EGFRvIII-Specific Chimeric Antigen Receptor T Cells Migrate to and Kill Tumor Deposits Infiltrating the Brain Parenchyma in an Invasive Xenograft Model of Glioblastoma

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and is uniformly lethal. T-cell-based immunotherapy offers a promising platform for treatment given its potential to specifically target tumor tissue while sparing the normal brain. However, the diffuse and infiltrative nature of these tumors in the brain parenchyma may pose an exceptional hurdle to successful immunotherapy in patients. Areas of invasive tumor are thought to reside behind an intact blood brain barrier, isolating them from effective immunosurveillance and thereby predisposing the development of "immunologically silent" tumor peninsulas. Therefore, it remains unclear if adoptively transferred T cells can migrate to and mediate regression in areas of invasive GBM. One barrier has been the lack of a preclinical mouse model that accurately recapitulates the growth patterns of human GBM in vivo. Here, we demonstrate that D-270 MG xenografts exhibit the classical features of GBM and produce the diffuse and invasive tumors seen in patients. Using this model, we designed experiments to assess whether T cells expressing third-generation chimeric antigen receptors (CARs) targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, would localize to and treat invasive intracerebral GBM. EGFRvIII-targeted CAR (EGFRvIII⁺ CAR) T cells demonstrated in vitro EGFRvIII antigen-specific recognition and reactivity to the D-270 MG cell line, which naturally expresses EGFRvIII. Moreover, when administered systemically, EGFRvIII⁺ CAR T cells localized to areas of invasive tumor, suppressed tumor growth, and enhanced survival of mice with established intracranial D-270 MG tumors. Together, these data demonstrate that systemically administered T cells are capable of migrating to the invasive edges of GBM to mediate antitumor efficacy and tumor regression.     

Impact of Carotid Atherosclerosis Combined with Hypercholesterolemia on Cerebral Microvessels and Brain Parenchyma in a New Complex Rat Model

Most previous investigations about stroke caused by carotid atherosclerosis have focused on thromboembolism. There is a lack of knowledge about pathophysiology of the brain before ischemic insults. The objective of this study was to develop a new model of hypercholesterolemia plus carotid injury and to investigate the impact of carotid atherosclerosis combined with hypercholesterolemia in the rat brain. The complex rat model was developed by carotid injury induced by an air-drying endothelial denudation method after high cholesterol diet for 2 weeks. Plasma cholesterol, carotid pathomorphology, oxidative stress and inflammation in cerebral microvessels and brain parenchyma were measured at 7, 14 and 28 days after carotid surgery. The results showed that plasma concentrations of total cholesterol and low density lipoprotein-cholesterol were significantly increased, and severe carotid atherosclerosis and stenosis was observed in the complex rat model at 14 and 28 days after carotid surgery. The activity of superoxide dismutase was decreased, while the content of malondialdehyde was increased in cerebral microvessels and brain parenchyma. The levels of tumor necrosis factor-α and interleukin-1β were elevated in brain tissues of this model. Almost all above changes were more severe than those in either hypercholesterolemia alone group or carotid injury alone group. These results suggest that this complex rat model may more resemble human disease than the classic acute ischemic insult model for assessing the impact of carotid atherosclerosis as a preexisting disease on cerebral microcirculation and brain tissue.     

Mathematical Modelling of a Brain Tumour Initiation and Early Development: A Coupled Model of Glioblastoma Growth,Pre-Existing Vessel Co-Option,Angiogenesis and Blood Perfusion

