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European Urology

Volume 57, issue 2, pages 179-362, February 2010

Prostate Cancer

Stem Cell Characteristics in Prostate Cancer Cell Lines

Minja J. Pfeiffer, Jack A. Schalken lowast .

Accepted 8 January 2009, pages 246 - 255

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Abstract

Background

Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population.

Objective

Six established prostate cancer (PCa) cell lines—DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3—were examined for their stem cell properties in vitro.

Design, settings, and participants

The colony-forming efficiency and self-renewal ability of morphologically distinguishable holoclones and paraclones were tested with low-density plating and serial passaging. Expression of the putative stem cell marker CD133 and breast cancer resistance protein (BCRP) was examined with flow cytometry, and immunohistochemical stainings were made for CD133, α2-integrin, nestin, BCRP, cytokeratin 5 (CK5), and cytokeratin 18 (CK18).

Results and limitations

Five out of six cell lines formed clear holo-, mero-, and paraclones. Unlike paraclones, we can maintain DU145 holoclones in culture for several passages, which is indicative of self-renewal ability. Using fluorescence-activated cell sorting (FACS) analysis only in DU145 cells, a small fraction (0.01%) of CD133+ cells was detected. CD133+ cells; however, like DU145 BCRP+ (0.15%) cells, they were not more clonogenic, and they did not show more holoclone formation than the marker-negative cells or unselected cells. Immunohistochemistry revealed α2-integrin and BCRP as potential stem cell markers and CK5 with the combination of CK18 to distinguish transient amplifying cells.

Conclusions

These results indicate the possible presence of stem-like cells in several established PCa cell lines. CD133 selection does not enrich for stem-like cells in PCa cell lines.

Take-Home Message

Several prostate cancer cell lines contain cancer stem or cancer-initiating cells that are shown as the presence of self-renewing holoclones that express putative stem cell markers. CD133 is excluded as a cancer stem cell marker in the examined cell lines.

Keywords: Cancer stem cell, CD133, Holoclone, Prostate cancer.

Article Outline

1. Introduction

The concept that solid cancers are hierarchically organised, with cancer stem or cancer-initiating cells at the top of the organisation, gains acceptance and is based on experimental support. Thus, cancers, like normal organs, consist of cells with different potentials for proliferation and give rise to progeny, consequently establishing a hierarchical organisation of stem cells, transient amplifying cells, and terminally differentiated cells. In fact, cancer can be considered a caricature of normal development. The cancer cells that are able to self-renew give rise to differentiated nonclonogenic progeny and are capable of reconstituting the whole tumour upon transplantation and can thus be called cancer-initiating or cancer stem cells (CSC). The first CSCs were isolated from patients with leukaemia [1], and [2]. Since then, CSCs have also been isolated and characterised in solid tumours such as those in the breast [3] and brain [4]. There is also accumulating evidence for CSCs in colon [5], and [6], liver [7], pancreatic [8], skin [9], and prostate [10], [11], and [12] tumours.

Because CSCs resemble normal stem cells in many of their properties, methods similar to those that have been used to identify normal stem cells can be applied to identify and to enrich CSCs. CSCs have a combination of properties such as renewal ability, colony formation in semisolid media, and dye-exclusion, and they are tumorigenic at low cell numbers. Specific sets of surface markers are an essential part of most stem cell–enrichment procedures.

At low density, nontumorigenic cells generate colonies with different morphologies in vitro that are classified as holoclones, meroclones, and paraclones. These morphologies are believed to derive from stem cells, early progenitor cells, and late progenitor cells, respectively. The holoclones are round colonies with tightly packed, generally small cells, whereas paraclones are highly irregular in shape and contain more flattened and scattered cells. Meroclones are a mixture of the previous two colony types [13]. Only holoclones can be repeatedly passaged, which is in agreement with the existence of self-renewing cells in those colonies [14].

