Glial Tumor Cells Research Articles

Characterization and Histological Examination of a Rare Giant Cell Glioblastoma

Glioblastoma multiforme (GBM) is the most common malignant glial cell tumor of the brain. GBM typically occurs in the cerebral hemispheres and is characterized as a grade IV neoplasm due to its highly invasive nature. GBM can be subdivided into two subtypes, gliosarcoma and giant cell (GC) glioblastoma. While there are similarities between the subtypes, the biggest differences are the rate of occurrence with GC accounting for only 1% of cases, and the tendency of GC to occur more commonly in younger aged patients. In this case study, a GC neoplasm is documented in a 68-year-old male cadaver.

Diffuse midline glioma of the cervical spinal cord with H3 K27M genotype phenotypically mimicking anaplastic ganglioglioma: a case report and review of the literature

Here, we report on a 28-year old male patient presenting with neck and shoulder pain, dysesthesia of all four limbs and hypesthesia of both hands, without motor deficits. Magnetic resonance imaging showed an intradural, intramedullary mass of the cervical spinal cord of 6.4 cm length and 1.7 cm diameter. The patient underwent surgical resection. Histological and immunohistochemical evaluation showed pleomorphic glial tumor cells, mitoses, calcifications, and atypical ganglioid cells compatible with the morphology of anaplastic ganglioglioma (WHO Grade III). Extensive molecular workup revealed H3F3A K27M, TERT C228T and PDGFRα Y849C mutations indicating poor prognosis. The H3F3A K27M mutation assigned the tumor to the molecular group of diffuse midline glioma (WHO Grade IV). Epigenome-wide methylation profiling confirmed the methylation class of diffuse midline glioma. Thus, this is a very rare case of malignant glioma with H3 K27M genotype phenotypically mimicking anaplastic ganglioglioma. This case emphasizes the importance of comprehensive morphological and molecular workup including methylome profiling for advanced patient care.

In Vivo-like model of glial tumors: Creation of primary cell lines.

e14515 Background: The choice of cell source for 3D bioprinting of in vivo-like models of glial tumors is crucial and must take into account the ability to proliferation and stable metabolism. Oral administration of 5-aminolevulinic acid (5-ALA) in patients prior to surgery increases the fluorescent contrast between tumor and surrounding tissue, but the effect of contrast agents on cells in vitro is unknown. The aim of the study was obtaining viable glial tumor tissues using 5-ALA, as well as the development of a stable primary cell culture for 3D bioprinting. Methods: Tumor tissue was obtained from patients with glioblastoma during surgery under visual control using the Opmi Pentero Blue E400 microscope and 5-ALA. Material was disaggregated on a BD Machine using Medicons 50 μm (BD). Glioblastoma cells were cultured in DMEM/F12 medium with L-glutamine (Gibco) containing 10% fetal bovine serum (Biolot, Russia), 1% non-essential amino acids (NEAA, Sigma-Aldrich) and 0.5% penicillin-streptomycin (Biolot) at 37C. Glial cell lines were characterized immunohistochemically using antibodies to the glial fibrillary acidic protein (GFAP) and proliferation index (Ki-67). Microsatellite analysis was performed using three dinucleotide repeat markers D2S123, D17S250, D5S346 and five mononucleotide loci BAT25, BAT26, NR21, NR24 and NR27. Results: The positive expression of GFAP on the cell processes of the star-like shape was clearly visualized, indicating a morphological feature of glial tumors. The Ki-67 labeling index was 70%. Changes were observed at the D17S250 locus (148-148/148-152) for the glial tumor primary cells after the sixth passage. Microsatellite instability was not observed in the primary cell culture. Conclusions: The accumulation of porphyrins from 5-ALA in glial tumor cells does not prevent the in vitro creation of a cell culture from tumor tissue. Microsatellite analysis showed that the obtained glioblastoma cell lines remain stable for at least 10 passages. Material obtained during resection using 5-ALA is a reliable source of stable glial tumor cell lines.

