Oligodendroglioma cell lines containing t (1; 19)(q10; p10)

Oligodendroglioma cell lines containingt(1;19)(q10;p10)

John J. P. Kelly†, Michael D. Blough†, Owen D. M. Stechishin, Jennifer A. W. Chan,Desiree Beauchamp, Marco Perizzolo, Doug J. Demetrick, Lisa Steele, Roland N. Auer,Walter J. Hader, Morgan Westgate, Ian F. Parney, Robert Jenkins,J. Gregory Cairncross†, and Samuel Weiss†

Hotchkiss Brain Institute, Clark Smith Brain Tumor Centre, Department of Cell Biology and Anatomy (J.J.P.K,

O.D.M.S., D.B., S.W.), Department of Clinical Neurosciences (M.D.B., W.J.H., M.W., J.G.C.), Department of

Pathology and Laboratory Medicine, University of Calgary (J.A.W.C.), Department of Pathology and

Laboratory Medicine (M.P., D.J.D., R.N.A), University of Calgary, Calgary, Alberta, Canada; Department of

Neurosurgery (I.F.P.), Department of Pathology and Laboratory Medicine (R.J.), Mayo Clinic, Rochester,

Minnesota

Investigating the biology of oligodendroglioma andits characteristic combined deletion of chromosomalarms 1p and 19q, mediated by an unbalanced transloca-tion, t(1;19)(q10;p10), has been hampered by the lackof cell lines that harbor these traits. We grew cellsfrom 2 anaplastic oligodendrogliomas in serum-freeconditions. Serial propagation and expansion ledto the establishment of permanent cell lines thatmaintained the genetic signature of the parent oligoden-drogliomas and displayed features of brain tumorstem cells in vitro. One line was established from atreatment-naı̈ve tumor and the other from a temozolo-mide resistant recurrent tumor. These lines may beimportant tools for understanding the biology ofoligodendrogliomas and the function of their defininggenetic traits.

Keywords: 19q, 1p, brain tumor stem cell, IDH1,oligodendroglioma, translocation.

Oligodendrogliomas, a subtype of diffusely infil-trating glioma, are slowly growing primarybrain cancers that typically occur in young or

middle-aged adults. Histologically, they containsmall, round cells with uniform nuclei, which

resemble mature oligodendrocytes.1 They were thefirst gliomas for which specific molecular predictorsof chemotherapeutic response and overall survivalwere identified.2 Combined allelic loss of chromoso-mal arms 1p and 19q (1p/19q) constitutes the earliestknown molecular alteration in 50%–70% of oligo-dendrogliomas and results from a recurring unba-lanced translocation, t(1;19)(q10;p10).3,4 In both aretrospective and prospective series of anaplastic oli-godendrogliomas (WHO grade III), radiographic andclinical response to chemotherapy, long progression-free survival after chemotherapy or radiotherapy,and long overall survival have been associated withthe loss of 1p/19q alleles.5 Critically important ques-tions about oligodendrogliomas and their defininggenetic abnormality remain unanswered. Does codele-tion of chromosomes 1p and 19q or t(1;19)(q10;p10)contribute to the genesis of oligodendrogliomas, if so,how? Does the codeletion or translocation explain theunusual chemosensitivity and radiosensitivity of oli-godendrogliomas, or are these anomalies simply bio-markers of a variant of human glioma that issensitive to DNA damaging therapies for other unre-lated reasons? Addressing these questions has beendifficult in part because there have been nooligodendroglioma cell lines that possess itssignature genetic alteration in which to explore thebiology of this disease. Here, we describe 2 oligoden-droglioma cell lines generated from 1p/19qco-deleted anaplastic oligodendrogliomas usingserum-free conditions that contain a codeletion ofchromosomes 1p and 19q and the unbalanced trans-location, t(1;19)(q10;p10).

†These authors contributed equally to this work.

Corresponding Author: Samuel Weiss, PhD, Faculty of Medicine,

Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta,

Canada, T2N 4N1 ([email protected]).

Received November 8, 2009; accepted January 29, 2010.

Neuro-Oncology 12(7):745–755, 2010.doi:10.1093/neuonc/noq031 NEURO-ONCOLOGYAdvance Access publication April 13, 2010

# The Author(s) 2010. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rightsreserved. For permissions, please e-mail: [email protected].

