO2 overexpressed the stemness genes in the adherent culture and WO1O showed mostly comparable expression levels (Fig. 1D). This lack of clear advantage for the WT spheres led us to further examine WT cells in adherent cultures.

Table 1.  Patient and tumour characteristics
Patient Code GenderAgePatternHistologyRemarks
W002Female4 yearsTriphasicFavourable histologyLung metastasis
W003Male10 yearsBlastemalUnfavourable histologyRecurrent with diffused anaplasia
W004Female6 yearsTriphasicFavourable histologyBilateral
W005Male2 yearsTriphasicFavourable histology
W006Male2 yearsTriphasicFavourable histologyFocal anaplasia
W007Female3 yearsTriphasicFavourable histologyRecurrent WT with focal anaplasia
W009Male2 yearsTriphasicUnfavourable histologyRecurrent with diffused anaplasia
W010Female1 yearsTriphasicFavourable histology
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Figure 1. Primary Wilms’ tumour (WT) cell cultures. (A) Pictures by confocal microscope of six primary WTs in culture, showing mostly mesenchymal cell morphology. (magnification ×10); (B) Morphologic plasticity of cells from the same primary WT (WOO2) after an equal number of passages, showing different morphologic cell structures in culture – (a) – cobblestone shaped cells, (b) – spindle shaped cells. (magnification ×10); (C) Primary WTs of different histologic subtypes form sphere-like clusters in low attachment conditions. Photomicrographs of cultured WT cells (magnification ×20) at 5 days after plating the cells in ultra-low attachment plates with medium suitable for growing embryoid bodies from hESC [30]; (i) WOO2, (ii) WOO3, (iii), WOO4 and (iv) WOO5; (magnification ×20). (D) Quantitative RT-PCR analysis for the expression of WT-stem signature (nephric-progenitor- WT1, SIX2, Sall1, polycomb group- EZH2, BMI1, Wnt pathway-FZD7, β-catenin and ESC – nanog, Oct4) genes in WT cells grown in adherence promoting conditions (open bars) in comparison to WT spheres (grey bars). Shown are experiments in cells derived from four different WTs (WOO2, WOO4, WOO5 and WO1O). The value for the spheres 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 of at least three separated experiments. Quantitative transcript levels were normalized to expression of β-actin or GAPDH.

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Phenotypical analysis of low-passage cultures of WT

We initially examined unsorted populations of low-passage WT cells by flow cytometry in order to identify sub-populations expressing putative stem cell markers. Table 2 summarizes immunophenotyping data of primary WT cells. Each surface marker was analysed in at least five different WTs. Figure 2 presents the results of FACS analysis grouped into early blastemal markers, haematopoietic and mesenchymal stem cell markers, markers that were overexpressed in the stem-like WT xenografts and other non-specific stem cell markers (MDR1-cancer, CD24-pluripotent embryonic). A representative FACS analysis for each marker of the various groups and detailed analysis of NCAM, FZD7 and CD133 in the remaining five tumours with the corresponding isotype controls are shown in Fig. 2, a–f and Fig. 3, a–c, respectively. Four main immunophenotypes with regard to the various cell markers were identified in the primary heterogeneous cultures: constant low expression, <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 as other known stem cell markers.

Table 2.  Immunophenotype of primary WTs
Marker groupsSurface markers Primary Wilms’ Tumours
WOO2WOO3WOO4WOO5WOO6WOO7
  1. +0.5–10% of the cells express the marker; ++ 11%-50% of the cells express the marker; +++ >50% of the cells express the marker; – none of the cells express the marker; ND not determined.

Early blastemal markerCadherin-11++++++
Haematopoietic stem/progenitor cell markersCD133++++++++
C-KIT
CD34++++++
Mesenchymal stem cell markerCD105ND
CD90++++++++++++ND+++
Markers that were high in the WT stem like XnNCAM++++++++++
ACVRIIB+++++ND
GPR39++++ND
FZD7++++++++
FZD2++++++++?
Additional normal and cancer stem cell markersCD24++++++++++
MDR1++++++++++ND
Pan haematopoietic markerCD45
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Figure 2. Flow cytometric analyses of primary Wilms’ tumours. A representative FACS analysis for each marker of the various groups: (A) Blastemal markers, (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 are representative of no less than two separate experiments for each marker in at least five tumours.

