ABC transporter activity linked to radiation resistance and molecular subtype in pediatric medulloblastoma
© Ingram et al.; licensee BioMed Central Ltd. 2013
Received: 26 September 2013
Accepted: 30 September 2013
Published: 4 October 2013
Resistance to radiation treatment remains a major clinical problem for patients with brain cancer. Medulloblastoma is the most common malignant brain tumor of childhood, and occurs in the cerebellum. Though radiation treatment has been critical in increasing survival rates in recent decades, the presence of resistant cells in a substantial number of medulloblastoma patients leads to relapse and death.
Using the established medulloblastoma cell lines UW228 and Daoy, we developed a novel model system to enrich for and study radiation tolerant cells early after radiation exposure. Using fluorescence-activated cell sorting, dead cells and cells that had initiated apoptosis were removed, allowing surviving cells to be investigated before extensive proliferation took place.
Isolated surviving cells were tumorigenic in vivo and displayed elevated levels of ABCG2, an ABC transporter linked to stem cell behavior and drug resistance. Further investigation showed another family member, ABCA1, was also elevated in surviving cells in these lines, as well as in early passage cultures from pediatric medulloblastoma patients. We discovered that the multi-ABC transporter inhibitors verapamil and reserpine sensitized cells from particular patients to radiation, suggesting that ABC transporters have a functional role in cellular radiation protection. Additionally, verapamil had an intrinsic anti-proliferative effect, with transient exposure in vitro slowing subsequent in vivo tumor formation. When expression of key ABC transporter genes was assessed in medulloblastoma tissue from 34 patients, levels were frequently elevated compared with normal cerebellum. Analysis of microarray data from independent cohorts (n = 428 patients) showed expression of a number of ABC transporters to be strongly correlated with certain medulloblastoma subtypes, which in turn are associated with clinical outcome.
ABC transporter inhibitors are already being trialed clinically, with the aim of decreasing chemotherapy resistance. Our findings suggest that the inhibition of ABC transporters could also increase the efficacy of radiation treatment for medulloblastoma patients. Additionally, the finding that certain family members are associated with particular molecular subtypes (most notably high ABCA8 and ABCB4 expression in Sonic Hedgehog pathway driven tumors), along with cell membrane location, suggests ABC transporters are worthy of consideration for the diagnostic classification of medulloblastoma.
KeywordsMedulloblastoma Radiation resistance ABC transporter ABCG2 ABCA1 Stem Brain tumor Verapamil Reserpine Sonic hedgehog
Tumors of the central nervous system (CNS) are the leading cause of cancer related childhood death and disability in developed countries . Of the malignant pediatric brain tumors, medulloblastoma is the most common and treatment typically involves surgery, radiotherapy and chemotherapy . Age of onset, degree of surgical removal and presence of metastatic disease provide useful criteria to stratify patients into “standard” and “high risk” groups, however a subset of patients in both groups are prone to treatment resistance and relapse. Although recent studies show medulloblastoma patients cluster into at least four distinct groups based on gene expression signatures, it is not yet possible to determine which individuals will display treatment resistance .
Evidence suggests that increased resistance to both chemotherapy and radiation is a feature of Cancer Stem-like Cells (CSCs): the subset of cells within a tumor that have the ability to drive tumor re-growth or to initiate a metastatic lesion [4–7].
Resistance to radiation can arise from a variety of mechanisms, including enhanced DNA repair, altered apoptotic responses, tolerance of genomic instability and dormancy . A key mechanism by which cells resist chemotherapy drugs is via ATP-binding cassette (ABC) transporters. The ABC transporter gene family has 50 members in humans, encoding membrane-bound pumps that transport an assortment of substrates . Four members of this family have notable roles in multi-drug resistance: ABCG2 (also known as BCRP), ABCB1 (Pgp/MDR1), ABCC1 (MRP1) and ABCC2 (MRP2) [10, 11]. ABCG2 is of special interest, as it marks stem cells in a wide range of normal tissues, including brain, and may have a functional role in maintaining a non-differentiated state . ABCG2 is also implicated as a CSC marker in diverse malignancies [13, 14].
While definitive markers of CSCs and resistant cells remain elusive, genes other than ABCG2 are also expressed in tumor sub-populations enriched for stem-like behavior. These include other cell surface molecules (e.g. prominin 1 (CD133), FUT4 (CD15)); filaments (e.g. nestin); regulators of DNA architecture (e.g. BMI1) and mediators of developmental signaling networks (e.g. genes involved in the NOTCH, WNT and Sonic Hedgehog (SHH) pathways) [15, 16]. Despite mounting evidence for the existence of CSCs in brain tumors, markers in medulloblastoma remain poorly understood [17, 18].
Evidence of increased radiation resistance in brain CSCs has come mainly from studies of glioma and atypical teratoid/rhabdoid tumors (AT/RT) for which experimental isolation depended on CD133 [19–23]. Selection of CD133 positive cells enriches for CSCs in some tumors, but is not without controversy [17, 24, 25]. Nonetheless, activation of the DNA damage response and chromatin remodeling appear to mediate radiation tolerance in glioma [19, 20, 22]. A glioma study using a non-CD133 driven experimental approach found Hedgehog pathway signaling important for maintaining radiation tolerant CSCs . However, radiation resistance and its relationship to stem-like behavior has been much less studied in medulloblastoma. CD133 positive cells from the Daoy line are reported to have increased radiation tolerance, while in vivo work has shown Nestin expressing medulloblastoma cells to have enhanced survival after irradiation [27, 28].
We undertook the reverse approach of many studies to examine radiation tolerant medulloblastoma cells. Rather than isolating cells with a putative marker and then investigating resistance, we turned the problem around by selecting surviving cells functionally, followed by a candidate gene approach to see if putative stem cell markers were associated with the radiation tolerance phenotype. This gave us the potential to identify characteristics present before radiation was encountered, as well as responses that might be preferentially up-regulated by cells after radiation exposure. This approach proved fruitful, with the discovery of several genes (including ABCG2), that were elevated in surviving cells. Furthermore, functional experiments indicated that ABC transporters may play a previously undiscovered role in protecting brain tumor cells from radiation induced death.
Development of a model system for early enrichment of radiation tolerant tumor cells
Firstly, established medulloblastoma cell lines (Daoy and UW228) and early passage cultures of resected medulloblastoma tissue (R001M and R026M) were irradiated to generate dose response curves. This was done with the Clonogenic Colony Forming Assay (CCFA) when cells had low motility (Figure 1B), and in other cases by total viability assay (Figure 1C). CCFAs give more complete data than viability based assays, because cells must be both alive and proliferative to be counted. Differences in results from the two types of assay were observed (for early passage cultures in particular), reflecting the presence of non-proliferative cells that were damaged by radiation but that had not yet died in viability assay data. Both assays are informative for comparing the overall health of cell populations before and after radiation exposure. Our aim was to determine a dose at which between 1% and 5% of treated cells would remain viable and proliferative, such that the treatment was harsh but would provide enough survivor cells for meaningful analysis. After receiving 10 Gy, Daoy, UW228 and R001M gave 1.3%, 3.8% and 3.2% of cells (respectively) that were able to survive and form colonies (Figure 1B; R026M cells were unsuitable for CCFA).
The time course of phosphatidylserine (PS) externalization, an early step in apoptosis initiation , was profiled in Daoy and UW228 lines after 10 Gy irradiation, the dose chosen for resistant cell isolation (Daoy data shown Figure 1D, UW228 results similar). While high levels of external PS were readily detected after 1 day, peak levels were not reached until two days and were still significant at three days post-irradiation. For resistant cell isolation, 10 to 20 million cells were irradiated and then incubated for three days (i.e. until after the apoptotic peak). Surviving cells were then isolated by eliminating both dead (Propidium Iodide (PI) positive) cells and “doomed to die” (Annexin V positive) cells by Fluorescence-Activated Cell Sorting (FACS). Non-irradiated cells were sorted in parallel to provide a comparable control population, in which spontaneous apoptotic cells (notable in Figure 1D) were eliminated.
Radiation tolerant medulloblastoma cells display stem behavior and are tumorigenic
Daoy and UW228 cells surviving 10 Gy radiation were able to proliferate long term in tumorsphere cultures (Figure 1E). Daoy tumorsphere cells descended from radiation survivors were assessed in an intracranial xenograft model and found to be tumorigenic in vivo. This confirmed that the isolated post-irradiation population included cells with cancer stem cell properties (the ability to initiate and recapitulate a tumor; Figure 1F).
Certain stem-associated genes are elevated in medulloblastoma cells surviving high dose radiation
Flow cytometry showed ABCG2 is also elevated in radiation survivors at the protein level (Figure 2C). Our focus then turned to this and the three related genes that play major roles in chemotherapy resistance. An additional member, ABCA1, was investigated as its elevation after irradiation has been reported in glioma . The ABCA1 protein transports lipids and has important roles in cholesterol homeostasis and macrophage engulfment . Significant mRNA changes were not observed for ABCB1, ABCC1 or ABCC2 in Daoy or UW228 radiation survivors; however the increase in ABCA1, as for ABCG2, was striking in both lines (Figures 2D and 3B).
An alternate model involving long-term fractionated irradiation of UW228 corroborates findings of elevated ABCA1 and ABCG2 in surviving cells
We used FACS to isolate ABCA1 and ABCG2 expressing and non-expressing sub-populations from radiation naive UW228 and Daoy cultures, to examine whether expression of ABC transporters prior to irradiation is a marker for cells that are more likely to display radiation tolerance. Interestingly, the percentage of cells expressing either gene was very low in both lines (0.3% for ABCG2 (Figure 2C) and 1.5% for ABCA1), and positive cells did not show increased resistance over negative cells by CCFA (data not shown). Thus there may be differences in the way these genes are controlled in resistant cells in response to radiation, rather than being intrinsic markers prior to exposure.
Verapamil mediated sensitization suggests ABC transporters may have a functional role in radiation protection
To test the hypothesis that ABC transporter proteins may provide a functional advantage to cells surviving radiation damage, we investigated a range of available ABC transporter inhibitors to see if these altered the sensitivity of Daoy or UW228 cells.
Verapamil is a drug with complex effects. In addition to inhibiting ABC transporters, it also blocks L-type calcium channels [11, 33]. Exposing cells to other L-type calcium channel blockers (diltiazem and nifedipine) had no effect on radiation sensitivity, showing the sensitization to radiation is unlikely to be due to calcium channel effects (Figure 6A). Optical isomers of verapamil have different potencies as inhibitors, making them a useful research tool (Figure 6C). The “S” isomer possesses calcium channel blocking activity that is an order of magnitude more potent than that of the “R” isomer (dexverapamil), while both are strong ABC transport inhibitors [34–36]. Both isomers were similarly effective at sensitizing Daoy cells to radiation, further suggesting the radiation sensitizing effect is independent of calcium channel inhibition (Figure 6A).
Transient exposure to verapamil in vitro reduces the tumorigenicity of medulloblastoma cells in vivo
Reserpine mediated sensitization also suggests a functional role for ABC transporters in radiation protection
ABC transporters are expressed in medulloblastoma in variable patterns and some family members are associated with particular molecular subtypes
As molecular sub-typing data was unavailable for the majority of our patient cohort, we turned to a large and well annotated public medulloblastoma dataset for further investigations . As in our dataset, ABC transporter genes tended to be expressed at varying levels for these 62 cases (results shown in Additional files 1, 2, 3). Notable patterns were discovered in the expression of several ABC transporters when tumors were clustered by subtype. Of the five genes focused on in our study, ABCA1 was of note, as high expression was significantly associated with group A (WNT associated) tumors and low expression with groups B (SHH associated) and E. ABCB1 tended to be more highly expressed in group B, C and D than in the other two groups (refer Additional file 1; Note that groups C and D in this dataset are equivalent to “group 4” in the current consensus nomenclature ). The most striking associations however were with other family members. Our initial analysis identified a number of ABC transporters that show significant subtype-related expression trends, with the highest significance discriminators being ABCA8, ABCB4, ABCC8 and ABCD2 (refer Additional files 2 and 3).