Materials and Methods

Oligodendroglioma Cell Culture

Fresh tissue samples were obtained from 2 adult patientsduring resection of primary intra-axial brain neoplasms.Cell culture was performed using the neurosphereassay.6 Briefly, tumor tissue was washed in sterile 1 ×phosphate buffered saline (PBS) containing penicillinand streptomycin. Each specimen was finely mincedthen placed in a serum-free culture medium (SFM) con-taining DMEM/F12 (1:1) with 5 mM HEPES buffer,0.6% glucose, 3 mM sodium bicarbonate, 2 mM gluta-mine, 25 mg/mL insulin, 100 mg/mL transferrin, 20 nMprogesterone, 10 mM putrescine, and 30 nM selenite,supplemented with epidermal growth factor (EGF,20 ng/mL, Peprotech), fibroblast growth factor 2(FGF2, 20 ng/mL, R&D Systems), and heparin sulfate(HS, 2 mg/mL; R&D Systems) (SFM-EF). Once inSFM-EF, specimens were manually dissociated. A single-cell suspension was obtained by performing a series ofmechanical dissociations followed by filtration (40 mm).Red blood cells were removed by hypotonic lysis.Finally, tumor cells were resuspended in SFM, countedusing Trypan blue to exclude dead cells, and plated at adensity of 20 000 viable cells/mL in SFM or SFM sup-plemented with EGF (20 ng/mL, Peprotech), FGF2(20 ng/mL, R&D Systems), and HS (2 mg/mL; R&DSystems) (SFM-EF). Cultures were fed weekly by remov-ing half the media and replacing with an equal volumeof fresh media. Neurospheres were evident approximately2 weeks following plating and were grown for anadditional 2 weeks until they reached a size adequatefor plating, differentiation, and serial passage.

Oligodendroglioma Sphere Self-Renewaland Differentiation

Self-renewal capacity was examined by dissociatingprimary spheres into single cells using Accumax(Innovative Cell Technologies). Single cells were replatedat a density ≤20 000 cells/mL in either 24-well plates(uncoated, Nunc) or 25-cm2 flasks (uncoated, Nunc) inSFM or SFM-EF. Cultures were fed and observed weeklyfor secondary sphere formation. Primary and secondarysphereswereplatedontopoly-L-ornithine-coatedglass cov-erslips and allowed to differentiate for 7 days in SFM in thepresence of 1% fetal bovine serum (FBS; Invitrogen).After differentiation, coverslips were fixed for 20 minutesin 4% paraformaldehyde, washed 3 times with 1× PBS,and prepared for immunocytochemical analysis.

Immunocytochemical Staining of OligodendrogliomaSpheres and Cells

Triple immunostaining of differentiated oligodendro-glioma spheres was performed for glial fibrillary acidicprotein (GFAP) (rabbit anti-GFAP, 1:400, BTI),b-III-tubulin (mouse-anti-b-III-tubulin, 1:1000, Sigma),and O4 (mouse IgM anti-O4, 1:10, Chemicon).

Immunolabeling of oligodendroglioma cells for nestinwas performed using mouse IgG anti-nestin (1:100,Chemicon). Nuclei were labeled with Hoechst 33 258in all cases. The number of nestin-expressing cells wasquantified by counting the total number of Hoechst+nuclei per high-powered field (HPF; 40×) and thenumber that co-labeled with nestin in 10 HPFs.

Flow Cytometry for CD133

Primary and passaged oligodendroglioma cells werewashed and resuspended in PBS + 0.5% BSA (PBS–BSA) and then incubated with either a monoclonalCD133/1-phycoerytherin-conjugated antibody or aphycoerytherin-conjugated mouse IgG1 isotype controlantibody (both, 1:10, Miltenyi Biotec, according to themanufacturer’s instructions) for 60 min at 48C. Cellswere analyzed using a BD FACScalibur flow cytometerand analyzed using ModFit LTTM software (BectonDickinson) and FlowJo software (Tree Star, Inc.).

Molecular Diagnostic Methods

Tumor cells were evaluated for deletions of 1p/19q byfluorescent in situ hybridization (FISH) as previouslydescribed.7 For loss of heterozygosity (LOH) analysisby PCR, 6 independent loci on both 1p and 19q wereevaluated in each DNA sample. Tumor DNA wasextracted from paraffin blocks and sections using theQiagen QIAmp DNA Micro kit and from cell linesusing the DNAeasy kit (Qiagen). Normal DNA wasextracted from blood using the Qiagen QIAmp DNAMini kit. DNA (10 ng) was amplified for 35 cycles(948C, 30 seconds/608C, 1 minute 30 seconds/728C,30 seconds) using the Qiagen Multiplex Master Mixwith 4 multiplex mixes as follows: 1pA[D1S226;D1S209;D1S468]; 1pB[D1S2734;D1S438;D1S457]; 19qA[D19S926;D19S208;D19S223]; and 19qD[D19S426;D19S112;D19S596]. Each of these 4 multiplexes con-tained 3 sets of primers targeted at 3 microsatellitemarkers. For each microsatellite marker, the CA strandprimer was labeled with Licor IRD dye and used at afinal concentration of 0.2 mM; the GT strand primerswere unlabeled and used at a final concentration of0.4 mM. Following PCR, the samples were diluted1:140 with Li-Cor loading dye and the dilution (1 mL)was loaded on an Li-Cor IR2 DNA sequencer.Electrophoresis was performed on the captured gelimage using GeneImagR software (Scanalytics). LOHscores were calculated based on the intensities ofthe peak height of alleles between normal and tumorsamples.