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Figure 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 all tumours examined. Upper panels represent isotype control antibody analysis of the corresponding stained cells in bottom panels.

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Immunostaining of human foetal kidney

We chose to further analyse the NCAM and FZD7 markers, which were overexpressed in the stem-like WT xenografts (at the gene level) and were now found in primary WTs to be constantly expressed in low-to-moderate levels likely to contain the stem cell fraction. CD133, a widespread cancer stem cell marker [15, 17, 18] which showed a similar expression pattern in the primary WT cultures, was also tested. In human foetal kidney, the nephrogenic mesenchyme is the region containing the normal kidney stem cells. Taking into account both the presence of these normal renal stem cells within this region and the close relations of WT with normal nephrogenesis [1], we determined the in situ localization of these markers in mid-gestation human foetal kidneys (Fig. 4). Both NCAM and FZD7 were intensely expressed by cells of the nephrogenic mesenchyme (Fig. 4A, B), suggestive of cell origin within this tissue. We could not achieve CD133 staining in the human foetal kidney. Nevertheless, CD133 was abundantly expressed in tubular epithelial cells of the adult human kidney, whereas in WT it localized predominantly to the tumour vasculature (Fig. 4C), staining in both cases differentiated rather than un-differentiated cell types.

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Figure 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) in the 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.

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

We next sorted WT cell sub-populations according to FZD7, CD133 or NCAM expression, in order to test whether these cell fractions possess stem cell potential. Cell sorting with an anti-FZD7 antibody resulted in extensive cell death (as was shown by trypan blue staining of the sorted cells) and precluded us from obtaining a positive cell 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 at the 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 by either compound (Fig. 5A, a and b, P < 0.05). In addition, we analysed apoptosis and cell death in WT cells treated with anti-FZD7 by 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 annexin+ 7AAD (early apoptotic cells) and annexin+7AAD+ (late apoptotic/necrotic) cells and decrease in annexin7AAD viable cells (Fig. 5Bb). Thus, application of anti-FZD7 reduces WT cell survival and increases WT cell death and apoptosis, limiting the use of FZD7 for functional cell sorting.

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Figure 5. Exclusion of FZD7 and CD133 as markers for the isolation of WT stem/progenitor cells. (A) Cell viability was determined by XTT cell proliferation assay (black bars, WT cells treated with either sFRP1 or anti-FZD7 antibody; white bars, untreated WT cells), which measures mitochondrial respiratory 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 decreased survival rate in all preparations by either compound in comparison to untreated controls. Data are means ± S.E.M derived from two independent experiments with triplicate wells per condition, P < 0.05; (B) Enhanced WT Cell death after exposure to 5 μg of anti FZD7 antibody for 12 hrs. (a) Flow cytometric analysis of WT cells incubated overnight with (treated) or without (untreated) anti-FZD7 antibody. Cell number is plotted as a function of the intensity 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 untreated control. Data presented are representative of three independent experiments. (C) Clonogenicity assays of the sorted CD133+ and CD133 WT sub-populations performed on three different WTs. Columns represent the mean number of colonized wells. Limiting dilutions followed by plating of a single cell/well in 96-well plates were performed in order to compare the clonogenic capabilities between WT CD133+ and CD133 cell fractions. No significant difference in 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, β-catenin and self-renewal/multipoteniality- OCT4) in CD133+ and CD133 WT sorted cells from at least three different tumours, demonstrates no difference between the cell fractions or higher expression in the CD133 in comparison to the CD133+ sub-population. The value for the CD133+ was used as the calibrator (therefore = 1) and all other values were calculated with respect to it. Results are the mean ± S.E.M of four separate experiments, *P < 0.05. Quantitative transcript levels were normalized to expression of β-actin.