Stem cells express distinct molecular markers that can be used for enrichment of these cells. A surface molecule, CD133 (prominin), has been identified as a haematopoietic [15] and neuronal [16] stem cell marker and has been used to enrich brain tumour–initiating cells [4]. Also, for prostate cancer (PCa) CD133, co-immunophenotyping proved to be of potential utility. In PCa, a CD44+/α2β1-integrin high/CD133+ (0.1–0.3%) subpopulation demonstrated a higher proliferation potential and self-renewal ability in vitro than the negative cells. This population may thus represent prostate CSCs [10]. Another potential marker for CSCs is the breast cancer resistance protein (BCRP), a member of the ATP-binding cassette (ABC) protein family that mediates the resistance to various classes of chemotherapeutic agents [17]. Based on this ability, populations of candidate stem cells have been isolated in various tissues and tumours [18]. Also, the cytoskeletal intermediate filament protein nestin, which was identified as a marker for normal neuroepithelial stem/progenitor cells in the brain [19], is a subject of interest. Since the discovery, expression of nestin has been found in several central nervous system (CNS) tumours [20], and [21], and it is strongly expressed in aggressive PCa specimens [22]. Additionally, in prostate epithelial cells, cytokeratin 5 (CK5) presents a marker for basal and, in particular, transient amplifying cells [23]. Several other prostate epithelial basal markers may prove important in identifying the CSC phenotype, such as c-Met and Net [24], and [25]. Yet, it is important to note that the characterisation of prostate CSCs is still in the early phase of research.

The use of cancer cell lines that contain stem-like cells would also facilitate the study of the molecular pathways and the behaviour of CSCs in vitro. Recent studies suggest the presence of cells with stem-like behaviour, as dye-exclusion and higher clonogeneity, in several human epithelial cancer cell lines [26], [27], [28], [29], and [30], including some PCa cell lines [26], and [27]. Despite these findings, it has to be kept in mind that the established cell lines do not completely resemble the original tissue. Changes may have occurred during clonal evolution and culturing in in vitro conditions.

In this study, we examined six PCa cell lines for their expression of the putative stem cell marker CD133 and the presence of self-renewing holoclones. The colonies from the DU145 cell line were further characterised with the differentiation marker CK18; a transient amplifying cell marker, CK5; and stem cell markers CD133, α2-integrin, nestin, and BCRP. We show evidence for cells with stem-like properties within the studied PCa cell lines that can be used in the functional studies and characterisation of prostate CSCs.

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2. Materials and methods

2.1. Cell culture

All the cell lines were derived from prostate carcinomas. DU145, PC-3, LNCaP, and 22Rv1 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). DuCaP cells were kindly provided by Dr Ken Pienta (University of Michigan, MI, USA) and LAPC-4 cells by Dr Rob Reiter (University of California, CA, USA). The cell lines were grown in RPMI-1640 supplemented with 10% foetal calf serum (FCS), 2 mM glucose, and 2 mM antibiotics, except for LAPC-4, which was cultured in Iscove's Modified Dulbecco's Media (IMDM) supplemented with 7.5% FCS, 10 nM R1881, and 2 mM antibiotics. The passage numbers used for experiments were DU145 P91-125, PC-3 P47-68, LNCaP P50-76, 22Rv1 P9-16/30-38/50-64, DuCaP P41-58, and LAPC-4 P33-55.

2.2. Flow cytometric analysis

Fluorescence-activated cell sorting (FACS) analysis was used to detect CD133+ in all cell lines and to isolate CD133+ and BCRP+ cells in DU145. Five million cells were labelled with CD133/2-PE (Miltenyi Biotec, Auburn, CA, USA) or bcrp1/ABCG2-PE antibody (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions. A nonspecific Opticlone immunoglobulin G1 (IgG1)-FITC/IgG1-PE/IgG1-PE-CY5 antibody (Beckman Coulter, Fullerton, CA, USA) was used as a negative control. Samples were analysed with Epics Altra, Epics Elite, or Epics XL flow cytometers (Beckman Coulter) with a band pass filter at 575 nm. At least four independent experiments were performed for each cell line. DU145 cells were sorted with Epics Altra or Epics Elite flow cytometers.

2.3. 2.3.Colony-forming assay

To determine different colony morphologies, 1000 viable cells were plated as single cells onto a 10-cm petri dish (CELLSTAR, Greiner Bio-One, Frickenhausen, Germany) or 120 cells onto a 6-well plate well (Corning, New York, NY, USA). Conditioned medium was added (50%) to DuCaP and LNCaP cells to facilitate their growth. After 6–30 d depending on the cell line, the different colony morphologies were scored under a light microscope. Also, FACS sorted DU145 cells from four separate experiments were plated at low density: 90–1200 CD133+ (all selected cells), 100–1000 CD133−, and 120–1000 BCRP+ and BCRP− cells. Only colonies with >32 cells were scored.

