Intratumoral Delivery of STING Agonist Results in Clinical Responses in Canine Glioblastoma.

PURPOSE: Activation of STING (stimulator of interferon genes) can trigger a robust, innate antitumor immune response in immunologically "cold" tumors such as glioblastoma. PATIENTS AND METHODS: A small-molecule STING agonist, IACS-8779, was stereotactically administered using intraoperative navigation intratumorally in dogs with spontaneously arising glioblastoma. The phase I trial used an escalating dose design, ascending through four dose levels (5-20 µg). Treatment was repeated every 4-6 weeks for a minimum of two cycles. Radiographic response to treatment was determined by response assessment in neuro-oncology (RANO) criteria applied to isovoxel postcontrast T1-weighted MR images obtained on a single 3T magnet. RESULTS: Six dogs were enrolled and completed ≥1 cycle of treatment. One dog was determined to have an abscess and was removed from further analysis. One procedure-related fatality was observed. Radiographic responses were dose dependent after the first cycle. The first subject had progressive disease, whereas there was 25% volumetric reduction in one subject and greater than 50% in the remaining surviving subjects. The median progression-free survival time was 14 weeks (range: 0-22 weeks), and the median overall survival time was 32 weeks (range: 11-39 weeks). CONCLUSIONS: Intratumoral STING agonist (IACS-8779) administration was well tolerated in dogs with glioblastoma to a dose of 15 µg. Higher doses of IACS-8779 were associated with radiographic responses.

Photodynamic detection of a canine glioblastoma using 5-aminolevulinic acid.

Photodynamic detection using 5-aminolevulinic acid has been used to identify the surgical margins during resection of human primary brain tumours. Although there are some reports on its use in malignant tumours in veterinary medicine, it has never been used for primary brain tumours. Here we describe a canine glioblastoma that was detected at autopsy with protoporphyrin IX fluorescence induced by orally administered 5-aminolevulinic acid. The fluorescence was strongest towards the centre of the lesion and was absent in normal brain tissue. Moreover, the fluorescence findings were consistent with MRI and histopathological findings. Our findings suggest that photodynamic detection using 5-aminolevulinic acid might be useful for intraoperative fluorescence-guided resection of malignant gliomas in dogs.

Baseballs, tennis balls, livestock farm manure, the IDH1 mutation, endothelial cell proliferation and hypoxic pseudopalisading (granulomatous) necrosis: Mycobacterium avium subspecies paratuberculosis and the epidemiology, cellular metabolism and histology of diffuse gliomas, including glioblastoma.

An increased rate of diffuse gliomas, including glioblastoma, has been noted in livestock farmers in Western countries. Some researchers have suggested that a zoonotic virus or bacteria present in the livestock animal's feces or manure may be a possible etiologic factor. Mycobacterium avium subspecies paratuberculosis (MAP), the cause of a chronic enteropathy in domestic livestock and a probable zoonosis, is heavily excreted in an infected animal's feces or manure, contaminating soil and ground on the animal's farm. Once excreted in an animal's feces, MAP lasts indefinitely in a dormant but viable form, and easily spreads outside farms to the surrounding environment. MAP's presence throughout the soil in countries where MAP infection of domestic livestock is extensive and long-standing may explain the increased rates of glioblastoma in tennis and baseball players who handle balls coated with MAP-contaminated dirt. MAP infection is consistent with glioblastoma's two defining histopathologic characteristics: endothelial cell proliferation and pseudopalisading necrosis. MAP is a non-tuberculous or atypical mycobacterium, which can cause hypoxic necrotizing granulomas, granulomas that resemble areas of pseudopalisading necrosis. There are known bacterial causes of endothelial cell proliferation. Almost unique amongst intracellular bacteria, MAP's variant isocitrate dehydrogenase 1 (IDH1) enzyme, a type 2-oxoglutarate ferredoxin oxidoreductase, can use a host cell's cytosolic α-ketoglutarate in its own Krebs or tricarboxylic acid cycle. MAP's ability to use a host cell's α-ketoglutarate may explain the survival advantage of the cytosolic IDH1 enzyme mutation for patients with diffuse gliomas including glioblastoma, astrocytoma, and oligdendroglioma, a mutation that results in a reduced supply of cytosolic α-ketoglutarate. MAP may therefore be one possible infectious cause of glioblastoma and the other histologic categories of diffuse glioma.

