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

Abstract

Investigating the biology of oligodendroglioma and its characteristic combined deletion of chromosomal arms 1p and 19q, mediated by an unbalanced translocation, t(1;19)(q10;p10), has been hampered by the lack of cell lines that harbor these traits. We grew cells from 2 anaplastic oligodendrogliomas in serum-free conditions. Serial propagation and expansion led to the establishment of permanent cell lines that maintained the genetic signature of the parent oligodendrogliomas and displayed features of brain tumor stem cells in vitro. One line was established from a treatment-naïve tumor and the other from a temozolomide resistant recurrent tumor. These lines may be important tools for understanding the biology of oligodendrogliomas and the function of their defining genetic traits.

Oligodendrogliomas, a subtype of diffusely infiltrating glioma, are slowly growing primary brain cancers that typically occur in young or middle-aged adults. Histologically, they contain small, round cells with uniform nuclei, which resemble mature oligodendrocytes.¹ They were the first gliomas for which specific molecular predictors of chemotherapeutic response and overall survival were identified.² Combined allelic loss of chromosomal arms 1p and 19q (1p/19q) constitutes the earliest known molecular alteration in 50%–70% of oligodendrogliomas and results from a recurring unbalanced translocation, t(1;19)(q10;p10).^3,4 In both a retrospective and prospective series of anaplastic oligodendrogliomas (WHO grade III), radiographic and clinical response to chemotherapy, long progression-free survival after chemotherapy or radiotherapy, and long overall survival have been associated with the loss of 1p/19q alleles.⁵ Critically important questions about oligodendrogliomas and their defining genetic abnormality remain unanswered. Does codeletion 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 the unusual chemosensitivity and radiosensitivity of oligodendrogliomas, or are these anomalies simply biomarkers of a variant of human glioma that is sensitive to DNA damaging therapies for other unrelated reasons? Addressing these questions has been difficult in part because there have been no oligodendroglioma cell lines that possess its signature genetic alteration in which to explore the biology of this disease. Here, we describe 2 oligodendroglioma cell lines generated from 1p/19q co-deleted anaplastic oligodendrogliomas using serum-free conditions that contain a codeletion of chromosomes 1p and 19q and the unbalanced translocation, t(1;19)(q10;p10).

Materials and Methods

Oligodendroglioma Cell Culture

Fresh tissue samples were obtained from 2 adult patients during resection of primary intra-axial brain neoplasms. Cell culture was performed using the neurosphere assay.⁶ Briefly, tumor tissue was washed in sterile 1 × phosphate buffered saline (PBS) containing penicillin and streptomycin. Each specimen was finely minced then placed in a serum-free culture medium (SFM) containing DMEM/F12 (1:1) with 5 mM HEPES buffer, 0.6% glucose, 3 mM sodium bicarbonate, 2 mM glutamine, 25 µg/mL insulin, 100 µg/mL transferrin, 20 nM progesterone, 10 µM 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 µg/mL; R&D Systems) (SFM-EF). Once in SFM-EF, specimens were manually dissociated. A single-cell suspension was obtained by performing a series of mechanical dissociations followed by filtration (40 µm). Red blood cells were removed by hypotonic lysis. Finally, tumor cells were resuspended in SFM, counted using Trypan blue to exclude dead cells, and plated at a density of 20 000 viable cells/mL in SFM or SFM supplemented with EGF (20 ng/mL, Peprotech), FGF2 (20 ng/mL, R&D Systems), and HS (2 µg/mL; R&D Systems) (SFM-EF). Cultures were fed weekly by removing half the media and replacing with an equal volume of fresh media. Neurospheres were evident approximately 2 weeks following plating and were grown for an additional 2 weeks until they reached a size adequate for plating, differentiation, and serial passage.

Oligodendroglioma Sphere Self-Renewal and Differentiation

Self-renewal capacity was examined by dissociating primary spheres into single cells using Accumax (Innovative Cell Technologies). Single cells were replated at a density ≤20 000 cells/mL in either 24-well plates (uncoated, Nunc) or 25-cm² flasks (uncoated, Nunc) in SFM or SFM-EF. Cultures were fed and observed weekly for secondary sphere formation. Primary and secondary spheres were plated onto poly-l-ornithine-coated glass coverslips and allowed to differentiate for 7 days in SFM in the presence of 1% fetal bovine serum (FBS; Invitrogen). After differentiation, coverslips were fixed for 20 minutes in 4% paraformaldehyde, washed 3 times with 1× PBS, and prepared for immunocytochemical analysis.

