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CANCER STEM CELLS |
Departments of aPathology,
bNeurosurgery, and
cOncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA;
dDepartment of Biotechnology and Biosciences, University of Milan Bicocca, Milan, Italy;
eIstituto Nazionale Neurologico "Carlo Besta," Milan, Italy
Key Words. Hedgehog • Glioma • Stem cell
Correspondence: Charles G. Eberhart, M.D., Ph.D., Johns Hopkins University School of Medicine, Department of Pathology, 720 Rutland Avenue, Ross Building 558, Baltimore, Maryland 21205, USA. Telephone: 410-502-5185; Fax: 410-955-9777; e-mail: ceberha{at}jhmi.edu
Received on March 7, 2007;
accepted for publication on June 29, 2007.
First published online in STEM CELLS EXPRESS July 12, 2007.
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ABSTRACT |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Hh ligands are secreted glycoproteins that bind the cell surface receptor Patched (Ptch). Ligand binding to Ptch relieves its inhibition of Smoothened (Smo) and allows signaling to proceed. Smo activates the canonical Hh pathway through Gli-dependent transcription of multiple targets, including N-myc, cyclin D, Ptch, Gli1, and Gli2 [13, 14]. Gli proteins are large, multifunctional transcription factors, and their activities are tightly regulated (reviewed in [15, 16]). Hedgehog pathway blockade has emerged as a promising therapy for multiple types of cancer, including brain tumors and carcinomas of the prostate, digestive tract, and lung [17–21]. In these latter tumors, the Hh pathway is activated predominantly by ligands, rather than by the Ptch mutations seen in medulloblastoma.
Because Hh signaling plays a critical role in non-neoplastic stem cells, it has been suggested that stem-like neoplastic cells may also be susceptible to Hh pathway blockade. The nature of such cancer stem cells, and how one might target them therapeutically, has been the subject of several recent reviews [22–26]. In brief, the cancer stem cell hypothesis suggests that only the stem cell compartment in tumors is capable of unlimited self-renewal and that elimination of these cells will ultimately halt neoplastic expansion, as better-differentiated cells have limited mitogenic capacity and will not contribute to long-term tumor growth. The first well-documented example of cancer stem cells was in leukemia, where only a small fraction of the tumors identified by expression of stem-cell markers possessed the capacity for tumor propagation [27, 28]. Similar cells have subsequently been isolated from brain tumors using markers such as CD133 and side population, which were originally developed to study non-neoplastic stem cells [29–33]. The study of stem cells in gliomas also benefits from the capacity of these tumors to grow as neurospheres, allowing the clonogenic potential and differentiation capacity to be measured [33, 34]. In this study, we demonstrate that markers of Hh pathway activity are detected in malignant gliomas samples and that the pathway appears to regulate the stem cell fraction in GBM cell lines.
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MATERIALS AND METHODS |
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Cell Culture
The U87-MG, A172, U251, and SW1088 cell lines were obtained from American Type Culture Collection (ATCC) (Manassas, VA, http://www.atcc.org) and maintained in ATCC's recommended growth medium supplemented with 10% fetal bovine serum (FBS). The CJ-MG and BK-MG cell lines were a kind gift from Dr. Carol A. Kruse [53]. JHH-GBM2 was derived as follows: fresh tumor was chopped to small pieces using sterile razor blade and then mechanically triturated through a 21-gauge needle and filtered through a 40 µm cell strainer (BD Falcon; BD Biosciences, Bedford, MA, http://www.bdbiosciences.com). Cells were concentrated by centrifugation and then seeded in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with 10% FBS. The glioblastoma-derived neurosphere line HSR-GBM1 was derived and propagated as previously described [54]. 293T, 293T/SHH-N', and Light2 cells were the kind gift of Dr. Phil Beachy.
