- Email: [email protected]
Models of malignant glioma
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. 3, No. 2 2006
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK
Cancer
Models of malignant glioma Ken Aldape1,*, Howard Colman2, C. David James3 1
Department of Pathology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, United States Department of Neuro-Oncology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, United States 3 Division of Experimental Pathology, Mayo Clinic, 200 First St, Rochester, MN 55905, United States 2
Malignant gliomas are biologically and clinically aggressive neoplasms with a high mortality rate. Recent therapeutic advances have been modest and incremen-
Section Editors: Nikolina Vlatovic – University of Liverpool, Liverpool, UK Mark T. Boyd – University of Liverpool, Liverpool, UK
tal at best. An increased understanding of the factors, which promote progressive biologic and clinical behavior, is needed to improve upon current therapeutic modalities. Model systems of glioma, which accurately reflect the biology of the human disease, show promise as a means to accomplish this goal. Introduction Primary brain tumors, especially malignant gliomas, are among the most aggressive of human tumors. Among the gliomas, the infiltrating glial neoplasms are nearly universally fatal, owing to a combination of their infiltrative nature and relative resistance to existing therapeutic modalities. The infiltrating gliomas are subdivided into the astrocytomas and oligodendrogliomas, according to the morphologic resemblance that the tumors exhibit relative to normal glia. Of these, the astrocytic tumors are more common. High-grade astrocytomas are among the most lethal neoplasms in humans. Glioblastoma (grade IV astrocytoma) carries a median survival time of 1 year, with less than 5% of patients alive 5 years after diagnosis. Model systems, especially those which closely mimic the human tumor, provide an important opportunity to investigate the biology of molecular genetic lesions, which confer the aggressive clinical behavior. In addition they provide a preclinical system to test new agents directed against these lesions. A recent exchange [1,2] debating the merits of xenografts of genetically engineered models
*Corresponding author: K. Aldape ([email protected]) 1740-6757/$ ß 2006 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2006.06.002
for preclinical cancer models has enlivened interest in this topic for gliomas. Because most glioma model systems are based in glioblastoma (GBM), this will be the subject of this review.
In vitro models of glioma Established cell lines Cell lines are well established as in vitro model systems of glioma. The simplest in vitro models of glioma are cultures of human or rodent tumors growing two-dimensionally on plastic. A large number of glioma lines are available, nearly all from the highest glioma grade, glioblastoma. Attempts at developing immortalized gliomas of lower grade have been of limited success. Long-term cultures of human gliomas are limited in their usefulness for our understanding of glioma biology use, owing to the requirement for artificial growth conditions, including high oxygen content and animal sera for growth. Serum is a particular problem when one considers the blood–brain barrier as a means to restrict access of glial cell to serum in vivo (although the blood–brain barrier tends to be lost in high-grade gliomas). A further limitation is that they lose glial or neuroepithelial characteristics (e.g. loss of GFAP expression) after several passages and acquire mesenchymal characteristics (e.g. acquisition of fibronectin and collagen expression). One large study found GFAP expression in only 6/60 glioma cell lines [3]. It is probable that even the GFAP-positive lines that do exist have acquired a significant mesenchymal character. This study further found that tumors derived from oligodendrogliomas did not express 191
Drug Discovery Today: Disease Models | Cancer
antigens reminiscent of these tumors. These problems render it very difficult to extrapolate from in vitro data to the human condition using established glioma cell lines. A finding of an in vitro phenomenon, using established glioma cell lines without additional evidence, cannot be assumed to be operative in human tumors. Better uses of glioma cell lines involve testing of hypotheses originated from more representative models, such as in vivo models, or ideally the human tumors themselves, where findings can be extended from mere association studies (e.g. identification of a biomarker associated with radioresistance) to causality, where addition or removal of the biomarker in a controlled experiment actually affects radioresistance in cultured cells. Given the heterogeneity of gliomas and cell lines derived from them, confirmation of findings in more than cell line is prudent. An alternative culture system of human glial neoplasia in vitro involves the use of genetically defined cultured astrocytes. Genes known or suspected to affect cell growth and/or tumorigenesis are introduced into astrocytes derived from normal CNS tissue. For example the addition of the human papilloma virus E6 and E7 proteins (known to inhibit p53 and pRb, respectively) plus the addition of the hTERT (telomerase) cDNA into normal human astrocytes (NHA) results in immortalization of the astrocytes. These immortalized NHA cells are nontumorigenic when introduced into immunocompromised mice, but become so on addition of activated Ras oncogene. Addition of constitutively active Akt to the NHA-E6-E7-hTERT-Ras cells render in more rapidly tumorigenic [4,5]. The NHA-E6-E7-hTERT cells can therefore be useful as an ‘indicator line’ when it is desired to test whether addition of a gene or genes promotes tumorigenesis. However, some of the limitations described above (e.g. acquisition of mesenchymal characteristics) apply as well to this system. Three-dimensional cultures using a system in which the cells do not attach to a surface have been employed [6]. Such ‘spheroids’ have the potential advantage that cell–cell contact is enhanced, and many of the tumor cells are not in direct contact with the serum (similar to tumors). Such spheroids have been used as confrontation cultures. In confrontation cultures, cells are grown in suspension, where they aggregate into spheroids. Spheroids from tumor cells are then place in wells in which aggregates of rodent brain cells have formed. Tumor spheroids attach and invade the rodent brain cell aggregates. This process can be assessed semiquantitatively as a measure of invasion [7].
