High levels of ubidecarenone (oxidized CoQ10) delivered using a drug-lipid conjugate nanodispersion (BPM31510) differentially affect redox status and growth in malignant glioma versus non-tumor cells.

Metabolic reprogramming in cancer cells, vs. non-cancer cells, elevates levels of reactive oxygen species (ROS) leading to higher oxidative stress. The elevated ROS levels suggest a vulnerability to excess prooxidant loads leading to selective cell death, a therapeutically exploitable difference. Co-enzyme Q10 (CoQ10) an endogenous mitochondrial resident molecule, plays an important role in mitochondrial redox homeostasis, membrane integrity, and energy production. BPM31510 is a lipid-drug conjugate nanodispersion specifically formulated for delivery of supraphysiological concentrations of ubidecarenone (oxidized CoQ10) to the cell and mitochondria, in both in vitro and in vivo model systems. In this study, we sought to investigate the therapeutic potential of ubidecarenone in the highly treatment-refractory glioblastoma. Rodent (C6) and human (U251) glioma cell lines, and non-tumor human astrocytes (HA) and rodent NIH3T3 fibroblast cell lines were utilized for experiments. Tumor cell lines exhibited a marked increase in sensitivity to ubidecarenone vs. non-tumor cell lines. Further, elevated mitochondrial superoxide production was noted in tumor cells vs. non-tumor cells hours before any changes in proliferation or the cell cycle could be detected. In vitro co-culture experiments show ubidecarenone differentially affecting tumor cells vs. non-tumor cells, resulting in an equilibrated culture. In vivo activity in a highly aggressive orthotopic C6 glioma model demonstrated a greater than 25% long-term survival rate. Based on these findings we conclude that high levels of ubidecarenone delivered using BPM31510 provide an effective therapeutic modality targeting cancer-specific modulation of redox mechanisms for anti-cancer effects.

Reversed metabolic reprogramming as a measure of cancer treatment efficacy in rat C6 glioma model.

BACKGROUND: Metabolism in tumor shifts from oxidative phosphorylation to inefficient glycolysis resulting in overproduction of lactate (Warburg effect), and cancers may be effectively treated if this imbalance were corrected. The aim of this longitudinal study of glioblastoma in a rat model was to determine whether the ratio of lactate (surrogate marker for glycolysis) to bicarbonate (for oxidative phosphorylation), as measured via in vivo magnetic resonance imaging of hyperpolarized 13C-labeled pyruvate accurately predicts survival. METHODS: C6 Glioma implanted male Wistar rats (N = 26) were treated with an anti-vascular endothelial growth factor antibody B20.4.1.1 in a preliminary study to assess the efficacy of the drug. In a subsequent longitudinal survival study, magnetic resonance spectroscopic imaging (MRSI) was used to estimate [1-13C]Lactate and [1-13C]Bicarbonate in tumor and contralateral normal appearing brain of glioma implanted rats (N = 13) after injection of hyperpolarized [1-13C]Pyruvate at baseline and 48 hours post-treatment with B20.4.1.1. RESULTS: A survival of ~25% of B20.4.1.1 treated rats was noted in the preliminary study. In the longitudinal imaging experiment, changes in 13C Lactate, 13C Bicarbonate and tumor size measured at baseline and 48 hours post-treatment did not correlate with survival. 13C Lactate to 13C Bicarbonate ratio increased in all the 6 animals that succumbed to the tumor whereas the ratio decreased in 6 of the 7 animals that survived past the 70-day observation period. CONCLUSIONS: 13C Lactate to 13C Bicarbonate ratio (Lac/Bic) at 48 hours post-treatment is highly predictive of survival (p = 0.003). These results suggest a potential role for the 13C Lac/Bic ratio serving as a valuable measure of tumor metabolism and predicting therapeutic response.

A proteomic clock for malignant gliomas: The role of the environment in tumorigenesis at the presymptomatic stage.