Glucosinolates and phenolic compounds rich broccoli extract: Encapsulation by electrospraying and antitumor activity against glial tumor cells

Gliomas, intracranial malignant tumors, are aggressive, asymptomatic and difficult to treat due to their degree of infiltration, alternatives are needed to treat the disease. In this sense, natural compounds from the specialized metabolism of plants can act to control the disease. Glucosinolates and phenolic compounds, present in broccoli, have a potential to promote tumor cell death, however due to the low stability of these compounds, encapsulation becomes an alternative for their preservation. The objective was to encapsulate the broccoli extract by electrospraying and to evaluate its cytotoxicity in the primary cell culture of astrocytes and gliomas. The capsules were produced and characterized by encapsulation efficiency, functional groups, thermal stability and morphology, the capsule that presented the best parameters was used for the evaluation of cell cytotoxicity and antitumor activity. Capsules with equal or less than 50 % extract showed high encapsulation efficiency, high thermal stability and uniform morphology due to non-saturation of the active zein sites, which allowed a complete encapsulation of the added extract, as well as a greater protection of the compounds. The capsule with 50 % of the extract showed good results of the efficiency, morphology and thermal stability and was used to evaluate the antitumor activity, since the addition of extract in proportions greater than 60 % promoted saturation of the active sites and lower encapsulation efficiency, and directly affects the morphology and thermal stability. The encapsulated and unencapsulated extracts showed strong selective antitumor effect against glial tumor cells without toxicity to non-tumor cells.

High expression of estrogen receptor alpha and aromatase in glial tumor cells is associated with gender-independent survival benefits in glioblastoma patients

IntroductionGlioblastoma multiforme (GBM) is a highly malignant glial tumor, affecting men more often than women. The reason for this gender-specific predominance remains unclear, raising the question whether these effects are subject to hormonal control. The purpose of this study was to examine the expression of estrogen receptor alpha (ERα) and aromatase in human GBM tissue samples in relation to patient survival and furthermore to investigate the effect of standard chemotherapy in combination with estradiol treatment on glioblastoma tumor cell lines in vitro.Methods60 tissue samples (31 male, 29 female) of GBM patients were analysed with immunohistochemistry for ERα and aromatase for survival analyses. The cell lines LN18 and LN229 were treated with 17β-estradiol (E2) in different dosing regimens and the cell viability was measured with MTT assay. After estradiol pre-treatment Temozolomide was added and tested again.ResultsHigh expression of ERα and aromatase in the GBM tissue samples was associated with significantly longer survival times of GBM patients, regardless of gender and body-mass-index. The treatment with high concentrations of estradiol resulted in lower tumor cell viability, compared to control. The cells significantly showed a stronger sensitivity against Temozolomid (TMZ) after estradiol pre-treatment.ConclusionERα-expression of glial tumour cells seems to play an important prognostic role as a biomarker in GBM, as well as the expression of the enzyme Aromatase. The combined treatment of GBM with standard chemotherapy and estradiol may be beneficial to patient’s survival.

ACTR-51. REMARKABLE RESPONSE OF A PATIENT WITH SECONDARY GBM TO A HISTONE DEACETYLASE INHIBITOR

Abstract Glioblastomas are highly aggressive, grade IV tumors of glial cells that arise either as de novo primary tumors or as secondary tumors, which malignantly transformed from lower grade gliomas. Secondary glioblastomas have a relatively low incidence making up 5–10% of all glioblastoma diagnoses and tend to occur in younger patients. However, these tumors are still quite aggressive with survival outcomes that do not differ substantially from primary glioblastomas. Secondary glioblastomas also often harbor mutation of isocitrate dehydrogenase (IDH) enzyme, which produces the oncometabolite 2-hydroxyglutarate (2-HG) from alpha-ketoglutarate (α-KG). The accumulation of 2-HG has several downstream effects due to its competitive inhibition of α-KG-dependent enzymes, including epigenetic modification via hypermethylation of histones. Histone deacetylase inhibitors (HDACi) are a class of molecules that inhibit histone deacetylation and have been shown to have anti-tumor effect in part due to this epigenetic modification. Thus, because of their shared targets with regard to histone modification, it is plausible that HDACi could counter the oncometabolite effects of accumulated 2-HG in IDH1 mutant tumors. Since belinostat is a pan-HDACi that has improved blood-brain barrier penetration compared to many other HDACis, we conducted a pilot study that enrolled 15 patients examining its upfront use for the treatment of glioblastomas and found that it was well tolerated. One patient in particular, likely with secondary glioblastoma harboring the typical IDH1 mutation, underwent treatment with standard chemoradiation as well as belinostat on this study. For this case, a remarkable improvement in tumor burden with very significant decrease in enhancing residual tumor and restoration of magnetic resonance spectroscopy (MRS)-detectable metabolism was noted. Furthermore, improved neurocognitition and quality-of-life were also observed in this patient during the 18-month follow-up period. Collectively, these outcomes potentially support the use of belinostat as an adjuvant therapy for patients with secondary glioblastoma that harbor a mutant IDH enzyme.