Cancer stem-like cells Many tumors, including gliomas, are composed of a heterogeneous set of cells. Analogous to the role of stem cells in the development of normal tissues, the cancer stem cell hypothesis proposes that many cancers might be initiated and maintained by a small subset of relatively quiescent cells with properties reminiscent of normal stem cells [8,9]. The 192
www.drugdiscoverytoday.com
Vol. 3, No. 2 2006
major stem cell properties are self-renewal, unlimited but tightly regulated replication and a broad range of differentiation potential. These stem cells have the ability to give rise to a hierarchy of progeny that ultimately comprise the bulk of the tumor. Data indicating that such cancer stem cells exist and comprise only a fraction of the total cells in a cancer that has come from number of neoplasms [10,11]. Glioma stem cells derived from human tumors have been successfully expanded in vitro using serum-free suspension cultures. These glioma stem cells have the ability to ‘differentiate’ into cells expressing both astrocytic and neuronal markers, and form tumors that recapitulate the morphology of the original tumor when injected into immune-deficient mice [12,13]. A subset of these cells purified by the cell-surface marker CD133 has been shown to be the tumor-initiating cells in a xenograft assay [14]. These findings suggest that the ability to isolate and purify tumor-initiating stem cell-like cells from human gliomas might provide a more biologically relevant model than existing adherent cell lines for studying the biology of these tumors in vitro and in xenograft models. In addition, several properties of cancer stem cells including high expression of drug transporters, active DNA repair, resistance to apoptosis and relative quiescence compared with other cells in the cancer indicate that the stem cells might also represent the more treatment resistant cells in a cancer. Although definitive data regarding these issues in glioma stem cells are still lacking, these observations suggest that better understanding of the mechanisms of resistance in the glioma stem cells might provide insights into one of the most vexing clinical problems in this disease, namely the almost universal ability of these tumors to recur, even from the minimal residual disease setting after radiation and chemotherapy, in a highly aggressive, resistant and lethal form.
In vivo models of glioma Xenograft and syngeneic models Most xenograft models of glioma employ the use of established human tumor cell lines injected into immunocompromised mice or rats. Potential advantages of using a wellestablished glioma cell line as an in vivo model include the ability to perform experiments longitudinally using the same cell line, as well as reproducible growth kinetics (i.e. a reproducible time between injection and tumor development). Xenograft experiments are commonly performed using either a subcutaneous injection or intracranial injection of tumor cells, which the latter being considered more ‘physiologic’. Although tumors created from cell lines within the rodent brain have a purpose for selected preclinical studies, they have important limitations. One is the compromised immune system of the host, rendering them of little use for immunologic studies. Additionally, they are limited both with respect to the long-term passage issues raised earlier (loss of glial markers an acquisition of mesenchymal
Vol. 3, No. 2 2006
markers), as well as phenotypic considerations. In particular, although a hallmark of diffuse glial neoplasms in single cell infiltration of brain parenchyma, established cell lines invariably do not show single cell infiltration of tumor cells. In fact, xenografts of established glioma lines are morphologically more similar to brain metastases, where infiltration is seen by groups of cells, along vessels. Syngeneic transplantable models (such as the C6 gliosarcoma model in Fischer rats) have an advantage in that the host immune system is not compromised, but otherwise suffer from considerations similar to xenograft models. An orthotopic GBM xenograft test panel approach that has been recently described [15] and applied to therapeutic testing [16], employs an unconventional means for sustaining tumor cell growth before harvesting of tumor cells for intracranial injection. Specifically, tumor cells are maintained as subcutaneous, heterotopic xenografts, rather than as continuous cell lines. Propagating human GBM in this way is now known to promote the retention of crucial GBM molecular and biologic characteristics that are lost or suppressed through sustained cell culturing. The subcutaneous propagation of GBM promotes the retention of an extremely important biologic property of GBM, namely tumor invasiveness (Fig. 1). By contrast, most intracranial tumors established from permanent cell lines [17–19] produce noninvasive lesions. The lack of EGFR-amplified GBM cell lines has additionally hindered the investigation of EGF receptor mutants, such as the commonly occurring EGFRvIII [20,21]. In contrast to the lack of EGFR mutations in GBM cell lines, mutant EGFR can be sustained through serial passaging of subcutaneous GBM xenografts [22,23]. The retention of GBM xenograft intracranial invasiveness following sustained subcutaneous passaging has been independently reported by two groups [15,24] thereby, lending good support of this being a reproducible characteristic. One of these groups has additionally shown that short-term culturing of subcutaneous explants does not have an apparent effect on tumor cell intracranial invasiveness, and provides an additional benefit of being able to harvest and deliver defined numbers of cell to multiple mice. Tumors established in this way show a consistent rate of growth, as indicated by the relatively narrow ranges in survival among mice receiving intracranial injection from a common explant culture [15]. Models, which utilize small pieces of flank tumor xenografts [24], might display less consistency. An additional key aspect associated with the subcutaneous serial propagation of GBM xenografts, concerns their retention of patient tumor genetic signature lesions. The retention of patient tumor EGFR amplification status has already been discussed, but in fact all common gene alterations that occur in patient tumors have been shown to be retained in serially passaged GBM xenografts [23,25,26]. Consequently, the
Drug Discovery Today: Disease Models | Cancer
Figure 1. Growth patterns of intracranial xenographs in full coronal section (left) and corresponding with morphologic features of neoplastic cells in areas of maximal tumor density (right). Tumors are maintained serially in vivo, without extensive passaging in vitro. Diffuse infiltration of tumor cells can be seen (for example A1 and D1). Histologic features including abnormal mitoses (B2, C2) and the pseudopalisading necrosis (C2) can be appreciated in this model system. Reprinted from Ref. [15] with permission from the author.
immunocompromised rodents used for the heterotopic propagation and serial passaging of GBMs can be viewed as reservoirs for providing a continuing supply of individual tumors, and the information derived from the molecular characterization of an established xenograft line should prove increasingly meaningful over time. Earlier methods using patient surgical material [27,28], and tissue spheroids [29,30], do not allow for propagation of a renewable tissue resource.
