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Glioblastoma is the most common primary brain tumor. It is highly malignant and has a correspondingly poor prognosis. Diagnosis and monitoring are mainly accomplished with MRI, but remain challenging in some cases. Therefore, complementary methods for tumor detection and characterization would be beneficial. Using Magnetic Resonance Elastography (MRE), we performed a longitudinal study of the biomechanical properties of intracranially implanted glioblastoma (GBM) in mice and compared the results to histopathology. The biomechanical parameters of viscoelastic modulus, shear wave speed and phase angle were significantly lower in tumors compared to healthy brain tissue and decreased over time with tumor progression. Moreover, some MRE parameters revealed sub-regions at later tumor stages, which were not easily detectable on anatomical MRI images. Comparison to histopathology showed that softer tumor regions contained necrosis and patches of viable tumor cells. In contrast, areas of densely packed tumor cells and blood vessels identified with histology coincided with higher values of viscoelastic modulus and shear wave speed. Interestingly, the phase angle was independent from these anatomical variations. In summary, MRE depicted longitudinal and morphological changes in GBM and may prove valuable for tumor characterization in patients.

High-grade gliomas are deadly cancers, and current standard-of-care has demonstrated limited success. The ability to specifically target glioma cells can allow for the development of safer and more efficacious brain cancer therapy strategies. Brevican (BCAN), a CNS-specific extracellular matrix protein is upregulated in glioma cells and its expression correlates with tumor progression. Particularly, a membrane-bound BCAN isoform lacking normal glycosylation, called B/bΔg is a unique glioma marker and is not expressed in non-cancerous tissues. Therefore, B/bΔg represents a valuable target for anti-cancer strategies. Here, we describe the utilization of state-of-the-art technologies that we have recently developed to discover novel “B/bΔg-Targeting Peptides” (BTP). Briefly, small magnetic beads displaying B/bΔg were used to screen a one-bead-one-compound combinatorial library, enabling high-throughput labeling of library beads presenting peptide candidates with high affinity for B/bΔg. The “hit” beads were rapidly sorted using a microfluidic magnetic-activated sorter of our own design. The hits were then, exposed to cells expressing B/bΔg, and beads with the highest cell association were isolated and sequenced. Characterization of the purified peptides through kinetic binding analysis and cell uptake studies revealed BTP-7 as the lead candidate for B/bΔg binding. BTP-7 displayed 260 nanomolar affinity for recombinant B/bΔg protein, and had little association with the fully glycosylated isoform of BCAN. Scrambling of the BTP-7 sequence led to complete abrogation of B/bΔg binding. For in vivo evaluation, GBM-6 tumors derived from human patients were established intracranially in nude mice, and tumor formation was verified by MRI. Upon systemic administration, BTP-7 displayed approximately 10x greater binding to GBM-6 tumors than the control, as well as 4x higher tumor uptake compared to normal brain tissue. We are currently working on conjugating BTP-7 to a variety of toxic payload to selectively target gliomas, with the goal of improving brain cancer therapeutic efficacy that can ultimately benefit patient.

Glioblastoma is the most prevalent and lethal primary intrinsic brain tumor with a median patient survival of less than two years, even with the optimal standard of care, namely, surgical resection followed by radiotherapy with adjuvant temozolomide chemotherapy. Long-term survival is extremely rare and there is a tremendous need for novel GBM therapies. Following our prior reports on the anticancer activity of osmium(VI) nitrido compounds and their effectiveness against cancer initiating cells, we investigated the efficacy of Os(VI) on GBM initiating cells in vitro and in vivo. Conventional MTT and 3D cytotoxicity assays revealed that patient-derived GBM models were sensitive to cisplatin, TMZ, and two Os(IV) derivatives. Rapid cell death occurred at low micromolar concentrations of the Os(IV) compounds. Cell cycle analysis, Os uptake studies, and cellular distribution experiments provided further insight into the anticancer properties of these compounds, indicating differential uptake for both compounds and a modest G2/M arrest after treatment. Moreover, in vivo experiments showed a significant increase in survival after a single intracranial chemotherapeutic injection, results that warrant further studies using this approach.

Background Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.

Developing therapeutics that target multiple receptor signaling pathways in tumors is critical as therapies targeting single specific biomarker/pathway have shown limited efficacy in patients with cancer. In this study, we extensively characterized a bi-functional molecule comprising of epidermal growth factor receptor (EGFR) targeted nanobody (ENb) and death receptor (DR) targeted ligand TRAIL (ENb-TRAIL). We show that ENb-TRAIL has therapeutic efficacy in tumor cells from different cancer types which do not respond to either EGFR antagonist or DR agonist monotherapies. Utilizing pharmacological inhibition, genetic loss of function and FRET studies, we show that ENb-TRAIL blocks EGFR signalling via the binding of ENb to EGFR which in turn induces DR5 clustering at the plasma membrane and thereby primes tumor cells to caspase-mediated apoptosis. In vivo, using a clinically relevant orthotopic resection model of primary glioblastoma and engineered stem cells (SC) expressing ENb-TRAIL, we show that the treatment with synthetic extracellular matrix (sECM) encapsulated SC-ENb-TRAIL alleviates tumor burden and significantly increases survival. This study is the first to report novel mechanistic insights into simultaneous targeting of receptor-mediated proliferation and cell death signaling pathways in different tumor types and presents a promising approach for translation into the clinical setting.