A Novel Highly Selective Cannabinoid CB2 Agonist Reduces in vitro Growth and TGF-beta Release of Human Glial Cell Tumors

Cannabinoid receptors have been detected in human gliomas and cannabinoids have been proposed as novel drug candidates in the treatment of brain tumors. To test the in vitro antitumor activity of COR167, a novel cannabinoid CB2-selective agonist displaying a high binding affinity for human CB2 receptors, on tumor cells isolated from human glioblastoma multiforme and anaplastic astrocytoma. Glioma cell cultures were established from two glioblastoma multiforme and two anaplastic astrocytomas. Proliferation was measured in the presence or absence of COR167 with a bromodeoxyuridine (BrdU) cell proliferation ELISA assay. CB2 receptor expression was detected by western blotting. Apoptosis was assessed with phycoerythrin (PE) annexin V flow cytometry kit. TGF-beta 1 and 2 levels were analyzed in culture supernatants with commercial ELISAs. COR167 was found to significantly reduce the proliferation of both glioblastoma and anaplastic astrocytoma in a dose-dependent manner at lower doses than other known, less specific CB2 agonists. This activity is independent of apoptosis and is associated with a significant reduction of TGF-beta 1 and 2 levels in supernatants of glioma cell cultures. These findings add to the role of cannabinoid CB2 receptor as a possible pharmacological target to counteract glial tumor growth and encourage further work to explore any other pharmacological effect of this novel CB2 agonist useful in the treatment of human gliomas.

Evaluation of VEGF, FGF and PDGF and Serum Levels of Inflammatory Cytokines in Patients with Glioma and Meningioma in Southern Iran.

Background:Meningioma and glioma are common central nervous system tumors. Hypoxic tumor cells secrete angiogenic cytokines, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) that stimulate neovascular formation and inflammatory cytokine, such as TNF-α and IL-1β. We measured these serum levels in patients with glial cell tumors and meningioma. Materials and Methods:This was a case-control study in 2014-2015 on patients diagnosed with meningioma/glioma. All demographic and clinical data were registered. The tumor volume and intraoperative bleeding were recorded. Serum levels of VEGF, PDGF, FGF, TNF-α and IL-1β were measured by ELISA methods. Results:Ninety-six patients were enrolled in this study, 32 in each group. Patients VEGF level with cranial tumor, glioma/meningioma had increased. VEGF level was highest among grade IV tumors, larger tumors, and in glioblastoma multiform. There was an upsurge in VEGF serum level as glioma grade increased. The highest VEGF levels were seen in parasagittal meningioma. In contrast to VEGF, PDGF was slightly elevated in glial cell tumors, which was significantly elevated in meningioma. Higher PDGF correlated with increased intraoperative bleeding, especially in meningioma cases. Oligodendroglial tumors expressed higher PDGF levels in contrast to other glial tumors. FGF level was not statistically significant. TNF-α and IL-1β expressions were significantly higher in the meningioma and glioma group in comparison to control group. Conclusion:We found increased VEGF and PDGF serum levels in CNS patient’s tumor. A different role for PDGF was found in the pathogenesis of neovascularization of meningioma, as well as oligodendroglioma. No significant result was found for FGF. TNF-α and IL-1β can serve as key prognostic biomarker in high-grade glioma and meningioma patients.

Aptamer-Anchored Rubrene-Loaded Organic Nanoprobes for Cancer Cell Targeting and Imaging

Aptamer-Anchored Rubrene-Loaded Organic Nanoprobes for Cancer Cell Targeting and Imaging