Genetically engineered mouse models A variety of both transgenic and knockout models of cancer in mice have been developed. Models, which utilize widespread changes in oncogenes and tumor suppressor genes www.drugdiscoverytoday.com
193
Drug Discovery Today: Disease Models | Cancer
Vol. 3, No. 2 2006
Table 1. Comparison of glioma models In vitro models
In vivo models
In silico models
Pros
Easily to grow Ideal to test effects of single gene changes Wide range of available assays
More ‘physiologic’, especially with spontaneous models and new short-term culture xenograft models
Wealth of data for mining to generate new hypotheses
Cons
Established cell lines have little similarity to the tumor from which they were derived Some key alterations (EGFRvIII) are not maintained in culture Tumor stem-like cells are difficult to characterize
Spontaneous models might require expensive (e.g. imaging) techniques Some xenograft models do not display features of human gliomas
Large amounts of data are difficult to pare down into specific questions Risk of ‘false positives’ when using high throughput systems
Best use of model
Testing effect of single genes on a specific phenotype; initial studies of novel therapeutics
Preclinical studies of drug efficacy; studies of gliomagenesis
Identification of novel biomarkers and therapeutic targets
Refs
[2–13]
[14–18,27,30–35]
[36–37]
often do not result in gliomas, even when such genes have been strongly associated with glioma formation (e.g. p53 loss). The reasons for this are not fully clear; although one factor could be that such mice might develop and die from other cancers before a glioma became evident. Several transgenic models have been described [31–36], which utilize a tissue-specific promoter (e.g. GFAP or S-100) to drive expression of an oncogene designed to induce or promote glioma formation. A model which utilizes trangenesis but offers somewhat more experimental flexibility involves a mouse, which expresses the RCAS viral receptor on the cell surface. Genes of interest are subcloned into an RCAS viral vector, and then injected into the brains of these transgenic mice [33].
In silico models of glioma Advances have been made in the molecular subclassification of glioma. Some progress has been made in the classification of glioblastoma, where recent reports suggest that molecular subclasses of this tumor exist [37,38]. These subclasses show different clinical behavior, and also suggest the concept that treatment for these might require optimization based on the molecular profile of the tumor. It is certain that data from these and future studies will be mined to identify such molecular subclasses and also suggest novel targets for therapy. The increasing availability of publicly accessible expression array data (required as a condition of acceptance in many journals) will facilitate data mining to identify new genes and pathways of importance. Web portals such as Oncomine are available which provide a central resource for data for this purpose (http://www.oncomine.org/main/ index.jsp).
Model comparison Each of the existing models has specific advantages while also exhibiting specific limitations (summarized in Table 1). In vitro models, based on established cell lines, are of particular use when testing hypotheses derived from ‘higher order’ models (or human tumors themselves), as well as initial 194
www.drugdiscoverytoday.com
screening of potential therapeutic agents. Their ease of manipulation allows for well-controlled experiments designed to tease out the mechanisms of specific phenomena. One must be cautious, however, on the use of these models as hypothesis generation tolls, because many established glioma lines bare little resemblance to the tumors from which they were derived. Studies using newer in vitro culture techniques, especially that of cancer stem-like cells are in their infancy and might show promise as a means to understand and target cells responsible for gliomagenesis. A niche remains for traditional xenografts of established cell lines, particularly in the early evaluation of the potential of key cancer genes to regulate tumorigenesis as well as in early preclinical drug screening. The reproducible latency of tumor take and animal survival increases the feasibility of early studies in this regard. Their inability of xenografts using established glioma lines to recapitulate the invasive behavior of human gliomas limits the interpretation, however, especially of agents, which might target this phenotype in gliomas. Newer xenograft methods using animal passaging of fresh human glioma samples show promise in this regard and might overcome this limitation. Spontaneous models, using genetically engineered approaches have distinct advantages, which include knowledge of the initiating genetic lesions, immunocompetence of the mice and recapitulation of the human disease on the morphologic level. Drawbacks include the potential for a long latency and heterogeneity with respect to tumor development, which can be challenging for preclinical studies.
Conclusions Models of human glioma have been used successfully to elucidate key features of molecular pathogenesis as well as identifying potential therapeutic targets. However, given that the relative lack of progress in the successful treatment of these tumors needs to be understood more about them. Standard in vitro models (established cell lines grown on plastic) provide limited information about tumors as a whole and are useful primarily for questions related to the function of specific candidate genes. Although traditional xenograph
Vol. 3, No. 2 2006
Drug Discovery Today: Disease Models | Cancer
References Links Brain Tumor Progress Review Group: http://www.ninds.nih.gov/ find_people/groups/brain_tumor_prg/index.htm.htm NCI-sponsored information on brain tumors: http:// www.cancer.gov/cancertopics/types/brain/ ABC2 (foundation dedicated to new therapies for glioma): http://www.abc2.org/statistics.shtml MD Anderson web site with brain tumor information: http://www.mdanderson.org/diseases/braincancer/ Oncomine, a web portal which compiles microarray data from cancer specimens: http://www.oncomine.org/main/index.jsp
models have disadvantages (e.g. lack of tumor infiltration into surrounding brain tissue), they remain useful for early preclinical studies. The recent identification of cancer stemlike cells opens up new investigative avenues, which might improve our ability to target these tumors effectively. However, the field of cancer stem-like cells is in its infancy, and needs to be understood more with regard to the characterization, propagation and biology of these cells. New techniques in the establishment of xenograph models include the use of fresh human tumor specimens, followed by a short time in culture before injection into a rodent brain. Further characterization of these novel xenograph models will probably lead to improved preclinical studies for the testing of novel targeted agents. Finally, the development of new spontaneous models of the glioma, using transgenic and gene knockout approaches will further enhance our understanding of specific molecular factors which cause and/or phenotypically modulate this disease. On a practical level the utilization of a model appropriate for the question at hand is prudent. The ease of manipulation of in vitro models (especially established cell lines) ensures their continued use, and despite their limitations, they have a role is addressing specific hypothesis-driven or mechanistic questions. Studies, which are more exploratory, address questions regarding gliomagenesis, and require systems that incorporate tumor–stromal interactions and require the use of models more akin to the human disease. Advances in methods to image the CNS of small animals to characterize neoplastic processes will help significantly to promote the incorporation of these models into biologic and preclinical studies of glioma.
Outstanding issues Generation of additional models, which recapitulate the human disease genetically and morphologically. Utilization of models for to test for target inhibition after treatment with novel small molecule inhibitors. Development of models for low-grade gliomas. Increased understand of key molecular genetic abnormalities responsible for resistance to radiation and alkylator-based chemotherapy.