Immunocytochemical Staining of Oligodendroglioma Spheres and Cells

Triple immunostaining of differentiated oligodendroglioma spheres was performed for glial fibrillary acidic protein (GFAP) (rabbit anti-GFAP, 1:400, BTI), β-III-tubulin (mouse-anti-β-III-tubulin, 1:1000, Sigma), and O4 (mouse IgM anti-O4, 1:10, Chemicon). Immunolabeling of oligodendroglioma cells for nestin was performed using mouse IgG anti-nestin (1:100, Chemicon). Nuclei were labeled with Hoechst 33 258 in all cases. The number of nestin-expressing cells was quantified by counting the total number of Hoechst+ nuclei per high-powered field (HPF; 40×) and the number that co-labeled with nestin in 10 HPFs.

Flow Cytometry for CD133

Primary and passaged oligodendroglioma cells were washed and resuspended in PBS + 0.5% BSA (PBS–BSA) and then incubated with either a monoclonal CD133/1-phycoerytherin-conjugated antibody or a phycoerytherin-conjugated mouse IgG1 isotype control antibody (both, 1:10, Miltenyi Biotec, according to the manufacturer's instructions) for 60 min at 4°C. Cells were analyzed using a BD FACScalibur flow cytometer and analyzed using ModFit LT™ software (Becton Dickinson) and FlowJo software (Tree Star, Inc.).

Molecular Diagnostic Methods

Tumor cells were evaluated for deletions of 1p/19q by fluorescent in situ hybridization (FISH) as previously described.⁷ For loss of heterozygosity (LOH) analysis by PCR, 6 independent loci on both 1p and 19q were evaluated in each DNA sample. Tumor DNA was extracted from paraffin blocks and sections using the Qiagen QIAmp DNA Micro kit and from cell lines using the DNAeasy kit (Qiagen). Normal DNA was extracted from blood using the Qiagen QIAmp DNA Mini kit. DNA (10 ng) was amplified for 35 cycles (94°C, 30 seconds/60°C, 1 minute 30 seconds/72°C, 30 seconds) using the Qiagen Multiplex Master Mix with 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 contained 3 sets of primers targeted at 3 microsatellite markers. For each microsatellite marker, the CA strand primer was labeled with Licor IRD dye and used at a final concentration of 0.2 µM; the GT strand primers were unlabeled and used at a final concentration of 0.4 µM. Following PCR, the samples were diluted 1:140 with Li-Cor loading dye and the dilution (1 µL) was loaded on an Li-Cor IR2 DNA sequencer. Electrophoresis was performed on the captured gel image using GeneImagR software (Scanalytics). LOH scores were calculated based on the intensities of the peak height of alleles between normal and tumor samples.

Relative gains and losses of chromosomal regions were evaluated by array comparative genomic hybridization (aCGH). For aCGH, DNA was isolated from patient tumor tissue and the oligodendroglioma cell lines (DNAeasy kit, Qiagen); all were analyzed at a core facility at the University of California at San Francisco (UCSF). Analysis was performed on a tiling Path Array (Hum32k) containing ∼32 000 human BAC clones spotted in singlets with continuous overlapping coverage of the genome (for details of the protocol as well as further information see CSF Helen Diller Family Comprehensive Cancer Center website at http://cancer.ucsf.edu/cores/index.php). Cells from the lines were harvested to prepare metaphase slides for G-banding and spectral karyotyping (SKY). Briefly, a probe cocktail containing 24 differentially labeled chromosome-specific painting probes and Cot-1 blocking DNA (SKY kit, Applied Spectral Imaging) was denatured and hybridized to the metaphase chromosome spreads as recommended by the manufacturer. After hybridization and washing, chromosome spreads were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride. Image acquisition, processing, and analysis were performed with SKY Vision software version 1.5, using a SD200 Spectracube system (Applied Spectral Imaging) mounted on a Zeiss Axiskop microscope with custom-designed optical filters (SKY-1, Chroma Technology) allowing for simultaneous excitation of all dyes and measurement of emission spectra. For each preparation, 5–10 metaphases were analyzed by SKY. Chromosomal designations followed the International System for Human Cytogenetic Nomenclature (2005).

For isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) sequencing, RNA was extracted from oligodendroglioma using RNeasy (Qiagen) according to the manufacturer's instructions. Five hundred nanograms of RNA was then reverse transcribed with the Superscript III First-Strand Synthesis System (Invitrogen) using poly-T primers. Two microliters of cDNA were then used in a 50 µL RT–PCR (Invitrogen) to amplify exon 4 of both the IDH1 and IDH2 genes as previously described.^8,9 RT–PCR products were then purified by agarose gel electrophoresis and isolated with the QIAquick Gel Extraction Kit (Qiagen). Automated DNA sequencing was performed at the University of Calgary Core DNA Services facility.

Chemosensitivity Testing In Vitro

The relative sensitivities of oligodendroglioma vs glioblastoma (GBM) lines were assessed after exposure to temozolomide (TMZ) alone (1–100 µg/mL; Schering Plough). Cell viability was monitored over 12 days using the alamarBlue^® viability assay, as instructed by the manufacturer (Medicorp). Each treatment group was repeated in duplicate and each experiment in triplicate. To facilitate the interpretation of these results, the methylation status of the O-6-methylguanine-DNA-methyltransferase (MGMT) gene promoter was assessed by MS-PCR.

Oligodendroglioma Cell Implantation into Immunocompromised Mice

Oligodendroglioma spheres were mechanically dissociated to single cells, washed twice in SFM, and viable cells were counted using Trypan blue exclusion. Viable cells (2 × 10⁵) were resuspended in 3 µL of SFM for stereotactic 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 up to 16 weeks and euthanized. Brains were removed and examined for morphological evidence of tumor formation. Each brain was cut in the coronal plane in order to obtain 2 segments, each containing part of the tumor. Both halves were fixed in formalin prior to immunohistochemistry and immunoflourescent immunohistochemistry.

Immunohistochemistry

For histological examination of tumors that arose in SCID mice following cell implantation, hematoxylin and eosin (H&E) staining was performed according to standard protocols. Immunohistochemistry was performed on paraffin-embedded sections using an indirect immunoperoxidase method. Immunohistochemistry performed on formalin fixed paraffin-embedded sections used the following primary antibodies: mouse anti-GFAP (1:400, R&D) and mouse-anti Olig2 (1:1500, BD Pharmingen). Sections were incubated with each specific primary antibody listed above overnight at 4°C 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 a brown reaction and hematoxylin was used as a blue nuclear counterstain.

Results

Diagnosis of Anaplastic Oligodendrogliomas with Codeletion of 1p/19q

A previously healthy 49-year-old woman developed focal seizures. Imaging studies revealed a right frontal, intra-axial tumor that enhanced following gadolinium administration (Fig. 1A). The mass was resected and tissue sent for both standard neuropathology evaluation and tissue culture after obtaining informed consent. The tumor contained 2 distinct histological areas: the first contained small, round cells with perinuclear halos and branching vasculature, features typical of oligodendroglioma (Fig. 1B), whereas the second area had higher cellularity with abundant mini-gemistocytes (Fig. 1C). Both contained cells that were GFAP positive (Fig. 1B). Nuclear pleomorphism and focally brisk Ki67 staining (Fig. 1C) led to a diagnosis of anaplastic oligodendroglioma (WHO grade III).¹⁰ The tumor harbored the codeletion of chromosomes 1p and 19q as assessed by FISH (Fig. 1D). The patient was treated successfully with TMZ chemotherapy and radiotherapy.

Fig. 1.

Diagnostic characteristics of an anaplastic oligodendroglioma. (A) A T1-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).

A previously healthy man developed seizures at age 33. Imaging revealed a right frontal, enhancing, intra-axial tumor. A WHO grade III oligodendroglioma was removed and treated with procarbazine, lomustine, and vincristine chemotherapy. Over the ensuing 17 years, multiple recurrences were treated with radiotherapy and several courses of TMZ with benefit. The tumor harbored the codeletion of chromosomes 1p and 19q (data not shown). Ultimately, resistance to TMZ and other chemotherapies led to a final surgical resection for symptom control. Tumor tissue was sent for neuropathology review and culture after informed consent was obtained.

Oligodendroglioma Cells Express Neural Stem Cell Markers and Grow As Multipotent Oligodendroglioma Spheres In Vitro

Brain tumor stem cells (BTSCs) have been isolated and continuously cultured from many different types of primary brain tumor using the neurosphere system,^11,12 but not from oligodendrogliomas. Using tissue obtained at the time of surgical resection, single-cell suspensions were prepared for phenotypic evaluation and culture. Since BTSCs have been shown to express the neural stem cell (NSC) markers CD133^12,13 and nestin,^12,14 the oligodendroglioma cell suspensions were immediately assessed for CD133 expression by flow 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 first tumor were nestin positive (Fig. 2B). The remaining primary oligodendroglioma cell suspensions were cultured in SFM alone¹⁵ and in SFM-EF as described in detail elsewhere.⁶ After 3 weeks, SFM-EF cultures from the first tumor contained phase bright, floating spheres (Fig. 2C), a pattern typically observed for NSCs⁶ and BTSCs.^11,12 No growth was obtained in the absence of mitogens. In the second case, after 3 weeks, both SFM and SFM-EF cultures contained phase bright, floating spheres that were identical in appearance to those of the first tumor (Fig. 4A).