The Gli reporter assay was performed in either U87-MG by transient transfection of the Gli firefly luciferase reporter construct or in NIH3T3 cells stably transfected with Gli firefly luciferase and Renilla luciferase reporter constructs (Light2), as previously described with minor modifications [55]. Briefly, Light2 cells were plated in a 24 well plates at 7 x 104 cells per well in DMEM supplemented with 10% FBS. When confluent (normally the next day), the monolayers were washed once each with phosphate-buffered saline (PBS) and DMEM supplemented with 0.5% FBS. Cells were incubated overnight in a humidified incubator kept at 37°C, 5% CO2, and then growth medium was removed and monolayers were overlaid with conditioned medium from 293T, 293T/Shh-N', or HSR-GBM1 and incubated for an additional 48 hours. Gli reporter activity was quantified using the dual-luciferase reporter assay system (Promega, Madison, WI, http://www.promega.com) and normalized to Renilla luciferase activity. In U87-MG, Gli reporter activity was quantified using the luciferase reporter assay system (Promega) and normalized to total protein.
For mRNA quantification and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays, cells were plated in six-well plates (RNA) or 96-well plated (MTS) and incubated overnight, and the monolayers were washed once with PBS, followed by overlay with low-serum (0.5% fetal bovine serum) media. The following day (approximately 16 hours after serum withdrawal), medium was replaced with medium supplemented with 0.5% FBS and either ethanol or cyclopamine (5 or 10 µM). RNA was extracted at various time points after cyclopamine addition using Qiagen RNeasy kits. MTS assays were performed at various time points using the CellTiter96 assay (Promega) according to the manufacturer's instructions.
To assay neurosphere differentiation, HSR-GBM1 cells were triturated and plated at 2 x 104 cells per cm2 in 10-cm2 cell culture dishes precoated with Matrigel (BD Biosciences) diluted 1:100 in Neurocult medium (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) without epidermal growth factor (EGF). After attachment of cells to the plate surface, medium was replaced with Neurocult medium lacking mitogens and heparin but supplemented with 1% bovine calf serum (HyClone, Logan, UT, http://www.hyclone.com). Cells were incubated for 5 days as before and then washed, and RNA was extracted as described above.
shRNA targeting Gli1 was purchased from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com). pLKO.1-puro was a kind gift from Dr. Bob A. Weinberg and Dr. Carl D. Novina [56]. U87-MG cells were plated at 2.5 x 105 cells per well or 7.5 x 105 cells per dish in six-well plates or 10-cm culture dishes, respectively. The next day, medium was replaced with 2 ml of transduction medium. Dishes were incubated overnight in humidified incubator at 37°C. RNA was extracted from cells grown in six-well plates 48 hours after infection. For cells grown in 10-cm dishes, medium was replaced 72 hours postinfection with fresh medium containing 10 µg/ml puromycin (Sigma-Aldrich). Cells were stained with 4,6-diamidino-2-phenylindole, and pictures of 10 random high-power fields were taken for each condition.
For Gli1 and Gli2 short interfering RNA (siRNA) experiments, 2 x 105 HSR-GBM1 cells were plated in duplicates in six-well plate format for RNA analysis and at 1.5 x 104 cells per well in a 48-well format for growth assays (MTS). Cells were incubated overnight and then transfected every other day according to the manufacturer's directions using RNAiFect (Qiagen) with 8 nM/well of a nonspecific siRNA (Dharmacon, Lafayette, CO, http://www.dharmacon.com) as negative control, Gli1 pooled siRNA, or a single Gli2 siRNA duplex (Dharmacon) with the following oligo sequences: Gli1 forward, 5'-GGAAAUGACUGGCAAUGCAUU-3', and Gli1 reverse, 5'-PUGCAUUGCCAGUCAUUUCCUU-3' for duplex 1; Gli1 forward, 5'-GCACUGGUCUGUCCACUCUUU-3', and Gli1 reverse, 5'-PAGAGUGGACAGACCAGUGCUU-3', for duplex 2; Gli1 forward, 5'-GUCCUCACUUGAACAUUAUU-3', and Gli1 reverse, 5'-PUAAUGUUAAGUCGAGGACUU-3', for duplex 3; Gli1 forward, 5'-AGGCUCAGCUUGUGUGUAAUU-3', and Gli1 reverse, 5'-PUUACACACAAGCUGAGCCUUU-3', for duplex 4; Gli2 forward, 5'-CGUCAACCCUGUCGCCAUUUU-3', and Gli2 reverse, 5'-PAAUGGCGACAGGGUUGACGUU-3'.
