ENU-induced Brain Tumors Research Articles

Abstract 385: Inhibition of recurrences of experimental brain tumors after irradiation by blocking the activity of SDF-1 (CXCL12) using the Spiegelmer® NOX-A12.

Abstract With current treatment of glioblastomas 85% of the tumors recur within the field of high dose radiation. This tumor recurrence requires formation of new blood vessels. We have developed a new therapeutic paradigm based on the dual origin of tumor blood vessels: Angiogenesis - the sprouting of endothelial cells from nearby blood vessels, and vasculogenesis - the formation of blood vessels from circulating cells. Because tumor irradiation abrogates local angiogenesis the tumor must rely on the vasculogenesis pathway for regrowth after irradiation. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas implanted intracranially in the mouse brain can be delayed or prevented by inhibiting circulating blood vessel forming cells by blocking the interaction of the chemokine receptor CXCR4 with its ligand SDF-1 (CXCL12)1. However, SDF-1 has a second receptor, CXCR7, which has been implicated in endothelial cell migration, is present on tumor vasculature, and is potentially also able to activate vasculogenesis. To block both receptors we have therefore used NOX-A12, an L-enantiomeric RNA oligonucleotide (Spiegelmer®), which inhibits SDF-1 with subnanomolar affinity. We have tested the effect of this inhibitor on the response to irradiation of the U251 human GBM in nude mice and on ENU-induced brain tumors in the Sprague-Dawley rat. In the mouse model we found that inhibition of SDF-1 post-irradiation was highly effective in enhancing tumor response to a single dose of 20 Gy whole brain irradiation. In the rat model the pups from ENU-treated pregnant mothers begin to die of brain tumors from approximately 120 days of age. We performed two studies with this model: In the first we delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age and began treatment with NOX-A12 immediately following irradiation and continued every 2 days with either 5 or 20 mg/kg injected subcutaneously for either 4 or 8 weeks. These doses and times were chosen as equivalent to human doses and times that based on existing data have been found to be safe and well tolerated in human volunteers and which are effective in inhibiting the action of SDF-1. We found a marked prolongation of life of the rats that was dependent both on drug dose and length of treatment. In the second study we measured the size of the tumors post-irradiation using MRI and again found a pronounced enhancement of the efficacy of irradiation that was superior to the addition of temozolomide at doses equivalent to those used with concomitant radiotherapy in treating GBM. We believe that these encouraging data justify a human trial in first line glioblastoma patients. 1. Kioi M, Vogel H, Schultz G, Hoffman RM, Harsh GR, Brown JM: Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice. J Clin Invest 120:694-705, 2010 Citation Format: Shie-Chau Liu, Reem Alomran, Jason Stafford, Milton Merchant, Taichang Jang, Stefan Zollner, Anna Kruschinski, Laurence Recht, J. Martin Brown. Inhibition of recurrences of experimental brain tumors after irradiation by blocking the activity of SDF-1 (CXCL12) using the Spiegelmer® NOX-A12. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 385. doi:10.1158/1538-7445.AM2013-385

Abstract 4382: Inhibition of SDF-1 (CXCL12) using the Spiegelmer NOX-A12 markedly delays the recurrence of ENU-induced rat brain tumors following irradiation