Objective: Cytomegalovirus (CMV) is a ubiquitous member of the herpes virus family with a prevalence of between 50-100% in the human population and lifelong persistence. While usually clinically benign, severe cases are observed in neonates and immunocompromised patients. CMV has been associated with various tumors, including glioblastoma (GBM). The aim of this study is to clarify the interrelationship between CMV and GBM, as to elucidate its contribution to GBM progression. Methods: Pericytes, proneural (PN) and mesenchymal (MES) human GBM cell lines were assessed via flow cytometry for permissiveness to CMV (Towne Strain) infection. The transcriptome of infected cells was assessed using RT-qPCR and RNA-Seq analysis. Transwell migration and endothelial tube formation were evaluated in co-culture experiments. C57BL/6 mice were infected with murine CMV (MCMV-Δm157) at P2 and syngeneic GL261 tumor cells were implanted at week 14 after latency was established. Tumor composition was analyzed using flow cytometry. Murine and human brain sections were analyzed by immunofluorescence staining. Results: CMV was found to closely co-localize with tumor vessels in patient derived GBM specimens. In vitro studies confirmed a high permissiveness of pericytes for CMV infection. GBM cells, permissive for CMV infection had a proneural, rather than a mesenchymal signature. The transcription of pro-angiogenic cytokines such as IL-6, TGF-beta and angiogenesis inducing receptor PAR-1 were up-regulated upon CMV infection. Transwell assays revealed increased migration towards CMV infected cells after 48h (GBM cells towards infected pericytes: 1.5-fold (p=0.07); pericytes towards infected GBM cells: 1.48-fold (p=0.009)). Further, co-culture of human brain microvascular endothelial cells (HBMEC) and infected PN cells led to the establishment of larger (160%, p=

Great interest persists in useful prognostic and therapeutic targets in glioblastoma. In this study, we report the definition of miRNA (miR)-148a as a novel prognostic oncomiR in glioblastoma. miR-148a expression was elevated in human glioblastoma specimens, cell lines, and stem cells (GSC) compared with normal human brain and astrocytes. High levels were a risk indicator for glioblastoma patient survival. Functionally, miR-148a expression increased cell growth, survival, migration, and invasion in glioblastoma cells and GSCs and promoted GSC neurosphere formation. Two direct targets of miR-148a were identified, the EGF receptor (EGFR) regulator MIG6 and the apoptosis regulator BIM, which rescue experiments showed were essential to mediate the oncogenic activity of miR-148a. By inhibiting MIG6 expression, miR-148a reduced EGFR trafficking to Rab7-expressing compartments, which includes late endosomes and lysosomes. This process coincided with reduced degradation and elevated expression and activation of EGFR. Finally, inhibition of miR-148a strongly suppressed GSC and glioblastoma xenograft growth in vivo. Taken together, our findings provide a comprehensive analysis of the prognostic value and oncogenic function of miR-148a in glioblastoma, further defining it as a potential target for glioblastoma therapy. Cancer Res; 74(5); 1541–53. ©2014 AACR.

BACKGROUND: Extracellular vesicles (EVs) are known for their important role in cancer progression and hold considerable potential as tumor biomarkers. However, purification of tumor-specific EVs from plasma is still a pressing need since contamination by normal host cell-EVs, results in compromised analytical sensitivity. Fatty acid synthase (FASN) was recently shown by us to be present on EVs from cultured glioma cells. Here we analyzed circulating patient EVs for the expression of FASN, CD9, CD81 and CD63. METHODS: Plasma EVs from patients with glioblastoma (n=24), anaplastic astrocytoma (n=6) and healthy controls (n=16) as well as EVs from early passage glioma stem cells (GSCs) were analyzed for FASN, CD9, CD81, CD63 by imaging flow cytometry (IFC). EVs were further investigated by nanoparticle tracking analysis (NTA), electron microscopy and immunoblotting. Glioblastomas, anaplastic astrocytomas and normal brain tissue specimens were analyzed for FASN by Western blotting and immunohistochemistry. RESULTS: FASN is elevated in glioblastoma tissue compared to noncancerous brain and FASN expression levels correlate with WHO grade (p+/CD81(+)and FASN(+)/CD63(+)EVs are present in plasma from patients with glioblastoma and anaplastic astrocytoma as determined by IFC (p+EVs and double positive (CD9(+)/CD63(+), CD9(+)/CD81(+)or CD63(+)/CD81(+)) EVs (p CONCLUSION: FASN expression is elevated on circulating EVs from patients with malignant gliomas and FASN is a promising marker for the identification and enrichment of glioma-derived plasma EVs, an important prerequisite for in-depth genetic, epigenetic and transcriptional analyses that could inform clinicians on molecular alterations and help monitoring treatment efficacy.

BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

Background: Oncolytic viruses (OV) have been a topic of great interest as therapeutic agents for indications such as glioblastoma multiforme (GBM), where current treatment options are poor and limited. Alongside engineering these viruses, finding useful pre-clinical models to elucidate the efficacy of the OV has been challenging. In particular, these viruses have been developed to overcome immune resistance, allowing for the reinvigoration of the immune contexture in the tumor microenvironment (TME). Current pre-clinical models, such as mouse models, are limited in their ability to recapitulate the TME fully. Thus, there is a need for more relevant pre-clinical models to study the efficacy of OV and downstream effects in the TME. Methods: rQNestin34.5v.1 herpes simplex virus (rQNestin) has previously been investigated in both in vitro and in vivo studies of GBM. To assimilate the effects of rQNestin on the TME, a more humanized system was employed; CANscriptTM. CANscript is an ex vivo human tumor model, that recapitulates the native, patient-autologous TME, incorporating autologous patient-derived peripheral blood mononucleated cells. We treated GBM tissue (n=10) with rQNestin, capable of expressing GFP, and profiled GFP expression as a proxy for replication efficiency. We coupled this analysis with gene expression of immune-related pathways to gauge the modulation of the immune contexture by rQNestin. In addition, we performed multiplex cytokine analysis and multiplex immunohistochemistry to investigate the impact of rQNestin on the spatial context and activity of the immune compartment in the TME, ex vivo. Results: Immunohistochemistry established that viral replication and tissue penetration was observed, ex vivo. Furthermore, RNA transcriptional profiling and cytokine analysis revealed that the oHSV-1 was capable of dynamically altering the tumor microenvironment, and dysregulating immune subsets within the tumor. Further stratification of tumor samples based on the pharmacodynamic profile identified subsets of patient tumors that induce adaptive immunity. Concluding remarks: Here, we report that CANscript, an ex vivo human tumor model, can be used to evaluate the effects of OV in the TME - not only viral replication but also the direct effect on the immune compartment. CANscript will be an invaluable tool to investigate the response and resistance to OV. This platform has the potential to enable better pre-clinical modeling of OV development. Citation Format: Munisha Smalley, Vidushi Kapoor, Douglas Best, Carmela Passaro, Michal O. Nowicki, Suniti K. Saha, Komal Prasad, E. Antonio Chiocca, Sean E. Lawler, Aaron Goldman. Translational efficacy of oncolytic HSV-1 in glioblastoma using a human autologous ex vivo platform, CANscript™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5009.

Cancer is associated with epigenetic (i.e., histone hypoacetylation) and metabolic (i.e., aerobic glycolysis) alterations. Levels of N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate, are reduced in glioma; yet, few studies have investigated acetate as a potential therapeutic agent. This preclinical study sought to test the efficacy of the food additive Triacetin (glyceryl triacetate, GTA) as a novel therapy to increase acetate bioavailability in glioma cells. The growth-inhibitory effects of GTA, compared to the histone deacetylase inhibitor Vorinostat (SAHA), were assessed in established human glioma cell lines (HOG and Hs683 oligodendroglioma, U87 and U251 glioblastoma) and primary tumor-derived glioma stem-like cells (GSCs), relative to an oligodendrocyte progenitor line (Oli-Neu), normal astrocytes, and neural stem cells (NSCs) in vitro. GTA was also tested as a chemotherapeutic adjuvant with temozolomide (TMZ) in orthotopically grafted GSCs. GTA-induced cytostatic growth arrest in vitro comparable to Vorinostat, but, unlike Vorinostat, GTA did not alter astrocyte growth and promoted NSC expansion. GTA alone increased survival of mice engrafted with glioblastoma GSCs and potentiated TMZ to extend survival longer than TMZ alone. GTA was most effective on GSCs with a mesenchymal cell phenotype. Given that GTA has been chronically administered safely to infants with Canavan disease, a leukodystrophy due to ASPA mutation, GTA-mediated acetate supplementation may provide a novel, safe chemotherapeutic adjuvant to reduce the growth of glioma tumors, most notably the more rapidly proliferating, glycolytic and hypoacetylated mesenchymal glioma tumors.

Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.

Background The Polycomb Repressor Complex (PRC) is an epigenetic regulator of transcription whose action is mediated by 2 protein complexes, PRC1 and PRC2. PRC is oncogenic in glioblastoma, where it is involved in cancer stem cell maintenance and radioresistance. Methods We used a set of glioblastoma patient samples, glioma stem cells, and neural stem cells from a mouse model of glioblastoma. We characterized gene/protein expression and cellular phenotypes by quantitative PCR/Western blotting and clonogenic, cell-cycle, and DNA damage assays. We performed overexpression/knockdown studies by lentiviral infection and microRNA/small interfering RNA oligonucleotide transfection. Results We show that microRNA-128 (miR-128) directly targets mRNA of SUZ12, a key component of PRC2, in addition to BMI1, a component of PRC1 that we previously showed as a target as well. This blocks the partially redundant functions of PRC1/PRC2, thereby significantly reducing PRC activity and its associated histone modifications. MiR-128 and SUZ12/BMI1 show opposite expression in human glioblastomas versus normal brain and in glioma stemlike versus neural stem cells. Furthermore, miR-128 renders glioma stemlike cells less radioresistant by preventing the radiation-induced expression of both PRC components. Finally, miR-128 expression is significantly reduced in neural stem cells from the brain of young, presymptomatic mice in our mouse model of glioblastoma. This suggests that loss of miR-128 expression in brain is an early event in gliomagenesis. Moreover, knockdown of miR-128 expression in nonmalignant mouse and human neural stem cells led to elevated expression of PRC components and increased clonogenicity. Conclusions MiR-128 is an important suppressor of PRC activity, and its absence is an early event in gliomagenesis.

Airway exposure levels of lipopolysaccharide (LPS) determine type I versus type II helper T cell induced experimental asthma. While high LPS levels induce Th1-dominant responses, low LPS levels derive Th2 cell induced asthma. The present paper develops a mathematical model of asthma development which focuses on the relative balance of Th1 and Th2 cell induced asthma. In the present work we represent the complex network of interactions between cells and molecules by a mathematical model. The model describes the behaviors of cells (Th0, Th1, Th2 and macrophages) and regulatory molecules (IFN- γ, IL-4, IL-12, TNF-α) in response to high, intermediate, and low levels of LPS. The simulations show how variations in the levels of injected LPS affect the development of Th1 or Th2 cell responses through differential cytokine induction. The model also predicts the coexistence of these two types of response under certain biochemical and biomechanical conditions in the microenvironment.

Purpose We investigated whether microRNA expression data from glioblastoma could be used to produce a profile that defines a bevacizumab responsive group of patients. Patients and methods TCGA microRNA expression data from tumors resected at first diagnosis of glioblastoma in patients treated with bevacizumab at any time during the course of their disease were randomly separated into training (n = 50) and test (n = 37) groups for model generation. MicroRNA-seq data for 51 patients whose treatment included bevacizumab in the BELOB trial were used as an independent validation cohort. Results Using penalized regression we identified 8 microRNAs as potential predictors of overall survival in the training set. We dichotomized the response score based on the most prognostic minimum of a density plot of the response scores (log-rank HR = 0.16, p = 1.2e−5) and validated the profile in the test cohort (one-sided log-rank HR = 0.34, p = 0.026). Analysis of the profile using all samples in the TCGA glioblastoma dataset, regardless of treatment received, (n = 473) showed that the prediction of patient benefit was not significant (HR = 0.84, p = 0.083) suggesting the profile is specific to bevacizumab. Further independent validation of our microRNA profile in RNA-seq data from patients treated with bevacizumab (alone or in combination with CCNU) at glioblastoma recurrence in the BELOB trial confirmed that our microRNA profile predicted patient benefit from bevacizumab (HR = 0.59, p = 0.043). Conclusion We have identified and validated an 8-microRNA profile that predicts overall survival in patients with glioblastoma treated with bevacizumab. This may be useful for identifying patients who are likely to benefit from this agent.