Malignant gliomas remain incurable with a poor prognosis despite of aggressive treatment. We have been studying the development of brain tumors in a glioma rat model, where rats develop brain tumors after prenatal exposure to ethylnitrosourea (ENU), and there is a sizable interval between when the first pathological changes are noted and tumors become detectable with MRI. Our aim to define a molecular timeline through proteomic profiling of the cerebrospinal fluid (CSF) such that brain tumor commitment can be revealed earlier than at the presymptomatic stage. A comparative proteomic approach was applied to profile CSF collected serially either before, at and after the time MRI becomes positive. Elastic net (EN) based models were developed to infer the timeline of normal or tumor development respectively, mirroring a chronology of precisely timed, "clocked", adaptations. These CSF changes were later quantified by longitudinal entropy analyses of the EN predictive metric. False discovery rates (FDR) were computed to control the expected proportion of the EN models that are due to multiple hypothesis testing. Our ENU rat brain tumor dating EN model indicated that protein content in CSF is programmed even before tumor MRI detection. The findings of the precisely timed CSF tumor microenvironment changes at presymptomatic stages, deviation from the normal development timeline, may provide the groundwork for the understanding of adaptation of the brain environment in tumorigenesis to devise effective brain tumor management strategies.

A small molecule TrkB/TrkC neurotrophin receptor co-activator with distinctive effects on neuronal survival and process outgrowth.

Neurotrophin (NT) receptors are coupled to numerous signaling networks that play critical roles in neuronal survival and plasticity. Several non-peptide small molecule ligands have recently been reported that bind to and activate specific tropomyosin-receptor kinase (Trk) NT receptors, stimulate their downstream signaling, and cause biologic effects similar to, though not completely overlapping, those of the native NT ligands. Here, in silico screening, coupled with low-throughput neuronal survival screening, identified a compound, LM22B-10, that, unlike prior small molecule Trk ligands, binds to and activates TrkB as well as TrkC. LM22B-10 increased cell survival and strongly accelerated neurite outgrowth, superseding the effects of brain-derived neurotrophic factor (BDNF), NT-3 or the two combined. Additionally, unlike the NTs, LM22B-10 supported substantial early neurite outgrowth in the presence of inhibiting glycoproteins. Examination of the mechanisms of these actions suggested contributions of the activation of both Trks and differential interactions with p75(NTR), as well as a requirement for involvement of the Trk extracellular domain. In aged mice, LM22B-10 activated hippocampal and striatal TrkB and TrkC, and their downstream signaling, and increased hippocampal dendritic spine density. Thus, LM22B-10 may constitute a new tool for the study of TrkB and TrkC signaling and their interactions with p75(NTR), and provides groundwork for the development of ligands that stimulate unique combinations of Trk receptors and activity patterns for application to selected neuronal populations and deficits present in various disease states.

Hyperpolarized (13)C-lactate to (13)C-bicarbonate ratio as a biomarker for monitoring the acute response of anti-vascular endothelial growth factor (anti-VEGF) treatment.

Hyperpolarized [1-(13)C]pyruvate MRS provides a unique imaging opportunity to study the reaction kinetics and enzyme activities of in vivo metabolism because of its favorable imaging characteristics and critical position in the cellular metabolic pathway, where it can either be reduced to lactate (reflecting glycolysis) or converted to acetyl-coenzyme A and bicarbonate (reflecting oxidative phosphorylation). Cancer tissue metabolism is altered in such a way as to result in a relative preponderance of glycolysis relative to oxidative phosphorylation (i.e. Warburg effect). Although there is a strong theoretical basis for presuming that readjustment of the metabolic balance towards normal could alter tumor growth, a robust noninvasive in vivo tool with which to measure the balance between these two metabolic processes has yet to be developed. Until recently, hyperpolarized (13)C-pyruvate imaging studies had focused solely on [1-(13)C]lactate production because of its strong signal. However, without a concomitant measure of pyruvate entry into the mitochondria, the lactate signal provides no information on the balance between the glycolytic and oxidative metabolic pathways. Consistent measurement of (13)C-bicarbonate in cancer tissue, which does provide such information, has proven difficult, however. In this study, we report the reliable measurement of (13)C-bicarbonate production in both the healthy brain and a highly glycolytic experimental glioblastoma model using an optimized (13)C MRS imaging protocol. With the capacity to obtain signal in all tumors, we also confirm for the first time that the ratio of (13)C-lactate to (13)C-bicarbonate provides a more robust metric relative to (13)C-lactate for the assessment of the metabolic effects of anti-angiogenic therapy. Our data suggest a potential application of this ratio as an early biomarker to assess therapeutic effectiveness. Furthermore, although further study is needed, the results suggest that anti-angiogenic treatment results in a rapid normalization in the relative tissue utilization of glycolytic and oxidative phosphorylation by tumor tissue.