Clonogenic Assays
Clonogenic assays were performed as previously described [57]. Briefly, U87-MG cells plated at 3 x 103 cells per well. Cells were treated with either ethanol (vehicle [V]) or cyclopamine (5 and 10 µM). Each condition was tested in triplicate, and each assay was repeated twice with similar results. Colony number and size were scored with the ChemiDoc-XRS imager, using the QuantityOne software package (Bio-Rad, Hercules, CA, http://www.bio-rad.com).
HSR-GBM1 cells were seeded in T75 cm2 culture dishes at 1 x 105 cells per dish. Cells were treated with either ethanol or cyclopamine (10 µM) in a final volume of 20 ml of Neurocult medium (Stem Cell Technologies) supplemented with 0.002% heparin, 10 ng/ml human EGF, and 10 ng/ml human fibroblast growth factor-b (Peprotech, Rocky Hill, NJ, http://www.peprotech.com). Dishes were incubated upright for 7 days in a humidified incubator with 5% CO2 at 37°C. Cells were retreated with 2 ml of medium containing the indicated concentration of drug on days 2, 4, and 6. Seven days after treatment, spheres were triturated and replated in T25 cm2 at 2 x 103 cells per dish. Subsequent sphere formation was monitored and scored by light microscopy after 8 days. Ten random fields were photographed for both vehicle and cyclopamine-treated conditions, and the number of spheres over 50 µm in size was scored.
RNA Analyses
RNA levels were analyzed by real-time PCR analysis performed in triplicate with SYBR Green reagents (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com), according to the manufacturer's instructions on an I-Cycler IQ real-time detection system (Bio-Rad) as previously described [58]. To minimize contaminating genomic DNA, a 15-minute on-column DNase step (RNase-free DNase kit; Qiagen) was included during RNA extraction. In addition, all primer sets amplify a single product of the appropriate size and were designed to cross intron-exon boundaries. Oligo sequences were previously described [52].
Xenograft and Side Population Assays
For side population analyses, U87-MG and C6 cells were treated for 7 days in low-serum (0.5% FBS) media containing either ethanol (V) or cyclopamine (5 or 10 µM), with medium changes every 2 days. HSR-GBM1 cells were treated in 10 ml of media, with 2 ml of additional medium containing drug or vehicle supplemented every 2 days. Side population analyses on treated cells were performed as previously described [57]. For xenograft studies, HSR-GBM1 cells were treated as described above, and an aliquot collected from each flask following treatment was scored for viability by Guava-PCA flow cytometry system and ViaCount reagent, according to the manufacturer's instructions (Guava Technologies, Hayward, CA, http://www.guavatechnologies.com). Next, viable cells were diluted with fresh medium and injected over 10 minutes into the right striatum of athymic (nu/nu) mice (Harlan, Indianapolis, IN, http://www.harlan.com). Mice were monitored daily and sacrificed at the first indication of tumor development (ataxia, seizure, lethargy, or cachexia). Brains were surgically removed and fixed immediately in formalin before submission for histological analysis, as previously described [58].
Radiation Treatment
HSR-GBM1 cells were plated at 5 x 103 cells per well (in triplicate) in 48-well culture plates and treated with either ethanol (0) or cyclopamine (5 or 10 µM). After 3 days of growth, the cultures were irradiated at either mock (0 Gy) or 10 Gy using a Gammacell 40 irradiator equipped with a 132cesium source (MDS Nordion, Ottawa, ON, Canada, http://www.mds.nordion.com). Cell mass was subsequently measured on day 7 using the MTS assay (Promega), and growth rate calculated from the first day of cyclopamine treatment.