Relative gains and losses of chromosomal regions wereevaluated by array comparative genomic hybridization(aCGH). For aCGH, DNA was isolated from patienttumor tissue and the oligodendroglioma cell lines(DNAeasy kit, Qiagen); all were analyzed at a core facilityat the University of California at San Francisco (UCSF).Analysis was performed on a tiling Path Array(Hum32k) containing �32 000 human BAC clones

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spotted in singlets with continuous overlapping coverageof the genome (for details of the protocol as well asfurther information see CSF Helen Diller FamilyComprehensive Cancer Center website at http://cancer.ucsf.edu/cores/index.php). Cells from the lines were har-vested to prepare metaphase slides for G-banding andspectral karyotyping (SKY). Briefly, a probe cocktail con-taining 24 differentially labeled chromosome-specificpainting probes and Cot-1 blocking DNA (SKY kit,Applied Spectral Imaging) was denatured and hybridizedto the metaphase chromosome spreads as recommendedby the manufacturer. After hybridization and washing,chromosome spreads were counterstained with4′,6-diamidino-2-phenylindole dihydrochloride. Imageacquisition, processing, and analysis were performedwith SKY Vision software version 1.5, using a SD200Spectracube system (Applied Spectral Imaging) mountedon a Zeiss Axiskop microscope with custom-designedoptical filters (SKY-1, Chroma Technology) allowingfor simultaneous excitation of all dyes and measurementof emission spectra. For each preparation, 5–10 meta-phases were analyzed by SKY. Chromosomal designa-tions followed the International System for HumanCytogenetic Nomenclature (2005).

For isocitrate dehydrogenase 1 (IDH1) and isocitratedehydrogenase 2 (IDH2) sequencing, RNA was extractedfrom oligodendroglioma using RNeasy (Qiagen) accord-ing to the manufacturer’s instructions. Five hundred nano-grams of RNA was then reverse transcribed with theSuperscript III First-Strand Synthesis System (Invitrogen)using poly-T primers. Two microliters of cDNA werethen used in a 50 mL RT–PCR (Invitrogen) to amplifyexon 4 of both the IDH1 and IDH2 genes as previouslydescribed.8,9 RT–PCR products were then purified byagarose gel electrophoresis and isolated with theQIAquick Gel Extraction Kit (Qiagen). AutomatedDNA sequencing was performed at the University ofCalgary Core DNA Services facility.

Chemosensitivity Testing In Vitro

The relative sensitivities of oligodendroglioma vs glio-blastoma (GBM) lines were assessed after exposure totemozolomide (TMZ) alone (1–100 mg/mL; ScheringPlough). Cell viability was monitored over 12 daysusing the alamarBluew viability assay, as instructed bythe manufacturer (Medicorp). Each treatment groupwas repeated in duplicate and each experiment in tripli-cate. To facilitate the interpretation of these results, themethylation status of the O-6-methylguanine-DNA-methyltransferase (MGMT) gene promoter was assessedby MS-PCR.

Oligodendroglioma Cell Implantationinto Immunocompromised Mice

Oligodendroglioma spheres were mechanically disso-ciated to single cells, washed twice in SFM, and viablecells were counted using Trypan blue exclusion. Viable

cells (2 × 105) were resuspended in 3 mL of SFM forstereotactic implantation into the right striatum of 6–8-week-old CB-17 NOD-SCID mice (JaxLabs).Coordinates for implantation were as follows: AP-1.0,ML 2.0, and DV 3.0. Mice were allowed to survive upto 16 weeks and euthanized. Brains were removed andexamined for morphological evidence of tumor for-mation. Each brain was cut in the coronal plane inorder to obtain 2 segments, each containing part ofthe tumor. Both halves were fixed in formalin priorto immunohistochemistry and immunoflourescentimmunohistochemistry.

Immunohistochemistry

For histological examination of tumors that arose inSCID mice following cell implantation, hematoxylinand eosin (H&E) staining was performed according tostandard protocols. Immunohistochemistry was per-formed on paraffin-embedded sections using an indirectimmunoperoxidase method. Immunohistochemistryperformed on formalin fixed paraffin-embedded sectionsused the following primary antibodies: mouseanti-GFAP (1:400, R&D) and mouse-anti Olig2(1:1500, BD Pharmingen). Sections were incubatedwith each specific primary antibody listed above over-night at 48C followed by a biotinylated horse anti-mouse/goat anti-rabbit antibody (Vector). Avidin–biotin peroxidase complexes were formed using an“ABC” kit (Vector). Peroxidase converted DAB to abrown reaction and hematoxylin was used as a bluenuclear counterstain.

Results

Diagnosis of Anaplastic Oligodendrogliomaswith Codeletion of 1p/19q

A previously healthy 49-year-old woman developedfocal seizures. Imaging studies revealed a rightfrontal, intra-axial tumor that enhanced followinggadolinium administration (Fig. 1A). The mass wasresected and tissue sent for both standard neuropathol-ogy evaluation and tissue culture after obtaininginformed consent. The tumor contained 2 distinct his-tological areas: the first contained small, round cellswith perinuclear halos and branching vasculature, fea-tures typical of oligodendroglioma (Fig. 1B), whereasthe second area had higher cellularity with abundantmini-gemistocytes (Fig. 1C). Both contained cells thatwere GFAP positive (Fig. 1B). Nuclear pleomorphismand focally brisk Ki67 staining (Fig. 1C) led to a diag-nosis of anaplastic oligodendroglioma (WHO gradeIII).10 The tumor harbored the codeletion of chromo-somes 1p and 19q as assessed by FISH (Fig. 1D).The patient was treated successfully with TMZ che-motherapy and radiotherapy.

A previously healthy man developed seizures at age33. Imaging revealed a right frontal, enhancing,intra-axial tumor. A WHO grade III oligodendroglioma

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was removed and treated with procarbazine, lomustine,and vincristine chemotherapy. Over the ensuing 17years, multiple recurrences were treated with radiother-apy and several courses of TMZ with benefit. The tumorharbored the codeletion of chromosomes 1p and 19q(data not shown). Ultimately, resistance to TMZ andother chemotherapies led to a final surgical resectionfor symptom control. Tumor tissue was sent for neuro-pathology review and culture after informed consentwas obtained.

Oligodendroglioma Cells Express Neural Stem CellMarkers and Grow As Multipotent OligodendrogliomaSpheres In Vitro

Brain tumor stem cells (BTSCs) have been isolated andcontinuously cultured from many different types ofprimary brain tumor using the neurospheresystem,11,12 but not from oligodendrogliomas. Usingtissue obtained at the time of surgical resection, single-cell suspensions were prepared for phenotypic evalu-ation and culture. Since BTSCs have been shown toexpress the neural stem cell (NSC) markers CD13312,13

and nestin,12,14 the oligodendroglioma cell suspensionswere immediately assessed for CD133 expression byflow cytometry: the first tumor contained �14%CD133+ cells (Fig. 2A) and the second �0.34%CD133+ cells. In addition, 68+2% of cells in the firsttumor were nestin positive (Fig. 2B). The remainingprimary oligodendroglioma cell suspensions were cul-tured in SFM alone15 and in SFM-EF as described indetail elsewhere.6 After 3 weeks, SFM-EF culturesfrom the first tumor contained phase bright, floatingspheres (Fig. 2C), a pattern typically observed forNSCs6 and BTSCs.11,12 No growth was obtained in theabsence of mitogens. In the second case, after 3 weeks,both SFM and SFM-EF cultures contained phasebright, floating spheres that were identical in appearanceto those of the first tumor (Fig. 4A).

A fundamental characteristic of NSCs is multipo-tency.16 Primary oligodendroglioma spheres from indi-vidual tumor cells from both tumors were multipotent.Those from the first tumor differentiated into cells thatexpressed astrocyte, oligodendrocyte, and neuronalmarkers (Fig. 2C), whereas those from the second differ-entiated into cells that expressed oligodendrocyte andneuronal markers only (data not shown). Another fun-damental characteristic shared by NSCs and BTSCs isthe ability to self-renew.12 To test this property,primary oligodendroglioma spheres from both tumorswere dissociated to single cells and replated in SFMwith EGF + FGF2; secondary oligodendrogliomaspheres arose from individual cells within 3 weeks andretained the capacity for multilineage differentiation(Fig. 2D). Subsequent generations of spheres have beensuccessfully maintained in culture for 26 and 15months, respectively. These data demonstrate thatboth of these oligodendrogliomas contained cellsthat possess properties of both NSCs and BTSCsin vitro, and propagation of these cells has led tothe development of stable oligodendroglioma celllines. These cell lines are now referred to as BT054and BT088.

BT054 and BT088 Harbor Codeletion of Chromosomes1p and 19q and t(1;19)(q10;p10)

To determine whether cells from BT054 and BT088maintained the characteristic genetic alteration of oligo-dendrogliomas, codeletion of 1p/19q, we performedPCR-based LOH analysis. As shown for the firstpatient (Fig. 3A), comparison of normal, tumor, and

Fig. 1. Diagnostic characteristics of an anaplastic oligodendroglioma.

(A) AT1-weighted axial MR image demonstrates a large mass lesion in

the right frontal lobe (white arrow) that enhances heterogeneously

following administration of gadolinium (yellow arrow), leading to

the presumptive diagnosis of malignant glioma. (B) Histopathologic

evaluation of resected tissue from the lesion in (A) demonstrates

characteristic features of oligodendroglioma including the presence

of small, round cells with uniform nuclei, perinuclear halos and fine

branching vasculature. This area of tumor also contains GFAP-

positive neoplastic astrocytes and proliferating cells that express

Ki67. (C) A second histologic pattern containing GFAP-positive

mini-gemistocytes was present within the neoplasm. Ki67

immunostaining revealed a brisk mitotic rate in this area, confirming

the diagnosis of anaplastic oligodendroglioma (WHO grade III). (D)

FISH analysis of the primary tumor demonstrates relative loss of

chromosomal arms 1p (green FISH probes in the 1p image) and 19q

(red probes in the 19q image).