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In contrast to FZD7, an enriched viable fraction of CD133 expressing cells could be readily obtained and further subjected to clonogenicity assays from single cells (i.e. limiting dilution) and qRT-PCR analysis. No difference in clonogenicity was observed between the sorted CD133+ and CD133 WT cell fractions of three different tumours examined (Fig. 5B). We further compared the cell fractions for the expression of ‘tumour-progenitor’ genes (Fig. 5C, a). These genes were previously found to be overexpressed in the progressive WT stem-like xenografts [3] and comprise of Wnt pathway (FZD7, β-catenin), polycomb group (BMI-1, EZH2) and nephric-progenitor (SIX2, WT1) genes. In addition, both Wnt pathway and polycomb group of gene have been implicated in self-renewal of normal and cancer stem cells [12, 20, 21, 34–37] and OCT4 in pluripotentiality [38]. These WT-stem signature and stemness genes were inconsistent in their expression pattern between the CD133 and CD133+ cell fractions showing either similar expression levels or elevated expression in the CD133 cell fraction. Thus, both clonogenicity assays and real-time RT-PCR suggest that the 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 positive cell 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+ cell fraction to overexpress the ‘tumour-progenitor’ gene set compared to the negative fraction, i.e. nephric-progenitor (WT1, SIX2), polycomb group (EZH2, BMI-1), Wnt pathway (FZD7, β-catenin) and the embryonic stem cell pluripotency and self-renewal (nanog) genes. Thus, as opposed to CD133, NCAM expressing cells are highly clonogenic and overexpress a set of WT ‘stemness’ genes.

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Figure 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 three different donors, demonstrating the NCAM+ to be highly clonogenic compared to the NCAM WT cells. (C) Quantitative RT-PCR analysis of the WT-stem signature genes (WT1, SIX2, EZH2, BMI1, FZD7, β-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 the negative 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 the mean ± 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 adherent cultures. NCAM expression in WT cells follows the expression pattern of the stenmess genes, shown in Fig. 1D, regardless of the culturing method employed. 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 (therefore=1) and all other values were calculated with respect to it. Results are presented as the mean ± S.E.M of three separate experiments. Quantitative transcript levels were normalized to expression of β-actin or GAPDH.

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Analysis of the epithelial elements of WTs suggests that these represent regions of metanephric blastema that have attempted to undergo a normal process of mesenchyme (Vimentin+)-to-epithelial (E-cadherin+) transition (MET) but failed to proceed to nephron formation [39]. We therefore examined the differentiation status of the cell fractions by analysing expression of Vimentin and E-cadherin. High Vimentin and low E-cadherin expression indicated that the NCAM+ fraction is likely to originate from early stages of mesenchymal to epithelial transition, characteristic of early kidney development (Fig. 6C, a and b).

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

TOP2A is overexpressed in the NCAM+ cell fraction

To begin investigating the clinical relevance of the NCAM+ cells we sought to determine whether TOP2A, a molecular marker known to be associated with poor prognosis of WT [40–43] is elevated in this cell fraction. Real-time PCR demonstrated TOP2A to be significantly elevated (P < 0.05) in NCAM+ compared to NCAM cells obtained from five independent WTs (Fig 7A). These data complement our results showing overexpression of WT1 and EZH2 in the NCAM+ cells (Fig. 6C), as both of these markers are considered poor prognostic markers in WT [40, 44]. Having established the elevation of TOP2A in the NCAM cell fraction, we next determined whether in vitro treatment of heterogeneous WT cells with the topoisomerase inhibitors, Etoposide and Irinotecan influences expression of both TOP2A and NCAM. This was compared to treatment with Cisplatin, used in refractory childhood solid tumours [45, 46] and to untreated WT cells. Etoposide, a topoisomerase II inhibitor had a dramatic effect on TOP2A expression with concomitant reduction in NCAM expression in two of the three tumours evaluated (WOO2 and WOO4, but not in WO1O) in respect to the untreated control. Interestingly, Irinotecan, a topoisomerase I inhibitor, also reduced both TOP2A and NCAM expression in the three tumours evaluated (Fig. 7B). In contrast, application of Cisplatin did not show a similar decrease and in some instances, up-regulation of marker levels was observed (Fig. 7B). Moreover, application of either one of the topoisomerase inhibitors (Etoposide or Irinotecan), but not of Cisplatin, on the WT cells, resulted in a reduction in WT1 expression in both of the tumours examined, suggesting additional coupling of TOP2A with WT1 (Fig. 7C).