Evaluation of four different small animal radiation plans on tumour and normal tissue dosimetry in a glioblastoma mouse model.

OBJECTIVE:: Small animal radiotherapy research platforms such as XStrahl's SARRP enable more precise irradiation of tumours and normal tissues in pre-clinical models of cancer. Using an orthotopic G7 glioblastoma xenograft model we studied the impact of four different radiotherapy plans on tumour and normal tissue dosimetry. METHODS:: Plans were created using four different approaches (single beam, parallel opposed pair, single plane arcs, couch rotation arcs) and dose volume histograms (DVH) for the tumour and the relevant organs at risk (OARs) (mouth, ipsilateral brain, contralateral brain, brain stem) were compared for a sample mouse subject. To evaluate the accuracy of delivery, treatment plans were recreated in solid-water phantoms and delivered to radiochromic film. RESULTS:: Favourable tumour dosimetry was achieved by all plans. DVH analysis showed that different plans could be used to spare specific OARs depending on the objectives of the study. The delivery accuracy of the various treatments was better than 2%/2mm (dose difference/distance to agreement) in terms of global γ analysis. CONCLUSION:: Small animal radiotherapy research platforms are an exciting addition to the pre-clinical research environment. Such systems improve the conformality of irradiation of tumours and OARs while maintaining a high degree of accuracy and enable investigators to optimise experiments in terms of tumour coverage and inclusion or exclusion of relevant OARs. ADVANCES IN KNOWLEDGE:: This study confirms the utility of the SARRP in terms of the accuracy of plan delivery, and informs decisions on treatment planning to optimise the clinical relevance and scientific value of experiments.

Canine brain tumours: a model for the human disease?

Canine brain tumours are becoming established as naturally occurring models of disease to advance diagnostic and therapeutic understanding successfully. The size and structure of the dog's brain, histopathology and molecular characteristics of canine brain tumours, as well as the presence of an intact immune system, all support the potential success of this model. The limited success of current therapeutic regimens such as surgery and radiation for dogs with intracranial tumours means that there can be tremendous mutual benefit from collaboration with our human counterparts resulting in the development of new treatments. The similarities and differences between the canine and human diseases are described in this article, emphasizing both the importance and limitations of canines in brain tumour research. Recent clinical veterinary therapeutic trials are also described to demonstrate the areas of research in which canines have already been utilized and to highlight the important potential benefits of translational research to companion dogs.

Stage-specific embryonic antigen: determining expression in canine glioblastoma, melanoma, and mammary cancer cells.

The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1- and SSEA-1+ cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines.

Glioblastoma Multiforme with Hypodipsic Hypernatremia in a Seven-Month-Old Golden Retriever.

Primary hypodipsic hypernatremia is a rarely reported disease in dogs. Reported underlying causes associated with this disease in dogs include congenital malformations, encephalitis, intracranial neoplasia, and pressure atrophy of the hypothalamus secondary to hydrocephalus. The dog in this report had an infiltrative neoplastic disorder, likely causing damage to the hypothalamic osmoreceptors responsible for the thirst generation. The neoplastic process was identified histopathologically as glioblastoma multiforme, an unusual tumor to occur in a dog this young. A tumor of the central nervous system causing physical destruction of the osmoreceptors has rarely been reported in dogs and none of the previously reported cases involved a glial cell tumor.

Glioblastoma with oligodendroglioma component in a ewe.

