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Antigen heterogeneity that results in tumor antigenic escape is one of the major obstacles to successful chimeric antigen receptor (CAR) T cell therapies in solid tumors including glioblastoma multiforme (GBM). To address this issue and improve the efficacy of CAR T cell therapy for GBM, we developed an approach that combines CAR T cells with inhibitor of apoptosis protein (IAP) antagonists, a new class of small molecules that mediate the degradation of IAPs, to treat GBM. Here, we demonstrated that the IAP antagonist birinapant could sensitize GBM cell lines and patient-derived primary GBM organoids to apoptosis induced by CAR T cell-derived cytokines, such as tumor necrosis factor. Therefore, birinapant could enhance CAR T cell-mediated bystander death of antigen-negative GBM cells, thus preventing tumor antigenic escape in antigen-heterogeneous tumor models

Bispecific T cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. We hypothesized that BiTE-secreting T cells could be a valuable therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity, representing a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III, and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the single chain variable fragments to generate new BiTE molecules. Compared with CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE-secreting bivalent T cells compared with bivalent CAR T cells in a glioma mouse model at early phase, but not in the long term. In summary, BiTEs secreted by mono- or multi-valent T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

Prior to adoptive transfer, CAR T cells are activated, lentivirally infected with CAR transgenes, and expanded over 9 to 11 days. An unintended consequence of this process is the progressive differentiation of CAR T cells over time in culture. Differentiated T cells engraft poorly, which limits their ability to persist and provide sustained tumor control in hematologic as well as solid tumors. Solid tumors include other barriers to CAR T cell therapies, including immune and metabolic checkpoints that suppress effector function and durability. Sialic acids are ubiquitous surface molecules with known immune checkpoint functions. The enzyme C. perfringens neuraminidase (CpNA) removes sialic acid residues from target cells, with good activity at physiologic conditions. In combination with galactose oxidase (GO), NA has been found to stimulate T cell mitogenesis and cytotoxicity in vitro. Here we determine whether CpNA alone and in combination with GO promotes CAR T cell antitumor efficacy. We show that CpNA restrains CAR T cell differentiation during ex vivo culture, giving rise to progeny with enhanced therapeutic potential. CAR T cells expressing CpNA have superior effector function and cytotoxicity in vitro. In a Nalm-6 xenograft model of leukemia, CAR T cells expressing CpNA show enhanced antitumor efficacy. Arming CAR T cells with CpNA also enhanced tumor control in xenograft models of glioblastoma as well as a syngeneic model of melanoma. Given our findings, we hypothesize that charge repulsion via surface glycans is a regulatory parameter influencing differentiation. As T cells engage target cells within tumors and undergo constitutive activation through their CARs, critical thresholds of negative charge may impede cell-cell interactions underlying synapse formation and cytolysis. Removing the dense pool of negative cell-surface charge with CpNA is an effective approach to limit CAR T cell differentiation and enhance overall persistence and efficacy.

Chimeric antigen receptor T (CAR-T) cells, an immunotherapy that demonstrates marked success in treatment of hematologic malignancies, are an emergent therapeutic for patients with glioblastoma (GBM). GBM CAR-T trials have focused on targeting well-characterized antigens in the pathogenesis of GBM. Early stage trials demonstrate initial success in terms of safety and tolerability. There is preliminary evidence of antitumor activity and localization of the CAR-T product to tumoral sites. There are mixed results regarding patient outcomes. Ongoing GBM CAR-T trials will target novel antigens, explore CAR-T combination therapy, design multivalent CAR constructs, and assess the impact of lymphodepletion before CAR-T delivery.

“Humanized” immunodeficient mice generated via the transplantation of CD34+ human hematopoietic stem cells (hHSC) are an important preclinical model system. The triple transgenic NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mouse line is increasingly used as recipient for CD34+ hHSC engraftment. NSGS mice combine the features of the highly immunodeficient NSG mice with transgenic expression of the human myeloid stimulatory cytokines GM-CSF, IL-3, and Kit ligand. While generating humanized NSGS (huNSGS) mice from two independent cohorts, we encountered a fatal macrophage activation syndrome (MAS)-like phenotype resulting from the transplantation of CD34+ hHSC. huNSGS mice exhibiting this phenotype declined clinically starting at approximately 10 weeks following CD34+ hHSC engraftment, with all mice requiring euthanasia by 16 weeks. Gross changes comprised small, irregular liver, splenomegaly, cardiomegaly, and generalized pallor. Hematological abnormalities included severe thrombocytopenia and anemia. Pathologically, huNSGS spontaneously developed a disseminated histiocytosis with infiltrates of activated macrophages and hemophagocytosis predominantly affecting the liver, spleen, bone marrow, and pancreas. The infiltrates were chimeric with a mixture of human and mouse macrophages. Immunohistochemistry suggested activation of the inflammasome in both human and murine macrophages. Active Epstein-Barr virus infection was not a feature. Although the affected mice exhibited robust chimerism of the spleen and bone marrow, the phenotype often developed in the face of low chimerism of the peripheral blood. Given the high penetrance and early lethality associated with the MAS-like phenotype here described, we urge caution when considering the use of huNSGS mice for the development of long-term studies.