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Figure 7. TOP2A is overexpresed by NCAM+ WT cells. (A) Quantitative RT-PCR analysis for the expression of TOP2A in the NCAM+ in comparison to the NCAM WT cells. The NCAM+ cells consistently overexpressed the WT poor prognostic factor, TOP2A. Shown are experiments performed on five different 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 presented 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 the topoisomerase inhibitors, either Etoposide or Irinotecan, mostly correlates with reduction of NCAM expressions in WT cells in comparison to untreated control. Shown are experiments performed on three different WTs. (C) Treatment of WT cells with either of the topoisomerase inhibitors reduces the expression of the WT1 gene in comparison to untreated control. Shown are experiments performed on two different WTs. The value for the untreated control 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 of at least three separate experiments for each of the tumours, *P < 0.05. Quantitative transcript levels were normalized to expression of either β-actin or GAPDH.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledegments
  8. References

Herein we wanted to investigate for the first time whether human WT could contain tumour cell subsets that have potential as cancer stem/progenitor or tumour initiating cells. This investigation is based on our previous findings on the intimate link between tumourigenesis and organ development in the kidney [3, 19]. We chose 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 a DNA microarray screen of progressive stem-like WT xenografts and human foetal kidneys [3]. To identify additional potential WT progenitor sub-populations for further investigation, we also studied the presence of cellular fractions within WTs that express markers consistent with stem cells of human haematopoietic, mesenchymal, neuronal and pluripotent embryonic origin. This tour de force of phenotypic characterization is critical because specific markers of human multi-potent renal embryonic progenitors that lead to formation of WT are unknown. Accordingly, we have detected sub-populations of WT cells that are immunoreactive for various stem cell markers and may contribute to our understanding of the WT cell of origin. For instance, lack of expression of markers such as c-Kit (CD117), CD45 or CD105, which strongly characterize haematopoietic and mesenchymal stem cells, respectively, indicates the specificity of WT cells in regard to their lineage. Of all protein markers analysed, those found to be expressed in low-to-moderate portions in all primary WT samples (see Table 2) are likely to contain the stem/progenitor cell fraction and could serve as initial markers for sorting of putative cell sub-populations, an approach which has not been previously carried out in WT. Based on that criteria, we chose to more closely examine NCAM, FZD7 and CD133. NCAM and FZD7 were among the most up-regulated genes in the WT stem-like xenografts, while CD133 has been found to be expressed by many other tumour initiating cells [8, 13, 15, 17, 18]. The demonstration of the presence of NCAM and FZD7 in the human nephrogenic mesenchyme, which contains the normal renal stem cells, may contribute to our understanding of the WT cell of origin.

Sorting experiments and functional assays revealed the difficulties in sorting WT cells according to FZD7 expression which resulted in loss of cell viability. FZD7 is one of the Wnt pathway receptors associated with stem cell self-renewal and proliferation [47]. The Wnt pathway plays critical roles in multiple cellular events that occur during the development of the mammalian kidney [48], including regulation of renal epithelial progenitors [49]. Indeed, blocking the Wnt pathway (specifically with the FZD7 antibody or with a general antagonist at its level) was shown to be detrimental for these cells. Thus, FZD7 is a functional receptor for WT cells and therefore is not optimal for cell sorting. Nevertheless, since 15% of WTs have activating mutations in β-catenin [50] that would render them unresponsive to Wnt ligands and are likely to remove the need for signalling through the β-catenin pathway, there might be cases in which FZD7+ cell isolation and characterization can be accomplished. Accordingly, future studies aimed at detecting such mutations and correlating their presence to FZD7+ cell viability and function are warranted.