Volumetric spiral chemical shift imaging of hyperpolarized [2-(13) c]pyruvate in a rat c6 glioma model.

PURPOSE: MRS of hyperpolarized [2-(13)C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the (13)C label is not lost with the conversion of pyruvate to acetyl-CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2-(13)C]pyruvate in glioma-bearing brain. METHODS: Spiral chemical shift imaging with spectrally undersampling scheme (1042 Hz) and a hard-pulse excitation was exploited to simultaneously image [2-(13)C]pyruvate, [2-(13)C]lactate, and [5-(13)C]glutamate, the metabolites known to be produced in brain after an injection of hyperpolarized [2-(13)C]pyruvate, without chemical shift displacement artifacts. A separate undersampling scheme (890 Hz) was also used to image [1-(13)C]acetyl-carnitine. Healthy and C6 glioma-implanted rat brains were imaged at baseline and after dichloroacetate administration, a drug that modulates pyruvate dehydrogenase kinase activity. RESULTS: The baseline metabolite maps showed higher lactate and lower glutamate in tumor as compared to normal-appearing brain. Dichloroacetate led to an increase in glutamate in both tumor and normal-appearing brain. Dichloroacetate-induced %-decrease of lactate/glutamate was comparable to the lactate/bicarbonate decrease from hyperpolarized [1-(13)C]pyruvate studies. Acetyl-carnitine was observed in the muscle/fat tissue surrounding the brain. CONCLUSION: Robust volumetric imaging with hyperpolarized [2-(13)C]pyruvate and downstream products was performed in glioma-bearing rat brains, demonstrating changes in mitochondrial metabolism with dichloroacetate.

Cerebrospinal fluid protein dynamic driver network: At the crossroads of brain tumorigenesis.

To get a better understanding of the ongoing in situ environmental changes preceding the brain tumorigenesis, we assessed cerebrospinal fluid (CSF) proteome profile changes in a glioma rat model in which brain tumor invariably developed after a single in utero exposure to the neurocarcinogen ethylnitrosourea (ENU). Computationally, the CSF proteome profile dynamics during the tumorigenesis can be modeled as non-smooth or even abrupt state changes. Such brain tumor environment transition analysis, correlating the CSF composition changes with the development of early cellular hyperplasia, can reveal the pathogenesis process at network level during a time before the image detection of the tumors. In our controlled rat model study, matched ENU- and saline-exposed rats' CSF proteomics changes were quantified at approximately 30, 60, 90, 120, 150 days of age (P30, P60, P90, P120, P150). We applied our transition-based network entropy (TNE) method to compute the CSF proteome changes in the ENU rat model and test the hypothesis of the critical transition state prior to impending hyperplasia. Our analysis identified a dynamic driver network (DDN) of CSF proteins related with the emerging tumorigenesis progressing from the non-hyperplasia state. The DDN associated leading network CSF proteins can allow the early detection of such dynamics before the catastrophic shift to the clear clinical landmarks in gliomas. Future characterization of the critical transition state (P60) during the brain tumor progression may reveal the underlying pathophysiology to device novel therapeutics preventing tumor formation. More detailed method and information are accessible through our website at http://translationalmedicine.stanford.edu.

Blockade of SDF-1 after irradiation inhibits tumor recurrences of autochthonous brain tumors in rats.