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 (β-III-tubulin) markers (nuclei counterstained with Hoechst 33 258). Scale bars B: 50 µm, C and D: phase contrast, 100 µm; immunofluorescence, 50 µm.

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.

A fundamental characteristic of NSCs is multipotency.¹⁶ Primary oligodendroglioma spheres from individual tumor cells from both tumors were multipotent. Those from the first tumor differentiated into cells that expressed astrocyte, oligodendrocyte, and neuronal markers (Fig. 2C), whereas those from the second differentiated into cells that expressed oligodendrocyte and neuronal markers only (data not shown). Another fundamental characteristic shared by NSCs and BTSCs is the ability to self-renew.¹² To test this property, primary oligodendroglioma spheres from both tumors were dissociated to single cells and replated in SFM with EGF + FGF2; secondary oligodendroglioma spheres arose from individual cells within 3 weeks and retained the capacity for multilineage differentiation (Fig. 2D). Subsequent generations of spheres have been successfully maintained in culture for 26 and 15 months, respectively. These data demonstrate that both of these oligodendrogliomas contained cells that possess properties of both NSCs and BTSCs in vitro, and propagation of these cells has led to the development of stable oligodendroglioma cell lines. These cell lines are now referred to as BT054 and BT088.

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

To determine whether cells from BT054 and BT088 maintained the characteristic genetic alteration of oligodendrogliomas, codeletion of 1p/19q, we performed PCR-based LOH analysis. As shown for the first patient (Fig. 3A), comparison of normal, tumor, and BT054 DNA revealed an identical pattern of LOH between the tumor and the BT054 cells that was not present in somatic DNA (Fig. 3A). An identical LOH pattern was seen with normal, tumor, and BT088 DNA (data not shown). Characteristically, codeletion of 1p/19q in oligodendrogliomas is associated with complete loss of these chromosomal arms. In BT054, aCGH revealed complete loss of 1p and 19q (Fig. 3B). It also revealed the amplification of chromosome 11q and the deletion of chromosomes 14q and 15q; these changes have been seen previously in oligodendroglioma tumor tissues.¹⁷

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).

Codeletion of 1p/19q in oligodendrogliomas appears to result from an unbalanced translocation, t(1;19)(q10;p10).^3,4 To determine whether codeletion of 1p/19q in BT054 and BT088 cells was the result of a translocation, G-banding karyotypic analysis followed by SKY was performed. In BT054, every metaphase by standard karyotyping and SKY revealed at least one t(1;19)(q10;p10) chromosome with the corresponding loss of 1p and 19q (Fig. 3C). The formal karyotype by G-banding and SKY was: 76-108,XXX,-X,+der(1;19)(q10;p10)x2-3, del(2)(p11.2p15),+3,+3,+5,+5,−6,+7,+8,−9,+10,+12,−15,16,+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 aneuploid with near-tetraploid (∼75%) and near-sexaploid (∼25%) variants. Every metaphase contained multiple other clonal numeric and structural abnormalities including deletion of 2p, relative gains of 3, 5, 20, and 22, and relative losses of 6, 9, and 16. In addition, BT054 cells contained several marker chromosomes that could not be unequivocally identified by SKY, including a translocation that may involve chromosomes 7 or 15 and chromosomes 1 or 11. FISH analysis using 4 colored probes confirmed the presence of t(1;19)(q10;p10) within multiple cells (Fig. 3D). A very chaotic pattern of chromosomal rearrangements accompanied by relative codeletion of 1p and 19q loss was also seen in BT088, whose formal karyotype by G-banding and SKY is shown in Fig. 4B. Every metaphase had at least one copy of t(1;19)(q10;p10), corresponding loss of 1p and 19q, and multiple other clonal numeric and structural abnormalities including relative gains and losses of other chromosomes and marker chromosomes that could not be fully identified by SKY. These data establish unequivocally that cell lines BT054 and BT088 contain t(1;19)(q10;p10), thus setting the stage for functional and in vivo studies.