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BT054 DNA revealed an identical pattern of LOHbetween the tumor and the BT054 cells that was notpresent in somatic DNA (Fig. 3A). An identical LOHpattern was seen with normal, tumor, and BT088DNA (data not shown). Characteristically, codeletionof 1p/19q in oligodendrogliomas is associated withcomplete loss of these chromosomal arms. In BT054,aCGH revealed complete loss of 1p and 19q (Fig. 3B).It also revealed the amplification of chromosome 11qand the deletion of chromosomes 14q and 15q; thesechanges have been seen previously in oligodendrogliomatumor tissues.17

Codeletion of 1p/19q in oligodendrogliomas appearsto result from an unbalanced translocation,

t(1;19)(q10;p10).3,4 To determine whether codeletionof 1p/19q in BT054 and BT088 cells was the result ofa translocation, G-banding karyotypic analysis followedby SKY was performed. In BT054, every metaphase bystandard karyotyping and SKY revealed at least onet(1;19)(q10;p10) chromosome with the correspondingloss of 1p and 19q (Fig. 3C). The formal karyotypeby G-banding and SKY was: 76-108,XXX,-X,+der(1;19)(q10;p10)x2-3, del(2)(p11.2p15),+3,+3,+5,+5,26,+7,+8,29,+10,+12,215,16,+20,+20,+21,+21,+22,+22,+der(?7or ?15)t(?7or?15;?1;?11)x4-7,+3-5mar[cp15]/126-154,idemx2[cp5]. Cells were highly aneu-ploid with near-tetraploid (�75%) and near-sexaploid(�25%) variants. Every metaphase contained multiple

Fig. 2. Anaplastic oligodendroglioma cells express neural stem cell markers, proliferate as multipotent spheres and self-renew. (A) A

flow-cytometric dot plot demonstrates that a subpopulation of primary oligodendroglioma cells express the NSC and BTSC marker

CD133. (B) A subpopulation of primary oligodendroglioma cells cultured for 7 days stain positive for nestin (nuclei were counterstained

with Hoechst 33 258). (C and D) Phase-contrast photomicrographs demonstrate the typical, floating oligodendroglioma spheres, grown

using the neurosphere culture system in SFM with EGF + FGF2. Primary (C) or secondary (D) oligodendroglioma spheres are multipotent

and differentiate into cells that stain positive for astrocytic (GFAP), oligodendroglial (O4), and neuronal (b-III-tubulin) markers (nuclei

counterstained with Hoechst 33 258). Scale bars B: 50 mm, C and D: phase contrast, 100 mm; immunofluorescence, 50 mm.

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other clonal numeric and structural abnormalitiesincluding deletion of 2p, relative gains of 3, 5, 20, and22, and relative losses of 6, 9, and 16. In addition,BT054 cells contained several marker chromosomesthat could not be unequivocally identified by SKY,including a translocation that may involve chromosomes7 or 15 and chromosomes 1 or 11. FISH analysisusing 4 colored probes confirmed the presence oft(1;19)(q10;p10) within multiple cells (Fig. 3D). A verychaotic pattern of chromosomal rearrangementsaccompanied by relative codeletion of 1p and 19q losswas also seen in BT088, whose formal karyotype byG-banding and SKY is shown in Fig. 4B. Every meta-phase had at least one copy of t(1;19)(q10;p10), corre-sponding loss of 1p and 19q, and multiple other clonalnumeric and structural abnormalities including relativegains and losses of other chromosomes and markerchromosomes that could not be fully identified bySKY. These data establish unequivocally that cell linesBT054 and BT088 contain t(1;19)(q10;p10), thussetting the stage for functional and in vivo studies.

BT054 and BT088 Cells Proliferate Slowly but DisplayDivergent Chemosensitivity In Vitro

Oligodendrogliomas that harbor 1p/19q codeletionhave 2 defining features in vivo: slow growth and chemo-sensitivity. Comparatively, GBMs, the most aggressivetype of glioma, typically do not harbor 1p/19q codele-tion, grow more rapidly, and are less sensitive to com-parable therapies. To assess whether BT054 andBT088 cells could be tools to investigate oligodendro-glioma biology, we determined whether BT054 andBT088 possessed these defining features of oligodendro-gliomas. We compared the growth rate of BT054 andBT088 cells in vitro to that of 2 GBM BTSC lines,BT012 and BT048, which were established in our lab-oratory using identical methods. We seeded 1 × 106

cells from each line in 25 cm2 flasks containingSFM-EF and total cell number was counted after 14days. Additional counts were performed based on theindividual growth characteristics for each line at 7, 18,21, and 28 days to generate growth curves. On day 14,BT054 yielded 0.29+0.02 × 106 cells per 25 cm2