Herein we describe a glioblastoma partially occupying the telencephalic portion of the left cerebral hemisphere of a Sardinian (syn. Sarda) breed ewe. Microscopically, the mass consisted of a pleomorphic spindle-shaped cell component organized as bundles and numerous small areas of round cells displaying an oligodendroglioma-like aspect. A high number of mitotic figures, large areas of necrosis surrounded by pseudopalisading glial cells, and multiple foci of dystrophic mineralization were also observed. The neoplasm was highly vascularized with glomerular vascular proliferation. Immunohistochemically, neoplastic cells proved to be strongly positive for nestin, vimentin, and olig-2, whereas they were invariably negative for synaptophysin. Few neoplastic cells and reactive astrocytes, mainly located at the edge of necrotic foci, proved to be positive for glial fibrillary acidic protein, whereas glomerular vascular proliferation was clearly positive for factor VIII and vascular endothelial growth factor. Gene sequencing analysis demonstrated homozygous p53 tumor suppressor gene (TP53) point mutations in the DNA-binding domain located in exon 8. The presence of round cells immunoreactive for olig-2 demonstrated that this tumor is a glioblastoma with oligodendroglioma component. Our pathologic, immunohistochemical, and molecular findings largely overlap those previously reported in humans and dogs.

Targeted cellular ablation based on the morphology of malignant cells.

Treatment of glioblastoma multiforme (GBM) is especially challenging due to a shortage of methods to preferentially target diffuse infiltrative cells, and therapy-resistant glioma stem cell populations. Here we report a physical treatment method based on electrical disruption of cells, whose action depends strongly on cellular morphology. Interestingly, numerical modeling suggests that while outer lipid bilayer disruption induced by long pulses (~100 µs) is enhanced for larger cells, short pulses (~1 µs) preferentially result in high fields within the cell interior, which scale in magnitude with nucleus size. Because enlarged nuclei represent a reliable indicator of malignancy, this suggested a means of preferentially targeting malignant cells. While we demonstrate killing of both normal and malignant cells using pulsed electric fields (PEFs) to treat spontaneous canine GBM, we proposed that properly tuned PEFs might provide targeted ablation based on nuclear size. Using 3D hydrogel models of normal and malignant brain tissues, which permit high-resolution interrogation during treatment testing, we confirmed that PEFs could be tuned to preferentially kill cancerous cells. Finally, we estimated the nuclear envelope electric potential disruption needed for cell death from PEFs. Our results may be useful in safely targeting the therapy-resistant cell niches that cause recurrence of GBM tumors.

High-grade astrocytoma (Glioblastoma Multiforme) in an Atlantic spotted dolphin (Stenella frontalis).

This report describes the gross, microscopical and immunohistochemical features of a high-grade astrocytoma (glioblastoma multiforme) in an adult male Atlantic spotted dolphin (Stenella frontalis). On necropsy examination, a 5 × 2.5 × 2 cm, poorly demarcated, red, friable and locally expansile mass effaced the thalamus and the left periventricular region and extended to the left lateral ventricle of the brain. Microscopically, the mass consisted of haphazardly arranged bundles and rows of interweaving polygonal to spindle-shaped cells. These often palisaded along serpentine foci of necrosis and were surrounded by prominent vessels. Immunohistochemically, the neoplastic cells expressed glial fibrillary acidic protein, but not vimentin, S100 protein, neuron-specific enolase or neurofilament protein. A diagnosis of high-grade astrocytoma was made and this represents the first description of a glioma in a cetacean species.

Suspected primary glioblastoma multiforme in the canine spinal cord.

An eight-year-old mixed-breed dog was presented with progressive paraparesis. Neurological examination revealed a painful diffuse lesion between spinal cord segments T3 and L3. Magnetic resonance images displayed multi-focal contrast enhancing spinal cord and meningeal lesions. Cytology of these lesions revealed a malignant tumour prompting euthanasia of the dog. Histopathology confirmed the cytological diagnosis and a final diagnosis of a glioblastoma multiforme was made based on immunohistochemistry.

Magnetic resonance imaging apparent diffusion coefficients for histologically confirmed intracranial lesions in dogs.