1 Becher, O.J. and Holland, E.C. (2006) Genetically engineered models have advantages over xenografts for preclinical studies. Cancer Res. 66, 3355– 3358 (discussion 3358–3359) 2 Sausville, E.A. and Burger, A.M. (2006) Contributions of human tumor xenografts to anticancer drug development. Cancer Res. 66, 3351–3354 (discussion 3354) 3 Kennedy, P.G. et al. (1987) Antigenic expression by cells derived from human gliomas does not correlate with morphological classification. Neuropathol. Appl. Neurobiol. 13, 327–347 4 Sonoda, Y. et al. (2001) Akt pathway activation converts anaplastic astrocytoma to glioblastoma multiforme in a human astrocyte model of glioma. Cancer Res. 61, 6674–6678 5 Sonoda, Y. et al. (2001) Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma. Cancer Res. 61, 4956–4960 6 Lund-Johansen, M. et al. (1989) Multicellular tumor spheroids in serumfree culture. Anticancer Res. 9, 413–420 7 Lund-Johansen, M. et al. (1990) Effect of epidermal growth factor on glioma cell growth, migration, and invasion in vitro. Cancer Res. 50, 6039–6044 8 Wang, J.C. and Dick, J.E. (2005) Cancer stem cells: lessons from leukemia. Trends Cell Biol. 15, 494–501 9 Zhang, M. and Rosen, J.M. (2006) Stem cells in the etiology and treatment of cancer. Curr. Opin. Genet. Dev. 16, 60–64 10 Galderisi, U. et al. (2006) Stem cells and brain cancer. Cell Death Differ. 13, 5–11 11 Singh, S.K. et al. (2004) Cancer stem cells in nervous system tumors. Oncogene 23, 7267–7273 12 Galli, R. et al. (2004) Isolation and characterization of tumorigenic, stemlike neural precursors from human glioblastoma. Cancer Res. 64, 7011– 7021 13 Yuan, X. et al. (2004) Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23, 9392–9400 14 Singh, S.K. et al. (2004) Identification of human brain tumour initiating cells. Nature 432, 396–401 15 Giannini, C. et al. (2005) Patient tumor EGFR and PDGFRA gene amplifications retained in an invasive intracranial xenograft model of glioblastoma multiforme. Neuro-oncol. 7, 164–176 16 Sarkaria, J.N. et al. (2006) Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response. Clin. Cancer Res. 12 (7 Pt 1), 2264–2271 17 Yamini, B. et al. (2004) Transcriptional targeting of adenovirally delivered tumor necrosis factor alpha by temozolomide in experimental glioblastoma. Cancer Res. 64, 6381–6384 18 Schmidt, N.O. et al. (2004) Antiangiogenic therapy by local intracerebral microinfusion improves treatment efficiency and survival in an orthotopic human glioblastoma model. Clin. Cancer Res. 10, 1255– 1262 19 Zhang, Y. et al. (2004) Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer. Clin. Cancer Res. 10, 3667–3677 20 Frederick, L. et al. (2000) Diversity and frequency of epidermal growth factor receptor mutations in human glioblastomas. Cancer Res. 60, 1383–1387 21 Aldape, K.D. et al. (2004) Immunohistochemical detection of EGFRvIII in high malignancy grade astrocytomas and evaluation of prognostic significance. J. Neuropathol. Exp. Neurol. 63, 700–707 22 Bigner, S.H. et al. (1990) Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts. Cancer Res. 50, 8017–8022 23 Pandita, A. et al. (2004) Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR. Genes Chromosomes Cancer 39, 29–36 24 Antunes, L. et al. (2000) Analysis of tissue chimerism in nude mouse brain and abdominal xenograft models of human glioblastoma multiforme: what does it tell us about the models and about glioblastoma biology and therapy? J. Histochem. Cytochem. 48, 847–858
www.drugdiscoverytoday.com
195
Drug Discovery Today: Disease Models | Cancer
25
26
27
28 29
30 31
196
Leuraud, P. et al. (2003) Correlation between genetic alterations and growth of human malignant glioma xenografted in nude mice. Br. J. Cancer 89, 2327–2332 Jeuken, J.W. et al. (2000) Genetic reflection of glioblastoma biopsy material in xenografts: characterization of 11 glioblastoma xenograft lines by comparative genomic hybridization. J. Neurosurg. 92, 652–658 Horten, B.C. et al. (1981) Xenograft of human malignant glial tumors into brains of nude mice. A histopatholgical study. J. Neuropathol. Exp. Neurol. 40, 493–511 Shapiro, W.R. et al. (1979) Human brain tumor transplantation into nude mice. J. Natl. Cancer Inst. 62, 447–453 Mahesparan, R. et al. (2003) Expression of extracellular matrix components in a highly infiltrative in vivo glioma model. Acta Neuropathol. (Berl.) 105, 49–57 Engebraaten, O. et al. (1999) Growth of precultured human glioma specimens in nude rat brain. J. Neurosurg. 90, 125–132 Weiss, W.A. et al. (2003) Genetic determinants of malignancy in a mouse model for oligodendroglioma. Cancer Res. 63, 1589–1595
www.drugdiscoverytoday.com
Vol. 3, No. 2 2006
32
33 34
35
36 37 38
Xiao, A. et al. (2002) Astrocyte inactivation of the pRb pathway predisposes mice to malignant astrocytoma development that is accelerated by PTEN mutation. Cancer Cell 1, 157–168 Holland, E.C. et al. (2000) Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat. Genet. 25, 55–57 Ding, H. et al. (2001) Astrocyte-specific expression of activated p21-ras results in malignant astrocytoma formation in a transgenic mouse model of human gliomas. Cancer Res. 61, 3826–3836 Chiu, I.M. et al. (2000) Tumorigenesis in transgenic mice in which the SV40 T antigen is driven by the brain-specific FGF1 promoter. Oncogene 19, 6229–6239 Weissenberger, J. et al. (1997) Development and malignant progression of astrocytomas in GFAP-v-src transgenic mice. Oncogene 14, 2005–2013 Freije, W.A. et al. (2004) Gene expression profiling of gliomas strongly predicts survival. Cancer Res. 64, 6503–6510 Phillips, H.S. et al. (2006) Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell 9, 157–173
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. 3, No. 2 2006
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK
Cancer
Models of malignant glioma Ken Aldape1,*, Howard Colman2, C. David James3 1
Department of Pathology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, United States Department of Neuro-Oncology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, United States 3 Division of Experimental Pathology, Mayo Clinic, 200 First St, Rochester, MN 55905, United States 2
