Characterization of primary wilms tumor cultures as an in vitro model

Authors


  • Supported by: Wilhelm Sander-Stiftung, DFG-Graduiertenkolleg 639, and BMBF Kompetenznetz “Paediatrische Onkologie und Haematologie.”

Abstract

Functional analysis of gene candidates and testing of novel therapeutics in Wilms tumors (WT) has been hampered by the lack of in vitro model systems. WT are characterized by a spectrum of histological appearances, but published cell lines are mostly derived from rare anaplastic variants or even non-WT. There has been some success in establishing primary cultures, but these are often poorly characterized or only derived from less frequent WT1 mutant tumors. We report the generation of a set of primary WT-cell cultures using a simple cultivation protocol. Our cultures could be established after preoperative chemotherapy and irrespective of histological subtypes or genetic alterations. The presence of tumor-specific genetic alterations validates these cultures as being tumor-derived. Genetic characterization is of utmost importance as some cultures with similar morphological appearance lacked such alterations and either represent clonal variants or normal cells. By immunohistochemistry, the cells are either epithelial or more mesenchymal, and the latter exhibiting a longer life span with 30 or more passages before undergoing senescence. This may be related to WT being embryonal tumors with a strong differentiation potential that may prevail in vitro. Telomeres progressively shorten with cultivation, but their length does not predict lifespan. hTERT transfection may partly allow establishment of immortalized lines, because 2/7 cultures avoid senescence even in later passages. Importantly, these cells can be efficiently manipulated by transfection, making them a useful model system for in vitro testing. © 2011 Wiley Periodicals, Inc.

INTRODUCTION

Wilms tumor (WT), or nephroblastoma, is the most common malignant renal tumor in childhood, affecting one in 10,000, particularly children under the age of 5 years. It arises from embryonal blastema and most frequently presents as a unilateral (90–95%) and sporadic (98–99%) tumor (Huff, 1998). WT are genetically heterogeneous and typically present with triphasic histology composed of blastemal, epithelial, and stromal elements, but individual cell types may be predominant in certain tumors. Frequently mutated genes accounting for one third of classical WT cases are the WT1 WT suppressor gene, CTNNB1 (β-catenin), a member of the WNT pathway, and WTX, participating in β-catenin turnover (Gessler et al., 1990; Koesters et al., 1999; Major et al., 2007). Efforts to identify additional causative genetic defects largely failed with few exceptions (e.g., TP53 mutations in the rare anaplastic variant or FBXW7 mutations associated with the epithelial subtype; Bardeesy et al., 1994; Williams et al., 2010). Furthermore, there is a plethora of genes and proteins shown to be deregulated or correlated with clinical or pathological parameters in different cohorts of WT, although most have failed replication (Zirn et al., 2006; Wittmann et al., 2008; Huang et al., 2009).

To investigate candidate genes for development and progression of WT or to test new treatment strategies, in vitro and in vivo models are needed. Xenografts of human tumors have been used as in vivo models capable of replicating the typical triphasic histology of WT (Garvin et al., 1987; Houghton et al., 2007). Recently, the first mouse model has been described, where combined Wt1 ablation and deregulation of imprinted Igf2 results in WT (Hu et al., 2011). However, mouse models are laborious and expensive. Thus, an in vitro cell-culture system is highly needed. Currently, there is no model that would represent the genetic and histological heterogeneity of WT.

Despite several attempts to establish WT cell lines, only few suitable cultures have been obtained. Earlier studies reported short-term culture from primary WT tumor material or mouse xenografts (Rousseau et al., 1974; Robinson et al., 1980; Brown et al., 1989). Efforts to establish cells representing the different components of WT yielded only a small number of short living cultures: for example, blastema-like cells with early epithelial differentiation from blastemal predominant xenografts (limited to <12 serial passages; Garvin et al., 1987) or skeletal muscle cells from stromal parts of WT (Garvin et al., 1985). In further attempts to culture the epithelial component of WT, primary material of 18 classical WT was used, but only two gave rise to proliferating epithelial cells. These cells were not immortal, but proliferated for up to 40–60 generations (Hazen-Martin et al., 1993), similar to cells from a diffuse anaplastic WT that grew for >50 passages (Hazen-Martin et al., 1994). Establishing immortal WT cultures appears to be difficult, however, as only one of 40 classical triphasic WT samples used gave rise to an immortal epithelial-like cell culture (Sens et al., 1999). Importantly, most cultures were not genetically characterized to firmly rule out cultivation of contaminating nontumor cells. Furthermore, few of these primary WT cultures are available today as they are short-lived with limited proliferative potential.