We propose a coupled mathematical modelling system to investigate glioblastoma growth in response to dynamic changes in chemical and haemodynamic microenvironments caused by pre-existing vessel co-option, remodelling, collapse and angiogenesis. A typical tree-like architecture network with different orders for vessel diameter is designed to model pre-existing vasculature in host tissue. The chemical substances including oxygen, vascular endothelial growth factor, extra-cellular matrix and matrix degradation enzymes are calculated based on the haemodynamic environment which is obtained by coupled modelling of intravascular blood flow with interstitial fluid flow. The haemodynamic changes, including vessel diameter and permeability, are introduced to reflect a series of pathological characteristics of abnormal tumour vessels including vessel dilation, leakage, angiogenesis, regression and collapse. Migrating cells are included as a new phenotype to describe the migration behaviour of malignant tumour cells. The simulation focuses on the avascular phase of tumour development and stops at an early phase of angiogenesis. The model is able to demonstrate the main features of glioblastoma growth in this phase such as the formation of pseudopalisades, cell migration along the host vessels, the pre-existing vasculature co-option, angiogenesis and remodelling. The model also enables us to examine the influence of initial conditions and local environment on the early phase of glioblastoma growth.     

Xenopus Embryos as a Model to Study the Genetic Mechanisms of Brain Development

The review considers the advantages of Xenopus embryos as an experimental model to study the molecular-genetic mechanisms of embryo development. The results are described that were obtained with this model in studies on the early brain development within the framework of the Russian program Human Genome.     

Tannic Acid Attenuates Peripheral and Brain Changes in a Preclinical Rat Model of Glioblastoma by Modulating Oxidative Stress and Purinergic Signaling

Neurochemical Research - Glioblastoma (GB) is a highly aggressive and invasive brain tumor; its treatment remains palliative. Tannic acid (TA) is a polyphenol widely found in foods and possesses...     

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.     

Intracranial Stereotaxic Cannulation for Development of Orthotopic Glioblastoma Allograft in Sprague-Dawley Rats and Histoimmunopathological Characterization of the Brain Tumor

Glioblastoma is the most common brain tumor that causes significant mortality annually. Limitations of the current therapeutic regimens warrant development of new techniques and treatment strategies in orthotopic animal model for better management of this devastating brain cancer. There are only a few experimental orthotopic models of glioblastoma for pre-clinical testing. In the present investigation, we successfully implanted rat C6 cells via intracranial stereotaxic cannulation in adult Sprague-Dawley rats for development and histoimmunopathological characterization of an advanced orthotopic glioblastoma allograft model, which could be useful for investigating the course of glioblastoma development as well as for testing efficacy of new therapeutic agents. The orthotopic glioblastoma allograft was generated by intracerebral injection of rat C6 cells through a guide-cannula system and after 21 post-inoculation days the brain tumor was characterized by histoimmunopathological experiments. Histological staining and immunofluorescent labelings for TERT, VEGF, Bcl-2, survivin, XIAP, and GFAP revealed the distinct characteristics of glioblastoma in C6 allograft, which could be useful as a target for treatment with emerging new therapeutic agents. Our investigation indicated the successful development of intracranial cannulated orthotopic glioblastoma allograft in adult Sprague-Dawley rats, making it as a useful animal model of glioblastoma for pre-clinical evaluation of various therapeutic strategies for the management of glioblastoma. Special issue in honor of Naren Banik.     

Label-Free Delineation of Brain Tumors by Coherent Anti-Stokes Raman Scattering Microscopy in an Orthotopic Mouse Model and Human Glioblastoma

Background

Coherent anti-Stokes Raman scattering (CARS) microscopy provides fine resolution imaging and displays morphochemical properties of unstained tissue. Here, we evaluated this technique to delineate and identify brain tumors.

Methods

Different human tumors (glioblastoma, brain metastases of melanoma and breast cancer) were induced in an orthotopic mouse model. Cryosections were investigated by CARS imaging tuned to probe C-H molecular vibrations, thereby addressing the lipid content of the sample. Raman microspectroscopy was used as reference. Histopathology provided information about the tumor''s localization, cell proliferation and vascularization.

Results

The morphochemical contrast of CARS images enabled identifying brain tumors irrespective of the tumor type and properties: All tumors were characterized by a lower CARS signal intensity than the normal parenchyma. On this basis, tumor borders and infiltrations could be identified with cellular resolution. Quantitative analysis revealed that the tumor-related reduction of CARS signal intensity was more pronounced in glioblastoma than in metastases. Raman spectroscopy enabled relating the CARS intensity variation to the decline of total lipid content in the tumors. The analysis of the immunohistochemical stainings revealed no correlation between tumor-induced cytological changes and the extent of CARS signal intensity reductions. The results were confirmed on samples of human glioblastoma.