Thirty-day readmission following glioblastoma (GBM) resection is not only correlated with decreased overall survival but also increasingly tied to quality metrics and reimbursement. This study aimed to determine factors linked with 30-day readmission to develop a simple risk stratification score. From 2005 to 2016, 666 unique resections (467 patients) of primary/recurrent tissue-confirmed glioblastoma were retrospectively identified. We recorded patient demographics and medical history, tumor characteristics, post-operative complications and 30-day readmission. Univariate and multivariate logistic regression, optimized using a genetic learning algorithm, were used to determine factors associated with readmission. The multivariate model was converted to a simple additive score. The 30-day readmission rate was 20.3% in our cohort of 666 unique resections (60.7% first resection). Lower pre/post-operative KPS, recurrent resection, surgical-site infection, post-operative VTE, post-operative VPS, and discharge to a rehabilitation facility were significantly associated with an increased readmission risk (p 0.05). MGMT methylation and chemoradiation were associated with decreased readmission risk (p 0.05). Medical co-morbidities and past medical history, location of tumor in eloquent areas of the brain, and length of ICU/hospital stay did not predict readmission. The Glioblastoma Readmission Risk Score, developed from the multivariate model, accounts for increased BMI, decreased pre-operative KPS, current smoking, post-operative complications, MGMT methylation, and post-operative radiation. This risk score can be routinely used to stratify risk and assist in clinical decision making and outcome analyses.

Background The artery of Wollschlaeger and Wollschlaeger is a tentorial branch of the superior cerebellar artery that is usually not visualized on conventional cerebral angiography, unless it is pathologically enlarged. It can be recruited as part of the blood supply to tentorial dural arteriovenous fistulas (AVFs), although this occurs infrequently. Case Description Here we report the clinico-radiologic evaluation and treatment of a 48-year-old man referred to our institution for hitherto workup negative progressive, relapsing quadriparesis. This represents the first reported case of cervical myelopathy caused by venous congestion from a type V dural AVF supplied by the artery of Wollschlaeger and Wollschlaeger. Conclusions The anatomic discrepancy between the symptomatic spinal cord lesion and the etiologic intracranial fistula frequently results in delayed care in cases of myelopathy due to intracranial dural AVFs. Familiarity with these disorders and of their pathophysiologic mechanisms is important to avoid unnecessary diagnostic delays.

Objective: The LACE + index (Length of stay, Acuity of admission, Charlson Comorbidity Index (CCI) score, and Emergency department visits in the past 6 months) is a tool utilized to predict 30–90 day readmission and other secondary outcomes. We sought to examine the effectiveness of this predictive tool in patients undergoing brain tumor surgery. Patients and methods: Admissions and readmissions for patients undergoing craniotomy for supratentorial neoplasm at a single, multi-hospital, academic medical center, were analyzed. Key data was prospectively collected with the Neurosurgery Quality Improvement Initiative (NQII)-EpiLog tool. This included all supratentorial craniotomy cases for which the patient was alive at 90 days after surgery (n = 238). Simple logistic regression analyses were used to assess the ability of the LACE + index and subsequent single variables to accurately predict the outcome measures of 30–90 day readmission, 30–90 day emergency department (ED) visit, and 30–90 day reoperation. Analysis of the model’s or variable’s discrimination was determined by the receiver operating characteristic curve as represented by the C-statistic. Results: The sample included admissions for craniotomy for supratentorial neoplasm (n = 238) from 227 patients, of which 50.00% were female (n = 119). The average LACE + index score was 53.48 ± 16.69 (Range 9–83). The LACE + index did not accurately predict 30–90 day readmissions (P = 0.127), 30–90 day ED visits (P = 0.308), nor reoperations (P = 0.644). ROC confirmed that the LACE + index was little better than random chance at predicting these events in this population (C-statistic = 0.51−0.58). However, a single unit increase in LACE + leads to a 0.97 times reduction in the odds of being discharged home with fair predictive accuracy (P Conclusion: The results of this study show that the LACE + index is ill-equipped to predict 30–90 day readmissions in the brain tumor population and further analysis of significant covariates or other prediction tools should be undertaken.