In two cases, longer-lived cultures from WT1-mutant WTs have been reported: the MZ128 cells carry a truncating mutation in exon 2 (Lobbert et al., 1998; Zirn et al., 2005a), and, more recently, five long living primary cell cultures with features of mesenchymal stem cells were derived specifically from WT1-mutant tumor samples (Royer-Pokora et al., 2010). The few spontaneously immortalized cell lines are poor models for WT as they were established from a rare subclass. The WiT-49 line is derived from a TP53-mutant lung metastasis (Alami et al., 2003), and WT-Pe.1 exhibits numerous chromosomal abnormalities (Faussillon et al., 2008). The success in establishing these lines may be due to the anaplastic nature of TP53-mutant tumors.

Other published WT cell lines have been found to be of different origin. G401 cells have been reclassified as rhabdoid tumor cells (Garvin et al., 1993), and the expression profile of SK-NEP1 suggested a Ewing sarcoma origin (Smith et al., 2008). Finally, WCCS-1 is derived from clear cell sarcoma of the kidney (Talts et al., 1993). Therefore, these established cell lines represent only a subgroup of WT (anaplastic) or unrelated kidney tumors and are not suitable as a general in vitro model for nephroblastoma.

Thus, there is an urgent need for additional WT-cell cultures that can be used to investigate the biology of typical WT and mechanisms leading to its formation. To address this problem, we generated a series of primary cultures from different histological subgroups of WT using simple protocols, and we characterized their biological properties.

MATERIALS AND METHODS

WT Samples and Clinical Data

Frozen tissue and fresh, viable tumor material, and corresponding control samples (blood or normal kidney) were obtained from hospitals participating in the SIOP2001/GPOH WT study. Clinical data and reference pathology are from the central GPOH study registry.

Primary WT-Cell Culture

Dulbecco's modified Eagle medium supplemented with 10% FCS and 1% penicillin/streptomycin (DMEM-10) was used for all cell cultures. Primary cultures were initiated from fresh tumor material shipped from participating institutions in PBS, cell-culture medium, or Liforlab (Onco Science, Wedel, Germany) overnight and processed immediately upon arrival. Representative sections were set aside for histological analysis and DNA extraction. Tissue specimens of 0.1–1 cm3 were washed with medium, minced with scalpels, and treated with 10 ml trypsin (0.25%)/EDTA for 10–15 min, followed by inactivation with 10 ml DMEM-10. Digested tumor material was pelleted by centrifugation and plated on 10-cm culture dishes in 2 ml DMEM-10 for 6 hr. After adhesion of tumor material, medium was added to 10 ml. Medium was changed every second day. Confluent cultures were split at a 1:3 ratio using trypsin/EDTA. After three passages, DNA and RNA were isolated for baseline molecular characterization. For cryopreservation, cells were frozen in DMEM containing 10% DMSO and 30% FCS and stored in liquid nitrogen.

Isolation of DNA and RNA

Total RNA and DNA from tumor tissue and cell cultures were isolated using QIAGEN or Macherey-Nagel kits (Qiagen, Hilden, Germany; Macherey-Nagel, Düren, Germany). Genomic DNA from kidney and blood samples was purified as described before (Wittmann et al., 2007).

Molecular Characterization

Loss of heterozygosity (LOH) analysis has been described in detail before (Wittmann et al., 2007). Primers and PCR conditions used are listed in Supporting Information Table S1. Details of mutation screening of CTNNB1 exon 3 have been described previously (Zirn et al., 2005b). Exons 1-10 of WT1 were amplified from genomic DNA individually and sequenced with primers specified in Supporting Information Table S1. Point mutations and deletion of WTX were analyzed as described (Wegert et al., 2009).

Immunohistochemical Characterization

Formalin fixed and paraffin-embedded tumor tissue and formalin fixed primary cells (in cell blocks) were used for immunohistochemical analysis. To prepare cell blocks from primary cells, cell suspensions were centrifuged, and pellets were resuspended in 500 μl fetal bovine serum and 10 ml 96% ethanol and centrifuged again. The viscous pellets were transferred into tissue caps and conventionally processed in a routine embedding system. All immunohistochemical analyses were performed according to standard protocols using Advance™ HRP Detection kit (K4068, Dako). Antibodies used are listed in Supporting Information Table S3.

Telomere Length Analysis

Telomere length was estimated as described by Cawthon (2002). Briefly, quantitative PCR was performed on genomic DNA using iQ™ SYBR® Green Supermix (Bio-rad) with primers tel-1b/tel-2b to amplify telomeric sequences, while the albumin gene (alb-u/alb-d) served as a reference (Supporting Information Table S1). Ct-values were measured using the iQ5-cycler (Bio-rad). To quantify telomere DNA, the ratio of Ct(tel)/Ct(alb) was calculated.

Senescence Associated β-Gal-Staining

Cells grown in 6-well cell dishes were washed with PBS (pH 7.2), fixed for 10 min with 0.5 % glutaraldehyde, and again washed with PBS (pH 7.2) + 1 mM MgCl2. About 1.2 ml of staining solution containing 1 mg/ml X-Gal, 0.12 mM K3Fe(CN)6, 0.12 mM K4Fe(CN)6, and 1 mM MgCl2 in PBS (pH 6.0) was added. After 3–10 hr incubation at 37°C staining was stopped by washing with PBS + 1 mM MgCl2 (pH 7.2).

Transfection and Transduction of Primary WT Cells

Plasmid pLL3.7 (Addgene) was transiently transfected using either TransPEI (Eurogentec, Köln, Germany), FugeneHD (Roche), Metafectene Pro (Biontex, Martinsried, Germany), or electroporation (Basic Nucleofector Kit for primary epithelial cells, Lonza) according to the manufacturer's instructions. Transfection was done in six-well plates at 70–80% confluency with 2 μg of pLL3.7.

Lentiviral particles were prepared in HEK293T cells (ATCC CRL-11268, validated by testing for SV40LT and neo genes) by TransPEI-mediated transfection. For a 10 cm dish of subconfluent HEK293T cells, 6 μg of pLL3.7, 4.5 μg of psPAX2, and 3 μg of pCMV-VSVg (all Addgene) were co-transfected. Sodium butyrate (10 mM) was added 12 hr after transfection. Virus-containing supernatants were harvested after 48 and 72 hr and passed through 0.22 μm filters. For the transduction of primary WT cells, supernatants were added to subconfluent cultures in the presence of 8 μg/ml polybrene. Expression of transgenic GFP was checked 24–48 hr after transfection or lentiviral transduction.

hTERT Transfer

A hTERT transposon vector for safe gene transfer was generated by transferring the hTERT-IRES-Blast expression cassette from pWZL-blast3-hTert (Venetsanakos et al., 2002) into pMiniTol2-CMVpA (Balciunas et al., 2006). The resulting pTol2-CMV-hTERT-blast plasmid and the CMV-driven TOL2 transposase expression vector were transfected by electroporation or with FugeneHD into primary WT-cell cultures. Selection was done with 5 μg/ml blasticidine.

Real time-PCR

About 2.5 μg of total RNA was used per cDNA synthesis reaction using the RevertAid First Strand cDNA synthesis kit (MBI Fermentas, St.Leon-Rot, Germany) with oligo dT primers. After cDNA synthesis, water was added to a final volume of 200 μl. Real time (RT)-PCR was conducted as described before with SybrGreen quantification (Wittmann et al., 2008). Primers and PCR conditions used are listed in Supporting Information Table S2. The housekeeping gene HPRT was used to normalize expression levels. All measurements were performed at least twice and mean values were calculated.

RESULTS

Establishment of Primary WT Cultures

Viable tumor material was available from 55 patients, with three of them having bilateral disease, resulting in a total of 58 tumors. Most of the tumors had been resected after preoperative chemotherapy; only two patients had been treated by primary surgery. Cultures were initiated from fresh tumor specimens immediately upon arrival, and cellular outgrowth from smaller aggregates was usually detected within 1–5 days. After 5–20 days, cells could be passaged and expanded. All cultures were grown in DMEM-10 medium. Initial attempts to improve efficiency of culture initation or subsequent growth through addition of conditioned media from the M15 metanephric cell line or G401 cells failed. The use of either reduced (5%) or increased (20%) serum concentrations (or hypoxia in one case) or a change to RPMI medium likewise did not improve growth or promote appearance of cells with alternative morphologies in a small series of experiments (data not shown).

Cultures were obtained from 46 tumor samples with different histological subtypes: 12 triphasic, 6 stromal, 5 blastemal, 3 epithelial, 3 diffuse anaplastic, 13 regressive, and 1 necrotic WT, as well as three tumors that were reclassified as either mesoblastic nephroma, nephroblastomatosis lesion, and rhabdoid tumor. In 12 cases, no cultures could be established (six regressive, one necrotic, two triphasic, two blastemal, and one non-WT). Thus, it is possible to derive primary cell cultures from different variants of WT, although the success rate is clearly reduced for regressive and necrotic tumors. There was no evidence that shipping medium or conditions strongly affected success.

Validation of Primary WT-Cell Cultures

Tumor samples were screened for mutations of CTNNB1 (exon 3), WT1, and WTX by direct sequencing and allele loss (LOH) analyses were performed for chromosome arms 7p, 9q, 11p, 11q, 14q, 16q, and Xq to detect tumor-specific alterations. Corresponding alterations present in cell cultures verify those as bona fide WT cells. Twenty-three tumors were informative for at least one of these parameters and sequence/LOH chromatograms suggested a complete presence of the respective alteration in the majority of cells (>90%). In 12 cases, tumor-specific genetic alterations were also found in DNA from primary cell cultures at passage three (Fig. 1A). In the remaining 11 cultures, only normal (wild type) sequences or allele status were detected, while the initial tumor material contained mutations and/or LOH. This suggests either cultivation of normal cells or in case of tumor evolution the cultures were at least not derived from the predominant tumor component. Classification of cultivated cells was not possible for tumor samples from 20 patients where no genetic alterations had been detected. These cultures may become classifiable later on with the development of additional tumor-specific markers. Thus, for more than 50% of informative tumor samples (12 of 23 patients), cultivation of bona fide tumor cells was successful.

Figure 1.

Establishment and morphology of primary WT-cell cultures. A: For almost half of the tumors for which primary cultures could be established, there was no specific alteration present in tumor tissue, precluding classification of cell cultures. For cultures with wild-type (nonmutant) allele status, it is not possible to distinguish between outgrowth of normal cells or a variant tumor cell population that lacks the genetic alterations prevalent in bulk tumor tissue. B–G: Morphology of cells. B: The initial plating of cell culture ws591 contained a mixture of different cell types, (C) one dominant cell type prevailed at passage 3 of culture ws591, (D) fibroblast-like cells of culture ws568li, (E) fibroblast-like (polygonal) cells with many protrusions of culture ws539A, (F) culture ws535-2tr with round cells, and (G) fibroblast-like (polygonal) cells of culture ws535-1, obtained from the same tumor sample. Scale bar: 100 μm.

In some instances, multiple cell cultures could be established from different regions of the same tumor and for two bilateral cases with distinct genetic alterations samples from both sides gave rise to primary cultures, yielding 28 independent cultures from 12 patients. For all subsequent experiments, only these validated WT-cell cultures listed in Table 1 were used.

Table 1. Characteristics of Primary Wilms Tumor Cultures
Case (ws)Age (m)SexaLateralityHistologyMutations, LOHbCell cultureGrowthcMax. passagesMorphology
  • a

    f, female; m, male.

  • b

    Hom, homozygous; het, heterozygous.

  • c

    Fast, subculturing at a ratio of 1:3 at least once a weak, slow, subculturing less frequent.

  • d

    No preoperative chemotherapy.

  • e

    CMN, congenital mesoblastic nephroma.

48336frightTriphasicΔWTX (het)ws483Slow7Spindle shaped
4899mLeftStromalCTNNB1 ΔS45 (het), WT1 R362X (hom), LOH 11pws489li-1Fast13Fibroblast-like
ws489li-2Fast12Fibroblast-like
RightTriphasicCTNNB1 T41A (het), WT1 R362X (hom), LOH 11pws489reFast13Fibroblast-like
50758mLeftDiffuse anaplasiaLOH 9q, 14qws507-1Slow5Fibroblast-like
ws507-2trSlow5Round (epith.)
520102fRightStromalCTNNB1 ΔS45 (het), LOH 11qws520-1Slow5Fibroblast-like
ws520-2trSlow5Fibroblast-like
53535fRightRegressiveLOH 11pws535-1Fast7Fibroblast-like
ws535-1BFast7Fibroblast-like
ws535-2Fast7Fibroblast-like
ws535-2trSlow5Round (epith.)
53953fLeftStromalLOH 7p, 11p, ΔWTX (het)ws539AFast30Fibroblast-like
ws539BFast28Fibroblast-like
565199mLeftTriphasicLOH 7pws565-1Fast9Fibroblast-like
ws565-2Fast8Fibroblast-like
ws565-3Fast8Fibroblast-like
ws565-4Fast9Fibroblast-like
5689fLeftStromalCTNNB1 S45F (het), WT1 Q184X (het), LOH 11pws568liFast34Fibroblast-like
RightTriphasicCTNNB1 ΔS45 (het), WT1 Q184X (het), LOH 11pws568reAFast30Fibroblast-like
ws568reBFast28Fibroblast-like
59162mRightStromalΔ WTXws591Fast17Fibroblast-like
592d2mRightCMNeLOH 11pws592Slow8Round (epith.)
60926fRightTriphasicLOH 11pws609-1Fast10Fibroblast-like
ws609-2AFast15Fibroblast-like
ws609-2BFast15Fibroblast-like
61311fRightTriphasicLOH 11pws613Fast7Round (epith.)
ws613BFast13Round (epith.)

Morphology of Primary WT Cells

Primary WT cells grew as adherent monolayers, but also as multilayers at high cell density. Cell size was in the range of 50–200 μm in length. There was a strong variation in morphology between different WT cultures, but individual cultures appeared rather homogeneous. At the beginning of cultivation, often a mixture of morphologically different cells grew out, but appearance became more uniform during cultivation (Figs. 1B–1G). Cytologically, two main groups could be distinguished: large round cells and more or less fibroblast-like cells. Clearly, the latter was more diverse: some cultures consisted of spindle-shaped cells with very few protrusions, while others comprised polygonal cells with numerous protrusions, but there were also intermediate phenotypes. Parallel cultures derived from a given tumor sample tended to be morphologically similar, but, in some instances, different cell types (round and fibroblast-like cells) could be obtained from the same tumor (e.g., ws535 or ws507).

Growth Characteristics and Senescence

There was large variation in growth rate and proliferative potential of primary cells (Fig. 2A). Fast growing cell cultures could be subcultivated 1:3 at least once a week. Some of the slow growing cells could be kept in culture for only few passages (e.g., ws483), while other long living cells could be passaged more than 30 times (e.g., ws539 and ws568).

Figure 2.

Growth characteristics of primary WT cultures. A: Growth rate and proliferative potential of primary cultures is highly variable. Confluent cultures were split 1:3. B: Senescence occurs upon long-term cultivation of primary WT cells. SA-β-Gal staining of ws568li cells: young cells at passage 5 were small and β-Gal negative (left panel), while most cells become β-Gal positive during cultivation (passage 30, middle). Senescent cultures showed a typical phenotype with multinucleated (arrow) and enlarged, flattened cells (right panel). Scale bar: 100 μm (left and middle); 10 μm (right panel). C: Telomere length of cultures at passage 3. Blood or normal kidney samples and HEK293T cells are shown as controls (light gray). The ratio Ct(tel)/Ct(alb) is given with long telomeres corresponding to smaller bars. D: Telomeres shorten upon long-term cultivation as shown by the increasing ratio Ct(tel)/Ct(alb) from early to late passages of primary WT cultures ws489li, ws539A, and ws568li. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

To analyze whether the observed growth arrest was associated with senescence, senescence-associated β-galactosidase (SA-β-Gal) staining was performed on young cell cultures at the beginning of cultivation and on older cell cultures after serial passages. As evident by the increasing fraction of β-Gal positive cells, primary WT-cultures become increasingly senescent upon cultivation. Senescence was also discernible by the typical phenotype: enlarged and flattened cells that sometimes were multinucleated. At the time of growth arrest, nearly every cell in a culture was senescent (example shown in Fig. 2B).