While incremental improvements to surgical, chemo-therapeutic and radiological procedures continue to increase survival for pediatric brain tumor patients, death rates and quality of life issues for those who remain in remission are significant. The key to overcoming limitations of existing treatments is to increase our understanding of tumor cell biology at the level of individual cells.
Towards this goal, we used a functional approach to investigate medulloblastoma cells displaying high radiation tolerance. FACS was employed to remove dead and apoptotic cells from irradiated cultures, so that surviving cells could be studied before extensive cell division took place. This “rapid isolation approach” allowed study of the tolerant cells or their early descendants, rather than distant progeny. The “survivor cells” displayed tumorigenic potential in vivo, showing that they were indeed “healthy” and retained stem-like behavior. The finding of elevated levels of ABCG2 in such cells from a subset of human patients, and of ABCA1 in all patient cultures, was of particular interest given the important roles ABC transporters play in chemo-resistance and stem cell biology. ABCG2 has also been implicated in cancer susceptibility, as a polymorphism in the human gene is associated with a change in incidence for several malignancies . In this study we chose to focus on a key subset of ABC transporters known to be strong drivers of drug resistance in human patients, or that play a known role in stem cell or brain tumor biology. It is anticipated that other members of this extensive gene family may also display increased expression in medulloblastoma cells after radiation treatment.
Elevated levels of ABC transporters may be observed in radiation tolerant cells for three reasons: they could reflect a generalized stress response, they could provide a functional advantage to irradiated cells, or their expression might be correlated with other genes that are functionally involved in resistance. The fact that both ABCA1 and ABCG2 were elevated in an independent model of radiation resistance in UW228 cells led us to further explore the role of the ABC transporter family. Findings with c-Myc and HIF2A in various patient cultures are also of interest, and are a focus of ongoing investigation.
ABCA1 and ABCG2 proteins were only expressed on a small proportion of cells within radiation naive medulloblastoma lines. Subsequent analysis showed their expression did not specifically mark cells that would better survive radiation exposure. This suggests cells with greater intrinsic resistance may exist in a “primed” state and have the ability to express such genes after radiation is experienced.
Difficulties were anticipated in using RNA-based knock-down technology to explore the possible functional relevance of expression changes, due to the vast number of ABC transporter genes in the human genome and their tendency for overlapping roles, and the fact that we wished to investigate early passage primary culture cells (which are challenging to transfect efficiently). Instead, we took advantage of the wide range of readily available compounds known to inhibit single or diverse ABC transporter proteins. Use of these led to our discovery that ABC transporters not only contribute to chemotherapy resistance, but also play a functional role in radiation protection. This finding is based on studies with ABC transporter inhibitors of two distinct molecular classes. Although the multi-ABC transporter blockers verapamil and reserpine enhanced radiation sensitivity, compounds specifically inhibiting just ABCG2 or ABCA1 had no detectable effect. This suggests overlapping function of genes, with multiple transporters having functions that together contribute to radiation resistance. Thus it appears combinations of transporters need to be blocked to prevent radio-protective effects. The fact that reserpine and verapamil sensitized cultures from different patients may be a reflection of the varied ABC transporter expression patterns observed between different medulloblastoma cases, in both our own and publicly available gene expression datasets.