Malignant gliomas are biologically and clinically aggressive neoplasms with a high mortality rate. Recent therapeutic advances have been modest and incremen-
Section Editors: Nikolina Vlatovic – University of Liverpool, Liverpool, UK Mark T. Boyd – University of Liverpool, Liverpool, UK
tal at best. An increased understanding of the factors, which promote progressive biologic and clinical behavior, is needed to improve upon current therapeutic modalities. Model systems of glioma, which accurately reflect the biology of the human disease, show promise as a means to accomplish this goal. Introduction Primary brain tumors, especially malignant gliomas, are among the most aggressive of human tumors. Among the gliomas, the infiltrating glial neoplasms are nearly universally fatal, owing to a combination of their infiltrative nature and relative resistance to existing therapeutic modalities. The infiltrating gliomas are subdivided into the astrocytomas and oligodendrogliomas, according to the morphologic resemblance that the tumors exhibit relative to normal glia. Of these, the astrocytic tumors are more common. High-grade astrocytomas are among the most lethal neoplasms in humans. Glioblastoma (grade IV astrocytoma) carries a median survival time of 1 year, with less than 5% of patients alive 5 years after diagnosis. Model systems, especially those which closely mimic the human tumor, provide an important opportunity to investigate the biology of molecular genetic lesions, which confer the aggressive clinical behavior. In addition they provide a preclinical system to test new agents directed against these lesions. A recent exchange [1,2] debating the merits of xenografts of genetically engineered models
*Corresponding author: K. Aldape ([email protected]) 1740-6757/$ ß 2006 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2006.06.002
for preclinical cancer models has enlivened interest in this topic for gliomas. Because most glioma model systems are based in glioblastoma (GBM), this will be the subject of this review.
In vitro models of glioma Established cell lines Cell lines are well established as in vitro model systems of glioma. The simplest in vitro models of glioma are cultures of human or rodent tumors growing two-dimensionally on plastic. A large number of glioma lines are available, nearly all from the highest glioma grade, glioblastoma. Attempts at developing immortalized gliomas of lower grade have been of limited success. Long-term cultures of human gliomas are limited in their usefulness for our understanding of glioma biology use, owing to the requirement for artificial growth conditions, including high oxygen content and animal sera for growth. Serum is a particular problem when one considers the blood–brain barrier as a means to restrict access of glial cell to serum in vivo (although the blood–brain barrier tends to be lost in high-grade gliomas). A further limitation is that they lose glial or neuroepithelial characteristics (e.g. loss of GFAP expression) after several passages and acquire mesenchymal characteristics (e.g. acquisition of fibronectin and collagen expression). One large study found GFAP expression in only 6/60 glioma cell lines [3]. It is probable that even the GFAP-positive lines that do exist have acquired a significant mesenchymal character. This study further found that tumors derived from oligodendrogliomas did not express 191
Drug Discovery Today: Disease Models | Cancer
antigens reminiscent of these tumors. These problems render it very difficult to extrapolate from in vitro data to the human condition using established glioma cell lines. A finding of an in vitro phenomenon, using established glioma cell lines without additional evidence, cannot be assumed to be operative in human tumors. Better uses of glioma cell lines involve testing of hypotheses originated from more representative models, such as in vivo models, or ideally the human tumors themselves, where findings can be extended from mere association studies (e.g. identification of a biomarker associated with radioresistance) to causality, where addition or removal of the biomarker in a controlled experiment actually affects radioresistance in cultured cells. Given the heterogeneity of gliomas and cell lines derived from them, confirmation of findings in more than cell line is prudent. An alternative culture system of human glial neoplasia in vitro involves the use of genetically defined cultured astrocytes. Genes known or suspected to affect cell growth and/or tumorigenesis are introduced into astrocytes derived from normal CNS tissue. For example the addition of the human papilloma virus E6 and E7 proteins (known to inhibit p53 and pRb, respectively) plus the addition of the hTERT (telomerase) cDNA into normal human astrocytes (NHA) results in immortalization of the astrocytes. These immortalized NHA cells are nontumorigenic when introduced into immunocompromised mice, but become so on addition of activated Ras oncogene. Addition of constitutively active Akt to the NHA-E6-E7-hTERT-Ras cells render in more rapidly tumorigenic [4,5]. The NHA-E6-E7-hTERT cells can therefore be useful as an ‘indicator line’ when it is desired to test whether addition of a gene or genes promotes tumorigenesis. However, some of the limitations described above (e.g. acquisition of mesenchymal characteristics) apply as well to this system. Three-dimensional cultures using a system in which the cells do not attach to a surface have been employed [6]. Such ‘spheroids’ have the potential advantage that cell–cell contact is enhanced, and many of the tumor cells are not in direct contact with the serum (similar to tumors). Such spheroids have been used as confrontation cultures. In confrontation cultures, cells are grown in suspension, where they aggregate into spheroids. Spheroids from tumor cells are then place in wells in which aggregates of rodent brain cells have formed. Tumor spheroids attach and invade the rodent brain cell aggregates. This process can be assessed semiquantitatively as a measure of invasion [7].
