Temozolomide (TMZ) is the standard of care chemotherapy drug for treating glioblastomas (GBMs), the most aggressive cancer that affects people of all ages. However, its therapeutic efficacy is limited by the drug resistance mediated by a DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), which eliminates the TMZ-induced DNA lesions. