Taichang Jang

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 brains 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 diseases centrifugal point would be the subependymal layer. The implications of such a model are discussed.

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.

Metabolic response of glioma to dichloroacetate measured in vivo by hyperpolarized 13C 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.

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.

Metabolite kinetics in C6 rat glioma model using magnetic resonance spectroscopic imaging of hyperpolarized [1‐13C]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 [1‐13C]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). Magn Reson Med, 2012.

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

MRS of hyperpolarized [2‐13C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the 13C label is not lost with the conversion of pyruvate to acetyl‐CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2‐13C]pyruvate in glioma‐bearing brain.

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 gliomas 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.

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 [1‐13C]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 13C‐pyruvate imaging studies had focused solely on [1‐13C]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 13C‐bicarbonate in cancer tissue, which does provide such information, has proven difficult, however. In this study, we report the reliable measurement of 13C‐bicarbonate production in both the healthy brain and a highly glycolytic experimental glioblastoma model using an optimized 13C MRS imaging protocol. With the capacity to obtain signal in all tumors, we also confirm for the first time that the ratio of 13C‐lactate to 13C‐bicarbonate provides a more robust metric relative to 13C‐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. Copyright

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.

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.