Cancer stem-like cells Many tumors, including gliomas, are composed of a heterogeneous set of cells. Analogous to the role of stem cells in the development of normal tissues, the cancer stem cell hypothesis proposes that many cancers might be initiated and maintained by a small subset of relatively quiescent cells with properties reminiscent of normal stem cells [8,9]. The 192
www.drugdiscoverytoday.com
Vol. 3, No. 2 2006
major stem cell properties are self-renewal, unlimited but tightly regulated replication and a broad range of differentiation potential. These stem cells have the ability to give rise to a hierarchy of progeny that ultimately comprise the bulk of the tumor. Data indicating that such cancer stem cells exist and comprise only a fraction of the total cells in a cancer that has come from number of neoplasms [10,11]. Glioma stem cells derived from human tumors have been successfully expanded in vitro using serum-free suspension cultures. These glioma stem cells have the ability to ‘differentiate’ into cells expressing both astrocytic and neuronal markers, and form tumors that recapitulate the morphology of the original tumor when injected into immune-deficient mice [12,13]. A subset of these cells purified by the cell-surface marker CD133 has been shown to be the tumor-initiating cells in a xenograft assay [14]. These findings suggest that the ability to isolate and purify tumor-initiating stem cell-like cells from human gliomas might provide a more biologically relevant model than existing adherent cell lines for studying the biology of these tumors in vitro and in xenograft models. In addition, several properties of cancer stem cells including high expression of drug transporters, active DNA repair, resistance to apoptosis and relative quiescence compared with other cells in the cancer indicate that the stem cells might also represent the more treatment resistant cells in a cancer. Although definitive data regarding these issues in glioma stem cells are still lacking, these observations suggest that better understanding of the mechanisms of resistance in the glioma stem cells might provide insights into one of the most vexing clinical problems in this disease, namely the almost universal ability of these tumors to recur, even from the minimal residual disease setting after radiation and chemotherapy, in a highly aggressive, resistant and lethal form.
In vivo models of glioma Xenograft and syngeneic models Most xenograft models of glioma employ the use of established human tumor cell lines injected into immunocompromised mice or rats. Potential advantages of using a wellestablished glioma cell line as an in vivo model include the ability to perform experiments longitudinally using the same cell line, as well as reproducible growth kinetics (i.e. a reproducible time between injection and tumor development). Xenograft experiments are commonly performed using either a subcutaneous injection or intracranial injection of tumor cells, which the latter being considered more ‘physiologic’. Although tumors created from cell lines within the rodent brain have a purpose for selected preclinical studies, they have important limitations. One is the compromised immune system of the host, rendering them of little use for immunologic studies. Additionally, they are limited both with respect to the long-term passage issues raised earlier (loss of glial markers an acquisition of mesenchymal
Vol. 3, No. 2 2006
markers), as well as phenotypic considerations. In particular, although a hallmark of diffuse glial neoplasms in single cell infiltration of brain parenchyma, established cell lines invariably do not show single cell infiltration of tumor cells. In fact, xenografts of established glioma lines are morphologically more similar to brain metastases, where infiltration is seen by groups of cells, along vessels. Syngeneic transplantable models (such as the C6 gliosarcoma model in Fischer rats) have an advantage in that the host immune system is not compromised, but otherwise suffer from considerations similar to xenograft models. An orthotopic GBM xenograft test panel approach that has been recently described [15] and applied to therapeutic testing [16], employs an unconventional means for sustaining tumor cell growth before harvesting of tumor cells for intracranial injection. Specifically, tumor cells are maintained as subcutaneous, heterotopic xenografts, rather than as continuous cell lines. Propagating human GBM in this way is now known to promote the retention of crucial GBM molecular and biologic characteristics that are lost or suppressed through sustained cell culturing. The subcutaneous propagation of GBM promotes the retention of an extremely important biologic property of GBM, namely tumor invasiveness (Fig. 1). By contrast, most intracranial tumors established from permanent cell lines [17–19] produce noninvasive lesions. The lack of EGFR-amplified GBM cell lines has additionally hindered the investigation of EGF receptor mutants, such as the commonly occurring EGFRvIII [20,21]. In contrast to the lack of EGFR mutations in GBM cell lines, mutant EGFR can be sustained through serial passaging of subcutaneous GBM xenografts [22,23]. The retention of GBM xenograft intracranial invasiveness following sustained subcutaneous passaging has been independently reported by two groups [15,24] thereby, lending good support of this being a reproducible characteristic. One of these groups has additionally shown that short-term culturing of subcutaneous explants does not have an apparent effect on tumor cell intracranial invasiveness, and provides an additional benefit of being able to harvest and deliver defined numbers of cell to multiple mice. Tumors established in this way show a consistent rate of growth, as indicated by the relatively narrow ranges in survival among mice receiving intracranial injection from a common explant culture [15]. Models, which utilize small pieces of flank tumor xenografts [24], might display less consistency. An additional key aspect associated with the subcutaneous serial propagation of GBM xenografts, concerns their retention of patient tumor genetic signature lesions. The retention of patient tumor EGFR amplification status has already been discussed, but in fact all common gene alterations that occur in patient tumors have been shown to be retained in serially passaged GBM xenografts [23,25,26]. Consequently, the
Drug Discovery Today: Disease Models | Cancer
Figure 1. Growth patterns of intracranial xenographs in full coronal section (left) and corresponding with morphologic features of neoplastic cells in areas of maximal tumor density (right). Tumors are maintained serially in vivo, without extensive passaging in vitro. Diffuse infiltration of tumor cells can be seen (for example A1 and D1). Histologic features including abnormal mitoses (B2, C2) and the pseudopalisading necrosis (C2) can be appreciated in this model system. Reprinted from Ref. [15] with permission from the author.
immunocompromised rodents used for the heterotopic propagation and serial passaging of GBMs can be viewed as reservoirs for providing a continuing supply of individual tumors, and the information derived from the molecular characterization of an established xenograft line should prove increasingly meaningful over time. Earlier methods using patient surgical material [27,28], and tissue spheroids [29,30], do not allow for propagation of a renewable tissue resource.
