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Recent publications on the molecular characterization of malignant glioma have had profound impact on the appreciation of tumoral heterogeneity within and between patients. Both these phenomena are implicated in the variability in clinical outcome between patients, as well as the inevitable recurrence of these tumors after conventional treatment. The advent of selective cell culture protocols for the propagation of patient-derived glioma stem-like cells (GSCs) provides researchers the ability to selectively study the cells that could be at the root of tumor proliferation and resistance to therapy. As these techniques are widely applied in contemporary studies and becoming the preferred in vitro model, molecular characterization of GSCs is considered pivotal for the identification and advancement of novel therapies for this devastating disease. This review aims to provide an overview of canonical molecular alterations defining subtypes of malignant glioma as derived from genotypic, transcriptomic and epigenetic profiling in relation to their representation in GSC models. The distribution of these hallmark alterations as found in characterization studies of GSCs is compared between publications. Finally, conclusions of these studies with respect to coverage of driving alterations and translational relevance are provided. By doing so, we provide a contemporary overview of scientific results derived from GSC models and hopefully create appreciation of the advantages and caveats of utilizing these models for studying malignant glioma.

Serum-free culture methods for patient-derived primary glioma cultures, selecting for glioma stem-like cells (GSCs), are becoming the gold standard in neurooncology research. These GSCs can be implemented in drug screens to detect patient-specific responses, potentially bridging the translational gap to personalized medicine. Since numerous compounds are available, a rapid and reliable readout for drug efficacies is required. This can be done using approaches that measure viability, confluency, cytotoxicity, or apoptosis. To determine which assay is best suitable for drug screening, 10 different assays were systematically tested on established glioma cell lines and validated on a panel of GSCs. General applicability was assessed using distinct treatment modalities, being temozolomide, radiation, rapamycin, and the oncolytic adenovirus Delta24-RGD. The apoptosis and cytotoxicity assays did not unequivocally detect responses and were excluded from further testing. The NADH- and ATP-based viability assays revealed comparable readout for all treatments; however, the latter had smaller standard deviations and direct readout. Importantly, drugs that interfere with cell metabolism require alternative techniques such as confluency monitoring to accurately measure treatment effects. Taken together, our data suggest that the combination of ATP luminescence assays with confluency monitoring provides the most specific and reproducible readout for drug screening on primary GSCs.

Glial tumors consist of a heterogeneous group of primary CNS neoplasms. While the clinical outcome of these tumors varies substantially between different grades (World Health Organization [WHO] grades I–IV), only grade I tumors can be treated curatively.1 In the last decade, substantial effort has been put into characterizing the pathogenic mechanisms underlying this complex group of glial cell–derived tumors. These efforts have led to the establishment of several molecular subclasses of glioma, which differentiate types of gliomas based on their intrinsic molecular distinctions and similarities, rather than the conventional characteristics used for histology-based grading.2–5 Indeed, gene expression–based clustering of glioma from several grades within the WHO grading system proved more predictive of survival than the histological classification.4, 6 Thus far, however, this gain of insight has not led to improved clinical outcome for patients, since there are no specific treatment strategies designed to target specific subtypes of glioma as of yet. Consequently, the development of preclinical models that accurately reflect the molecular heterogeneity of glioma is imperative for both translationally relevant drug screening programs and the advancement of patient-tailored treatment options. Commercially available cell cultures of glioma have been demonstrated to poorly mimic the molecular aberrations found in patient samples.7,8 Furthermore, the xenograft models derived from these cultures do not sufficiently recapitulate the histological hallmarks of glioma.9 With the advent of the cancer stem cell hypothesis, several groups have implemented serum-free (SF) cell culture regimens, originally developed for neural stem cell propagation, to establish glioma stem-like cell (GSC) cultures from fresh tumor tissue.10,11 By analyzing gene expression profiles of GSCs and serum supplemented (SS) cultures, a distinct separation between the aforementioned and the parental tumors was revealed by unsupervised clustering.7 Several groups have reported GSC cultures to be superior with regard to retaining the original patient gene expression signature and histological phenotype in xenografts.7,11,12 This has led to a wide variety of applications for these cell culture assays, ranging from inquiries into fundamental hypotheses13,14 to preclinical testing of novel agents.15–17 Several contradicting publications have since been published on the optimal cell culture methodology,18,19 mandatory molecular aberrations for successful propagation,20–22 and positive selection for gene expression signatures related to specific molecular subtypes in vitro.14, 23 Most of these previous publications have focused on the characterization of successfully propagated specimens of glioblastoma multiforme (GBM; WHO grade IV) from adults. Reports on the culture success of grade II and grade III gliomas are sparse.20,24 Therefore, we undertook a characterization study of a large cohort (N = 261), which addresses the distribution of glioma from all histological entities for the outcome of GSC culture attempt. Within equal WHO grades, correlations between cell culture outcome and patient overall survival were assessed. Tumor samples of both successful and unsuccessful cultures (n = 46 in total) were also subjected to molecular analysis, and a number of molecular traits that influence cell culture success rate were identified, as well as genes that may play a role in this process. These results emphasize the need for, and provide leads to, the development of improved culture protocols supporting growth of all subtypes of glioma. This is essential for implementation of this model in drug screening programs for personalized treatment strategies.