BT054 and BT088 Cells Proliferate Slowly but Display Divergent Chemosensitivity In Vitro

Oligodendrogliomas that harbor 1p/19q codeletion have 2 defining features in vivo: slow growth and chemosensitivity. Comparatively, GBMs, the most aggressive type of glioma, typically do not harbor 1p/19q codeletion, grow more rapidly, and are less sensitive to comparable therapies. To assess whether BT054 and BT088 cells could be tools to investigate oligodendroglioma biology, we determined whether BT054 and BT088 possessed these defining features of oligodendrogliomas. We compared the growth rate of BT054 and BT088 cells in vitro to that of 2 GBM BTSC lines, BT012 and BT048, which were established in our laboratory using identical methods. We seeded 1 × 10⁶ cells from each line in 25 cm² flasks containing SFM-EF and total cell number was counted after 14 days. Additional counts were performed based on the individual 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 flask (n = 3) and BT088 cultures yielded 0.35 ± 0.03 × 10⁶ cells per 25 cm² flask (n = 3). By comparison, the GBM cultures BT048 and BT012 yielded 1.67 ± 0.08 × 10⁶ cells (n = 3) and 2.63 ± 0.22 × 10⁶ cells (n = 3), respectively (Fig. 5A), and needed passage on day 14. BT054 and BT088 cells proliferated at a slower rate and needed passage at day 18 and 28, respectively (Fig. 5A).

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 = 3 experiments per culture; data expressed as mean ± SEM] (B) BT054 viability is reduced by treatment with a clinically relevant dose of TMZ (5 µg/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 µg/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 = 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.

Next, we examined the sensitivity of BT054 and BT088 to the DNA methylating agent, TMZ. Using the alamarBlue viability assay, the responses of BT054 and BT088 were compared with the 2 GBM BTSC lines, BT012 and BT048. BT054 was significantly more sensitive to TMZ than either BT088 or BT012, which were comparably resistant. Dose–response curves showed a 50% reduction in viability at 10 µg/mL for BT054 vs 50 µg/mL for BT088 and BT012 (Fig. 5B). Of note, the BT054 and the GBM line BT048 were equally sensitive to TMZ (Fig. 5B). Because methylation of the MGMT gene promoter silences MGMT expression, rendering GBM tumors more sensitive to TMZ¹⁸ and may also predict sensitivity in 1p/19q codeleted oligodendrogliomas,¹⁹ we examined MGMT methylation status in these lines. MGMT was methylated in BT054, BT088, and BT048 and unmethylated in BT012 (Fig. 5C). In 3 of the 4 lines, response and methylation status were congruent: BT054 and BT048 were methylated 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 oligodendroglioma and presumably had accumulated other genetic alterations that might explain TMZ resistance. Indeed, review of BT088-metaphase spreads revealed frequent loss of chromosome 2, the site of MSH6.

BT054 Cells Harbor an IDH1 Mutation

Mutations in either the IDH1 gene or less commonly the IDH2 gene have been demonstrated to occur in the majority of anaplastic oligodendrogliomas.⁸ To date, no oligodendroglioma or glioma cell lines exist that contain an IDH1 mutation and investigation of this mutation requires its introduction into glioma cells that endogenously express wild-type IDH. To provide further evidence for the clinical relevance of these oligodendroglioma cell lines, we set out to determine whether they harbor either IDH1 or IDH2 gene mutations. Evaluation of the IDH1 and IDH2 mutational status by DNA sequencing revealed the most frequently observed mutation of IDH1, which occurs in exon 4 at codon R132, in the BT054 cell line (Fig. 5D). The cell line BT088 did not contain mutations of either IDH1 (Fig. 5D) or IDH2 (data not shown). The BT054 cell line will be a valuable tool for investigating the role of IDH1 mutation in glioma and may aid the development of novel therapeutics that target the mutant form of IDH1.