BACKGROUND: Brain metastases are the most common central nervous system (CNS) tumors, occurring in 300,000 people per year in the US. The benefit of surgical resection, over radiosurgery, for dominant lesions remains unclear. METHODS: The University of Pennsylvania Health System database was retrospectively reviewed for patients presenting with multiple brain metastases from 1/1/16 to 8/31/18 with one dominant lesion > 2 cm in diameter, who underwent initial treatment with either resection of the dominant lesion or Gamma Knife radiosurgery (GKS). Inclusion criteria were age > 18, >1 brain metastasis, and presence of a dominant lesion (>2 cm). We analyzed factors associated with mortality. RESULTS: 129 patients were identified (surgery=84, GKS=45). The median number of intracranial metastases was 3 (IQR: 2-5). The median diameter of the largest lesion was 31 mm (IQR: 25-38) in the surgery group vs 21 mm (IQR: 20-24) in the GKS group (pCONCLUSION: In our institution, patients with multiple brain metastases and one symptomatic dominant lesion demonstrated similar survival after GKS when compared with up-front surgical resection of the dominant lesion.

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems.

Background and purpose In treating glioblastoma (GB), surgical and chemotherapeutic treatment guidelines are, for the most part, independent of tumor location. In this work, we compiled imaging data from a large cohort of GB patients to create statistical atlases illustrating the disease spatial frequency as a function of patient demographics as well as tumor characteristics. Materials and methods Two-hundred-six patients with pathology-proven glioblastoma were included. Of those, 65 had pathology-proven recurrence and 113 had molecular subtype and genetic information. We used validated software to segment the tumors in all patients and map them from patient space into a common template. We then created statistical maps that described the spatial location of tumors with respect to demographics and tumor characteristics. We applied a chi-square test to determine whether pattern differences were statistically significant. Results The most frequent location for glioblastoma in our patient population is the right temporal lobe. There are statistically significant differences when comparing patterns using demographic data such as gender (p = 0.0006) and age (p = 0.006). Small and large tumors tend to occur in separate locations (p = 0.0007). The tumors tend to occur in different locations according to their molecular subtypes (p, Highlights • Atlases of spatial distribution of glioblastoma are created. • Atlases are specific to patient characteristics such as age and gender. • Atlases are specific to tumor characteristics such as size and molecule subtype. • Atlases are specific to tumor genetics. • Spatial location should be considered when evaluating a patient with glioblastoma.

Summary Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.

Chimeric antigen receptor (CAR) T cells are a form of autologous immunotherapy that has changed the therapeutic landscape of hematologic malignancies. CAR T cell therapy involves collection of a patient's T cells by apheresis, ex vivo genetic modification of the T cells to encode a synthetic receptor that binds a specific tumor antigen, and infusion of the T cells back into the patient. With the unprecedented success of CAR T cells in leukemia and lymphoma, a growing number of preclinical studies and clinical trials have focused on translating this treatment to solid tumors. In non-hematologic malignancies, however, response rates have been much less favorable. Some of the most significant challenges for CAR T cell immunotherapy in solid cancers include a paucity of unique tumor target antigens, limited CAR T cell trafficking to tumor sites, tumor heterogeneity and antigen loss, and the severely immunosuppressive tumor microenvironment. This review article provides an update on completed and ongoing clinical trials of CAR T cells for solid tumors, as well as an overview of strategies to improve CAR T cell efficacy in non-hematologic malignancies.