BACKGROUND: Tumor irradiation blocks local angiogenesis, forcing any recurrent tumor to form new vessels from circulating cells. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas in the brains of nude mice can be delayed or prevented by inhibiting circulating blood vessel-forming cells by blocking the interaction of CXCR4 with its ligand stromal cell-derived factor (SDF)-1 (CXCL12). In the present study we test this strategy by directly neutralizing SDF-1 in a clinically relevant model using autochthonous brain tumors in immune competent hosts. METHODS: We used NOX-A12, an l-enantiomeric RNA oligonucleotide that binds and inhibits SDF-1 with high affinity. We tested the effect of this inhibitor on the response to irradiation of brain tumors in rat induced by n-ethyl-N-nitrosourea. RESULTS: Rats treated in utero with N-ethyl-N-nitrosourea began to die of brain tumors from approximately 120 days of age. We delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age, began treatment with NOX-A12 immediately following irradiation, and continued with either 5 or 20 mg/kg for 4 or 8 weeks, doses and times equivalent to well-tolerated human exposures. We found a marked prolongation of rat life span that was dependent on both drug dose and duration of treatment. In addition we treated tumors only when they were visible by MRI and demonstrated complete regression of the tumors that was not achieved by irradiation alone or with the addition of temozolomide. CONCLUSIONS: Inhibition of SDF-1 following tumor irradiation is a powerful way of improving tumor response of glioblastoma multiforme.

Visualization of rodent brain tumor angiogenesis and effects of antiangiogenic treatment using 3D ΔR2-µMRA.

Understanding of structural and functional characteristics of the vascular microenvironment in gliomas and the impact of antiangiogenic treatments is essential for developing better therapeutic strategies. Although a number of methods exist in which this process can be studied experimentally, no single noninvasive test has the capacity to provide information concerning both microvascular function and morphology. The purpose of present study is to demonstrate the feasibility of using a novel three-dimensional ΔR2-based microscopic magnetic resonance angiography (3D ΔR2-µMRA) technique for longitudinal imaging of tumor angiogenesis and monitoring the effects of antiangiogenic treatment in rodent brain tumor models. Using 3D ΔR2-µMRA, a generally consistent early pattern of vascular development in gliomas was revealed, in which a single feeding vessel was visualized first (arteriogenesis), followed by sprouting angiogenesis. Considerable variability of the tumor-associated vasculature was then noted at later stages of tumor evolution. ΔR2-µMRA revealed that anti-vascular endothelial growth factor treatment induced a rapid and significant alteration of the intratumoral angiogenic phenotype. In summary, 3D ΔR2-µMRA enables high-resolution visualization of tumor-associated vessels while simultaneously providing functional information on the tumor microvasculature. It can serve as a useful tool for monitoring both the temporal evolution of tumor angiogenesis and the impact of antiangiogenic therapies.

Metabolic response of glioma to dichloroacetate measured in vivo by hyperpolarized (13)C magnetic resonance spectroscopic imaging.

BACKGROUND: The metabolic phenotype that derives disproportionate energy via glycolysis in solid tumors, including glioma, leads to elevated lactate labeling in metabolic imaging using hyperpolarized [1-(13)C]pyruvate. Although the pyruvate dehydrogenase (PDH)-mediated flux from pyruvate to acetyl coenzyme A can be indirectly measured through the detection of carbon-13 ((13)C)-labeled bicarbonate, it has proven difficult to visualize (13)C-bicarbonate at high enough levels from injected [1-(13)C]pyruvate for quantitative analysis in brain. The aim of this study is to improve the detection of (13)C-labeled metabolites, in particular bicarbonate, in glioma and normal brain in vivo and to measure the metabolic response to dichloroacetate, which upregulates PDH activity. METHODS: An optimized protocol for chemical shift imaging and high concentration of hyperpolarized [1-(13)C]pyruvate were used to improve measurements of lactate and bicarbonate in C6 glioma-transplanted rat brains. Hyperpolarized [1-(13)C]pyruvate was injected before and 45 min after dichloroacetate infusion. Metabolite ratios of lactate to bicarbonate were calculated to provide improved metrics for characterizing tumor metabolism. RESULTS: Glioma and normal brain were well differentiated by lactate-to-bicarbonate ratio (P = .002, n = 5) as well as bicarbonate (P = .0002) and lactate (P = .001), and a stronger response to dichloroacetate was observed in glioma than in normal brain. CONCLUSION: Our results clearly demonstrate for the first time the feasibility of quantitatively detecting (13)C-bicarbonate in tumor-bearing rat brain in vivo, permitting the measurement of dichloroacetate-modulated changes in PDH flux. The simultaneous detection of lactate and bicarbonate provides a tool for a more comprehensive analysis of glioma metabolism and the assessment of metabolic agents as anti-brain cancer drugs.