BT088 Cells Proliferate In Vivo and Initiate Tumors in Immunocompromised Mice

Our in vitro cell culture findings suggested that sphere forming cells isolated from 2 anaplastic oligodendrogliomas were BTSCs. However, tumor formation in vivo is the critical test of BTSC identity;²⁰ hence, we assessed whether cells isolated from oligodendroglioma spheres were tumorigenic in vivo. Oligodendroglioma cells were implanted into the brains of NOD-SCID mice (2 × 10⁵ cells per animal, n = 5 animals for each of BT054 and BT088). Illness with weight loss was evident within 5 weeks postimplantation in the BT088-inoculated group and all animals developed brain tumors within 10 weeks (Fig. 6A). No animal implanted with BT054 became ill. Following euthanasia, brains were removed and examined; those implanted with BT054 looked normal, whereas those implanted with BT088 showed gross morphological changes. H&E staining demonstrated hypercellular tumors in all animals implanted with BT088 (Fig. 6B). No abnormalities could be identified in BT054-injected brains. BT088 gave rise to infiltrating tumors that closely resembled anaplastic oligodendrogliomas. Tumor cells infiltrated the brain parenchyma extensively (Fig. 6B and C), and the Ki67 proliferative index was high (Fig. 6D). Further, these tumors were composed of cells that resembled mature oligodendrocytes and contained small, round nuclei with perinuclear halos (Fig. 6C). A branching or “chicken-wire” vascular pattern was also evident in these tumors. Immunohistochemical analysis revealed that all tumors contained cells that expressed Olig2 (Fig. 6E), but not GFAP (Fig. 6F). Thus, BT088 cultures contain BTSCs that give rise to oligodendrogliomas in vivo.

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).

Discussion

This paper describes the first human oligodendroglioma cell lines, BT054 and BT088, that contain complete loss of chromosomal arms 1p and 19q, mediated by a centromeric translocation t(1;19)(q10;p10), which together constitute the signature genetic change in oligodendrogliomas.^1,3 Understanding how this characteristic genetic abnormality of oligodendrogliomas relates to their fundamental biology has been impeded by the lack of a cell system that closely mimics the disease. In addition, BT054 is the first cell line to contain the recently described IDH1 mutation that frequently occurs in oligodendrogliomas and a subgroup of glioblastomas. Although oligodendroglioma cell lines have been alluded to by others,^21,22 both their t(1;19)(q10;p10) status and IDH1 status is either unknown or not reported. Furthermore, these previously reported oligodendroglioma lines were generated by supplementing tissue culture medium with high concentrations of fetal calf serum, a method that has been demonstrated to fundamentally alter the properties of BTSCs.²³ By utilizing the neurosphere culture system with defined, serum-free growth conditions, 2 cell lines have been generated that appear to be true phenocopies of the parental anaplastic oligodendrogliomas, which in turn, had the classic features of the disease.

Recent studies have pointed to NSCs and lineage-restricted progenitors as the probable cells of origin for several types of primary brain tumors, especially GBM.²⁴ For decades, oligodendrogliomas, a related cancer of the brain, have been postulated to arise from mature oligodendrocytes, although in point of fact, their actual cell of origin has never been rigorously identified.¹ By employing the neurosphere culture system, we demonstrate that oligodendrogliomas harbor a subset of cells that possess the properties of BTSCs in vitro and in vivo.¹² The novel finding that these oligodendroglioma-derived cells (i) proliferate under serum-free culture conditions in the presence of EGF + FGF2, (ii) self-renew, (iii) give rise to multipotent oligodendroglioma spheres, and (iv) give rise to intracerebral tumors that are indistinguishable from oligodendrogliomas (BT088 only), raises the distinct possibility that NSCs or lineage-restricted progenitor cells are candidate cells of origin of oligodendrogliomas.

It is possible that the codeletion of chromosomes 1p and 19q or t(1;19)(q10;p10) directly mediate slow growth and chemosensitivity of oligodendrogliomas; however, a mechanistic relationship has not been established. Functional assays performed in this study yielded 2 interesting results. First, the MGMT-methylated GBM line BT048 and the methylated oligodendroglioma line, BT054, were comparably sensitive to TMZ. This finding raises the possibility that MGMT promoter methylation, not 1p/19q codeletion or t(1;19)(q10;p10), may be a principal mechanism of chemosensitivity in early stage co-deleted oligodendrogliomas. Secondly, the MGMT-unmethylated GBM line, BT012, and the methylated oligodendroglioma, BT088, were equally resistant to TMZ. This interesting observation raises the further possibility that TMZ exposure selects for a non-MGMT-based mechanism of drug resistance in advanced tumors. Further experimentation will be required to fully understand these findings, but at the very least BT054 and BT088 are new research tools that may help us discover how 1p/19 codeletion and t(1;19)(q10;p10) are related to the unique microscopic appearance, growth characteristics, and response to therapies of oligodendrogliomas. Perhaps most importantly, these cell lines, and others that will follow, provide a model system for developing new therapies for this disease.

Funding

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