Conclusions

CARS imaging enables label-free, rapid and objective identification of primary and secondary brain tumors. Therefore, it is a potential tool for diagnostic neuropathology as well as for intraoperative tumor delineation.     

Semaphorin-3D and Semaphorin-3E Inhibit the Development of Tumors from Glioblastoma Cells Implanted in the Cortex of the Brain

Class-3 semaphorins are secreted axon guidance factors. Some of these semaphorins have recently been characterized as suppressors of tumor progression. To determine if class-3 semaphorins can be used to inhibit the development of glioblastoma-multiforme tumors, we expressed recombinant sema-3A, 3B, 3D, 3E, 3F or 3G in U87MG glioblastoma cells. Sema3A and sema3B expressing cells contracted and changed shape persistently while cells expressing other semaphorins did not. Sema3A and sema3F differed from other semaphorins including sema3B as they also inhibited the proliferation of the cells and the formation of soft agar colonies. With the exception of sema3G and sema3B, expression of these semaphorins in U87MG cells inhibited significantly tumor development from subcutaneously implanted cells. Strong inhibition of tumor development was also observed following implantation of U87MG cells expressing each of the class-3 semaphorins in the cortex of mouse brains. Sema3D and sema3E displayed the strongest inhibitory effects and their expression in U373MG or in U87MG glioblastoma cells implanted in the brains of mice prolonged the survival of the mice by more then two folds. Furthermore, most of the mice that died prior to the end of the experiment did not develop detectable tumors and many of the mice survived to the end of the experiment. Most of the semaphorins that we have used here with the exception of sema3D were characterized previously as inhibitors of angiogenesis. Our results indicate that sema3D also functions as an inhibitor of angiogenesis and suggest that the anti-tumorigenic effects are due primarily to inhibition of tumor angiogenesis. These results indicate that class-3 semaphorins such as sema3D and sema3E could perhaps be used to treat glioblastoma patients.     

Wall Development in Apple Fruits: a Study of the Life History of a Parenchyma cell

The development of the walls of the parenchyma cells of applefruit cortex has been followed throughout two seasons underconditions which allow an assessment of wall structure as afactor involved in the keeping quality of apples. Changes inchemical compositions, in physical organization, and in cellsize have been monitored throughout.Unexpectedly rapid wallsynthesis occurs during cell extension but is followed, beforeharvest, by a period during which some pectic substances arereassimilated and in which some lability of wall componentsis indicated. There is a marked thinning of the wall at thisstage, for which subsequent secondary thickening fails to compensatefully.Differences in cell-wall structure between apples fromtrees receiving different manurial treatments are most markedduring cell extension, when cells from apples which keep wellshow symptoms consistent with a lower nitrogen level. Thesedifferences have largely disappeared by harvest, but reasonsare given to indicate that the earlier stages of fruit developmentmay have a considerable influence on potential storage life.     

Sleep and Rhythm Changes at the Time of Trypanosoma brucei Invasion of the Brain Parenchyma in the Rat

Human African trypanosomiasis (HAT), or sleeping sickness, is a severe disease caused by Trypanosoma brucei (T.b.). The disease hallmark is sleep alterations. Brain involvement in HAT is a crucial pathogenetic step for disease diagnosis and therapy. In this study, a rat model of African trypanosomiasis was used to assess changes of sleep-wake, rest-activity, and body temperature rhythms in the time window previously shown as crucial for brain parenchyma invasion by T.b. to determine potential biomarkers of this event. Chronic radiotelemetric monitoring in Sprague-Dawley rats was used to continuously record electroencephalogram, electromyogram, rest-activity, and body temperature in the same animals before (baseline recording) and after infection. Rats were infected with T.b. brucei. Data were acquired from 1 to 20 d after infection (parasite neuroinvasion initiates at 11–13 d post-infection in this model), and were compared to baseline values. Sleep parameters were manually scored from electroencephalographic-electromyographic tracings. Circadian rhythms of sleep time, slow-wave activity, rest-activity, and body temperature were studied using cosinor rhythmometry. Results revealed alterations of most of the analyzed parameters. In particular, sleep pattern and sleep-wake organization plus rest-activity and body temperature rhythms exhibited early quantitative and qualitative alterations, which became marked around the time interval crucial for parasite neuroinvasion or shortly after. Data derived from actigrams showed close correspondence with those from hypnograms, suggesting that rest-activity could be useful to monitor sleep-wake alterations in African trypanosomiasis. (Author correspondence: giuseppe.bertini@univr.it)     