PET Imaging and Glioblastoma

Abstract INTRODUCTION Patients who present with Glioblastoma have a median survival of around 15 mo, even when treated with the most aggressive methods available. Imaging of glial cell tumours is faced with multiple challenges, including the accurate delineation of abnormal tissue and the identification of low-grade gliomas. PET imaging depicts aspects of tissue activity in Vivo and thus, helps us improve our understanding of the tumor's size and behavior. While functional imaging is already an established modality in other fields of oncology, the appropriate tracer for the imaging of glial cancers remains under investigation since FDG, the traditional oncological tracer, proves suboptimal due to the high physiological glucose uptake of the cerebral grey matter. METHODS In this literature review, I discuss the physiological characteristics as well as the opportunities for clinical application of the tracers FDG, C-MET, FET, FDOPA and FLT. RESULTS Although PET imaging remains expensive and availability of tracers is limited by their mode of production and decaying nature, integration of PET scanning into the treatment pathway offers clear patient benefits. While sensitivity of the PET tracers C-MET and FET exceeds that of traditional imaging modalities, application in screening is limited by associated expenses. Combining amino-acid PET tracers with traditional modalities has shown significant benefits in biopsy and radiotherapy planning. CONCLUSION All amino acid tracers show good results in distinguishing treatment response from early recurrence, a task where MRI, the current gold standard, is lacking in reliability. FET shows especially good results in monitoring residual tumour mass.

A Human iPSC-derived 3D platform using primary brain cancer cells to study drug development and personalized medicine

A high throughput histology (microTMA) platform was applied for testing drugs against tumors in a novel 3D heterotypic glioblastoma brain sphere (gBS) model consisting of glioblastoma tumor cells, iPSC-derived neurons, glial cells and astrocytes grown in a spheroid. The differential responses of gBS tumors and normal neuronal cells to sustained treatments with anti-cancer drugs temozolomide (TMZ) and doxorubicin (DOX) were investigated. gBS were exposed to TMZ or DOX over a 7-day period. Untreated gBS tumors increased in size over a 4-week culture period, however, there was no increase in the number of normal neuronal cells. TMZ (100 uM) and DOX (0.3 uM) treatments caused ~30% (P~0.07) and ~80% (P < 0.001) decreases in the size of the tumors, respectively. Neither treatment altered the number of normal neuronal cells in the model. The anti-tumor effects of TMZ and DOX were mediated in part by selective induction of apoptosis. This platform provides a novel approach for screening new anti-glioblastoma agents and evaluating different treatment options for a given patient.

Quantification of Macrophages in High-Grade Gliomas by Using Ferumoxytol-enhanced MRI: A Pilot Study.

Purpose To investigate ferumoxytol-enhanced MRI as a noninvasive imaging biomarker of macrophages in adults with high-grade gliomas. Materials and Methods In this prospective study, adults with high-grade gliomas were enrolled between July 2015 and July 2017. Each participant was administered intravenous ferumoxytol (5 mg/kg) and underwent 3.0-T MRI 24 hours later. Two sites in each tumor were selected for intraoperative sampling on the basis of the degree of ferumoxytol-induced signal change. Susceptibility and the relaxation rates R2* (1/T2*) and R2 (1/T2) were obtained by region-of-interest analysis by using the respective postprocessed maps. Each sample was stained with Prussian blue, CD68, CD163, and glial fibrillary acidic protein. Pearson correlation and linear mixed models were performed to assess the relationship between imaging measurements and number of 400× magnification high-power fields with iron-containing macrophages. Results Ten adults (four male participants [mean age, 65 years ± 9 {standard deviation}; age range, 57-74 years] and six female participants [mean age, 53 years ± 12 years; age range, 32-65 years]; mean age of all participants, 58 years ± 12 [age range, 32-74 years]) with high-grade gliomas were included. Significant positive correlations were found between susceptibility, R2*, and R2' and the number of high-power fields with CD163-positive (r range, 0.64-0.71; P < .01) and CD68-positive (r range, 0.55-0.57; P value range, .01-.02) iron-containing macrophages. No significant correlation was found between R2 and CD163-positive (r = 0.33; P = .16) and CD68-positive (r = 0.24; P = .32) iron-containing macrophages. Similar significance results were obtained with linear mixed models. At histopathologic analysis, iron particles were found only in macrophages; none was found in glial fibrillary acidic protein-positive tumor cells. Conclusion MRI measurements of susceptibility, R2*, and R2' (R2* - R2) obtained after ferumoxytol administration correlate with iron-containing macrophage concentration, and this shows their potential as quantitative imaging markers of macrophages in malignant gliomas. © RSNA, 2018 Online supplemental material is available for this article.