Background: The EGF-receptor variant III (EGFRvIII) is expressed in approximately 30% of all primary glioblastoma (GBM). Although several anti-EGFRvIII strategies have already been tested, the importance of EGFRvIII expression on treatment response is still under debate. Our aim was to clarify the relevance of EGFRvIII for GBM treatment using uni- and multivariate analysis of clinical data and preclinical studies. Methods: We performed retrospective analysis of 338 IDH1 wild type GBM patients treated either at the University of Pennsylvania or North Bristol NHS Trust and included data from the TCGA database. Survival was stratified according to MGMT promoter methylation status, EGFRvIII expression and EGFR gene amplification. The effect of EGFRvIII expression was also analyzed at the cellular and tumor level using isogenic pairs of EGFRvIII-negative and -positive GBM cells and xenograft tumors. Findings: EGFRvIII expression was associated with a significantly prolonged median overall survival but only for GBM patients with MGMT promoter methylated tumors. Importantly, this effect was independent of EGFR gene amplification. We also observed increased temozolomide (TMZ) sensitivity of EGFRvIII positive cells, which translated into improved survival in xenograft experiments. This increase in TMZ sensitivity was associated with an elevated DNA damage induction accompanied by an increased expression of DNA mismatch repair (MMR) proteins. In line with this finding, knock down of MMR proteins or EGFRvIII restored TMZ resistance. Interpretation: EGFRvIII expression is of high relevance for the prediction of GBM therapy outcome. Due to EGFRvIII-dependent upregulation of MMR standard of care treatment is most efficient for GBM displaying both MGMT promoter methylation and EGFRvIII expression. Funding Statement: This work was supported by the Brigitte and Dr. Konstanze Wegener-Stiftung (M.K.), Wilhelm-Sander-Stiftung (M.K.), Forschungsforderungsfonds des Fachbereichs Medizin des Universitatsklinikums Hamburg-Eppendorf (N.S.) and the Federal Ministry of Education and Research (BMBF grant 02NUK032; M.K. K.R.), Cancer Research UK (CRUK/21600; S.C.S, T.B.), The Brain Tumour Charity (TBTC13/192; S.C.S., T.B.), Medical Research Council (MR/K015214/1; S.C.S., T.B.), University of Leeds Academic Fellowship (L.F.S), The Templeton Family Research Fund (Z.A.B) and the National Institutes of Health (NIH 2R01-NS042645-11A1; D.O.R.). U.S. is supported by the Fordergemeinschaft Kinderkrebs-Zentrum Hamburg Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Animal Use: All work was performed in accordance with the United Kingdom Animals (Scientific Procedures) Act (1986), under the authority of project licence 70/7340.

Radiation necrosis is the most significant complication associted with Gamma Knife radiosurgery. It typically becomes manifest s a necrotic white matter lesion 3 or more months following treatent [3]. Treatment volume and radiation dose are the two most mportant predictors of radiation necrosis. Once radiation necrois has become clinically apparent treatment has historically been imited to corticosteroids. Antiplatelet agents, anticoagulants and yperbaric oxygen have been studied but there is currently minimal igh quality evidence to support their use in routine clinical pracice [6]. If the radiation necrosis fails to respond to steroid treatment herapeutic options are limited. Recent trials have highlighted a potential role for bevacizumab, a onoclonal antibody directed against vascular endothelial growth actor (VEGF) in the treatment of radiation necrosis from convenional radiation therapy [14]. We present a case of symptomatic adiation necrosis following Gamma Knife radiosurgery for muliple atypical WHO grade II meningiomas initially unresponsive o steroid treatment and ultimately responsive to bevacizumab herapy.

N-(4-hydroxyphenyl) retinamide (4-HPR, fenretinide) a synthetic retinoid is in clinical trials for the treatment of several malignancies. However, its biological effects and therapeutic value in childhood brain tumor medulloblastoma (MB) has not been investigated. In this study, we report for the first time that fenretinide (2.5–10 μM) induces apoptotic cell death in human MB cells. We observed significant inhibition of cell survival in four MB cell lines (D425MED, D458MED, D283MED and D341MED) as determined by MTT assays. These results were further supported by inhibition of anchorage-independent colony formation in soft agar. Fenretinide-induced decrease in cell viability was in part due to activation of caspase-3 dependent cell death, which was further supported by the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1), a caspase-3 substrate. Cell death was partially prevented by the antioxidant, l -ascorbic acid suggesting that free radical intermediates might be involved in fenretinide effects. These results suggest that pharmacologically achievable concentrations of fenretinide are effective in killing MB cells and thus show its therapeutic potential to treat human MB.