Alterations in cerebrospinal fluid proteins in a presymptomatic primary glioma model.

BACKGROUND: Understanding the early relationship between brain tumor cells and their environment could lead to more sensitive biomarkers and new therapeutic strategies. We have been using a rodent model of neurocarcinogenesis in which all animals develop brain tumors by six months of age to establish two early landmarks in glioma development: the appearance of a nestin(+) cell at thirty days of age and the appearance of cellular hyperplasia between 60 and 120 days of age. We now report an assessment of the CSF proteome to determine the changes in protein composition that occur during this period. MATERIALS AND METHODS: Nestin(+) cell clusters and microtumors were assessed in 63 ethylnitrosourea-exposed rats on 30, 60, and 90 days of age. CSF was obtained from the cisterna magna from 101 exposed and control rats at 30, 60, and 90 days and then analyzed using mass spectrometry. Differentially expressed peaks were isolated and identified. RESULTS: Nestin(+) cells were noted in all ethylnitrosourea-exposed rats assessed pathologically. Small microtumors were noted in 0%, 18%, and 67% of 30-, 60-, and 90-day old rats, respectively (p<0.05, Chi square). False Discovery Rate analysis of peak intensities showed that the number of true discoveries with p<0.05 increased markedly with increasing age. Isolation and identification of highly differentially detected proteins at 90 days of age revealed increases in albumin and a fragment of α1 macroglobulin and alterations in glutathionylated transthyretin. CONCLUSIONS: The presence of increased albumin, fragments of cerebrospinal fluid proteins, and glutathione breakdown in temporal association with the development of cellular hyperplasia, suggests that, similar to many other systemic cancers, inflammation and oxidative stress is playing an important early role in the host's response to brain tumor development and may be involved in affecting the early growth of brain tumor.

Metabolite kinetics in C6 rat glioma model using magnetic resonance spectroscopic imaging of hyperpolarized [1-(13)C]pyruvate.

In addition to an increased lactate-to-pyruvate ratio, altered metabolism of a malignant glioma can be further characterized by its kinetics. Spatially resolved dynamic data of pyruvate and lactate from C6-implanted female Sprague-Dawley rat brain were acquired using a spiral chemical shift imaging sequence after a bolus injection of a hyperpolarized [1-(13)C]pyruvate. Apparent rate constants for the conversion of pyruvate to lactate in three different regions (glioma, normal appearing brain, and vasculature) were estimated based on a two-site exchange model. The apparent conversion rate constant was 0.018 ± 0.004 s(-1) (mean ± standard deviation, n = 6) for glioma, 0.009 ± 0.003 s(-1) for normal brain, and 0.005 ± 0.001 s(-1) for vasculature, whereas the lactate-to-pyruvate ratio, the metabolic marker used to date to identify tumor regions, was 0.36 ± 0.07 (mean ± SD), 0.24 ± 0.07, and 0.12 ± 0.02 for glioma, normal brain, and vasculature, respectively. The data suggest that the apparent conversion rate better differentiate glioma from normal brain (P = 0.001, n = 6) than the lactate-to-pyruvate ratio (P = 0.02).

5'-AMP-activated protein kinase activity is elevated early during primary brain tumor development in the rat.