MicroRNAs are proving to be potent tumour suppressors in cancer. Many microRNAs are in pre-clinical and clinical validation.

Importance Oncolytic viruses (OVs) are emerging as important agents in cancer treatment. Oncolytic viruses offer the attractive therapeutic combination of tumor-specific cell lysis together with immune stimulation, therefore acting as potential in situ tumor vaccines. Moreover, OVs can be engineered for optimization of tumor selectivity and enhanced immune stimulation and can be readily combined with other agents. The effectiveness of OVs has been demonstrated in many preclinical studies and recently in humans, with US Food and Drug Administration approval of the oncolytic herpesvirus talimogene laherparepvec in advanced melanoma, a major breakthrough for the field. Thus, the OV approach to cancer therapy is becoming more interesting for scientists, clinicians, and the public. The main purpose of this review is to give a basic overview of OVs in clinical development and provide a description of the current status of clinical trials. Observations In 2016 approximately 40 clinical trials are recruiting patients, using a range of OVs in multiple cancer types. There are also many more trials in the planning stages. Therefore, we are now in the most active period of clinical OV studies in the history of the field. There are several OVs currently being tested with many additional engineered derivatives. In OV clinical trials, there are a number of specific areas that should be considered, including viral pharmacokinetics and pharmacodynamics, potential toxic effects, and monitoring of the patients’ immune status. Clinical development of OVs is increasingly focused on their immune stimulatory properties, which may work synergistically with immune checkpoint inhibitors and other strategies in the treatment of human cancer. Conclusions and Relevance Oncolytic viruses are an active area of clinical research. The ability of these agents to harness antitumor immunity appears to be key for their success. Combinatorial studies with immune checkpoint blockade have started and the results are awaited with great interest.

Molecular targeting using ligands specific to disease markers has shown great promise for early detection and directed therapy. Bead-based combinatorial libraries have served as powerful tools for the discovery of novel targeting agents. Screening platforms employing magnetic capture have been used to achieve rapid and efficient identification of high-affinity ligands from one-bead-one-compound (OBOC) libraries. Traditional manual methodologies to isolate magnetized “hit” beads are tedious and lack accuracy, and existing instruments to expedite bead sorting tend to be costly and complex. Here, we describe the design and construction of a simple and inexpensive microfluidic magnetic sorting device using standard photolithography and soft lithography approaches to facilitate high-throughput isolation of magnetized positive hit beads from combinatorial libraries. We have demonstrated that the device is able to sort magnetized beads with superior accuracy compared to conventional manual sorting approaches. This chip offers a very convenient yet inexpensive alternative for screening OBOC libraries.

After more than two decades of research and development, oncolytic herpes viruses (oHSVs) are moving into the spotlight due to recent encouraging clinical trial data. oHSV and other oncolytic viruses function through direct oncolytic cancer cell-killing mechanisms and by stimulating antitumor immunity. As further viruses are developed and optimized for the treatment of various types of cancer, appropriate predictive preclinical models will be of great utility. This review will discuss existing data in this area, focusing on the mouse tumor models that are commonly used.

Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma.

Peripheral neuropathic pain is one of the most common and debilitating complications of diabetes. Several genes have been shown to be effective in reducing neuropathic pain in animal models of diabetes after transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)1-based vectors, yet there has never been a comparative analysis of their efficacy. We compared four different HSV1-based vectors engineered to produce one of two opioid receptor agonists (enkephalin or endomorphin), or one of two isoforms of glutamic acid decarboxylase (GAD65 or GAD67), alone and in combination, in the streptozotocin-induced diabetic rat and mouse models. Our results indicate that a single subcutaneous hindpaw inoculation of vectors expressing GAD65 or GAD67 reduced diabetes-induced mechanical allodynia to a degree that was greater than daily injections of gabapentin in rats. Diabetic mice that developed thermal hyperalgesia also responded to GAD65 or endomorphin gene delivery. The results suggest that either GAD65 or GAD67 vectors are the most effective in the treatment of diabetic pain. The vector combinations, GAD67 + endomorphin, GAD67 + enkephalin or endomorphin + enkephalin also produced a significant antinociceptive effect but the combination did not appear to be superior to single gene treatment. These findings provide further justification for the clinical development of antinociceptive gene therapies for the treatment of diabetic peripheral neuropathies.

Pro-inflammatory T cells mediate autoimmune demyelination in multiple sclerosis. However, the factors driving their development and multiple sclerosis susceptibility are incompletely understood. We investigated how micro-RNAs, newly described as post-transcriptional regulators of gene expression, contribute to pathogenic T-cell differentiation in multiple sclerosis. miR-128 and miR-27b were increased in naïve and miR-340 in memory CD4(+) T cells from patients with multiple sclerosis, inhibiting Th2 cell development and favouring pro-inflammatory Th1 responses. These effects were mediated by direct suppression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and interleukin-4 (IL4) expression, resulting in decreased GATA3 levels, and a Th2 to Th1 cytokine shift. Gain-of-function experiments with these micro-RNAs enhanced the encephalitogenic potential of myelin-specific T cells in experimental autoimmune encephalomyelitis. In addition, treatment of multiple sclerosis patient T cells with oligonucleotide micro-RNA inhibitors led to the restoration of Th2 responses. These data illustrate the biological significance and therapeutic potential of these micro-RNAs in regulating T-cell phenotypes in multiple sclerosis.