Fig. 3. BT054, established from an anaplastic oligodendroglioma, demonstrates the codeletion of chromosomes 1p and 19q and

t(1;19)(q10;p10). (A) Loss of heterozygosity analysis comparing DNA from the patient’s blood, primary tumor, and the

oligodendroglioma cell line demonstrates identical allelic loss for both the primary tumor and the oligodendroglioma cell line at

representative loci on chromosome arms 1p and 19q. Somatic DNA isolated from blood demonstrates the normal allelic complement at

representative loci on chromosomal arms 1p and 19q. (B) Array comparative genomic hybridization demonstrates that both the primary

tumor and the cell line derived from it harbor relative deletions of chromosomes 1 and 19 that encompass the entire arms 1p and 19q.

(C) Spectral karyotypic (SKY) analysis of a cell from the oligodendroglioma cell line demonstrates one copy of the translocation,

t(1;19)(q10;p10) and loss of 1p and 19q alleles. In the SKY image, t(1;19)(q10;p10) is present adjacent to a normal chromosome 1

(yellow) and contains a long arm of chromosome 1q (yellow) together with a short chromosomal arm 19p (green). Multiple other

chromosomal abnormalities are also evident in the karyotype of this cell. (D) Evaluation of the oligodendroglioma cell line using

metaphase FISH. Four colored probes identify chromosome 1p (aqua), centromeric region of chromosome 1 (pink), chromosome 19q

(gold), and centromeric region of chromosome 19 (green). In a single cell from BT054, this probe combination demonstrates 2 normal

copies of chromosome 1, 2 copies of chromosome 19, and 2 copies of t(1;19)(q10;p10).

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flask (n ¼ 3) and BT088 cultures yielded 0.35+0.03 ×106 cells per 25 cm2 flask (n ¼ 3). By comparison, theGBM cultures BT048 and BT012 yielded 1.67+0.08 × 106 cells (n ¼ 3) and 2.63+0.22 × 106 cells(n ¼ 3), respectively (Fig. 5A), and needed passage onday 14. BT054 and BT088 cells proliferated at aslower rate and needed passage at day 18 and 28,respectively (Fig. 5A).

Next, we examined the sensitivity of BT054 andBT088 to the DNA methylating agent, TMZ. Using thealamarBlue viability assay, the responses of BT054 andBT088 were compared with the 2 GBM BTSC lines,BT012 and BT048. BT054 was significantly moresensitive to TMZ than either BT088 or BT012, whichwere comparably resistant. Dose–response curvesshowed a 50% reduction in viability at 10 mg/mL forBT054 vs 50 mg/mL for BT088 and BT012 (Fig. 5B).Of note, the BT054 and the GBM line BT048 wereequally sensitive to TMZ (Fig. 5B). Because methylationof the MGMT gene promoter silences MGMTexpression, rendering GBM tumors more sensitive toTMZ18 and may also predict sensitivity in 1p/19q code-leted oligodendrogliomas,19 we examined MGMTmethylation status in these lines. MGMT was methylatedin BT054, BT088, and BT048 and unmethylated inBT012 (Fig. 5C). In 3 of the 4 lines, response and methyl-ation status were congruent: BT054 and BT048 weremethylated and sensitive to TMZ, and BT012 was

unmethylated and resistant. In the case of BT088,however, methylation status did not predict response:BT088 was methylated yet resistant to TMZ. Of note,BT088 was derived from a chemotherapy-resistant oligo-dendroglioma and presumably had accumulated othergenetic alterations that might explain TMZ resistance.Indeed, review of BT088-metaphase spreads revealed fre-quent loss of chromosome 2, the site of MSH6.

BT054 Cells Harbor an IDH1 Mutation

Mutations in either the IDH1 gene or less commonly theIDH2 gene have been demonstrated to occur in themajority of anaplastic oligodendrogliomas.8 To date, nooligodendroglioma or glioma cell lines exist that containan IDH1 mutation and investigation of this mutationrequires its introduction into glioma cells that endogen-ously express wild-type IDH. To provide further evidencefor the clinical relevance of these oligodendroglioma celllines, we set out to determine whether they harboreither IDH1 or IDH2 gene mutations. Evaluation ofthe IDH1 and IDH2 mutational status by DNA sequen-cing revealed the most frequently observed mutation ofIDH1, which occurs in exon 4 at codon R132, in theBT054 cell line (Fig. 5D). The cell line BT088 did notcontain mutations of either IDH1 (Fig. 5D) or IDH2(data not shown). The BT054 cell line will be a valuable

Fig. 3. Continued.

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tool for investigating the role of IDH1 mutation in gliomaand may aid the development of novel therapeutics thattarget the mutant form of IDH1.

BT088 Cells Proliferate In Vivo and Initiate Tumorsin Immunocompromised Mice

Our in vitro cell culture findings suggested thatsphere forming cells isolated from 2 anaplastic oligoden-drogliomas were BTSCs. However, tumor formationin vivo is the critical test of BTSC identity;20 hence, weassessed whether cells isolated from oligodendrogliomaspheres were tumorigenic in vivo. Oligodendrogliomacells were implanted into the brains of NOD-SCID mice(2 × 105 cells per animal, n ¼ 5 animals for each ofBT054 and BT088). Illness with weight loss was evidentwithin 5 weeks postimplantation in the BT088-inoculatedgroup and all animals developed brain tumors within 10weeks (Fig. 6A). No animal implanted with BT054became ill. Following euthanasia, brains were removedand examined; those implanted with BT054 lookednormal, whereas those implanted with BT088 showedgross morphological changes. H&E staining demon-strated hypercellular tumors in all animals implantedwith BT088 (Fig. 6B). No abnormalities could be ident-ified in BT054-injected brains. BT088 gave rise to infil-trating tumors that closely resembled anaplasticoligodendrogliomas. Tumor cells infiltrated the brainparenchyma extensively (Fig. 6B and C), and the Ki67proliferative index was high (Fig. 6D). Further, thesetumors were composed of cells that resembled mature oli-godendrocytes and contained small, round nuclei withperinuclear halos (Fig. 6C). A branching or “chicken-wire” vascular pattern was also evident in these tumors.Immunohistochemical analysis revealed that all tumorscontained cells that expressed Olig2 (Fig. 6E), but notGFAP (Fig. 6F). Thus, BT088 cultures contain BTSCsthat give rise to oligodendrogliomas in vivo.

Discussion

This paper describes the first human oligodendrogliomacell lines, BT054 and BT088, that contain completeloss of chromosomal arms 1p and 19q, mediated by acentromeric translocation t(1;19)(q10;p10), whichtogether constitute the signature genetic change in oligo-dendrogliomas.1,3 Understanding how this characteristicgenetic abnormality of oligodendrogliomas relates totheir fundamental biology has been impeded by thelack of a cell system that closely mimics the disease. Inaddition, BT054 is the first cell line to contain therecently described IDH1 mutation that frequentlyoccurs in oligodendrogliomas and a subgroup of glio-blastomas. Although oligodendroglioma cell lines

Fig. 4. BT088, established from a recurrent anaplastic

oligodendroglioma, demonstrates the codeletion of chromosomes

1p and 19q and t(1;19)(q10;p10). (A) Phase contrast

photomicrographs demonstrate floating oligodendroglioma

spheres that arose after culturing the primary cell suspension of

BT088 using the neurosphere culture system in SFM with EGF +FGF2. (B) Karyotypic analysis of one representative cell from the

oligodendroglioma cell line demonstrates one copy of the

translocation, t(1;19)(q10;p10), codeletion of 1p and 19q, and

many other numeric and structural abnormalities including other

chromosomal translocations. (C) Spectral karyotypic (SKY)

analysis of a cell from the oligodendroglioma cell line BT088

demonstrates one copy of the translocation, t(1;19)(q10;p10). In

this cell, t(1;19)(q10;p10) is present adjacent to a normal

chromosome 1 (yellow) and combines a long arm of

chromosome 1q (yellow) with a short arm of chromosome 19

(green). Multiple other abnormalities are also evident in this cell.

Kelly et al.: Oligodendroglioma cell lines with t(1;19)(q10;p10)

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have been alluded to by others,21,22 both theirt(1;19)(q10;p10) status and IDH1 status is eitherunknown or not reported. Furthermore, these previouslyreported oligodendroglioma lines were generated bysupplementing tissue culture medium with high concen-trations of fetal calf serum, a method that has beendemonstrated to fundamentally alter the properties of

BTSCs.23 By utilizing the neurosphere culture systemwith defined, serum-free growth conditions, 2 cell lineshave been generated that appear to be true phenocopiesof the parental anaplastic oligodendrogliomas, which inturn, had the classic features of the disease.

Recent studies have pointed to NSCs and lineage-restricted progenitors as the probable cells of origin

Fig. 5. Growth and chemotherapeutic response characteristics of BT054 and BT088. (A) BT054 and BT088 proliferate and expand at a much

slower rate than the GBM lines, BT012 and BT048. [n ¼ 3 experiments per culture; data expressed as mean+SEM] (B) BT054 viability is

reduced by treatment with a clinically relevant dose of TMZ (5 mg/mL) over 12 days. BT054 is more sensitive to TMZ than BT012 but

displays a response profile that is indistinguishable from BT048. BT088 and BT012 are equally resistant to TMZ. Dose–response curves

after exposure to TMZ (dose range: 1–100 mg/mL) are illustrated for all lines. BT054 and BT048 displayed similar reductions in viability

(ie, TMZ sensitivity) over the entire dose range, whereas BT088 and BT012 showed reduced viability only at very high doses of TMZ.