For cotreatment with cyclopamine and radiation, HSR-GBM1 cells were plated at 1 x 106 cells per T75 culture flask and treated with either ethanol (0) or cyclopamine (5 or 10 µM) at plating and every other day thereafter. Cells were irradiated 4 days after plating, and percentages of side and aldehyde dehydrogenase (ALDH)-positive populations were determined on day 7.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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The presence of nuclear Gli1 protein expression in formalin-fixed, paraffin-embedded tumor samples would also suggest that Hh signaling is active in gliomas. We therefore examined whether Gli1 is expressed in a nonrelated set of astrocytic tumors using a previously described tissue microarray [38]. All high-grade astrocytic tumors on our tissue microarray had at least trace levels of cytoplasmic Gli1 protein (Fig. 1C, top panel). In approximately half of the tumors, cytoplasmic staining was stronger, and Gli1 was also detected in the nucleus, suggesting that the pathway was active (Fig. 1C, bottom panel). Nuclear Gli1 was detected in cytologically atypical tumor cells but not in non-neoplastic cells, such as blood vessels. We did not observe a significant difference between the percentage of cases with nuclear Gli1 among the grade III anaplastic astrocytoma (10 of 20 positive) and grade IV glioblastoma (8 of 18 positive).
Cyclopamine Decreases Gli1 Expression and Inhibits Growth and Clonogenicity of Adherent GBM Cell Lines
We used the Hh pathway inhibitor cyclopamine to test the ongoing requirement for Hh signaling in glioma cells. All experiments using this inhibitor were performed in low (0.5%) serum. In U87-MG cells transfected with an Hh reporter construct, cyclopamine reduced pathway activity in a dose-dependent fashion, with 5 µM or higher levels required to effect significant pathway inhibition (Fig. 2A). Cyclopamine administration also inhibited endogenous Gli1 mRNA expression in U87-MG cells by 21% at 5 µM and 51% at 10 µM levels (data not shown). Gli1 mRNA expression was also inhibited by 30% or more in the A172 and SW1088 glioma lines, but not in a dose-dependent fashion (data not shown). Significantly, Hh pathway inhibition using cyclopamine resulted in reduced growth in lines with high baseline levels of Gli1 expression but not in cells lacking significant Hh activity. In the high-Gli U87-MG and A172 lines, growth was essentially abolished by 10 µM cyclopamine, whereas the increase in cell mass in the low-Gli CJ-MG and BK-MG lines was unaffected (Fig. 2B). The fact that growth inhibition was observed only in tumor lines expressing moderate or high levels of Gli1 mRNA suggests that the effects of cyclopamine treatment are Hh pathway-specific, rather than generally cytotoxic.
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To test whether the effect of cyclopamine is mediated by reducing levels of Gli1, rather than noncanonical effects on Hh signaling or nonspecific toxicity, we infected U87-MG cells with lentivirus carrying short hairpin against Gli1. Gli1 mRNA levels were reduced by 40%–50% 48 hours after virus transduction with shRNA 488, whereas a second construct (487) proved ineffective in reducing Gli1 levels (Fig. 2E, upper panel). To test the requirement for Gli1 only in cells containing the construct, we took advantage of the puromycin resistance gene in the pLKO.1-puro backbone. Consistent with an ongoing requirement for Gli1 in the glioma cells, introduction of Gli1 shRNA 488 and short-term puromycin selection significantly reduced the number of cells per high-power field, whereas virus carrying an empty vector or a nonfunctional Gli1 shRNA had no significant effect on viability (Fig. 2E, lower panel).
GBM-Derived Neurospheres Secrete and Respond to Shh Ligand
We used GBM-derived neurospheres to further examine the role of Hh in gliomas, including low-passage cells from tumors resected at Johns Hopkins Hospital and grown as neurospheres (JHH-GBM3) and a neurosphere line established at the Hospital San Raphael (HSR-GBM1), as previously described [34]. Such GBM-derived neurospheres demonstrate an infiltrative growth pattern similar to that of human gliomas in vivo and have improved karyotypic stability compared with adherent glioma lines [34, 39]. We detected expression of the transcription factors Gli1 and Gli2, as well as the receptor Smo and all three Ptch-receptor isoforms, in the GBM-derived neurospheres (Fig. 3A). Gli1 protein is also highly expressed in the infiltrating gliomas that arise when these cells are injected into athymic mice (Fig. 3B). Most of the immunopositive cells in this panel are found in or near the tumor mass and have the morphology of neoplastic astrocytes. However, some periventricular cells and scattered cells within the brain parenchyma are also positive, suggesting that some background staining of murine cells is also occurring. Interestingly, unlike the seven adherent glioma lines shown in Figure 1, the two glioma neurosphere cultures both expressed Shh mRNA, suggesting that cultured glioma cells are capable of generating this ligand when grown in this fashion (Fig. 3A).