Ionic Partition between Surface and Bulk Water in a Silica Gel: A Biological Model

Distribution of the biologically important ions between two aqueous phases of different structure has been used as a model for ionic distribution in living tissue. When other sources of specificity had been eliminated or corrected for, surface-oriented water in a silica gel was found to have increased solvent power for water-structure-breaking ions and decreased solvent power for water-structure-making ions; and the relative solubility of an ion in the phase of enhanced structure increased regularly with the water-structure-breaking powers of the ion. The ionic selectivity was decreased in the presence of urea. The selectivity of the gel water for potassium relative to sodium increased to a maximum when the gel surface was partially ionized so that distribution of cations was not linked to distribution of anions, and then decreased as the surface changed from a hydrogen bonding to an ionic surface. It is pointed out that the distribution of ions across most living cell membranes is qualitatively the same as that found in this silica gel, and it is suggested that the membrane separates two aqueous phases of different structure, and that the enhanced structure of cell water contributes to the observed ionic distributions.     

Photosynthetic Carbon Metabolism in the Palisade Parenchyma and Spongy Parenchyma of Vicia faba L

Palisade parenchyma cells and spongy parenchyma cells were isolated separately from Vicia faba L. leaflets. Extracts of the cell isolates were assayed for several enzymes involved in CO₂ fixation and photorespiration. When compared on a chlorophyll basis, the levels of enzyme activities either were equal in the different cell types or were greater in the spongy parenchyma; this difference is a reflection, perhaps, of the higher protein-chlorophyll ratio in the latter tissue. The distribution of radioactivity in the products of photosynthesis by each cell type was the same at various times after exposure to NaH¹⁴CO₃, and the kinetics of ¹⁴C incorporation into these compounds was similar. However, a larger percentage of radioactivity was incorporated by the cell isolates into the 80% ethanol-insoluble fraction and correspondingly less into the neutral fraction as compared to whole leaf. It was concluded that photosynthetic CO₂ fixation is similar in the different mesophyll tissues from which these cells were derived.     

Hyperthermic Laser Ablation of Recurrent Glioblastoma Leads to Temporary Disruption of the Peritumoral Blood Brain Barrier

Background

Poor central nervous system penetration of cytotoxic drugs due to the blood brain barrier (BBB) is a major limiting factor in the treatment of brain tumors. Most recurrent glioblastomas (GBM) occur within the peritumoral region. In this study, we describe a hyperthemic method to induce temporary disruption of the peritumoral BBB that can potentially be used to enhance drug delivery.

Methods

Twenty patients with probable recurrent GBM were enrolled in this study. Fourteen patients were evaluable. MRI-guided laser interstitial thermal therapy was applied to achieve both tumor cytoreduction and disruption of the peritumoral BBB. To determine the degree and timing of peritumoral BBB disruption, dynamic contrast-enhancement brain MRI was used to calculate the vascular transfer constant (K^trans) in the peritumoral region as direct measures of BBB permeability before and after laser ablation. Serum levels of brain-specific enolase, also known as neuron-specific enolase, were also measured and used as an independent quantification of BBB disruption.

Results

In all 14 evaluable patients, K^trans levels peaked immediately post laser ablation, followed by a gradual decline over the following 4 weeks. Serum BSE concentrations increased shortly after laser ablation and peaked in 1–3 weeks before decreasing to baseline by 6 weeks.