We found that adenosine 5'-monophosphate-activated protein kinase (AMPK), which is considered the "fuel sensor" of mammalian cells because it directly responds to the depletion of the fuel molecule ATP, is strongly activated by tumor-like hypoxia and glucose deprivation. We also observed abundant AMPK activity in tumor cells in vivo, using subcutaneous tumor xenografts prepared from cells transformed with oncogenic H-Ras. Such rapidly growing transplants of tumor cells, however, represent fully developed tumors that naturally contain energetically stressed microenvironments that can activate AMPK. Therefore, to investigate the induction of AMPK activity during experimental tumorigenesis, we used an established model of brain tumor (glioma) development in the offspring of rats exposed prenatally to the mutagen N-ethyl-N-nitrosourea. We observed that immunostaining for a specific readout of AMPK activity (AMPK-dependent phosphorylation of acetyl-CoA carboxylase) was prominent during N-ethyl-N-nitrosourea-initiated neurocarcinogenesis, from the occurrence of early hyperplasia (microtumors) to the emergence of large gliomas. Moreover, we observed that immunostaining for activating phosphorylation of AMPK correlated with the same stages of glioma development, notably in mitotic tumor cells in which the signal showed punctate as well as cytoplasmic patterns associated with spindle formation. Based on these observations, we propose that neurocarcinogenesis requires AMPK-dependent regulation of cellular energy metabolism.

A distinct phenotypic change in gliomas at the time of magnetic resonance imaging detection.

OBJECT: Although gliomas remain refractory to treatment, it is not clear whether this characteristic is fixed at the time of its origin or develops later. The authors have been using a model of neurocarcinogenesis to determine whether a time exists during a glioma's evolution during which it is detectable but still curable, thus providing a justification for exploring the clinical merits of an early detection and treatment strategy. The authors recently reported the presence of 2 distinct cellular subsets, 1 expressing nestin and the other both glial fibrillary acidic protein (GFAP) and osteopontin (OPN), within all examined gliomas that developed after in utero exposure to ethylnitrosourea. METHODS: In this study, the authors used magnetic resonance (MR) imaging to assess when these 2 subpopulations appeared during glioma evolution. RESULTS: Using T2-weighted and diffusion-weighted MR imaging, the authors observed that gliomas grew exponentially once detected at rates that were location-dependent. Despite large differences in growth rates, however, they determined by correlating histochemistry with imaging in a second series of animals, that all lesions initially detected on T2-weighted images contained both subsets of cells. In contrast, lesions containing only nestin-positive cells, which appeared on average 40 days before detection on MR images, were not detected. CONCLUSIONS: The sequential appearance of first the nestin-positive cells followed several weeks later by those expressing GFAP/OPN suggests that all gliomas arise through common early steps in this model. Furthermore, the authors hypothesize that the expression of OPN, a molecule associated with cancer aggressiveness, at the time of T2-weighted detection signals a time during glioma development when the lesion becomes refractory to treatment.

Osteopontin expression in intratumoral astrocytes marks tumor progression in gliomas induced by prenatal exposure to N-ethyl-N-nitrosourea.

To better study early events in glioma genesis, markers that reliably denote landmarks in glioma development are needed. In the present study, we used microarray analysis to compare the gene expression patterns of magnetic resonance imaging (MRI)-localized N-ethyl-N-nitrosourea (ENU)-induced tumors in rat brains with those of uninvolved contralateral side and normal brains. Our analysis identified osteopontin (OPN) as the most up-regulated gene in glioma. Using immunohistochemistry we then confirmed OPN expression in every tumor examined (n = 17), including those with diameters as small as 300 mum. By contrast, no OPN immunostaining was seen in normal brain or in brains removed from ENU-exposed rats before the development of glioma. Further studies confirmed that OPN was co-localized exclusively in intratumoral glial fibrillary acidic protein-expressing cells and was notably absent from nestin-expressing ones. In conjunction with this, we confirmed that both normal neurosphere cells and ENU-im-mortalized subventricular zone/striatal cells produced negligible amounts of OPN compared to the established rat glioma cell line C6. Furthermore, inducing OPN expression in an immortalized cell line increased cell proliferation. Based on these findings, we conclude that OPN overexpression in ENU-induced gliomas occurs within a specific subset of intratumoral glial fibrillary acidic protein-positive cells and becomes evident at the stage of tumor progression.

Isolation of immortalized, INK4a/ARF-deficient cells from the subventricular zone after in utero N-ethyl-N-nitrosourea exposure.