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Hedgehog Pathway Blockade Inhibits Glioma Neurosphere Formation and Growth and Targets Radioresistant Cells
To examine the effects of Hh inhibition on the formation and growth of glioma-derived neurospheres, we treated mechanically separated spheres for 4 days with cyclopamine. Treatment using 3 µM or higher concentrations inhibited Gli1 mRNA expression by 50%–75% compared with vehicle. Cyclopamine-mediated Hh pathway blockade inhibited the overall growth rate of the culture by 30%–75% in a dose-dependent fashion (Fig. 4A). This experiment was repeated three times with similar results. Microscopic examination of these cultures showed that few well-developed neurospheres had formed during the period of Hh blockade, suggesting that the cells required to initiate or sustain neurosphere growth had been inhibited or killed. Transient transfection of siRNA directed against Gli1 but not Gli2 into the neurospheres also significantly reduced cell mass after 7 days compared with control siRNA (Fig. 4B).
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Bao et al. have previously shown that stem-like CD133-positive tumor cells in GBM are resistant to radiation therapy [41]. However, radiation is clearly effective in eliminating at least a subset of cells in GBM clinically, and any patients receiving a novel chemotherapy will also likely have their tumor irradiated. We therefore examined the effects of Hh blockade given in combination with radiation. We treated HSR-GBM1 cultures with cyclopamine alone and in addition to 10 Gy of radiation. As expected, both radiation and cyclopamine significantly reduced the GBM neurosphere growth rate as monotherapies (Fig. 4E). However, we observed a significantly lower growth rate when 10 µM cyclopamine was given in combination with 10 Gy of radiation, as compared with radiation alone. Although 5 µM cyclopamine did not significantly reduce the growth rate, a trend in this direction was seen (p = .057, two-sided t-test). These data suggest that Hh blockade may target GBM cells not affected by radiation.
Cyclopamine Targets Stem-Like Glioma Cells and Blocks Tumor Engraftment
To quantitate the effects of Hh blockade at a single-cell level, we treated GBM neurospheres with 10 µM cyclopamine for 7 days and then used immunofluorescence to examine markers of stem/progenitor cells (nestin) and glial cells (GFAP). This revealed a significant decrease in the percentage of Nestin-positive cells and an increase of more than twofold in glial differentiation (Fig. 4F–4H). We also predicted that if Hh plays an important role in maintaining stem-like cells in glioma neurosphere cultures, markers of Hh activity would be reduced when such cultures are forced to differentiate. As previously described, when EGF, fibroblast growth factor, and heparin were removed and 1% serum added, tumor neurospheres adhered to the plastic culture dish and differentiated [34]. A reduction in the expression of the proliferation marker Ki67 was observed as cells differentiated and exited the cell cycle (Fig. 5A). In addition, we observed a 2.6-fold increase in the glial differentiation marker GFAP and a 2.2-fold increase in the neuronal marker microtubule-associated protein 2. Expression of the marker CD133 was reduced more than eightfold (Fig. 5A), indicating a loss of stem cells. As we predicted, the Hh pathway targets Ptch1b and Gli1 were downregulated sixfold and ninefold, respectively, in conjunction with tumor differentiation. Surprisingly, the expression of Bmi1, a gene that is involved in stem cell self-renewal, was increased almost twofold. One possible explanation for this observation is that Bmi1 expression has been reported to be maintained at high levels even in differentiation-inducing conditions [42].
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It has recently been reported by Bao et al. that radiation treatment does not effectively target stem/progenitor cells, as indicated by increased CD133-positive cell populations following radiation exposure [41]. Radiation alone also dramatically increased the percentages of side and ALDH-positive populations in our GBM neurospheres, supporting the concept that this standard therapy is not able to kill cancer stem cells (Fig. 5F). To test the ability of Hh blockade to augment radiation therapy, we treated HSR-GBM1 cells with cyclopamine for a total of 7 days. Four days after the beginning of cyclopamine treatment, we irradiated the cells with various doses. Side population and ALDH-positive population percentages were determined on the seventh day. When given alone, cyclopamine treatment reduced the side and ALDH-positive populations from 2.6% and 4.8% to 1.6% and 1.0% at 5 µM cyclopamine and to 0.4% and 0.4% at 10 µM cyclopamine (Fig. 5F). Strikingly, in cultures treated with 10 Gy radiation and cyclopamine, side and ALDH-positive populations were dramatically reduced from 7.0% and 12.3% to 1.1% and 2.1% at 5 µM cyclopamine and to 0.1% and 0.2% at 10 µM cyclopamine.
Collectively, these data suggest that cyclopamine targets glioma stem cells and might therefore prevent tumor engraftment and long-term growth. To test this, HSR-GBM1 cells were treated with vehicle or cyclopamine for 7 days in culture before intracerebral implantation of either 1 x 103 (n = 5 animals per group) or 1 x 104 (n = 3 animals per group) viable cells. Pretreatment with cyclopamine completely inhibited tumor engraftment and growth, as all animals survived for the duration of the experiment, and microscopic analysis of the injection site failed to identify any tumor deposits (Fig. 5G, 5H). In contrast, massive infiltrative gliomas were present in all mice implanted with vehicle-treated cells, resulting in death as early as 2 months following injection (Fig. 5G).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Our data suggest that the survival of stem-like side population cells in glioblastoma is dependent on ongoing Hh pathway activity. We first demonstrated that the components required to transduce the Hh signal are present in the majority of GBM samples and that mRNA expression of the pathway target Gli1 is relatively high in approximately one-quarter of cases. Forced differentiation of GBM neurospheres reduced both stem cell and Hh activity markers. Hh pathway blockade by cyclopamine reduced growth in glioma lines with elevated pathway activity but not in those with lower levels of Gli1, suggesting that when elevated Hh signaling is detected, it is required for ongoing tumor growth. Reduction of Gli levels using siRNA also reduced the viability and growth of glioma cells. Most significantly, cyclopamine therapy dramatically reduced the side and Aldefluor-positive populations present in GBM cells, resulting in cultures no longer able to form colonies in vitro or xenografts in vivo. We also show that the effects of cyclopamine and radiation on stem-like glioma cells are quite different, with only the former able to deplete this clinical critical population.
Our findings also point to expression of Shh ligand by GBM cells as a mechanism for Hh pathway activation. We detected Shh mRNA in many primary tumor samples, and the highest level of Shh expression was found in the tumor with the most elevated Gli1 level. In mRNA extracted from the primary tumor samples, it is not possible to determine whether the ligand is generated by tumor cells, entrapped non-neoplastic glia and neurons, or stromal elements such as vessels. It is therefore significant that we found that GBM cells grown in culture as neurospheres secreted even larger amounts of active Shh into their media than 3T3 cells genetically engineered to express the ligand. This suggests that some gliomas are capable of secreting their own ligand and are not dependent on exogenous sources.
Clement et al. have recently reported findings similar to ours in several ways [50]. They show that Hh signaling is active in both adherent and neurosphere-based glioma cultures and that pathway inhibition using either cyclopamine or siRNA reduced tumor growth, the self-renewal of glioma stem cells, and in vivo tumorigenicity. Our studies differ in the markers used to evaluate cancer stem cells, as they used CD133, whereas we examined side population and aldehyde dehydrogenase-expressing cells. We also demonstrate for the first time that malignant glioma cells are capable of secreting biologically active Shh ligand, suggesting antibody-mediated ligand depletion as a potential therapy. Also, our results are partially consistent with the report by Ehtesham et al., who found that Hedgehog activity is confined to progenitor cells within gliomas [51]. However, we detected Hedgehog pathway activity in grade IV gliomas, a finding that is in contrast to theirs. This discrepancy may be explained by the fact that gliomas are extremely heterogeneic, so differences in pathway activity may relate to sample location within the tumor. Taken together, our studies provide a strong rationale for testing Hh inhibitors in patients with malignant gliomas.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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