Abstract We have previously shown that the post-irradiation recurrence of human glioblastomas implanted intracranially in the mouse brain can be delayed or prevented by inhibiting the interaction of the chemokine receptor CXCR4 with its ligand SDF-1 (CXCL12)1. This effect is due to inhibition of the post-irradiation recovery of the tumor vasculature from circulating cells, a process known as vasculogenesis. However, SDF-1 has a second receptor, CXCR7, which has been implicated in endothelial cell migration2, is present on tumor vasculature2, and is potentially also able to activate vasculogenesis. Therefore we investigated the efficacy of the SDF-1 inhibitor NOX-A12, an L-enantiomeric RNA oligonucleotide (Spiegelmer), on brain tumor recurrences after irradiation. NOX-A12 inhibits SDF-1 with subnanomolar affinity and should therefore inhibit SDF-1 mediated activation of both receptors, CXCR4 and CXCR7. In this study we used ENU-induced brain tumors in the Sprague-Dawley rat, a model that has proved extremely resistant to anticancer therapy in prior studies by a variety of investigators. Pregnant rats were injected with the carcinogen ethylnitrosourea (50mg/kg) on day 17 of gestation. In this model the pups appear normal at birth but begin 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 and began treatment with NOX-A12 immediately following irradiation and continued every 2 days with either 5 or 20 mg/kg injected subcutaneously for either 4 or 8 weeks. These doses and times were chosen as equivalent to human doses and times that based on existing data have been found to be safe and well tolerated in human volunteers and which are effective in inhibiting the action of SDF-1. We found that neither 20 Gy nor NOX-A12 alone prolonged the lifespan of the tumor-bearing rats. However, the addition of NOX-A12 to 20 Gy prolonged the lifespan of the rats particularly at the highest dose and for the longer treatment period of 8 weeks (median lifespans of 20 Gy alone and 20 Gy + 5 and 20 mg/kg of NOX-A12 were 196, 291 and 349 days respectively with p values for NOX-A12 treated rats <0.05 vs radiation or controls alone). We believe that these encouraging data justify a human trial in first line glioblastoma patients. 1. Kioi M, Vogel H, Schultz G, Hoffman RM, Harsh GR, Brown JM: Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice. J Clin Invest 120:694-705, 2010 2. Miao Z, Luker KE, Summers BC, Berahovich R, Bhojani MS, Rehemtulla A, et al: CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature. Proc Natl Acad Sci U S A 104:15735-15740, 2007 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4382. doi:1538-7445.AM2012-4382

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.

Effects of a cell phone radiofrequency (860 MHz) on the latency of brain tumors in rats

The purpose of this study was to determine if an 860-MHz pulsed radiofrequency (PRF) shortened the latency of ethylnitrosourea (ENU)-induced brain tumors in rats. A total of 1080 Sprague–Dawley rats born of mothers given 6.3 or 10.0 mg/kg ENU intravenously on day 15 of gestation was randomized by ENU dose group into 18 groups of 60 rats (30 males and 30 females). Six groups were exposed to the PRF, 6 groups were sham exposed and 6 were cage controls. The PRF was delivered 6 h per day, 5 days per week beginning at 52±3 days of age. The specific absorption rate was 1±0.2 W/kg average to the brain. An equal number of rats from each exposure group was randomly selected for euthanasia and necropsy every 30 days from 828 rats between 172 and 322 days of age. A total of 252 rats were sacrificed because of the development of nervous disorders or they died spontaneously. There was no statistically significant difference between the incidence rates of brain tumors among PRF, sham or cage control groups in either the serial sacrifice or morbid groups. These results revealed no evidence that the PRF affects the incidence rate (or latency) of ENU-induced brain tumors in rats.

Gangliosides modulate the development of ethyl-nitrosourea induced brain tumors in rats

The possible effect of gangliosides as biological modifiers of the ethyl-nitrosourea (ENU) model of neurocarcinogenesis in rats is studied. Wistar rats that were exposed to ENU during fetal life, were treated with gangliosides at a postnatal age corresponding to a stage of pretumor or microtumor development. The results obtained suggest that postnatal administration of gangliosides can influence the development of ENU-induced brain tumors, delaying the appearance of symptomatic neoplasms and improving survival with respect to controls. This finding suggests the potential usefulness of gangliosides in the control of neuroectodermal tumors.