Diffusion-weighted imaging is an advanced magnetic resonance imaging technique that is well established in the diagnosis of nonhemorrhagic infarction in people. However, recent investigations into intracranial neoplastic and inflammatory disease in people have identified variable and overlapping results. In this retrospective study of 37 dogs with histologically confirmed intracranial disease, we investigated whether histogram analysis of quantitative apparent diffusion coefficients (ADC) can differentiate specific disease processes. Disease categories included: meningiomas, glial cell tumors, choroid plexus tumors, pituitary tumors, inflammatory brain diseases, acute nonhemorrhagic infarcts, chronic nonhemorrhagic infarcts, and hemorrhagic infarcts. A wide range of ADC value distributions were identified within the disease groups, and there were overlapping ADC values between most groups. Low ADC values indicating restricted diffusion of water were identified in acute nonhemorrhagic infarcts as expected, but were also seen in meningiomas, glial cell tumors, and granulomatous meningoencephalitis. Based on this preliminary data it is unlikely that singular quantitative ADC values can be used to determine the histological type of canine intracranial disease.

Giant cell glioblastoma in the cerebrum of a Pembroke Welsh corgi.

A 6-year-old, neutered female Pembroke Welsh corgi was presented with a 1-month history of ataxia and panting. The clinical signs progressed until the dog became anorexic, obtunded and exhibited circling to the left. At necropsy examination, a mass was detected in the left forebrain, impinging on the cribriform plate. Microscopically, the mass was composed of sheets of round to pleomorphic neoplastic cells with vacuolated cytoplasm. Nuclear atypia, anisocytosis and anisokaryosis were common. Numerous bizarre, multinucleated giant cells containing 60 or more nuclei and giant mononuclear cells were present. The matrix contained abundant reticulin. Immunohistochemistry revealed the neoplastic cells uniformly to express vimentin, and a small number of neoplastic cells expressed glial fibrillary acid protein. A diagnosis of giant cell glioblastoma was made. Although well recognized in man, this tumour has been documented rarely in the veterinary literature.

Identification of cancer stem cells in dog glioblastoma.

There is increasing evidence in some malignancies that the tumor clone is heterogeneous in regard to proliferation and differentiation. The cancer stem cell hypothesis implies that not all the cells in the tumor have the same capacity to proliferate and maintain the growth of the tumor. Only a relatively small fraction of cells in the tumor, termed cancer stem cells (CSCs), possess the ability to proliferate and self-renew extensively. In the past decade, several groups have reported the existence of a CSC population in different human brain tumors from both children and adults. We report here the identification of a CSC population from a Boxer dog with glioblastoma multiforme (GBM) that possesses a great capacity for proliferation, self-renewal, and differentiation. This cloned cell line is aneuploid, forms neurospheres in culture, possesses CSC markers, and reproduces the original dog GBM when inoculated into the nude mouse brain.

Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression.

Although rodent glioblastoma (GBM) models have been used for over 30 years, the extent to which they recapitulate the characteristics encountered in human GBMs remains controversial. We studied the histopathological features of dog GBM and human xenograft GBM models in immune-deficient mice (U251 and U87 GBM in nude Balb/c), and syngeneic GBMs in immune-competent rodents (GL26 cells in C57BL/6 mice, CNS-1 cells in Lewis rats). All GBMs studied exhibited neovascularization, pleomorphism, vimentin immunoreactivity, and infiltration of T-cells and macrophages. All the tumors showed necrosis and hemorrhages, except the U87 human xenograft, in which the most salient feature was its profuse neovascularization. The tumors differed in the expression of astrocytic intermediate filaments: human and dog GBMs, as well as U251 xenografts expressed glial fibrillary acidic protein (GFAP) and vimentin, while the U87 xenograft and the syngeneic rodent GBMs were GFAP(-) and vimentin(+). Also, only dog GBMs exhibited endothelial proliferation, a key feature that was absent in the murine models. In all spontaneous and implanted GBMs we found histopathological features compatible with tumor invasion into the non-neoplastic brain parenchyma. Our data indicate that murine models of GBM appear to recapitulate several of the human GBM histopathological features and, considering their reproducibility and availability, they constitute a valuable in vivo system for preclinical studies. Importantly, our results indicate that dog GBM emerges as an attractive animal model for testing novel therapies in a spontaneous tumor in the context of a larger brain.

Microarray analysis of differentially expressed genes of primary tumors in the canine central nervous system.