OBJECT: Brain tumors, including gliomas, develop several months after rats are exposed in utero to N-ethyl-N-nitroso-urea (ENU). Although pathological changes cannot be detected until these animals are several weeks old, the process that eventually leads to glioma formation must begin soon after exposure given the rapid clearance of the carcinogen and the observation that transformation of brain cells isolated soon after exposure occasionally occurs. This model can therefore potentially provide useful insights about the early events that precede overt glioma formation. The authors hypothesized that future glioma cells arise from stem/progenitor cells residing in or near the subventricular zone (SVZ) of the brain. METHODS: Cells obtained from the SVZ or corpus striatum in ENU-exposed and control rats were cultured in an epidermal growth factor (EGF)-containing, chemically defined medium. Usually, rat SVZ cells cultured in this manner (neurospheres) are nestin-positive, undifferentiated, and EGF-dependent and undergo cell senescence. Consistent with these prior observations, control SVZ cells undergo senescence by the 12th to 15th doubling (20 of 20 cultures). In contrast, three of 15 cultures of cells derived from the SVZs of individual ENU-treated rats continue to proliferate for more than 60 cell passages. Each of these nestin-expressing immortalized cell lines harbored a common homozygous deletion spanning the INK4a/ARF locus and was unable to differentiate into neural lineages after exposure to specific in vitro stimuli. Nevertheless, unlike the rat C6 glioma cell line, these immortalized cell lines demonstrate EGF dependence and low clonogenicity in soft agar and did not form tumors after intracranial transplantation. CONCLUSIONS: Data in this study indicated that immortalized cells may represent glioma precursors that reside in the area of the SVZ after ENU exposure that may serve as a reservoir for further genetic and epigenetic hits that could eventually result in a full glioma phenotype.

Aberrant nestin expression during ethylnitrosourea-(ENU)-induced neurocarcinogenesis.

Nestin is a unique intermediate filament protein. While it is robustly expressed in developing brain, postnatal expression is limited to the brain's subventricular zone (SVZ) and endothelial cells. Reexpression occurs, however, under several pathological conditions, including injury and neoplasia. We hypothesized that nestin would be a sensitive marker of early neoplasia after transplacental exposure of rats to ethylnitrosourea (ENU). Rats of various ages were administered bromodeoxyuridine (BudR) before sacrifice, and brain sections were examined for proliferative cells and several immunohistochemical markers, including nestin. Additional rats were examined after a stab wound injury to assess the expression of two of these markers, GFAP and nestin, in reactive astrocytes. All ENU-induced brain tumors (n = 9) were classified as gliomas (astrocytomas or oligoastrocytomas) based on their histology and immunophenotype. Nestin expression was noted in all tumors examined and was present in tumor cells as well as endothelial cells. During tumor development, we consistently noted nestin-expressing cells bearing multiple processes distributed throughout brain parenchyma. Both single cells and multiple cell clusters were observed as early as postnatal day 30 in all ENU-exposed brains examined (n = 11). Such distinctive nestin-expressing cells were not seen in nestin-stained control brains or ENU-exposed brains stained for GFAP or vimentin, nor was such a cell seen in a stab wound model used to assess reactive astrocytosis. While the number of these clusters was highly variable among rats, their size increased between 30 and 90 days. The data suggest that these nestin-expressing cells represent an early stage of the neoplastic process. It remains to be determined whether these cells become apparent at 30 days of age due to "dedifferentiation" of a local resident astrocyte or astrocyte precursor cell or migration of a relatively undifferentiated precursor/stem cell from the SVZ.

Neural stem cells and neuro-oncology: quo vadis?

Conventionally, gliomas are assumed to arise via transformation of an intraparenchymal glial cell that forms a mass that then expands centrifugally, eventually invading surrounding tissues. We propose an alternative model in which gliomas arise via initiation and promotion of cells within the brain's subependymal layer or subventricular zone, the source of a recently characterized pool of neural cells with the properties of self-renewal and multipotentiality (i.e., stem cells) that persists into adulthood. In this model, the particular histological subtype of glioma would represent the effects of temporal and spatial environmental influences rather than the particular cell of origin and the disease's centrifugal point would be the subependymal layer. The implications of such a model are discussed.