Genetically engineered mouse models A variety of both transgenic and knockout models of cancer in mice have been developed. Models, which utilize widespread changes in oncogenes and tumor suppressor genes www.drugdiscoverytoday.com
193
Drug Discovery Today: Disease Models | Cancer
Vol. 3, No. 2 2006
Table 1. Comparison of glioma models In vitro models
In vivo models
In silico models
Pros
Easily to grow Ideal to test effects of single gene changes Wide range of available assays
More ‘physiologic’, especially with spontaneous models and new short-term culture xenograft models
Wealth of data for mining to generate new hypotheses
Cons
Established cell lines have little similarity to the tumor from which they were derived Some key alterations (EGFRvIII) are not maintained in culture Tumor stem-like cells are difficult to characterize
Spontaneous models might require expensive (e.g. imaging) techniques Some xenograft models do not display features of human gliomas
Large amounts of data are difficult to pare down into specific questions Risk of ‘false positives’ when using high throughput systems
Best use of model
Testing effect of single genes on a specific phenotype; initial studies of novel therapeutics
Preclinical studies of drug efficacy; studies of gliomagenesis
Identification of novel biomarkers and therapeutic targets
Refs
[2–13]
[14–18,27,30–35]
[36–37]
often do not result in gliomas, even when such genes have been strongly associated with glioma formation (e.g. p53 loss). The reasons for this are not fully clear; although one factor could be that such mice might develop and die from other cancers before a glioma became evident. Several transgenic models have been described [31–36], which utilize a tissue-specific promoter (e.g. GFAP or S-100) to drive expression of an oncogene designed to induce or promote glioma formation. A model which utilizes trangenesis but offers somewhat more experimental flexibility involves a mouse, which expresses the RCAS viral receptor on the cell surface. Genes of interest are subcloned into an RCAS viral vector, and then injected into the brains of these transgenic mice [33].
In silico models of glioma Advances have been made in the molecular subclassification of glioma. Some progress has been made in the classification of glioblastoma, where recent reports suggest that molecular subclasses of this tumor exist [37,38]. These subclasses show different clinical behavior, and also suggest the concept that treatment for these might require optimization based on the molecular profile of the tumor. It is certain that data from these and future studies will be mined to identify such molecular subclasses and also suggest novel targets for therapy. The increasing availability of publicly accessible expression array data (required as a condition of acceptance in many journals) will facilitate data mining to identify new genes and pathways of importance. Web portals such as Oncomine are available which provide a central resource for data for this purpose (http://www.oncomine.org/main/ index.jsp).
Model comparison Each of the existing models has specific advantages while also exhibiting specific limitations (summarized in Table 1). In vitro models, based on established cell lines, are of particular use when testing hypotheses derived from ‘higher order’ models (or human tumors themselves), as well as initial 194
www.drugdiscoverytoday.com
screening of potential therapeutic agents. Their ease of manipulation allows for well-controlled experiments designed to tease out the mechanisms of specific phenomena. One must be cautious, however, on the use of these models as hypothesis generation tolls, because many established glioma lines bare little resemblance to the tumors from which they were derived. Studies using newer in vitro culture techniques, especially that of cancer stem-like cells are in their infancy and might show promise as a means to understand and target cells responsible for gliomagenesis. A niche remains for traditional xenografts of established cell lines, particularly in the early evaluation of the potential of key cancer genes to regulate tumorigenesis as well as in early preclinical drug screening. The reproducible latency of tumor take and animal survival increases the feasibility of early studies in this regard. Their inability of xenografts using established glioma lines to recapitulate the invasive behavior of human gliomas limits the interpretation, however, especially of agents, which might target this phenotype in gliomas. Newer xenograft methods using animal passaging of fresh human glioma samples show promise in this regard and might overcome this limitation. Spontaneous models, using genetically engineered approaches have distinct advantages, which include knowledge of the initiating genetic lesions, immunocompetence of the mice and recapitulation of the human disease on the morphologic level. Drawbacks include the potential for a long latency and heterogeneity with respect to tumor development, which can be challenging for preclinical studies.
Conclusions Models of human glioma have been used successfully to elucidate key features of molecular pathogenesis as well as identifying potential therapeutic targets. However, given that the relative lack of progress in the successful treatment of these tumors needs to be understood more about them. Standard in vitro models (established cell lines grown on plastic) provide limited information about tumors as a whole and are useful primarily for questions related to the function of specific candidate genes. Although traditional xenograph
Vol. 3, No. 2 2006
Drug Discovery Today: Disease Models | Cancer
References Links Brain Tumor Progress Review Group: http://www.ninds.nih.gov/ find_people/groups/brain_tumor_prg/index.htm.htm NCI-sponsored information on brain tumors: http:// www.cancer.gov/cancertopics/types/brain/ ABC2 (foundation dedicated to new therapies for glioma): http://www.abc2.org/statistics.shtml MD Anderson web site with brain tumor information: http://www.mdanderson.org/diseases/braincancer/ Oncomine, a web portal which compiles microarray data from cancer specimens: http://www.oncomine.org/main/index.jsp
models have disadvantages (e.g. lack of tumor infiltration into surrounding brain tissue), they remain useful for early preclinical studies. The recent identification of cancer stemlike cells opens up new investigative avenues, which might improve our ability to target these tumors effectively. However, the field of cancer stem-like cells is in its infancy, and needs to be understood more with regard to the characterization, propagation and biology of these cells. New techniques in the establishment of xenograph models include the use of fresh human tumor specimens, followed by a short time in culture before injection into a rodent brain. Further characterization of these novel xenograph models will probably lead to improved preclinical studies for the testing of novel targeted agents. Finally, the development of new spontaneous models of the glioma, using transgenic and gene knockout approaches will further enhance our understanding of specific molecular factors which cause and/or phenotypically modulate this disease. On a practical level the utilization of a model appropriate for the question at hand is prudent. The ease of manipulation of in vitro models (especially established cell lines) ensures their continued use, and despite their limitations, they have a role is addressing specific hypothesis-driven or mechanistic questions. Studies, which are more exploratory, address questions regarding gliomagenesis, and require systems that incorporate tumor–stromal interactions and require the use of models more akin to the human disease. Advances in methods to image the CNS of small animals to characterize neoplastic processes will help significantly to promote the incorporation of these models into biologic and preclinical studies of glioma.
Outstanding issues Generation of additional models, which recapitulate the human disease genetically and morphologically. Utilization of models for to test for target inhibition after treatment with novel small molecule inhibitors. Development of models for low-grade gliomas. Increased understand of key molecular genetic abnormalities responsible for resistance to radiation and alkylator-based chemotherapy.