The pathophysiologic similarities of many human and canine cancers support the role of the domestic dog as a model for brain tumor research. Here we report the construction of a custom canine brain-specific cDNA microarray and the analysis of gene expression patterns of several different types of canine brain tumor. The microarray contained 4000 clones from a canine brain specific cDNA library including 2161 clones that matched known genes or expressed sequence tags (ESTs) and 25 cancer-related genes. Our study included 16 brain tumors (seven meningiomas, five glial tumors, two ependymomas, and two choroid plexus papillomas) from a variety of different dog breeds. We identified several genes previously found to be differentially expressed in human brain tumors. This suggests that human and canine brain tumors share a common pathogenesis. In addition, we also found differentially expressed genes unique to either meningiomas or the glial tumors. This report represents the first global gene expression analysis of different types of canine brain tumors by cDNA microarrays and might aid in the identification of potential candidate genes involved in tumor formation and progression.

Recombinant Sendai virus vector induces complete remission of established brain tumors through efficient interleukin-2 gene transfer in vaccinated rats.

PURPOSE: Sendai virus (SeV), a murine parainfluenza virus type I, replicates independent of cellular genome and directs high-level gene expressions when used as a viral vector. We constructed a nontransmissible recombinant SeV vector by deleting the matrix (M) and fusion (F) genes from its genome (SeV/DeltaMDeltaF) to enhance its safety. We also estimated the therapeutic efficacy of the novel vector system against a rat glioblastoma model. EXPERIMENTAL DESIGN: We administered the recombinant SeV vector carrying the lacZ gene or the human interleukin-2 (hIL-2) gene into established 9L brain tumors in vivo simultaneous with peripheral vaccination using irradiated 9L cells. Sequential monitoring with magnetic resonance imaging was used to evaluate the therapeutic efficacy. RESULTS: We found extensive transduction of the lacZ gene into the brain tumors and confirmed sufficient amounts of interleukin 2 (IL-2) production by hIL2-SeV/DeltaMDeltaF both in vitro and in vivo. The magnetic resonance imaging study showed that the intracerebral injection of hIL2-SeV/DeltaMDeltaF brought about significant reduction of the tumor growth, including complete elimination of the established brain tumors. The (51)Cr release assay showed that significant amounts of 9L-specific cytotoxic T cells were induced by the peripheral vaccination. Immunohistochemical analysis revealed that CD4(+) T cells and CD8(+) T cells were abundantly infiltrated in the target tumors. CONCLUSION: The present results show that the recombinant nontransmissible SeV vector provides efficient in vivo gene transfer that induces significant regression of the established brain tumors and suggest that it will be a safe and useful viral vector for the clinical practice of glioma gene therapy.

Combined cimetidine and temozolomide, compared with temozolomide alone: significant increases in survival in nude mice bearing U373 human glioblastoma multiforme orthotopic xenografts.

OBJECT: Malignant gliomas consist of both heterogeneous proliferating and migrating cell subpopulations, with migrating glioma cells exhibiting less sensitivity to antiproliferative or proapoptotic drugs than proliferative cells. Therefore, the authors combined cimetidine, an antiinflammatory agent already proven to act against migrating epithelial cancer cells, with temozolomide to determine whether the combination induces antitumor activities in experimental orthotopic human gliomas compared with the effects of temozolomide alone. METHODS: Cimetidine added to temozolomide compared with temozolomide alone induced survival benefits in nude mice with U373 human glioblastoma multiforme (GBM) cells orthotopically xenografted in the brain. Computer-assisted phase-contrast microscopy analyses of 9L rat and U373 human GBM cells showed that cimetidine significantly decreased the migration levels of these tumor cells in vitro at concentrations at which tumor growth levels were not modified (as revealed on monotetrazolium colorimetric assay). Computer-assisted microscope analyses of neoglycoconjugate-based glycohistochemical staining profiles of 9L gliosarcomas grown in vivo revealed that cimetidine significantly decreased expression levels of endogenous receptors for fucose and, to a lesser extent, for N-acetyl-lactosamine moieties. Endogenous receptors of this specificity are known to play important roles in adhesion and migration processes of brain tumor cells. CONCLUSIONS: Cimetidine, acting as an antiadhesive and therefore an antimigratory agent for glioma cells, could be added in complement to the cytotoxic temozolomide compound to combat both migrating and proliferating cells in GBM.