Conclusions

The data from our pilot research support that disruption of the peritumoral BBB was induced by hyperthemia with the peak of high permeability occurring within 1–2 weeks after laser ablation and resolving by 4–6 weeks. This provides a therapeutic window of opportunity during which delivery of BBB-impermeant therapeutic agents may be enhanced.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01851733","term_id":"NCT01851733"}}NCT01851733     

Effects of Anti-Angiogenesis on Glioblastoma Growth and Migration: Model to Clinical Predictions

Glioblastoma multiforme (GBM) causes significant neurological morbidity and short survival times. Brain invasion by GBM is associated with poor prognosis. Recent clinical trials of bevacizumab in newly-diagnosed GBM found no beneficial effects on overall survival times; however, the baseline health-related quality of life and performance status were maintained longer in the bevacizumab group and the glucocorticoid requirement was lower. Here, we construct a clinical-scale model of GBM whose predictions uncover a new pattern of recurrence in 11/70 bevacizumab-treated patients. The findings support an exception to the Folkman hypothesis: GBM grows in the absence of angiogenesis by a cycle of proliferation and brain invasion that expands necrosis. Furthermore, necrosis is positively correlated with brain invasion in 26 newly-diagnosed GBM. The unintuitive results explain the unusual clinical effects of bevacizumab and suggest new hypotheses on the dynamic clinical effects of migration by active transport, a mechanism of hypoxia-driven brain invasion.     

Distribution of TRPC6 in the Cerebrospinal Fluid-Contacting Nucleus of Rat Brain Parenchyma and its Expression in Morphine Dependence and Withdrawal

To investigate the role of cerebrospinal fluid contacting nucleus (CSF-CN) and the changes of TRPC6 expression in morphine dependence and withdrawal. Male Sprague–Dawley rats (250 ± 50 g) were randomly divided into four groups: the normal group (N); the saline group (S); the morphine dependence group (D); the morphine withdrawal group (W). All animals in each group were tested the morphine withdrawal-like behavioral signs by total withdrawal scores. Double-labeled immunofluorescent technique and laser scanning confocal microscopy were used to identify the expression of TRPC6 in CSF-CN. TRPC6 labeled neurons were found in CSF-CN and the number of CB-HRP/TRPC6 double labeled neurons in CSF-CN significantly increased. TRPC6 existed in CSF-CN of the normal rats and its expression in morphine dependence and withdrawal increased. CSF-CN might participate in the development of morphine dependence and withdrawal by the up-regulated expression of TRPC6.     

Defective Craniofacial Development and Brain Function in a Mouse Model for Depletion of Intracellular Inositol Synthesis

myo-Inositol is an essential biomolecule that is synthesized by myo-inositol monophosphatase (IMPase) from inositol monophosphate species. The enzymatic activity of IMPase is inhibited by lithium, a drug used for the treatment of mood swings seen in bipolar disorder. Therefore, myo-inositol is thought to have an important role in the mechanism of bipolar disorder, although the details remain elusive. We screened an ethyl nitrosourea mutant mouse library for IMPase gene (Impa) mutations and identified an Impa1 T95K missense mutation. The mutant protein possessed undetectable enzymatic activity. Homozygotes died perinatally, and E18.5 embryos exhibited striking developmental defects, including hypoplasia of the mandible and asymmetric fusion of ribs to the sternum. Perinatal lethality and morphological defects in homozygotes were rescued by dietary myo-inositol. Rescued homozygotes raised on normal drinking water after weaning exhibited a hyper-locomotive trait and prolonged circadian periods, as reported in rodents treated with lithium. Our mice should be advantageous, compared with those generated by the conventional gene knock-out strategy, because they carry minimal genomic damage, e.g. a point mutation. In conclusion, our results reveal critical roles for intracellular myo-inositol synthesis in craniofacial development and the maintenance of proper brain function. Furthermore, this mouse model for cellular inositol depletion could be beneficial for understanding the molecular mechanisms underlying the clinical effect of lithium and myo-inositol-mediated skeletal development.