Ultrastructural Study of Capillary Permeability of Liposome-encapsulated Cisplatin in an Experimental Rat Brain Tumor Model

The anticancer chemotherapeutic agent cisplatin (cis-diamminedichloroplatinum) has several disadvantages, including extreme nephrotoxicity, rapid binding to plasma proteins, and poor penetration into the central nervous system. Liposomes would seem to be suitable as carriers of cisplatin to brain tumors. This ultrastructural study was undertaken to identify the capillary permeability of liposome-encapsulated cisplatin in a rat brain tumor model. Brain tumors were induced transplacentally with a single intravenous dose of 50 mg/kg of ethylnitrosourea (ENU). One ml of liposome-encapsulated cisplatin and ferritin prepared from phosphatidylcholine and cholesterol (molar ratio 7:2) was administered via the carotid artery to rats with ENU-induced brain tumors. The tumor platinum content and that of the contralateral hemisphere were assayed by flameless atomic absorption spectrometry. The presence of ferritin-bearing liposomes was demonstrated by electron microscopy of brain capillary endothelium as well as tumor cells. Thirty minutes after drug administration, the average dry-weight platinum concentrations were 3.98 +/- 0.50 micrograms/g within the intracerebral tumors and 0.72 +/- 0.24 micrograms/g in the contralateral hemispheres. Electron microscopic ultrastructural study, which can distinguish ferritin-bearing liposomes from cell materials, demonstrated the presence of liposomes containing ferritin in both cell surface folds and capillary endothelial cells and also within tumor cells. An enlarged electron micrograph of a liposome with a diameter of approximately 0.2 micron suggested the presence of ferritin molecules in the lamellar structure. Liposome-encapsulated cisplatin is apparently able to pass the blood-brain barrier and should be useful in the chemotherapy of glioma.

Morphology of early stages of ENU-induced brain tumors in rats

Tumors were induced in experimental animals in order to investigate early tumor growth with conventional histology and gliofibrillary acid protein (GFAP) demonstration with the peroxidase-antiperoxidase (PAP) method. In one experiment, the animals were killed after transplacental ENU administration, when microtumors were suspected; in the second experiment, the animals were followed up to their natural death and microtumors found at random were used for analysis. Conventional histology revealed 3 types of microtumors: growth restricted to the subventricular matrix, growth in the neighbourhood of the ventricles with obvious or probable connection to the ventricular zones and small tumors observed exclusively in the white matter. The latter tumors by conventional staining were composed of small round cells, considered to be oligodendrocytes. They did not contain GFA-protein positive cells within the tumor. The tumor in presumable and visible connection with the ventricular lining did contain GFAP-positive astrocytes. In the very small subventricular tumors, the small round cells (oligodendrocytes) were in continuous contact with the identical interfascicular glial cells, while GFAP positive astrocytes seemed to stem from the subventricular astrocytes (tanycytes, ependymoglia). The ependyma itself was always preserved. A twofold origin of these experimental tumors with probable development into one common cytological glial type is assumed.

Lysosomal isozyme patterns in ethylnitrosourea-induced brain tumors

Isozyme patterns for five acid hydrolases, acid phosphatase (AP), arylsulfatase (AS), β-glucoronidase (β-Glu), N-acetylglucosaminidase (NAG) and β-galactosidase (β-Gal), were studied in isolated lysosomes from ethylnitrosourea (ENU)-induced gliomas and compared with normal and newborn rat brains. With polyacrylamide gel electrophoresis (PAGE), AP was separated into three bands, acidic (A), intermediate (B) and basic (C). In tumors and newborn brains there was a decrease in A and C but a significant increase in B. For NAG the acidic form was elevated by 9–19% in tumors, while newborn brains showed a 19% decrease. Even though the band intensities of β-Glu in tumors and newborn brains were increased, the relative distribution remained similar to normal brain. With isoelectric focusing, five hydrolases were separated into four to five distinct forms. In ENU-induced gliomas the intensities of all peaks were considerably increased, but in most cases the number of isozymes remained the same. In tumors the isolectric points were shifted towards the acidic side and smaller peaks in the basic regions merged into more acidic peaks. This effect was especially evident for AP and Gal. In the cases of AS, β-Glu and NAG, consistently more activity was associated with acidic peaks than with the basic ones. Our data indicates that there is a significant increase in acidic forms of some of the lysosomal hydrolases studied in ENU-induced brain tumors.