The effect of celecoxib on the biological characteristics of glioma cells

Objective To investigate the effect of celecoxib on glioma cell proliferation, apoptosis, migration and invasion ability. Methods Methyl thiazol tetrazolium (MTT) assay was used to detect glial tumor cell proliferation of U251, T98G, U87 after treatment with 0, 20, 40 or 60 μmol/L celecoxib for 24, 48, 72 or 96 h. Flow cytometry was used to detect cell apoptosis after treatment with 40 μmol/L celecoxib for 48 h. Cell scratch test was used to detect cell migration after treatment with 40 μmol/L celecoxib for 48 h. Transwell was used to detect cell invasion after treatment with 40 μmol/L celecoxib for 48 h. The expression levels of cyclooxygenase-2 (COX-2), B cell lymphoma/leukemia-2 (bcl-2), B cell lymphoma/leukemia-2 associated X protein (bax), matrix metalloproteinase (MMP)-9, MMP-2 in cells was detected by Western blotting. Results Celecoxib can inhibit the proliferation of glioma cells and there is no obvious inhibitory effect on normal astrocytoma. The inhibitory effect of Celecoxib on glioma cell proliferation was depended on the reaction time and drug concentration. The apoptosis rate of glioma cell after 40μmol/L celecoxib was higher than the control group, and the difference was significant [(3.17±0.16)% vs. (16.08±1.57)%, (3.17±0.16)% vs. (16.08±1.57)%, t=14.169, P=0.000]. The migration rate of glioma cell after treatment with 40 μmol/L celecoxib was lower than the control group, the difference was significant . The invasion ability of glioma cell after 40 μmol/L celecoxib was lower than the control group, the difference was significant [(100.00±5.26)% vs. (41.28±5.84)%, t=12.940, P=0.000]. Glioma cells after celecoxib treatment in COX-2, bcl-2, MMP-9, MMP-2 was significantly decreased than control group [(229.5±11.36) vs. (97.32±7.95), t=16.512, P=0.000]. The bax protein of Glioma cells after celecoxib treatmentwas significantly higher than control group (t1=19.696, P1=0.000; t2=17.774, P2=0.000; t3=12.970, P3=0.000; t4=10.569, P4=0.001). Conclusion Celecoxib can inhibit glioma cell proliferation, migration and invasion ability, and promote the apoptosis of glioma cells, the mechanism of which was related to COX-2, bcl-2, bax, MMP-9, MMP-2. Key words: Glioma; Proliferation; Apoptosis; Invasion; Celecoxib

Outer Membrane Vesicles from Neisseria Meningitidis (Proteossome) Used for Nanostructured Zika Virus Vaccine Production

The increase of Zika virus (ZIKV) infections in Brazil in the last two years leaves a prophylactic measures on alert for this new and emerging pathogen. Concerning of our positive experience, we developed a new prototype using Neisseria meningitidis outer membrane vesicles (OMV) on ZIKV cell growth in a fusion of OMV in the envelope of virus particles. The fusion of nanoparticles resulting from outer membrane vesicles of N. meningitidis with infected C6/36 cells line were analyzed by Nano tracking analysis (NTA), zeta potential, differential light scattering (DLS), scan and scanning transmission eletronic microscopy (SEM and STEM) and high resolution mass spectometry (HRMS) for nanostructure characterization. Also, the vaccination effects were viewed by immune response in mice protocols immunization (ELISA and inflammatory chemokines) confirmed by Zika virus soroneutralization test. The results of immunizations in mice showed that antibody production had a titer greater than 1:160 as compared to unvaccinated mice. The immune response of the adjuvant and non-adjuvant formulation activated the cellular immune response TH1 and TH2. In addition, the serum neutralization was able to prevent infection of virus particles in the glial tumor cell model (M059J). This research shows efficient strategies without recombinant technology or DNA vaccines.

Glioblastoma cells labeled by robust Raman tags for enhancing imaging contrast.