The epidermal growth factor receptor (EGFR) has been shown to play an important role in brain development, including stem and precursor cell survival, proliferation, differentiation, and migration. To further examine the temporal and spatial requirements of erbB signals in uncommitted neural stem cells (NSCs), we expressed the ligand-independent EGF receptor, EGFRvIII, in C17.2 NSCs. These NSCs are known to migrate and to evince a tropic response to neurodegenerative environments in vivo but for which an underlying mechanism remains unclear. We show that enhanced erbB signaling via constitutive kinase activity of EGFRvIII in NSCs sustains an immature phenotype and enhances NSC migration.

Genetic suppressor element (GSE) methodology was applied to identify new genes controlling cell response to tumor necrosis factor (TNF). A retroviral library of randomly fragmented normalized cDNA from mouse fibroblasts was screened for GSEs capable of protecting NIH3T3 cells from TNF-induced apoptosis. The most abundant among isolated GSEs represented a fragment of cDNA encoding the C-terminal cytoplasmic region of the immunoglobulin family inhibitory receptor, SHPS-1 (mouse homologue of human SIRPalpha). Ectopic expression of this fragment (both from human and mouse versions) increased the NF-kappaB-dependent transcription in three cell lines tested; this effect could be reduced by the expression of full-length SIRPalpha, suggesting that the isolated GSE acts through a dominant negative mechanism. GSE-mediated activation of NF-kappaB depended on the presence of serum, was abrogated by wortmannin, and was associated with phosphorylation of PKB/Akt, suggesting that Akt mediates it. These data indicate that SIRPalpha/SHPS-1 is involved in negative regulation of NF-kappaB signaling.

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of beta-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed beta-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total beta-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2. 95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both beta-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.

Purpose: Glioblastomas often express high levels of vascular endothelial growth factor (VEGF), even under normoxic conditions. Genetic alterations that commonly occur in these tumors, including PTEN mutations and epidermal growth factor receptor (EGFR) overexpression, may contribute to this increased VEGF expression. Our previous work showed that, compared to parental U87MG human glioblastoma cells, VEGF mRNA levels are decreased in U87/T691, a derivative line in which EGFR signaling is inhibited by introduction of a truncated p185Neu protein (Cancer Research 60: 5879-5886, 2000). In that study, we also showed that the effect of EGFR activation on VEGF was mediated at the level of transcription via a PI3K dependent pathway. The purpose of the current study was to assess the role of PTEN, a negative regulator of PI3K, and its interaction with EGFR activation in regulating VEGF expression. Materials and Methods: Protein lysates were obtained from U87MG and U87/T691 cells for use in Western blotting for phosphorylated Akt. U87MG and U87/T691 cells were infected with adenovirus expressing either wildtype PTEN or phosphatase dead PTEN, then RNA was harvested for Northern blotting for VEGF. Nuclear extracts were also obtained from these cells for use in gel shift assays. Luciferase assays were performed with lysates of U87MG cells transfected with luciferase reporter constructs containing fragments of the VEGF promoter. Results: The level of phosphorylated Akt, a marker for PI3K activation, was lower in U87/T691 cells compared to U87MG cells, an expected result because of inhibition of the EGFR in the former. Treatment of U87/T691 cells with the PI3K inhibitor LY294002 further decreased the level of phosphorylated Akt and VEGF mRNA. Therefore, in spite of EGFR inhibition, U87/T691 cells contain residual PI3K activity that regulates VEGF expression. To determine whether the PTEN status of these cells influences VEGF mRNA levels, wildtype PTEN was introduced into U87MG and U87/T691 cells using adenovirus, This resulted in a decrease in both phosphorylated Akt and VEGF mRNA levels. Treatment of U87MG cells with LY294002, a PI3K inhibitor, or cotransfection with a vector expressing either wildtype PTEN or dominant negative PI3K decreased activity of a luciferase reporter containing −88/+54 bp of the VEGF promoter (+1 is the transcription start site). LY294002 also blocked induction of activity of this reporter by EGF stimulation. Gel shift assays were also performed using nuclear lysates of U87MG cells infected with either wildtype or phosphatase dead PTEN adenovirus. In these assays, the radioactively labeled probe used was an oligonucleotide spanning −88 to −65. These assays showed that introduction of wildtype PTEN decreased binding of a protein complex to this region in the promoter. Conclusion: In human glioblastoma cells, PTEN mutation can cooperate with EGFR activation to increase VEGF mRNA levels by transcriptionally upregulating the proximal VEGF promoter (−88/+54) via a PI3K-dependent pathway. Furthermore, PI3K activation leads to increased binding of a protein complex to a segment of the promoter located between −88 and −65.