2.4. Single-cell cloning

Colonies with different morphologies were plated again as single cells to test their renewal ability. Single cells from DU145 and PC-3 colonies were seeded onto 96-well plates in a density of 1–2 cells per well with conditioned medium (50%). Morphologies of the established colonies were scored and counted under a light microscope.

2.5. Immunohistochemistry

The expression of several markers in the DU145 colonies was examined by immunohistochemistry. Cells were plated at low density on glass slides and left to grow for 8 d until colonies formed. Colonies were fixed with ice-cold acetone for 10 min or with 4% paraformaldehyde at room temperature for 20 min. Acetone-fixed colonies were stained with monoclonal antibodies: CK18 (clone DC10, DAKO, Glostrup, Denmark), CK5 (clone RCK103, Euro-Diagnostica, Arnhem, the Netherlands), BCRP (Millipore, Billerica, MA, USA), and CD49b (α2-integrin, clone Gi-14, Dr S Santoso, Giessen, Germany). In addition, 4% paraformaldehyde-fixed colonies were stained with CD133/2 (Miltenyi Biotec) and nestin (Millipore). CK18 (6 ng/μl), CK5 (culture supernatant); CD49b (2.5 ng/μl) antibodies were incubated for 1 h; CD133/2 (0.25 ng/μl) antibodies were incubated for 2 h at room temperature; and antibodies against nestin (5 ng/μl) and BCRP (2.5 ng/μl) were incubated overnight at 4 °C.

Detection of the antibody binding was performed using Powervision poly-HRP-goat anti-mouse/rabbit/rat IgG (ImmuniLogic, Duiven, the Netherlands) as the secondary antibody and 3′3-diaminobenzidine (Power DAB, ImmunoLogic) to observe the peroxidase activity. The nuclei were counterstained with haematoxylin. To evaluate the specificity of the antibodies, known positive tissues or cells were used as controls. Normal human prostate tissue was used as a positive control for CK18, CK5, and α2-integrin; human hippocampus tissue was used to control the nestin staining; and Caco-2 cells were used as a positive control for CD133 and BCRP. In negative controls, only secondary (not primary) antibody was used. The immunohistochemical stainings were studied under a light microscope.

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3. Results

3.1. CD133 is expressed only in DU145

From six PCa cell lines, DU145 showed expression for CD133 in a small proportion (0.01 ± 0.01%) of the cells (Fig. 1). Because the amount of positive cells was very low, a reanalysis of the flow cytometry–selected cells could not be performed. In DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3 cell lines, CD133 expression was not detected in FACS analysis. A small population (0.15 ± 0.20%) of BCRP+ DU145 cells was detected cells. Other cell lines were not tested.

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Fig. 1 Fluorescence-activated cell sorting (FACS) analysis for the CD133 stem cell marker in DU145 prostate cancer cell line; (A) isotype control; (B) DU145 cells showing a small detectable population (0.01%) of CD133+ cells (50 000 cells were analysed).

3.2. Prostate cancer cell lines show different colony morphologies

When plated in vitro at low density, five (DU145, 22Rv1, LAPC-4, DuCaP, and LNCaP) of six cell lines show all three colony morphologies: holoclones, meroclones, and paraclones. PC-3 cells did not form tightly packed, round holoclones. Fig. 2 shows light microscope pictures of typical colonies from DU145 (Fig. 2A–2C), 22Rv1 (Fig. 2D–2F), LAPC-4 (Fig. 2G–2I), DuCaP (Fig. 2J–2L), LNCaP (Fig. 2M–2O), and PC-3 (Fig. 2P–2Q).

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Fig. 2 Observed colony morphologies in prostate cancer cell lines: DU145 (6 d; A–C), 22Rv1 (13 d; D–F), LAPC-4 (8 d; G–I), DuCaP (35 d; J–L), and LNCaP (12 d; M–O) formed three morphologically different colonies: holoclones, meroclones, and paraclones. In PC-3 (11 d; P–O), a typical holoclone phenotype was not observed; only meroclones and paraclones were detected. The holoclones are generally more round and tightly packed, whereas paraclones are irregular in composition and often contain more elongated or flattened cells. The colonies with an intermediate phenotype are meroclones. Colonies with at least 32 cells (five cell doublings) were considered colonies. Bar indicates 200 μm.

Depending on the cell line, growth kinetics (number of colonies) was assessed after 1–3 wk. In some cell lines, the cell morphology differed between the colony types, where holoclones contained generally small and tightly packed cells and paraclones consisted of round or flattened, more irregularly shaped and loosely packed cells. In DuCaP, LAPC-4, and 22Rv1, many of the holoclones and meroclones started to grow three-dimensionally when the cell number in the colonies increased with culturing time, while the paraclones stayed as a monolayer. The colony-forming efficiency of each cell line and the proportion of holo-, mero-, and paraclones in at least three independent experiments are depicted in Fig. 3.

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Fig. 3 Colony forming efficiency and the proportion of each colony type in prostate cancer cell lines DU145, 22Rv1, LAPC-4, DuCaP, LNCaP, and PC-3 shown as box plots. The cells were plated at low density as single cells; after 1–4 wk, the colonies that contained >32 cells were counted under a light microscope. The data were analysed using the Statistical Package for the Social Sciences. Median value (horizontal line), outliers (open circle), and extremes (asterisk) are shown.
CFE = colony forming efficiency; HOLO = holoclones; MERO = meroclones; PARA = paraclones.

We noticed in DU145 that when there is no selective pressure in terms of plating density on the cell line (ie, with passing the cells in a standard dilution [1:10]), more paraclones start to form when the passage number progresses. Instead, when cells are plated at low density (a high dilution, such as 1:30), the proportion of holoclones increases. In other words, in stable conditions, the stem-like cells that form holoclones are in a more quiescent state but start to proliferate when grown at low density. This observation supports the evidence that DU145 holoclones indeed contain the more stem-like cells because their proportion increases with environmental pressure.

3.3. CD133 or breast cancer resistance protein–positive cells are not more clonogenic than the negative cells in DU145

Because Collins et al [10] showed that the CD133+ primary PCa cells were more clonogenic than the negative population, we assayed the colony-forming efficiency of these two populations in the DU145 cell line. If CD133 marked the CSC population, one would expect that CD133+ cells would generate more colonies—primarily holoclones. Yet, FACS-isolated CD133+ DU145 cells were not able to generate more colonies or more holoclones than CD133− at low density. The colony-forming efficiency varied strongly between and within experiments. Also, BCRP+ cells did not form significantly more holoclones or any colonies (data not shown). Statistical significance was calculated using a student t test.

3.4. Holoclones contain cells that show self-renewal

DU145 holoclones can be maintained in culture for several (at least four) passages, showing renewal ability, whereas paraclones can be kept in culture for one to two passages (Table 1). Cells isolated from holoclones and plated as single cells produced mainly holoclones (80–100%), but meroclones and paraclones were also observed. Cells from paraclones were able to generate only paraclones and occasional meroclones, depending on the morphologic purity of the plated colony. Instead, colonies from PC-3 cell line could be maintained in culture up to four passages (the end point of the study), independent of the morphology. The colonies showed great plasticity, being able to produce different colony morphologies regardless of the original morphology.

Table 1 The ability to passage DU145 colonies. After plating single cells at low density (passage 0), some of the formed holoclones and paraclones were isolated and plated again as single cells. The replating of the colonies was continued until the cells were unable to form new colonies. Holoclones could be maintained in culture for at least four passages (the end point of the study), whereas paraclones vanished within one or two passages

Passage No. 0 1 2 3 4
Paraclone + + + − −
Holoclone + + + + +
3.5. Stem cell and differentiation markers are expressed in DU145 colonies

To support the observation that holoclones contain more stem-like cells, immunohistochemical staining of stem cell and differentiation markers was assayed in the various colonies. DU145 cells express the putative stem cell markers α2-integrin, nestin, and BCRP as well as CK5 and cytokeratin 18 (CK18), but there was no detectable staining for the stem cell marker CD133. The staining of each marker was compared among holoclones and paraclones. Representative pictures of each staining are shown in Fig. 4.

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Fig. 4 Immunohistochemical staining of DU145 colonies. Eight-day-old DU145 holoclones and paraclones were stained for the putative stem cell markers CD133, α2-integrin, nestin, and breast cancer resistance protein as well as for the differentiation markers CK5 and CK18. Bar indicates 200 μm.

The quantity of α2-integrin protein staining was similar in holoclones and paraclones in a lower passage (P91), where three-quarters of all colonies were 95–100% positive for the marker. In a later passage (P107), the α2-integrin staining in the holoclones stayed the same but was reduced in paraclones, where most of the colonies showed no or little staining for the marker. It must be noted that some of the holoclones were negative (0–5% of the cells within a colony stained for the marker) and some paraclones were positive (95–100%). The level of the BCRP protein staining was similar in holoclones and paraclones, with the difference that the staining in holoclones was mostly in the cytoplasm or cell membrane, whereas in paraclones, the localisation of the protein was restricted mainly to the nucleus. In contrast, the staining of the marker for transient amplifying cells, CK5, was more intense in paraclones and less intense in holoclones, although completely negative paraclones were also observed, whereas in most of the holoclones at least some cells were stained. All DU145 cells stained strongly cytoplasmic for the luminal cell marker CK18 and weakly (0–100%) for the stem cell marker nestin regardless of the colony type. A later passage contained generally more nestin-stained colonies than the earlier passage.

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4. Discussion

The aim of this study was to determine whether in the commonly used PCa cell lines cells with stem cell characteristics could be identified. There are previous reports of stem-like cells in cell lines based on the dye-exclusion capacity, revealing a side population in FACS; however, Patrawala et al did not detect a side population in the PCa cell lines DU145, PC-3m and LNCaP [31]. Putative prostate CSCs in cell lines have been found by utilising the cell surface markers CD44 [26] and CD133 [32]. Based on previous studies with PCa cells, we decided to look at the CD133 expression and the colony formation at low density in PCa cell lines DU145, 22Rv1, LAPC-4, DuCaP, LNCaP, and PC-3.

In five out of six PCa cell lines, there was no detectable CD133+ population measured by FACS. Collins et al found a subpopulation of CD44+/α2β1high/CD133+ cells in PCa specimens that show stem cell characteristics in vitro [10]. There is evidence that CD133 is a stem cell marker in the normal prostate [33]. If CD133 marks the CSCs in primary PCa specimens, the lack of CD133 in cell lines may be explained by the long-term in vitro culturing, during which the conditions have altered the protein expression. In the DU145 cell line, we were able to find a small population (0.01%) of CD133+ cells.

To examine the properties of the CD133+ and CD133− DU145 cells further, the colony-formation efficiency of FACS-selected positive and negative populations and unselected cells was compared. However, there was no difference in the ability of these three populations to form colonies. If the CD133+ population is enriched with stem-like/progenitor cells, one would expect these cells to form more colonies. Also, the proportion of holoclones (formed by cells with stem cell properties) was not higher, and the proportion of paraclones (formed by more differentiated cells) was not lower in the CD133+ population than in the CD133− or unselected population. The colony-forming assay was also performed with cells selected with CD133-labelled magnetic beads (MACS, Miltenyi Biotec), but the results were similar (data not shown). Altogether, from these observations we can conclude that the CD133+ population was not enriched with stem-like cells and is not a marker for CSCs in PCa cell lines. However, this does not exclude the possibility that CD133 is a stem cell marker in the cancerous tissue in vivo.

Colony-formation assays performed with DU145 BCRP+ (0.15%) and BCRP− cells did not show significant differences between these populations. It has to be noted that the FACS-sorted CD133+ and BCRP+ cells formed in fact a shift from the main population, not a clear separate population. Thus, the division into two populations might have been fairly artificial, and positive cells were not truly detectable in FACS.

Interestingly, five (DU145, 22Rv1, LAPC-4, DuCaP, and LNCaP) of the examined six PCa cell lines formed holoclones, meroclones, and paraclones at low density. These are thought to derive from stem cells, early progenitors, and late progenitors, respectively [13]. The number of colonies and the proportional number of holo-, mero-, and paraclones may, however, change depending on the culturing conditions. An important factor is the extracellular matrix, which affects cell proliferation and survival. Culturing these cancer cell lines in a friendlier environment, on coated plates, might yield a larger number of colonies.

We did not detect morphologically typical holoclones in the PC-3 cell line, because the colonies that were initially considered as possible holoclones rapidly changed their morphology. It was described earlier that PC3 cells have an intrinsic impaired cell–cell adhesion through E-cadherin because of the lack of alpha-catenin expression (homozygous gene deletion) [34]; thus, they are not able to form tightly packed colonies. However, Li et al [35] showed recently that PC-3 cells formed holoclones that contain cells with stem-like properties. We did not observe those round, tightly packed colonies in the PC-3 line. In all of the cell lines, the definition of the three colony morphologies differs somewhat from each other, and there are no strict borderlines between the colony types, which makes the grading fairly subjective. In fact, a continuous gradient of different colony types exists made the colonies difficult to distinguish from each other. Based on our results and taking the previous studies with different colony forms [13], and [14] into consideration, we can deduce that at least DU145, 22Rv1, LAPC-4, DuCaP, and LNCaP may contain cells with the hierarchical organisation of stem, transient amplifying, and differentiated cells. In addition, the PCa cell lines used in this study are able to grow anchorage independently in soft agar (data not shown), which is a character of stem/progenitor cells.