Oncolytic adenoviral vectors are a promising alternative for the treatment of glioblastoma. Recent publications have demonstrated the advantages of shielding viral particles within cellular vehicles (CVs), which can be targeted towards the tumor microenvironment. Here, we studied T-cells, often having a natural capacity to target tumors, for their feasibility as a CV to deliver the oncolytic adenovirus, Delta24-RGD, to glioblastoma. The Jurkat T-cell line was assessed in co-culture with the glioblastoma stem cell (GSC) line, MGG8, for the optimal transfer conditions of Delta24-RGD in vitro. The effect of intraparenchymal and tail vein injections on intratumoral virus distribution and overall survival was addressed in an orthotopic glioma stem cell (GSC)-based xenograft model. Jurkat T-cells were demonstrated to facilitate the amplification and transfer of Delta24-RGD onto GSCs. Delta24-RGD dosing and incubation time were found to influence the migratory ability of T-cells towards GSCs. Injection of Delta24-RGD-loaded T-cells into the brains of GSC-bearing mice led to migration towards the tumor and dispersion of the virus within the tumor core and infiltrative zones. This occurred after injection into the ipsilateral hemisphere, as well as into the non-tumor-bearing hemisphere. We found that T-cell-mediated delivery of Delta24-RGD led to the inhibition of tumor growth compared to non-treated controls, resulting in prolonged survival (p = 0.007). Systemic administration of virus-loaded T-cells resulted in intratumoral viral delivery, albeit at low levels. Based on these findings, we conclude that T-cell-based CVs are a feasible approach to local Delta24-RGD delivery in glioblastoma, although efficient systemic targeting requires further improvement.

Lessons from the history of cancer therapy inform that a multimodal approach offers the best potential to eradicate the tumor mass and prevent the recurrence after therapy. One of the most promising experimental therapies for cancer is the use of oncolytic adenoviruses as therapeutic agents. These biologic agents exert their function by directly infecting and killing tumor cells. The new progeny generated after the first infection round will spread, generating a therapeutic wave that will optimally eliminate every cancer cell. Recently, modifications in the virus genome have allowed for the designing of viruses that infect with more potency than wild-type adenoviruses, replicate exclusively in tumor cells, selectively target cellular receptors or molecular defects in the cancer cells, and deliver prodrug genes. In addition, these last-generation viruses can be combined with chemotherapy and other forms of cancer therapies to enhance the tumor killing capability. With the steady and fast pace of the progress in our knowledge of the genetics of tumors, we may soon fulfill our hope of reaching the final objective of an adenovirus-based personalized medicine for cancer.

Objective: A high percentage of grade II and III gliomas have mutations in the gene encoding isocitrate dehydrogenase (IDH1). This mutation is always a heterozygous point mutation that affects the amino acid arginine at position 132 and results in loss of its native enzymatic activity and gain of alternative enzymatic activity (producing D-2-hydroxyglutarate). The objective of this study was to investigate the cellular effects of R132H mutations in IDH1.Methods: Functional consequences of IDH1(R132H) mutations were examined among others using fluorescence-activated cell sorting, kinome and expression arrays, biochemical assays, and intracranial injections on 3 different (glioma) cell lines with stable overexpression of IDH1(R132H).Results: IDH1(R132H) overexpression in established glioma cell lines in vitro resulted in a marked decrease in proliferation, decreased Akt phosphorylation, altered morphology, and a more contact-dependent cell migration. The reduced proliferation is related to accumulation of D-2-hydroxyglutarate that is produced by IDH1(R132H). Mice injected with IDH1(R132H) U87 cells have prolonged survival compared to mice injected with IDH1(wt) or green fluorescent protein-expressing U87 cells.Interpretation: Our results demonstrate that IDH1(R132H) dominantly reduces aggressiveness of established glioma cell lines in vitro and in vivo. In addition, the IDH1R132H-IDH1(wt) heterodimer has higher enzymatic activity than the IDH1(R132H)-IDH1(R132H) homodimer. Our observations in model systems of glioma might lead to a better understanding of the biology of IDH1 mutant gliomas, which are typically low grade and often slow growing. ANN NEUROL 2011;69:455-463