Human cytomegalovirus (HCMV) is highly prevalent, and like other herpes viruses, can persist for life in its host in a latent state. However, HCMV can be severely pathogenic in immunocompromised individuals. Interestingly, HCMV proteins and nucleic acids have been identified in up to 90% of patients with the incurable brain tumor glioblastoma (GBM) as well as some other cancers. Accumulating data supports the clinical relevance of HCMV in GBM, with some encouraging responses reported with HCMV-targeted immunotherapies. Although various HCMV proteins increase cell proliferation and invasion, a mechanistic link between HCMV and cancer in vivo has not been established, and the role of HCMV in GBM remains a subject of debate. In the current report we show that perinatal murine CMV (MCMV) infection induces a pro-angiogenic secretome, increasing tumor growth, pericyte accumulation, angiogenesis and tumor blood flow in a murine GBM model. Specifically, we identify platelet-derived growth factor-d (PDGF-D) as a CMV-induced factor essential for tumor growth. In our model, MCMV can be seen in tumor cells and vascular pericytes, a finding that we confirm in human GBM specimens. The anti-viral drug cidofovir improves survival in MCMV-infected mice, inhibiting MCMV activation, PDGF-D expression, pericyte recruitment and tumor angiogenesis. Together these data provide the first mechanistic explanation of how CMV potentiates GBM growth in vivo, identify PDGF-D as a potential therapeutic target, and support the application of anti-viral approaches for GBM therapy.

Recent years have seen an intense period of research on the functions of miRNAs, recently discovered key regulators of gene expression that act through suppression of translation of target mRNAs. Several hundred miRNAs have been identified in humans, and these show characteristic expression patterns, depending on tissue type, cell type or environmental stimuli. Like other types of cancer, the brain tumor glioblastoma shows a distinct miRNA expression signature, and a number of recent studies have linked these miRNA alterations to key hallmarks of glioblastoma including proliferation, survival, invasion, angiogenesis and stem cell-like behavior. These studies have opened the door to the possibility of utilizing miRNAs or miRNA antagonists as therapeutic agents for the treatment of brain tumors.

To sustain tumor growth, cancer cells must be able to adapt to fluctuations in energy availability. We have identified a single microRNA that controls glioma cell proliferation, migration, and responsiveness to glucose deprivation. Abundant glucose allows relatively high miR-451 expression, promoting cell growth. In low glucose, miR-451 levels decrease, slowing proliferation but enhancing migration and survival. This allows cells to survive metabolic stress and seek out favorable growth conditions. In glioblastoma patients, elevated miR-451 is associated with shorter survival. The effects of miR-451 are mediated by LKB1, which it represses through targeting its binding partner, CAB39 (MO25 alpha). Overexpression of miR-451 sensitized cells to glucose deprivation, suggesting that its downregulation is necessary for robust activation of LKB1 in response to metabolic stress. Thus, miR-451 is a regulator of the LKB1/AMPK pathway, and this may represent a fundamental mechanism that contributes to cellular adaptation in response to altered energy availability.

The microtubule-associated protein tau (MAPT) and alpha-synuclein (SNCA) genes play central roles in neurodegenerative disorders. Mutations in each gene cause familial disease, whereas common genetic variation at both loci contributes to susceptibility to sporadic neurodegenerative disease. Here, we demonstrate exquisite gene regulation of the human MAPT and SNCA transgene loci and functional complementation in neuronal cell cultures and organotypic brain slices using the herpes simplex virus type 1 (HSV-1) amplicon-based infectious bacterial artificial chromosome (iBAC) vector to express complete loci >100 kb. Cell cultures transduced by iBAC vectors carrying a 143 kb MAPT or 135 kb SNCA locus expressed the human loci similar to the endogenous gene. We focused on analysis of the iBAC-MAPT vector carrying the complete MAPT locus. On transduction into neuronal cultures, multiple MAPT transcripts were expressed from iBAC-MAPT under strict developmental and cell type-specific control. In primary neurons from Mapt(-/-) mice, the iBAC-MAPT vector expressed the human tau protein, as detected by enzyme-linked immunosorbent assay and immunocytochemistry, and restored sensitivity of Mapt(-/-) neurons to Abeta peptide treatment in dissociated neuronal cultures and in organotypic slice cultures. The faithful retention of gene expression and phenotype complementation by the system provides a novel method to analyze neurological disease genes.

MicroRNAs (miR) show characteristic expression signatures in various cancers and can profoundly affect cancer cell behavior. We carried out miR expression profiling of human glioblastoma specimens versus adjacent brain devoid of tumor. This revealed several significant alterations, including a pronounced reduction of miR-128 in tumor samples. miR-128 expression significantly reduced glioma cell proliferation in vitro and glioma xenograft growth in vivo. miR-128 caused a striking decrease in expression of the Bmi-1 oncogene, by direct regulation of the Bmi-1 mRNA 3′-untranslated region, through a single miR-128 binding site. In a panel of patient glioblastoma specimens, Bmi-1 expression was significantly up-regulated and miR-128 was down-regulated compared with normal brain. Bmi-1 functions in epigenetic silencing of certain genes through epigenetic chromatin modification. We found that miR-128 expression caused a decrease in histone methylation (H3K27me3) and Akt phosphorylation, and up-regulation of p21CIP1 levels, consistent with Bmi-1 down-regulation. Bmi-1 has also been shown to promote stem cell self-renewal; therefore, we investigated the effects of miR-128 overexpression in human glioma neurosphere cultures, possessing features of glioma “stem-like” cells. This showed that miR-128 specifically blocked glioma self-renewal consistent with Bmi-1 down-regulation. This is the first example of specific regulation by a miR of a neural stem cell self-renewal factor, implicating miRs that may normally regulate brain development as important biological and therapeutic targets against the “stem cell–like” characteristics of glioma. [Cancer Res 2008;68(22):9125–30]

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma. [Cancer Res 2007;67(19):9398–406]

Oncolytic viral (OV) therapy, which uses genetically engineered tumor-targeting viruses, is being increasingly used in cancer clinical trials due to the direct cytolytic effects of this treatment that appear to provoke a robust immune response against the tumor. As OVs enter tumor cells, intrinsic host defenses have the potential to hinder viral replication and spread within the tumor mass. In this report, we show that histone deacetylase 6 (HDAC6) in tumor cells appears to alter the trafficking of post-entry OVs from the nucleus toward lysosomes. In glioma cell lines and glioma-stem-like cells, HDAC6 inhibition (HDAC6i) by either pharmacologic or genetic means substantially increased replication of oncolytic herpes simplex virus type 1 (oHSV). Moreover, HDAC6i increased shuttling of post-entry oHSV to the nucleus. In addition, electron microscopic analysis revealed that post-entry oHSVs are preferentially taken up into glioma cells through the endosomal pathway rather than via fusion at the cell surface. Together, these findings illustrate a mechanism of glioma cell defense against an incoming infection by oHSV and identify possible approaches to enhance oHSV replication and subsequent lysis of tumor cells.