In DU145, only holoclones can be maintained in serial culture, thus providing proof of self-renewal—a crucial property of stem cells. In contrast, paraclones contain cells that have only a limited capacity to proliferate, because they died out within two passages. Additionally, cells from paraclones could form only paraclones, never holoclones. In contrast, the replated holoclones produced mainly holoclones, but some occasional meroclones and paraclones were also detected. This gives us proof that some of the cells in holoclones have undergone differentiation in the hierarchical ladder from stem-like cells into more differentiated early and late progenitors. Different colonies of PC-3 cells were all able to stay in culture with morphologically variable daughter colonies, suggesting that the strict morphologic criteria used to identify DU145 colonies cannot be directly applied to PC-3. In fact, many PC-3 colonies of undefined morphology may contain stem-like cells.

The differences in the expression of a set of markers were studied in DU145 holoclones and paraclones. There was no expression of the putative stem cell marker CD133. The expression of α2-integrin was higher in the holoclones than in paraclones of an older passage, but not in a lower passage. This is in agreement with an earlier study [10] that suggests that α2-integrin is a marker for stem cells and transient amplifying cells. Culturing the cells might push the majority towards differentiation but maintaining a stem-like subset of α2-integrin+ cells that keep forming holoclones. The expression of BCRP did not differ in the intensity between holoclones and paraclones, but the localisation of the protein was restricted in holoclones mostly to the cytoplasm or cell membrane and in paraclones mainly to the nucleus. Because the BCRP actively effluxes cytotoxins from the cell [17], we can assume that the protein has to be present outside the nucleus to contribute to the toxin-recessive phenotype of the stem cells. Therefore, it can be speculated that the BCRP on the cell membrane might be a marker for more stem-like cells. However, there was no significant difference in colony formation between FACS-selected DU145 BCRP+ and BCRP− cells. The expression of the neural stem cell marker nestin in DU145 colonies was mostly very weak, ranging between 0% and 100% positive cells in any colony type. At least in PCa cell lines, nestin does not seem to be a useful marker for defining CSCs. The presence of CK5− cells indicates that there must be cells that are only positive for the luminal marker CK18, because all the cells were CK18+. Also, CK5+/CK18+ cells must exist that present the intermediate (transiently amplifying) basal–luminal cells that are the progenitors of the differentiated CK5−/CK18+ luminal cells [23].

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5. Conclusions

Most of the examined cancer cell lines did not express the potential stem cell marker CD133, implicating that it is not a marker for stem cells in PCa cell lines. Instead, the cell lines DU145, 22Rv1, LAPC-4, LNCaP, and DuCaP did contain cells that can form holoclones. Holoclones contain self-renewing cells and express the putative stem cell markers α2-integrin and BCRP. We suggest that PCa cell lines may contain stem-like cells and should be characterised and studied further.

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Author contributions: Minja J. Pfeiffer had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Pfeiffer, Schalken.

Acquisition of data: Pfeiffer.

Analysis and interpretation of data: Pfeiffer.

Drafting of the manuscript: Pfeiffer.

Critical revision of the manuscript for important intellectual content: Schalken.

Statistical analysis: Pfeiffer.

Obtaining funding: Schalken.

Administrative, technical, or material support: None.

Supervision: Schalken.

Other (specify): None.

Financial disclosures: I certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None.

Funding/Support and role of the sponsor: None.

Acknowledgement statement: The authors acknowledge Rob Woestenenk at the central haematology laboratory for performing the flow cytometric sorting. This work is part of the Cancer Cure Early Stage Research Training (CANCURE) project funded by the European Commission (MEST-CT-2005-020970).

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Footnotes

Department of Urology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands

lowast Corresponding author. 267 Experimental Urology, Radboud University Nijmegen Medical Centre, PO Box 9101, NL-6500HB Nijmegen, the Netherlands. Tel. +31 24 3614146; Fax: +31 24 3541222.

Article information

PII: S0302-2838(09)00028-1
DOI: 10.1016/j.eururo.2009.01.015
© 2009 European Association of Urology. Published by Elsevier B.V.

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