To investigate whether senescence was associated with telomere shortening in cultivated cells, telomerase expression, and telomere length were measured in early and late passage primary cells. Genomic DNA of patient blood samples and normal kidney tissue as well as of HEK293T cells with a known telomere length of about 6 kb (Canela et al., 2007) were used as control. None of the cell cultures analyzed showed expression of telomerase, neither in early nor late passages. Analysis of telomere length using quantitative PCR revealed a shortening of telomeres during long-term cultivation of primary cells (Figs. 2C and 2D). However, telomere length at the beginning of cultivation did not correlate with the life span of individual cell cultures.

Immunohistochemical Characterization

To investigate whether only a single histological component of a tumor sample gave rise to viable cell cultures and to determine from which component the cultured cells arise, we performed immunohistochemical analyses on tumor tissue and corresponding primary cultures from passages 3 to 5.

All cell cultures analyzed lacked significant WT1 and PAX5 expression, but they were uniformly positive for vimentin and exhibited a cytoplasmatic localisation of β-catenin (Supporting Information Table S4). CD56 (NCAM), which has been proposed to be specific for the blastemal compartment of WT (Roth et al., 1988; Muir et al., 2001), was not observed in any WT culture, while all primary cells expressed CITED1, another proposed blastemal marker (Lovvorn et al., 2007).

Cytokeratin staining (Cam5.2), which is specific for the epithelial component of WT tissue, distinguished two types of WT cultures (Fig. 3A): primary cultures ws483, ws507-2tr, ws520-2tr, ws535-2tr, ws592, and ws613 were completely cytokeratin positive, while Cam5.2 was absent in all other cultures. This pattern was consistent with cell morphology: round WT cells were Cam5.2 positive, while all cytokeratin-negative cells looked fibroblast-like. It is worth noting that even for cytokeratin-negative cultures, there were positive epithelial structures present in the original tumor, but these apparently did not contribute to the cultures or cells lost expression upon cultivation.

Figure 3.

Immunohistochemical staining of WT tissue and corresponding primary cultures. A: Cases ws568li and ws613 are shown as examples. Cam5.2 specifically stains epithelial cells of WT tissue. Ws568li gave rise to cytokeratin negative fibroblast-like cultures that expressed the mesenchymal stem-cell marker CD105. Cells from ws613 cultures with a large, round morphology expressed cytokeratin but not CD105. B: Classification of primary cultures by Cam5.2 and CD105 staining. Different cultures derived from one tumor sample are marked by asterisk. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

CD105 (endoglin) and CD90, which are often used as mesenchymal stem cell markers, showed almost the opposite pattern. The majority of our cultures consisted of CD105 positive cells, where 80–100% of cells were strongly stained (Fig. 3B). Exceptions were the previously described round Cam5.2 positive cells (ws507-2tr, ws535-2tr, ws592, ws613, and ws613B). Only two cultures were different: the spindle-shaped ws483 cells were positive for Cam5.2 and CD90/CD105, but they lacked the typical cytokeratin staining of the plasma membrane and exhibited a weak spotted intracellular staining instead. The fibroblast-like cells of ws520-1 were both Cam5.2+ and CD105+.

Ki67 staining corresponded well with proliferation rate, that is, faster growing cultures contained higher numbers of Ki67 positive cells. Endosialin stained almost all primary cells, but did not help to subclassify these cultures.

Gene Expression Data

To further characterize the cell cultures, we analyzed the expression of genes that is known to be differentially expressed in tumors classified by clinical parameters (DKK1, EGR1, HEY2, NMYC, TOP2A, and TRIM22; Wittmann et al., 2008). Furthermore, genes mutated in WTs (WT1, CTNNB1) and muscle markers typically expressed in the stromal component of WT (CASQ2, DPT, MB, MYL2, and PITX2) were analyzed by RT-PCR in early and late passages of primary WT-cell cultures.

Expression of CTNNB1, DKK1, EGR1, HEY2 (at low level), TOP2A, and TRIM22 could be demonstrated on the RNA level in all primary cultures tested. Even parallel cultures from the same tumor (e.g., ws535 and ws565) showed variation in relative expression levels at baseline (P3). Upon long-term cultivation, TRIM22 and DKK1 were upregulated (4–20-fold) in most of the cultures, while TOP2A was downregulated (2–250–fold; Fig. 4). In contrast, no expression of WT1 (as also seen in IHC) and NMYC could be detected in WT cultures. There was almost no expression of muscle marker genes in primary WT cells. Only for CD105 expressing cells that are presumably less differentiated and mesenchymal a weak signal could be detected in some cases.

Figure 4.

Expression level of DKK1, TRIM22, and TOP2A as measured by real time (RT)-PCR in early (P3) and late passages of primary WT-cell cultures. Relative expression level compared to mean is given.

The gene expression pattern of cultured WT cells was different from the one seen in the original WT tissue samples, which is likely due to artificial conditions of cell culture in general. Even RNA levels at passage 3 of primary cultures did not correspond well to values obtained in tumor samples.

Transfection of Primary WT Cells

Primary tumor-cell cultures should ideally be amenable to genetic manipulation to serve as an in vitro model system. To test this, we used expression vectors for green fluorescent protein to mark successfully altered cells. Gene transfer was possible using different reagents or protocols: transfection using polyethyleneimine (PEI) was only marginally efficient (<1% green cells); FugeneHD (nonliposomal) achieved a high transfection rate (50% of cells strongly positive), while Metafecten (Biontex) showed similarly high efficiency, but was lethal for all cultures tested. Electroporation using the Amaxa Nucleofector device (Basic Nucleofector Kit for primary epithelial cells with protocols X-005, L-017, S-005, and V-001) showed a very high transfection rate of up to 95 % (Supporting Information Fig. S1).

Transfection is often used for transient assays only and may result in multicopy integration. Viral transduction has the advantage of single- or low-copy integration that is more useful for regulated expression. WT cells could be transduced with lentiviral particles to generate (stable) cultures with sufficient expression of the transgene (Supporting Information Fig. S1). Although transient DNA transfection led to unequal expression levels of the transgene with a broad range of GFP fluorescence signals in individual cells, lentiviral transduction resulted in a more evenly distributed, but weaker GFP expression.

Telomerase Expression in Primary WT Cells

The finite lifespan of primary cells makes them a limited resource. Derivation of immortalized WT cell lines would certainly benefit molecular dissection of signaling pathways and gene pathology by providing a standardized source of cells. The gradual loss of telomeric repeats in our cultures suggested that telomere shortening may represent a critical step limiting proliferation. To test this hypothesis, we ectopically expressed the catalytic subunit of hTERT. Cell cultures obtained from two different tumors (ws539 and ws568li) were stably transfected with pTol2-CMV-hTERT-blast leading to CMV-promotor driven expression of hTERT (Fig. 5). All cells expressed telomerase at similarly high levels, which indeed stabilized and even elongated telomeres in most cases as shown by qPCR analysis of telomere length. Nevertheless, five of seven cultures reduced proliferation after serial passaging due to senescence (50–90%), regardless of the level of telomerase expression. However, two cultures (ws539A-hTERT#4 and ws568li-hTERT#3) presented with less than 10% of SA-β-Gal positive cells and continue to grow since more than 8 months. These cells are now in long-term culture since 52 and 32 passages (i.e., ∼80 and 50 population doublings), and they can thus be viewed as immortalized. The cells grow vigorously and do not show morphologic changes or striking differences in gene expression (e.g., for CDKN2A and BIRC). This suggests that telomere length is perhaps not the only critical factor, but telomerase expression may support long-term cultivation and immortalization. In preliminary experiments, we also obtained robustly proliferating cultures after transfection with vectors expressing inducible SV40 large T-antigen (not shown). This is clearly a more dramatic alteration, but it emphasizes that multiple long-term proliferating cultures may be obtainable via different approaches.

Figure 5.

Transgenic expression of telomerase in primary WT cells. Primary cultures ws539A (A–D) and ws568li (E, F) were stably transfected with pTol2-CMV-hTERT-blast expressing the catalytic subunit of hTERT. A, E: RT-PCR analysis showed that only transfected cells express hTERT. B, F: Several of the cultures stop or even reversed telomere shortening (ws539A-hTERT #4 and ws568li-hTERT #3). Controls are as in Figure 3. C, G: Growth rate of parental and hTERT expressing cells. D, H: Only ws539A-hTERT#4 and ws568li-hTERT#3 exhibit low levels of senescent cells (blue) at higher passages. Scale bar: 100 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

DISCUSSION

To study molecular principles of WT formation and progression and to test new therapeutic approaches, a suitable in vitro model is necessary that recapitulates typical nephroblastoma. As there are very few established WT cell lines available and these only represent rare subtypes of WT, there is a need for the generation of well-characterized WT-cell cultures that more closely resemble classical WT. In our study, we have established a set of 28 different primary tumor-cell cultures from 14 specimens obtained from 12 patients and we have characterized their biological properties.