SummaryIn aggressive, rapidly growing solid tumors such as glioblastoma multiforme (GBM), cancer cells face frequent dynamic changes in their microenvironment, including the availability of glucose and other nutrients. These challenges require that tumor cells have the ability to adapt in order to survive periods of nutrient/energy starvation. We have identified a reciprocal negative feedback loop mechanism in which the levels of microRNA-451 (miR-451) are negatively regulated through the phosphorylation and inactivation of its direct transcriptional activator OCT1 by 5′ AMP-activated protein kinase (AMPK), which is activated by glucose depletion-induced metabolic stress. Conversely, in a glucose-rich environment, unrestrained expression of miR-451 suppresses AMPK pathway activity. These findings uncover miR-451 as a major effector of glucose-regulated AMPK signaling, allowing tumor cell adaptation to variations in nutrient availability in the tumor microenvironment.

Gene therapy is a potentially useful approach in the treatment of human brain tumors, which are notoriously refractory to conventional approaches. Most human clinical trials to date have been unsuccessful in terms of improving patient outcome. Recent improvements in viral vectors, the development of stem cell technology, and increased understanding of the mechanism of action of therapeutic transgenes provide hope that the next generation of gene therapeutics may show increased efficacy in treatment of this devastating disease.

INTRODUCTION Glioblastoma multiforme remains one of the most difficult to treat cancers due to the highly infiltrative nature of cancer cells. Targeting the invasive properties of glioblastoma presents a novel treatment avenue. Recently, specific GSK-3 beta (GSK3b) inhibitors such as Bio-Indirubin (BIO), have been described that inhibit the migratory activity of glioma cells. In this study, the GSK-3b downstream targets were investigated.

The cellular dispersion and therapeutic control of glioblastoma, the most aggressive type of primary brain cancer, depends critically on the migration patterns after surgery and intracellular responses of the individual cancer cells in response to external biochemical and biomechanical cues in the microenvironment. Recent studies have shown that a particular microRNA, miR-451, regulates downstream molecules including AMPK and mTOR to determine the balance between rapid proliferation and invasion in response to metabolic stress in the harsh tumor microenvironment. Surgical removal of main tumor is inevitably followed by recurrence of the tumor due to inaccessibility of dispersed tumor cells in normal brain tissue. In order to address this multi-scale nature of glioblastoma proliferation and invasion and its response to conventional treatment, we propose a hybrid model of glioblastoma that analyses spatio-temporal dynamics at the cellular level, linking individual tumor cells with the macroscopic behaviour of cell organization and the microenvironment, and with the intracellular dynamics of miR-451-AMPK-mTOR signaling within a tumour cell. The model identifies a key mechanism underlying the molecular switches between proliferative phase and migratory phase in response to metabolic stress and biophysical interaction between cells in response to fluctuating glucose levels in the presence of blood vessels (BVs). The model predicts that cell migration, therefore efficacy of the treatment, not only depends on oxygen and glucose availability but also on the relative balance between random motility and strength of chemoattractants. Effective control of growing cells near BV sites in addition to relocalization of invisible migratory cells back to the resection site was suggested as a way of eradicating these migratory cells. Publisher PDF

Epigenetic deregulation is a hallmark of glioblastoma (GBM), causing tumor cells to be refractory to differentiation, hyperproliferative and resistant to multiple genotoxic insults, including radiation and TMZ-induced DNA damage. Analysis of TCGA data revealed a highly significant inverse correlation between the expression of a group of microRNAs belonging to the neural lineage, downregulated in GBM, and several target proteins with epigenetic functions, including EZH2, BMI1 and LSD1, known oncogenes upregulated in the tumor. Consequently, we engineered an artificial DNA sequence encoding 5 of the above microRNAs arranged as a genetic cluster, and delivered to human GBM stem cells by lentiviral infection. The artificial transgene correctly produced all 5 mature microRNAs, and resulted in profound downregulation not only of putative targets, but also of other non-target oncogenes with epigenetic functions like DNMT1 and MYC, suggesting a synergistic effect caused by the combination of microRNAs. This combination determined a much more marked phenotypic effect than each single microRNA, both in vitro, measured as cell proliferation, induction of differentiation and clonogenic capacity, and in vivo, resulting in more than double survival time in mice with intracranial GBM xenografts. After temozolomide treatment, 90% of GBM cells overexpressing the multi-microRNA cluster underwent apoptotic cell death, as compared to 10–15% observed with single microRNA upregulation. Importantly, this effect was independent of MGMT expression status. Finally, co-culture experiments showed that all 5 overexpressed microRNAs were actively transferred from transduced cells to non-transduced ones, retaining their biological function in the acceptor cells. This suggests the potential for a vector-mediated delivery of clustered microRNAs in the clinical setting. In conclusion, we show a new microRNA-based gene therapy approach for GBM, which takes advantage of small size, ease of genetic manipulation, broad molecular targeting and cell-to-cell diffusivity proper of microRNAs.