Complete removal of a glioblastoma multiforme (GBM), a highly malignant brain tumor, is challenging due to its infiltrative characteristics. Therefore, utilizing imaging agents such as fluorophores to increase the contrast between GBM and normal cells can help neurosurgeons to locate residual cancer cells during image guided surgery. In this work, Raman tag based labeling and imaging for GBM cells in vitro is described and evaluated. The cell membrane of a GBM adsorbs a substantial amount of functionalized Raman tags through overexpression of the epidermal growth factor receptor (EGFR) and "broadcasts" stronger pre-defined Raman signals than normal cells. The average ratio between Raman signals from a GBM cell and autofluorescence from a normal cell can be up to 15. In addition, the intensity of these images is stable under laser illuminations without suffering from the severe photo-bleaching that usually occurs in fluorescent imaging. Our results show that labeling and imaging GBM cells via robust Raman tags is a viable alternative method to distinguish them from normal cells. This Raman tag based method can be used solely or integrated into an existing fluorescence system to improve the identification of infiltrative glial tumor cells around the boundary, which will further reduce GBM recurrence. In addition, it can also be applied/extended to other types of cancer to improve the effectiveness of image guided surgery.

Reproductive epidemiology of glial tumors may reveal novel treatments: high-dose progestins or progesterone antagonists as endocrino-immune modifiers against glioma.

Female gender, contraceptives, and menopausal hormone replacement treatments containing progesterone analogues associate with higher risk of meningiomas yet with lower risk of gliomas. Progesterone receptor (PR) expression and mifepristone treatment was highly discussed for meningiomas. However, much less is known in regard to progesterone actions in gliomas despite PR expression strongly correlates with their grade. Meningiomas and gliomas may grow faster during gestation; but paradoxically, parousity reduces lifetime risk of gliomas which can be explained with dichotomous cell growth-stimulating and inhibitory actions of progesterone at low versus high levels. Progesterone levels gradually increase in gestation up to 200-fold and the incidence of highly angiogenic brain tumors decreases in the last trimester. Indeed, progesterone stimulates glial tumor cell growth at low doses (10nM) while induces cell kill at higher doses. During gestation, some immune pathways are activated to protect the mother and the fetus against microbial pathogens. In parallel, high-dose medroxyprogesterone acetate (MPA) used in treatment of endometrial carcinoma decreases tumoral expression of PR-B and increases infiltration of cytotoxic T lymphocytes and natural killer cells. MPA also synergies with IL-2 in clinical treatment of renal cancer. In both glioma and meningioma, the dominant cytosolic PR is PR-B which increases cell growth, while PR-A limits cell growth. This seems also paradoxical at the first glance due to opposite behavior of these tumors in diverse endocrine conditions. High-dose progestins may inhibit brain tumor growth by downregulating PR-B, yet the dosage thresholds may differ between glial and meningeal tumors due to higher total PR expression in meningiomas. Supporting this proposal, certain progestins were reported to stimulate meningioma growth in anecdotal reports, but same agents at much higher doses reduced meningioma cell proliferation in pilot clinical studies. PR antagonist mifepristone reduced meningioma growth in some clinical studies, but lacked efficacy in others. In fact, mifepristone also has partial PR agonist efficacy and acts in synergy with MPA to block EC growth. Hence, a similar mechanism of receptor downregulation may also account for mifepristone. Both MPA and mifepristone also harbor myeloprotective features against chemotherapy. Ulipristal is another contraceptive PR antagonist and exerts promising anticancer activity on drug-resistant ovarian cancer and BRCA1-mutant breast cancer cells, which can be tested in animal glioblastoma models. We propose that progestins strongly deserve to be investigated in experimental models of glioblastoma alone and in combination with immunostimulating agents.

SOX2 immunity and tissue resident memory in children and young adults with glioma.

Therapies targeting immune checkpoints are effective in tumors with a high mutation burden that express multiple neo-antigens. However, glial tumors including those seen in children carry fewer mutations and there is an unmet need to identify new antigenic targets of anti-tumor immunity. SOX2 is an embryonal stem cell antigen implicated in the biology of glioma initiating cells. Expression of SOX2 by pediatric glial tumors and the capacity of the immune system in these patients to recognize SOX2 has not been previously studied. We examined the expression of SOX2 on archived paraffin-embedded tissue from pediatric glial tumors. The presence of T-cell immunity to SOX2 was examined in both blood and tumor-infiltrating T-cells in children and young adults with glioma. The nature of tumor-infiltrating immune cells was analyzed with a 37-marker panel using single-cell mass cytometry. SOX2 is expressed by tumor cells but not surrounding normal tissue in pediatric gliomas of all grades. T-cells against this antigen can be detected in blood and tumor tissue in glioma patients. Glial tumors are enriched for CD8/CD4 T-cells with tissue resident memory (TRM; CD45RO+, CD69+, CCR7-) phenotype, which co-express multiple inhibitory checkpoints including PD-1, PD-L1 and TIGIT. Tumors also contain natural killer cells with reduced expression of lytic granzyme. Our data demonstrate immunogenicity of SOX2, which is specifically overexpressed on pediatric glial tumor cells. Harnessing tumor immunity in glioma will likely require the combined targeting of multiple inhibitory checkpoints.