Results obtained with verapamil and reserpine need to be interpreted with care, since their channel inhibition effects extend further than the ABC transporter family. Verapamil also possesses anti-L-type calcium channel activity and influences potassium transport. Our finding that the R-verapamil optical isomer mediates radiation sensitization at a level indistinguishable from that of S-verapamil, even though the former only possesses weak calcium/potassium channel activity, is important given both are similarly potent in blocking ABC transporters [34–36, 45, 46]. This suggests the radiation protection mechanism is ABC transporter driven, although it is possible that a residual level of anti-cation channel activity could saturate a sensitive system. Our second line of evidence suggests this is not the case, as deliberate addition of other inhibitors of L-type calcium/potassium activity did not have any observable effect on radiation sensitivity. While such experiments implicate ABC transporters, this does not preclude verapamil having effects on other undiscovered cellular targets that may also protect cells from radiation. However, observation of a similar radiation sensitizing effect with the independent ABC transporter inhibitor reserpine further strengthens the case for ABC transporter involvement.
Reserpine is used extensively in research due to its potent inhibition of ABC transporters [11, 37]. While verapamil and reserpine overlap in their inhibition of ABCB1, their effects on the majority of other individual family members remain unstudied . In addition to ABC transporters, reserpine also inhibits the vesicular monoamine transporter (VMAT), which is involved in neurotransmitter movement in the CNS . Although less likely in light of the verapamil result, it remains possible that the dose dependent sensitization to radiation observed with reserpine was due to neurotransmitter rather than ABC transporter effects. If this is the case, it would raise the intriguing possibility that reserpine may influence radiation responses by directly manipulating the stem-like properties of cells, since inhibitors of neurotransmitter pathways are known to reduce neural stem cell populations .
The anti-proliferative effect of verapamil observed in medulloblastoma lines supports earlier reports of this phenomenon in various neoplastic cells [49–53]. Though initially presumed to be due to calcium effects, there is now evidence that the anti-cancer properties of verapamil involve non-calcium dependent mechanisms [52, 54]. Verapamil is also anti-tumorigenic when given to live animals harboring meningioma xenografts ; however our study is the first to show transient exposure to verapamil in vitro has long lasting effects when tumorigenicity is subsequently assessed in vivo. This innate anti-tumorigenic effect may provide a therapeutic bonus (in addition to chemo- and radiotherapy sensitization), if new generation drugs with structures similar to verapamil are used in the treatment of future patients.
Increased expression of ABC transporters, particularly ABCB1, has been reported after irradiation in several cell types [56–58]. The exact mechanism by which this is controlled remains largely unexplored, although a number of ABC transporters are known to be up-regulated in response to xenobiotics or stimuli that indicate harsh conditions. One such condition is hypoxia, which is known to influence expression via the binding of Hypoxia Inducible Factor (HIF) complexes to ABC transporter promoters . Oxidative stress is also known to stimulate ABC transporter expression, for example activation of the ABCG2 promoter can be mediated by the transcription factor NRF2 under such conditions . It is possible that this, or alternate transcription systems that detect oxidative stress or cellular damage, may be responsible for up-regulating ABC transporter family members after irradiation in medulloblastoma.
The fact that observed gene expression changes upon irradiation in R001 and R026 cells were more similar to each other than to other investigated cells led us to investigate the subtype classification for these patients. Both were found to represent “Group 3” tumors by 22 gene signature nanoString nCounter analysis ( and data not shown). Both UW228 and Daoy have previously been analyzed by other researchers , and found to be of the SHH subtype. This may in part explain the variability observed between the responses, particularly since elevation of ABCG2 in response to radiation was observed for UW228 and Daoy but not for R001 or R026. The transcription of certain ABC transporter genes, including ABCG2, is known to be directly influenced by activation of the Hedgehog signaling pathway [63–65].
While ABC transporter related responses to drugs have been investigated by numerous groups, a functional role for ABC transporters in radiation protection remained largely unexplored prior to our studies. However, a hypothetical radio-protective role has previously been proposed , and a precedent for the transport of molecules as a mechanism of radiation protection does exist, with the non-ABC transporter RLIP76 known to play a key role in glutathione-mediated radiation protection in non-cancerous cells . ABC transporters may potentially protect cells from radiation via direct transport of toxic radiation by-products from cells, by moderating glutathione balance, or by actively removing glutathione-bound conjugates. Alternatively, the effect may be more indirect, such as ABC transporters shuttling post-irradiation “messenger molecules” between compartments or cells. Such effects could in turn trigger DNA repair or modulate the apoptotic response in order to enhance cell survival. There is evidence from leukemia cells suggesting that verapamil-reversible drug resistance can be driven by glutathione based systems, rather than drug efflux . Additionally, over-expression of ABCB1 in hematopoietic cells appears to reduce apoptosis . ABC transporters may contribute to radiation resistance via similar means. Elucidation of the exact mechanism awaits further study.