Mutation and LOH analyses were used to classify cultures as being tumor derived. As expected from the limited number of genetic alterations typically found in WT, it was not possible to define tumor-specific alterations in almost half of the initial cases. Nevertheless, in more than 50% of the remaining cases, a clear classification of cells as being tumor-derived was possible, while the others may either represent clonal variants in case of heterogeneous tumors or contaminating normal cells. This represents the first extended set of WT-cell culture experiments where such rigorous typing has been used.

Our cultures are from tumors with a variety of alterations such as WT1, WTX, and CTNNB1 mutations or LOH at chromosome 11 as well as more unusual LOH for other chromosomes. Cultures with specific alterations always shared the entire set of LOH/mutation characteristics with the original tumor. There was no evidence that the type of genetic alterations influences the success rate of cultivation. Thus, these cultures reflect the molecular heterogeneity seen in WT. Of note, almost all cultures were derived from postchemotherapy samples. These apparently still contain sufficient numbers of viable cells that readily proliferate and give rise to expanding cultures. It will be interesting to see if such cultures are derived from cells that exhibit enhanced resistance to the agents used (vincristine and actinomycin D) and whether they may help to predict susceptibility in postoperative treatment.

Two different classes of cells could be distinguished by morphology and immunohistochemistry: epithelial cells with slow growth and restricted in vitro life span and more mesenchymal (stromal) cells that lack the epithelial marker cytokeratin, but express the mesenchymal stem-cell markers CD105 and CD90. Culture was possible in standard medium and did not require special media or mutant WT1 as suggested previously (Royer-Pokora et al., 2010). Cultivation of epithelial or stromal cells has been reported before, but blastemal cells have rarely been obtained and were still described as showing epithelial characteristics (Garvin et al., 1987). Similarly, none of our cultures exhibited blastemal morphology or expressed NCAM (CD56), proposed to be blastemal-specific, and most of the initial cultures obtained from blastemal tumors were of wild-type allele status. The lack of true blastemal cells in all cultures reported to date may be due to specific cultivation requirements that have yet to be identified. This could similarly be true for the almost complete absence of WT1 expression, a phenomenon that has been reported by others before (Hazen-Martin et al., 1994; Faussillon et al., 2008; Royer-Pokora et al., 2010). The consistent changes seen over time in the expression of DKK1, TRIM22, and TOP2A may also hint at ongoing adaptation to culture condition or dynamic changes towards differentiation and/or senescence.

Our epithelial cultures possessed only a short life span, while some of the fibroblast-like cultures grew for many passages. Nevertheless, all exhibited a restricted proliferation potential and finally became senescent. Telomerase expression was absent and telomeres became progressively shorter during cultivation. Forced expression of hTERT stabilized or even increased telomere length in most cultures, but this did not reproducibly lead to immortalization. Most cultures still became senescent, but we also obtained cultures that do not show increased senescence even at higher passages. It appears that these cells are truly immortalized suggesting that telomerase expression may support immortalization in such cells. Telomere length of early passages was not predictive of subsequent life span, suggesting that alternative functions of hTERT beyond telomere lengthening may be relevant (Stewart et al., 2002).

An important feature of the primary cultures established here is that they can be genetically altered efficiently by different means. This makes them ideal substrates to test functions of genes known to be deregulated or mutated in WT and to test candidates for therapeutic agents. Transfer of primary cultures into nude mice may help to unravel as to what extent these epithelial or stromal like cells may be able to transdifferentiate into other cell types or even recapitulate the mixed histological picture seen in classical triphasic WT.

Acknowledgements

We thank B. Klamt, S. Wittmann, and B. Zirn for help during the starting phase of this project and H. Augustin for providing the endosialin antibody. We gratefully acknowledge the efforts of all clinicians, nurses, and patients of the SIOP2001/GPOH study who made it possible to collect the tumor specimens.

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