In contrast, highly enriched populations of WT NCAM or CD133 expressing cells could be isolated. Their analysis revealed a fundamental difference: While NCAM+ cells are highly clonogenic and overexpress the WT ‘stemness genes’ (nephric-progenitor, Wnt pathway and polycomb group genes), previously observed in the ‘WT-stem’ signature set of the progressive WT xenografts [3] and in independent real-time PCR verifications of these stem-like tumours [19], CD133+ do not possess such characteristics. CD133+ cells previously isolated from adult kidney cancer were shown to contribute to tumour vascularization, rather than being tumour-initiating cells [19, 51]. Similarly, WT CD133+ cells are not likely to represent the epithelial malignant renal progenitor that drives WT initiation.

Our new functional data of the sorted WT NCAM+ population integrates with previous data which showed by immunostaining that NCAM is predominantly localized to WT blastema as well as in the de-differentiated cells of WTs [52]. In addition, it has been demonstrated that following senescence and crisis of primary WT cultures, surviving cells were all shown to be NCAM+, supporting it as a marker of self-renewing cells in WT [53]. Collectively, these data suggest NCAM as a marker for the malignant renal progenitor population. We emphasize that future in vivo xenotransplantation studies of the NCAM+ cell population are required to determine its tumour-initiating capabilities. Nevertheless, while WT xenografts are readily formed via implantation of fresh surgical samples into immunodeficient hosts [3], WT are notorious for their inability to establish xenografts after tumours have been processed into single cell suspension and especially from primary WT cultures [54]. Taking into account these inherent limitations of WT and the fact that cell sorting and heterotransplantation in immunodeficient host animals require large numbers of the rather rare clinical WTs for calibration of both implantation site and cell dosage, makes these experiments beyond the scope of this paper.

Others and we have very recently demonstrated a similar phenomenon in which progressive xenografts of human tumours adopting a highly infiltrative and stem-like phenotype, down-regulate angiogenesis genes and expand independent of angiogenesis [19]. From a practical point of view, cancer treatment strategies of WT recurrence aimed at angiogenic targets might therefore not suffice and there is a need to pursue the invasive stem-like cancer cells. Since WT stem cells are likely to be defined by a combination of markers including NCAM as the initial marker, we propose, at this stage, that WT patients may benefit from targeting the NCAM molecule. Even in the case of WT being a model for tumour arising from self-renewing progenitor cells that divide rapidly and are sensitive to the conventional chemotherapies along with the tumour bulk, targeting NCAM could in turn reduce the late adverse effects resulting from current therapeutics. NCAM has been implicated with both adverse prognosis and metastatic behaviours of several tumours [55, 56] Furthermore, WT recurrence and metastasis is associated the appearance of a blastemal phenotype in the tumour [57, 58]. In this regard, our demonstration of high levels of the poor prognostic marker TOP2A in the NCAM+ cell fraction and the down-regulation of both TOP2A and NCAM following Etoposide or Irinotecan but not Cisplatin treatment of WT cells is especially important. These preliminary data suggest that the NCAM cell population might be especially susceptible to topoisomerase inhibitors. Nevertheless, un-coupling of TOP2A and NCAM reduction after applying Etoposide in one of the three tumours examined (WO1O) and their strong connection in the same tumour following Irinotecan treatment, suggests a more complicated mechanism for the operation of the topoisomerase inhibitors. Future in vitro and in vivo studies that combine the two topoisomerase inhibitors (already in trials for small cell lung cancer showing high affectivity and relatively low toxicity [59, 60]) with anti-NCAM immunotherapy for targeting the cancer initiating cells and possibly preventing WT recurrence with limited drug complications are warranted. Immunotherapy directed against NCAM in adult tumours is under intensive investigation and several newly developed reagents are readily available [61].

Acknowledegments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledegments
  8. References

We thank the Kahn Family Foundation for supporting our research. This work was supported by ‘Talpiot’ Medical Leadership Award, the Israel Science Foundation (ISF) Project Grant, the Israel Cancer Research Foundation Clinical Career Development Award (ICRF), The Schreiber and Breteler 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 the Djerassi Chair in Oncology at the Tel Aviv University.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledegments
  8. References