[n ¼ 3 experiments per time point per cell line; data presented as mean+SEM] (C) BT054, BT088, and BT048 have a methylated

MGMT promoter, whereas BT012 is unmethylated. (D) Nucleotide base positions numbered 625–635 from the start of the IDH1 gene,

that include codon 132 of exon 4, are displayed demonstrating that BT054 harbors a somatic IDH1 mutation at codon 132 resulting in

the alteration R132H. IDH1 in BT088 is wild-type.

Kelly et al.: Oligodendroglioma cell lines with t(1;19)(q10;p10)

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for several types of primary brain tumors, especiallyGBM.24 For decades, oligodendrogliomas, a relatedcancer of the brain, have been postulated to arise frommature oligodendrocytes, although in point of fact,their actual cell of origin has never been rigorouslyidentified.1 By employing the neurosphere culturesystem, we demonstrate that oligodendrogliomas harbora subset of cells that possess the properties of BTSCs invitro and in vivo.12 The novel finding that theseoligodendroglioma-derived cells (i) proliferate underserum-free culture conditions in the presence of EGF +FGF2, (ii) self-renew, (iii) give rise to multipotent oligo-dendroglioma spheres, and (iv) give rise to intracerebral

tumors that are indistinguishable from oligodendroglio-mas (BT088 only), raises the distinct possibility thatNSCs or lineage-restricted progenitor cells are candidatecells of origin of oligodendrogliomas.

It is possible that the codeletion of chromosomes 1pand 19q or t(1;19)(q10;p10) directly mediate slowgrowth and chemosensitivity of oligodendrogliomas;however, a mechanistic relationship has not been estab-lished. Functional assays performed in this study yielded2 interesting results. First, the MGMT-methylated GBMline BT048 and the methylated oligodendroglioma line,BT054, were comparably sensitive to TMZ. This findingraises the possibility that MGMT promoter methylation,

Fig. 6. BT088 oligodendroglioma cells initiate oligodendrogliomas in immunocompromised mice. (A) A Kaplan–Meier survival curve

demonstrates that animals implanted with BT088 cells have significantly shorter survival than those implanted with an identical number of

BT054 cells. Animals implanted with BT054 cells did not develop tumors. (B) H&E staining demonstrates the presence of a highly cellular,

infiltrating mass lesion within the brain resulting from the implantation of BT088 cells. (C) H&E staining at higher magnification demonstrates

that BT088 tumors contain small, round cells with uniform nuclei and thus have the characteristic appearance of oligodendroglioma. (D)

Brain tumors that formed in NOD-SCID mice following implantation of BT088 cells were highly proliferative lesions demonstrated by the

high number of mitotically active cells that expressed Ki67 and as such resemble anaplastic oligodendroglioma (WHO grade III). (E and F)

Expression of markers indicating oligodendroglial (Olig2) (E) differentiation was observed in tumors that grew in the brains of NOD-SCID

mice following BT088 cell implantation; markers of astroglial differentiation (GFAP) were not observed in these tumors (F).

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not 1p/19q codeletion or t(1;19)(q10;p10), may be aprincipal mechanism of chemosensitivity in early stageco-deleted oligodendrogliomas. Secondly, the MGMT-unmethylated GBM line, BT012, and the methylatedoligodendroglioma, BT088, were equally resistant toTMZ. This interesting observation raises the furtherpossibility that TMZ exposure selects for a non-MGMT-based mechanism of drug resistance in advancedtumors. Further experimentation will be required to fullyunderstand these findings, but at the very least BT054 andBT088 are new research tools that may help us discoverhow 1p/19 codeletion and t(1;19)(q10;p10) are relatedto the unique microscopic appearance, growth character-istics, and response to therapies of oligodendrogliomas.Perhaps most importantly, these cell lines, and othersthat will follow, provide a model system for developingnew therapies for this disease.

Acknowledgments

We thank Ms Kari Hafner and Ms Jamie Randolph(Mayo Clinic) for karyotype and SKY analysis and

Dr Arie Perry (Washington University School ofMedicine) for neuropathology review, FISH analysis,and images from the first tumor.

Conflict of interest statement. None declared.

Funding

This work was also made possible by operating grantsfrom the Canadian Institutes of Health Research andthe Stem Cell Network of Canada (S.W.), the AlbertaCancer Foundation Chair in Brain Tumor Research(J.G.C.), the Natural Sciences and EngineeringResearch Council of Canada Studentship (O.D.M.S.),the Alberta Heritage Foundation for Medical ResearchAwards—Clinical Fellowship (J.J.P.K.), ClinicalInvestigator (I.F.P.) and Scientist (S.W.), and theAlberta Cancer Board Translational Research Award(M.D.B.).

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