In light of our findings supporting a functional role for ABC transporters in radiation protection, two previous studies are of note. In 2009 a stem-like sub-population of bladder cancer cells with high ABC transporter activity was shown to be sensitized to radiation in the presence of verapamil . This study did not attempt to determine if this was mediated by cation channel effects or ABC transporter blockade. Our findings show pediatric medulloblastoma behaves similarly, and that the mechanism is likely to be independent of cation effects. Further evidence supporting our finding of ABC transporters as mediators of radiation resistance comes from a study of breast cancer patients. A single nucleotide polymorphism screen has previously shown that patients harboring an amino acid change in ABCA1 tend to suffer radiation induced dermatitis during therapy , implying ABCA1 is a contributor to cellular radio-protection, at least in some cell types. While the exact combination of members in this large family that play a role in the radiation tolerance phenotype is yet to be determined, our finding that certain multi-ABC transporter inhibitors can sensitize cells to radiation whilst single transporter specific agents lack an effect, indicates that a number of ABC transporter proteins are involved, probably with redundant functions.
We utilized a large publicly available dataset , to complement our initial qRT-PCR study and provide the first family-wide survey of ABC transporter expression in medulloblastoma. The observed variation in expression patterns contributes to our finding that ABC transporter inhibitors give differing effects on radiation tolerance depending on the patient that cell cultures are derived from.
Our analysis of publicly available microarray data showed that the expression level for several ABC transporter family genes is strongly associated with particular medulloblastoma subtypes. This was particularly evident with ABCA8, ABCC8 ABCD2 and ABCB4. Potential correlations between ABC transporter expression and relapse/clinical progression will be explored as our cohort matures. This information was not available for the dataset used for the discovery of subtype related associations, however evidence from multiple groups has shown robust correlation between subtype and clinical outcome (in particular a relatively favorable outcome for WNT driven tumors and a poorer than average outcome for “Group 3” medulloblastoma) [39, 71, 72].
The fact that certain ABC transporters show distinct patterns of expression for particular subtypes, and that the proteins are membrane localized, makes them attractive candidates as novel diagnostic markers (particularly for discriminating between SHH and non-SHH pathway driven tumors). In will be interesting in future studies to see if this association also extends to other tumor types.
Whether the suite of ABC transporters that play a role in radiation protection overlap with the family members that show strong subtype dependent expression patterns is also of particular interest for future study. Further study of ABC transporters stands to provide key insights into the biology of how certain cancer cells evade radiation-induced death.
Our work provides proof of principle for a novel method of enrichment for radiation tolerant medulloblastoma cells isolated early after gamma ray exposure. We further demonstrate that these tolerant cells retain tumor initiating properties. Success with an initial candidate set has provided insight into the expression of genes related to stem-like biology in cells that survive radiation exposure. The observed elevation of ABC transporter expression in these cells has implications for the order in which treatments are delivered to patients, as increased efflux pump activity may reduce the efficacy of chemotherapeutic drugs that are delivered after radiotherapy.
For the first time, functional data is presented suggesting that ABC transporter proteins contribute to cellular radiation protection mechanisms. Our in vitro studies suggest that multiple ABC transporter proteins likely act in concert to contribute to radiation tolerance in human medulloblastoma cells. The fact that broad spectrum inhibitors significantly sensitize cells, whilst those targeting individual ABC transporters do not, suggests an overlap in the roles of the various family members involved in this newly detected mechanism for radiation protection.
Although clinical trials of ABC transport inhibiting compounds aimed at circumventing chemotherapy resistance initially hit obstacles, promising “new generation” anti-ABC transporter drugs are now at various stages of pre-clinical and clinical assessment [9, 73, 74]. Our work suggests such compounds, if they inhibit multiple transporters, could potentially provide additional benefits for patients if they are delivered during radiation as well as chemotherapy treatment.
Most importantly we also show, through analysis of publically available gene expression data sets, that certain ABC transporters contribute to gene expression signatures for particular molecular subtypes of medulloblastoma, a finding which could be exploited in future prognostic tests.
Materials and methods
Medulloblastoma patient tissue, lines and culture conditions
Human medulloblastoma patients for which tumor tissue was used for gene expression and/or culture experiments
Pathology at diagnosis
Medulloblastoma, poorly differentiated classic
Reported by independent pathologists as 1) Medulloblastoma and 2) unusual AT/RT-like tumor (without INI1 loss)
Medulloblastoma, classic, with metastatic nodule nearby
Medulloblastoma, with extensive nodularity
Cells were seeded and allowed to adhere for two to five hours prior to radiation delivery via a Gammacell 40 irradiator (Nordion). For survivor cell enrichment, cells were exposed to a single 10 Gy dose (except for R026M for which 60 Gy was used) and FACS performed three days after radiation exposure. Populations undergoing repeated weekly radiation exposure were rested for one week after the final dose, before gene expression analysis was undertaken. This delay was to ensure that any potential “stress effects” of the radiation treatment itself would not confound analysis of the surviving populations.
Immunofluorescence and flow cytometry
PS externalization was detected using Annexin V conjugated to Alexa 488 or Pacific Blue (#A13201 or #A35122, Invitrogen). Dead cells were detected with PI or 7-ADD (#P21493 or #A1310, Invitrogen). Immunofluorescence was performed using standard methods after fixation with 4% paraformaldehyde. A FACS ARIA Cell Sorter was used for cell isolation and a FACS Canto II for general flow cytometry (both BD Biosciences). ABCG2 was detected with an Alexa Fluor 700 labeled antibody (#332016; BioLegend) and ABCA1 with a DyLight 488 conjugate (#NB400-105G; Novus Biologicals).
Treatment with channel blocking compounds
Verapamil (R and S mixture), R-verapamil and S-verapamil were from Sigma-Aldrich (#V4629, #V106 and #V105 respectively). Other inhibitors were also obtained from Sigma-Aldrich, except for the ABCG2 specific blockers fumitremorgin C (FTC) and Ko143, which were from Enzo Life Sciences (#ALX-350-127-C250 and #BML-EI396-0025) . Stocks were prepared in ethanol or dimethyl sulfoxide (DMSO) as appropriate, and the carrier used as a control. Compounds were used at 50 μM unless otherwise stated, with the exception of FTC, Ko143 and reserpine, which were routinely used at 10 μM. Drugs were added 1 to 2 hours before radiation treatment for inhibitor screening. Media was changed post-irradiation and fresh drug added, to circumvent potential damage to compounds from radiation itself, and fresh media/drug was again added after 24 hours.
Assessment of cell viability and colony forming ability
Total cellular viability assays were performed in 96 well plates using the resazurin based “CellTiter-Blue” assay (#G8081, Promega). A minimum of six replicate assays were performed per treatment and cells were incubated for five to nine days post-treatment, until control wells were 90% confluent. Fluorescence was measured using a Fluorocount reader (Perkin Elmer).
Colony formation was assessed using the Clonogenic Colony Forming Assay (CCFA). Cells were treated after seeding at low density then incubated (six days for UW228, seven days for Daoy and 28 days for R001). A minimum of five 25 cm2 flasks were used per treatment, such that at least 2500 cells were assessed. Colonies were counted and sized from high resolution whole flask digital photographs with automated image analysis software (Analysis LS Research, Olympus). Clusters were not counted as colonies unless cells had achieved five to six population doublings (minimum mean diameter of 0.4 mm) .