SOX2 as a target for immunotherapy of pediatric gliomas.

e22012 Background: New treatments are needed to improve outcomes for pediatric gliomas. Immune checkpoint inhibitors are effective therapies in tumors with a high mutation burden that express multiple neo-antigens. However, for pediatric tumors that carry few mutations, there is a need to identify new antigenic targets of anti-tumor immunity. SOX2 is an embryonal stem cell antigen implicated in the biology of glioma initiating cells. Expression of SOX2 by pediatric glial tumors, and the capacity of the immune system in these patients to recognize SOX2, has not been studied. Methods: We examined the expression of SOX2 on paraffin-embedded tissue from pediatric glial tumors (n = 30). The presence of T cell immunity to SOX2 was examined in both blood and tumor-infiltrating T cells using antigen-dependent cytokine and T cell proliferation assays (n = 15). The nature of tumor-infiltrating immune cells in glial tumors (n = 4) was analyzed using single cell mass cytometry. Results: SOX2 is expressed by tumor cells but not surrounding normal tissue in all low grade gliomas (n = 15), high grade gliomas (n = 7), ependymomas (n = 3) and in 60% of oligodendrogliomas (n = 5). T cells against SOX2 can be detected in blood and tumor tissue in 33% of patients. CD4 and CD8 tumor infiltrating T-cells display a higher proportion of PD-1 expression compared to circulating T cells (p &lt; 0.05). Glial CD4 and CD8 T cells are enriched for tissue resident memory phenotype (TRM; CD45RO+, CD69+, CCR7-) and the expression of PD-1 is primarily on these TRM cells (p &lt; 0.05). A subset of CD4 and CD8 TRM cells also co-express multiple inhibitory checkpoints including PD-L1 and TIGIT. Glial tumors also contain NK cells with reduced expression of lytic granzyme (p &lt; 0.05). Conclusions: Our data demonstrate in vivo immunogenicity of SOX2, which is specifically overexpressed on pediatric glial tumor cells. Our data also suggest that the TRM subset of tumor-infiltrating T cells may be key targets for immune checkpoint blockade, and harnessing tumor immunity will likely require the combined targeting of multiple inhibitory checkpoints. Future efforts to target SOX2 with dendritic cell vaccines combined with immune checkpoint blockade could provide effective tumor immunity and improve outcomes in pediatric brain tumors.

P08.31 Periostin is expressed by pericytes and promotes angiogenesis under the control of glial tumor cells

P08.31 Periostin is expressed by pericytes and promotes angiogenesis under the control of glial tumor cells

Glioblastoma spheroids produce infiltrative gliomas in the rat brainstem.

Diffuse intrinsic pontine glioma (DIPG) is universally fatal without proven therapy other than radiation therapy for palliation. Representative animal models will play an essential role in the preclinical stage of future therapy development. To address the shortage of representative models, we created a novel infiltrative brainstem glioma model in rats based on glioblastoma spheroids. Cells dissociated from glioblastoma spheroids grown from surgical specimens were implanted into the brainstem of NIH nude rats. Animals were serially assessed clinically and radiographically with magnetic resonance imaging (MRI). Tumors were further characterized using histology, immunohistochemistry, and cytogenetics. Tumor generation was successful in all animals receiving glioblastoma spheroid cells. The rats survived 17-25weeks before severe symptoms developed. The tumors showed as diffuse hyperintense lesions on T2-weighted images. Histologically, they demonstrated cellular heterogeneity, and infiltrative and invasive features, with cells engorging vascular structures. The tumors were shown to comprise immature human origin glial tumor cells, with human epidermal growth factor receptor (EGFR) gene amplification and gain. This study showed that cells from glioblastoma spheroids produced infiltrative gliomas in rat brainstem. The rat brainstem gliomas are radiographically and histologically accurate compared to DIPG. These tumors develop over several months that would allow sequential clinical and radiographic assessments of therapeutic interventions. This study demonstrated in principle the feasibility of developing patient-specific animal models based on putative cancer stem cells from biopsy or resection samples.