1 Becher, O.J. and Holland, E.C. (2006) Genetically engineered models have advantages over xenografts for preclinical studies. Cancer Res. 66, 3355– 3358 (discussion 3358–3359) 2 Sausville, E.A. and Burger, A.M. (2006) Contributions of human tumor xenografts to anticancer drug development. Cancer Res. 66, 3351–3354 (discussion 3354) 3 Kennedy, P.G. et al. (1987) Antigenic expression by cells derived from human gliomas does not correlate with morphological classification. Neuropathol. Appl. Neurobiol. 13, 327–347 4 Sonoda, Y. et al. (2001) Akt pathway activation converts anaplastic astrocytoma to glioblastoma multiforme in a human astrocyte model of glioma. Cancer Res. 61, 6674–6678 5 Sonoda, Y. et al. (2001) Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma. Cancer Res. 61, 4956–4960 6 Lund-Johansen, M. et al. (1989) Multicellular tumor spheroids in serumfree culture. Anticancer Res. 9, 413–420 7 Lund-Johansen, M. et al. (1990) Effect of epidermal growth factor on glioma cell growth, migration, and invasion in vitro. Cancer Res. 50, 6039–6044 8 Wang, J.C. and Dick, J.E. (2005) Cancer stem cells: lessons from leukemia. Trends Cell Biol. 15, 494–501 9 Zhang, M. and Rosen, J.M. (2006) Stem cells in the etiology and treatment of cancer. Curr. Opin. Genet. Dev. 16, 60–64 10 Galderisi, U. et al. (2006) Stem cells and brain cancer. Cell Death Differ. 13, 5–11 11 Singh, S.K. et al. (2004) Cancer stem cells in nervous system tumors. Oncogene 23, 7267–7273 12 Galli, R. et al. (2004) Isolation and characterization of tumorigenic, stemlike neural precursors from human glioblastoma. Cancer Res. 64, 7011– 7021 13 Yuan, X. et al. (2004) Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23, 9392–9400 14 Singh, S.K. et al. (2004) Identification of human brain tumour initiating cells. Nature 432, 396–401 15 Giannini, C. et al. (2005) Patient tumor EGFR and PDGFRA gene amplifications retained in an invasive intracranial xenograft model of glioblastoma multiforme. Neuro-oncol. 7, 164–176 16 Sarkaria, J.N. et al. (2006) Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response. Clin. Cancer Res. 12 (7 Pt 1), 2264–2271 17 Yamini, B. et al. (2004) Transcriptional targeting of adenovirally delivered tumor necrosis factor alpha by temozolomide in experimental glioblastoma. Cancer Res. 64, 6381–6384 18 Schmidt, N.O. et al. (2004) Antiangiogenic therapy by local intracerebral microinfusion improves treatment efficiency and survival in an orthotopic human glioblastoma model. Clin. Cancer Res. 10, 1255– 1262 19 Zhang, Y. et al. (2004) Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer. Clin. Cancer Res. 10, 3667–3677 20 Frederick, L. et al. (2000) Diversity and frequency of epidermal growth factor receptor mutations in human glioblastomas. Cancer Res. 60, 1383–1387 21 Aldape, K.D. et al. (2004) Immunohistochemical detection of EGFRvIII in high malignancy grade astrocytomas and evaluation of prognostic significance. J. Neuropathol. Exp. Neurol. 63, 700–707 22 Bigner, S.H. et al. (1990) Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts. Cancer Res. 50, 8017–8022 23 Pandita, A. et al. (2004) Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR. Genes Chromosomes Cancer 39, 29–36 24 Antunes, L. et al. (2000) Analysis of tissue chimerism in nude mouse brain and abdominal xenograft models of human glioblastoma multiforme: what does it tell us about the models and about glioblastoma biology and therapy? J. Histochem. Cytochem. 48, 847–858
www.drugdiscoverytoday.com
195
Drug Discovery Today: Disease Models | Cancer
25
26
27
28 29
30 31
196
Leuraud, P. et al. (2003) Correlation between genetic alterations and growth of human malignant glioma xenografted in nude mice. Br. J. Cancer 89, 2327–2332 Jeuken, J.W. et al. (2000) Genetic reflection of glioblastoma biopsy material in xenografts: characterization of 11 glioblastoma xenograft lines by comparative genomic hybridization. J. Neurosurg. 92, 652–658 Horten, B.C. et al. (1981) Xenograft of human malignant glial tumors into brains of nude mice. A histopatholgical study. J. Neuropathol. Exp. Neurol. 40, 493–511 Shapiro, W.R. et al. (1979) Human brain tumor transplantation into nude mice. J. Natl. Cancer Inst. 62, 447–453 Mahesparan, R. et al. (2003) Expression of extracellular matrix components in a highly infiltrative in vivo glioma model. Acta Neuropathol. (Berl.) 105, 49–57 Engebraaten, O. et al. (1999) Growth of precultured human glioma specimens in nude rat brain. J. Neurosurg. 90, 125–132 Weiss, W.A. et al. (2003) Genetic determinants of malignancy in a mouse model for oligodendroglioma. Cancer Res. 63, 1589–1595
www.drugdiscoverytoday.com
Vol. 3, No. 2 2006
32
33 34
35
36 37 38
Xiao, A. et al. (2002) Astrocyte inactivation of the pRb pathway predisposes mice to malignant astrocytoma development that is accelerated by PTEN mutation. Cancer Cell 1, 157–168 Holland, E.C. et al. (2000) Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat. Genet. 25, 55–57 Ding, H. et al. (2001) Astrocyte-specific expression of activated p21-ras results in malignant astrocytoma formation in a transgenic mouse model of human gliomas. Cancer Res. 61, 3826–3836 Chiu, I.M. et al. (2000) Tumorigenesis in transgenic mice in which the SV40 T antigen is driven by the brain-specific FGF1 promoter. Oncogene 19, 6229–6239 Weissenberger, J. et al. (1997) Development and malignant progression of astrocytomas in GFAP-v-src transgenic mice. Oncogene 14, 2005–2013 Freije, W.A. et al. (2004) Gene expression profiling of gliomas strongly predicts survival. Cancer Res. 64, 6503–6510 Phillips, H.S. et al. (2006) Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell 9, 157–173