RNA and gene expression analysis
RNA was prepared with RNeasy columns (#74104, Qiagen). Synthesis of cDNA was carried out with Superscript III (#18080-085, Invitrogen) and qRT-PCR was performed with an ABI 7900HT system using TaqMan gene expression assay primer-probe sets (Applied Biosystems). Relative expression was determined using ΔCts, from triplicate or quadruplicate reactions, using multiplexed GAPDH as the endogenous reference.
Subgroup affiliations for tumors from patients R001 and R026 were determined using nanoString-based targeted gene expression profiling as previously described . Normalization of nanoString data and subgroup prediction were performed using the R statistical environment (v 2.15), as previously described .
The publicly available medulloblastoma gene expression array data used for initial analysis was obtained from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/; identifier GSE10327 ) and analyzed using the NCBI GEO2R package. Significance testing was performed conservatively using the Benjamini and Hochberg multiple-testing correction, to minimize the false discovery rate .
Validation data from three further cohorts (“Boston” , “Toronto”  and “Heidelberg” [42, 43]), were analyzed using R2 (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi). The KEGG geneset annotated as “ABC transporters” was visualized using heat maps according to subgroup affiliation and in normal tissue if applicable. One-way ANOVA and post-hoc comparisons were used to determine gene expression differences across subgroups and between subgroups, respectively. P-values below 0.05 were considered significant.
Murine xenograft models for in vivo human tumor growth
Orthotopic cerebellar xenografts were performed using a stereotaxic platform and micro-drill to make a burr hole 1 mm lateral to the sagittal suture and 1 mm posterior to the lambdoidal suture. 100,000 cells were then injected 3 mm below the skull surface using a Hamilton syringe (delivered over three minutes and needle withdrawn slowly after a further minute). For in vivo tumorigenicity testing after in vitro verapamil exposure, 50 μM of drug was added to cells in culture and media changed and fresh drug added after 24 hours. The cells were then incubated for a further four days before trypsinization and delivery of 200,000 live cells per injection. Cells were delivered subcutaneously in the lateral mid-flank, in a 200 μl volume with 50% matrigel (#356234, BD Biosciences). Mice were examined for presence of palpable tumors twice per week thereafter. All experiments used female severe combined immunodeficiency mice. Animal studies were performed in accordance with approvals obtained from the Health Sciences Animal Ethics Committee of The University of Queensland (approval #RCH/118/09).
Significance testing of large scale gene expression data is outlined above. For other experiments, statistical analysis was performed using GraphPad Prism5 software. For qRT-PCR data (presented as the level of expression for the gene of interest relative to GAPDH), significance was assessed using the ratio t-test (two-tailed paired t-test on log transformed data). Error bars on CCFA and viability assay graphs represent the standard deviation (SD) for the numerator (treated) and denominator (control) components of each survival calculation, combined (as percentages of the associated means) using the “square root of the sum of squares” technique. Tumor formation in mice over time was assessed using the log-rank (Mantel-Cox) test. For all other experiments, p-values were derived using two-tailed t-tests.
Clonogenic colony forming assay
Central nervous system
Cancer stem-like cell
Dulbecco's modified eagle medium/nutrient mixture F-12
Fluorescence-activated cell sorting
Fetal bovine serum
Quantitative real-time PCR
This work was supported by the Royal Children’s Hospital Foundation (Brisbane, Australia), the Brain Foundation (Australia) and the Clive and Vera Ramaciotti Foundation (Australia). Specimens were obtained via the Queensland Children’s Tumour Bank (Brisbane, Australia). MDT is supported by funds from the Garron Family Chair in Childhood Cancer Research at The Hospital for Sick Children and The University of Toronto, and operating funds from the Canadian Institutes of Health Research, The Terry Fox Research Institute, the National Institutes of Health (R01CA159859 and R01CA148699) and the Pediatric Brain Tumor Foundation. MR is supported by a fellowship from the Mildred Scheel Cancer Foundation. We thank Prof. James Olson (Fred Hutchinson Cancer Research Center, Seattle) for additional medulloblastoma samples, Prof. Brent Reynolds (University of Florida) for guidance on tumorsphere culture and Patricia Lusby for assistance with sample preparation.
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