Programmed cell death protein-1 (PD1) expressed on T cells is a key mediator of immune evasion via mediating the inhibition of T-cell function. This occurs via its binding to ligands PD-L1 and PD-L2 on tumor cells and other cells such as macrophages and microglia. Immune checkpoint blockade using anti-PD1 antibodies is of clinical benefit in multiple cancer types and is under investigation in glioblastoma. Tumor PD-L1 expression is currently used to screen for eligibility for anti-PD1 treatment, suggesting that only PD-L1 positive tumors will be included in clinical trials. However, the role of PD-L2 in immune evasion in glioblastoma has not been well studied. To assess the roles of PD-L1 and PD-L2 in glioblastoma, their expression was analyzed in TCGA and IVY Atlas RNAseq data. Genes whose expression was correlated with either PD-L1 or PD-L2 were assessed for pathway enrichment. To compare their effects on patient outcome, Cox regression models for PD-L1 and PD-L2 were fitted. To confirm these findings, western blot and qPCR gene expression was conducted of glioma-patient derived cell cultures (n=19) and fresh tumor samples (n=9). Here we show that PD-L2 is present in glioblastoma at higher levels than PD-L1, and that the level of PD-L2 in the tumor impacts patient survival. Furthermore PD-L2 expression negatively correlated with KPS rho=-0.13 (p=0.007). We show that PD-L2 correlates with retinoic acid receptor and viral induction of apoptosis pathways in glioblastoma. Western blot and gene expression analysis of patient tumor samples and patient-derived cell lines shows that PD-L2 is robustly expressed, while PD-L1 expression is variable. PD-L2 expression was also inducible by IFNy in certain cell lines. These results suggest that PD-L2 should be considered as a biomarker for eligibility of patients in clinical trials for anti-PD1 therapies and widens the net for patient recruitment in these trials.

Background: Extracellular Vesicles (EVs) shed by tumor cells have recently been demonstrated to act as a major conduit in cell-cell communication. Increasing knowledge of the effect of EVs on infiltrating lymphocytes within the tumor microenvironment shows the immune modulation capacity of EVs. In this study we investigated the potential role of PD-L1 on EVs in immune escape in heterogeneous glioblastoma (GBM). Methods: Peripheral blood mononuclear cells (PBMCs) (n=8) were stimulated by IL-2 or anti-CD3±anti-CD28±anti-PD1 treatment. Magnetic cell sorting was used to isolate CD3+ cells. Activation levels of CD3+CD4+, CD3+CD8+ and CD3-CD56+ were monitored by flow cytometry of the activation markers CD69, CD25, PD1 and TIM3. Concurrent eFluor staining was used to measure proliferation. EVs derived from four different glioblastoma stem-like cell lines (GSCs) from either the mesenchymal (M) or proneural (P) subtype were used in this study. PD-L1 expression was validated by immunoblotting and electron microscopy (EM). EV binding was visualized by PALM-tdTomato and PD-L1_RFP positive EVs and tested on plate-bound PD1. RNA-seq from TCGA and Immunohistochemistry (IHC) was used to correlate PD-L1 expression with CD3 infiltration. In addition, circulating EVs from GBM patients (n=22) and controls (n=5) were used to determine PD-L1 content via digital droplet-PCR. Results: PD-L1 was expressed on the surface of M GSC EVs. EVs were able to bind to the outer surface of CD3+ cells. In whole PBMCs EV treatment led to a significant, PD-L1 dependent, reduction of CD3+CD8+ and CD3+CD4+ T-cell activation as well as decreased proliferation in the M subtype. These effects were also observed in CD3+ sorted cells, indicating a direct effect of EVs on T cells. Furthermore PD-L1 on EVs was capable of binding directly to PD1. Using RNA-seq and IHC we show a correlation of CD3 infiltration and PD-L1 expression. In addition, circulating EVs from GBM patients show enriched PD-L1 DNA cargo that correlates with tumor volume. CONCLUSION: Our findings demonstrate the immunosuppressive potential of GSC-derived EVs in a PD-L1 dependent manner. Furthermore we show that PD-L1 on EVs can directly bind PD1 and is capable of hindering T cell activation locally and at distant sites and show how immune checkpoint blockade may systemically enhance immunity against GBM.

BACKGROUND: Small molecule inhibitors of the mitotic kinases, including the Aurora kinase inhibitor MLN8237, are being investigated as novel therapeutic agents for the treatment of solid cancers including malignant brain tumours. However, resistance has been observed and anti-apoptotic proteins of the BCL-2 family have been shown to promote cell survival following treatment. In this study, we investigated the effect of disrupting anti-apoptotic protein function in medulloblastoma and pediatric glioblastoma (pGBM) models using BH3-mimetics in combination with MLN8237. METHODS: The effects of single agent and combination treatment on cell viability were determined by MTT/WST-1 assays and flow cytometry. Effects on cell phenotype, cell cycle progression and cellular ploidy were determined by live cell imaging and DNA content analysis. Induction of apoptosis was monitored in real-time, by time-lapse microscopy, using a fluorescent, polarity-sensitive phosphatidylserine probe, and confirmed by measuring caspase-3/7 activity. Knockdown of BAX/BAK was used to confirm these effects. Results: ABT-263 in combination with MLN8237 reduced mitotic slippage and polyploidy, significantly reduced cell viability and promoted the elimination of mitotically defective cells. This occurred through a BAX/BAK-dependent, caspase-mediated apoptotic pathway. The BCL-xL antagonist, WEHI-539, which targets BCL-2, significantly augmented tumour cell killing when used in combination with MLN8237. It also sensitized resistant brain tumour cells to a novel BAX activator, SMBA1. In addition, siRNA mediated knockdown of BCL-xL sensitized pGBM and medulloblastoma cells to MLN8237, and mimicked the effect of combination drug treatment. CONCLUSION: Combination treatment using a pro-apoptotic agent enhanced the cytotoxicity of MLN8237 in paediatric tumours and may overcome an important resistance mechanism.

Early clinical trial data show that blockade of PD-1 signaling leads to significant anticancer responses in a subset of patients in certain cancer types. While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics in brain tumors has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune checkpoint inhibitors block the body's natural checkpoint response, the combination of these two approaches offers a potentially advantageous interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD-1) on T-cells that recognizes its ligand PD-L1. AdV-TK is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene (TK) followed by administration of an anti-herpetic prodrug (ganciclovir GCV) and recently showed encouraging results in a Phase II trial in glioblastoma (Wheeler et al., 2016). The immunological component results from the delivery vehicle being a virus, the mode of cell death, through both necrosis and apoptosis, and the pro-immunogenic properties of the TK protein. We confirm that this approach induces glioblastoma cell death and a consistent anti-tumor immune stimulation. Not surprisingly, however, this immune stimulation also leads to increase in cell surface of immune checkpoint inhibitory ligands on tumor cells, including PD-L1, detected by flow cytometry and immunohistochemistry. We show that GMCI induces a type-I interferon response, and using IFN decoy we demonstrated that the release of IFNβ in vitro is at least partially responsible for autocrine/paracrine PD-L1 up-regulation both in human and mouse glioblastoma cell lines. In vivo studies using an intracranial GL261 model showed high numbers of long term survivors in the GMCI/PD-1 combination (11/14), compared with GMCI (6/16), anti-PD-1 (5/12) and untreated (0/11). In addition, long term survival mice were no longer able to form tumors after rechallenge indicating the establishment of anti-tumor immunity. Finally, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and IFNγ+ cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD-1+ /TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD-1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients. Citation Format: Maria Carmela Speranza, Franz Ricklefs, Carmela Passaro, Sarah R. Klein, Kazue Kasai, Johanna Kaufmann, Hiroshi Nakashima, Bronisz Agnieszka, Estuardo Aguilar-Cordova, Brian W. Guzik, Gordon J. Freeman, David A. Reardon, Patrick Wen, E. Antonio Chiocca, Sean E. Lawler. Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B84.

MicroRNA expression can be exploited to define tumor prognosis and stratification for precision medicine. It remains unclear whether prognostic microRNA signatures are exclusively tumor grade and/or molecular subtype-specific, or whether common signatures of aggressive clinical behavior can be identified. Here, we defined microRNAs that are associated with good and poor prognosis in grade III and IV gliomas using data from The Cancer Genome Atlas. Pathway analysis of microRNA targets that are differentially expressed in good and poor prognosis glioma identified a link to oligodendrocyte development. Notably, a microRNA expression profile that is characteristic of a specific oligodendrocyte precursor cell type (OP1) correlates with microRNA expression from 597 of these tumors and is consistently associated with poor patient outcome in grade III and IV gliomas. Our study reveals grade-independent and subtype-independent prognostic molecular signatures in high-grade glioma and provides a framework for investigating the mechanisms of brain tumor aggressiveness.

INTRODUCTION MicroRNAs, single-stranded non-coding RNAs that function by reducing translation or causing degradation of target mRNAs, have been shown to play roles in multiple hallmarks of GBM, suggesting they may be of therapeutic importance. METHODS: The miRIDIAN mimic library (Dharmacon) that encompasses all human microRNAs annotated in miRBase v16.0 was used with a high-throughput imaging platform (Operetta) to identify microRNAs with potent effects on GBM cell proliferation and survival. Screens were performed in duplicate on U251 (adult) and KNS42 (paediatric) GBM cell lines. Cell number (nuclei count) was assessed 72h post-transfection and expressed as a z-score for each microRNA. MicroRNAs were considered significant candidates if their mean z-score was below zero and if it differed from that of the negative control by at least two standard deviations. Validation included RTqPCR, imaging and flow cytometry based assays. RESULTS: The functional screens resulted in approximately 100 candidates per cell line with 70% overlap between them. Based on available knowledge and potential relevance for GBM therapy, seven initial microRNAs candidates were chosen for further validation in a panel of four adult and two paediatric GBM cell lines: miR-34a-5p, miR-34b-5p, miR-34c-5p, miR-449a-5p, miR-449b-5p which all share a common seed sequence; miR-340-5p and miR-X. Of these candidates miR-X was identified as having the most potent, robust effects. MiR-X consistently caused a G2M cell cycle arrest followed by apoptosis in all cell lines and has predicted targets involved in the induction of the cytoskeleton rearrangement and non-canonical Wnt pathways following the G2M phase transition. This phenotype was specific to mimic-miR-X transfected cells. CONCLUSION: We have identified a novel potent pro-apoptotic microRNA in GBM that may be relevant for therapy. Work is ongoing to characterise the mechanism responsible for this effect and test for efficacy in vivo.

BACKGROUND: There have been several reports that have linked human cytomegalovirus (CMV) to gliomas. Although clinical trials of immunotherapy against CMV antigens found in gliomas have commenced in multiple institutions, the data linking these tumors to this virus remains controversial. METHODS: To study the controversial role of cytomegalovirus (CMV) in glioblastoma, we assessed the effects of murine CMV (MCMV) perinatal infection in a GFAP-cre; Nf1loxP/+ ; Trp53-/+ genetic mouse model of glioma (Mut3 mice). RESULTS: Early on after infection, MCMV antigen was predominantly localized in CD45+ lymphocytes in the brain with active viral replication and local areas of inflammation, but by 7 weeks there was a generalized loss of MCMV in brain, confirmed by bioluminescent imaging. MCMV-infected Mut3 mice exhibited a shorter survival time from their gliomas, when compared to control Mut3 mice, perinatally infected with mock or with a different neurotropic virus. Animal survival was also significantly shortened when orthotopic gliomas were implanted in mice perinatally infected with MCMV vs. controls. MCMV infection increased phosphorylated STAT3 (P-STAT3) levels in neural stem cells (NSCs) harvested from Mut3 mice SVZ and in vivo there was increased P-STAT3 in NSCs in MCMV-infected compared to control mice. Of relevance, human CMV (HCMV) also increased P-STAT3 and proliferation of patient-derived glioblastoma neurospheres, while a STAT3 inhibitor reversed this effect in vitro and in vivo. CONCLUSIONS: These findings thus associate CMV infection to a STAT3-dependent modulatory role in glioma formation/progression in the context of tumor suppressor mutations in mice and possibly in humans. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.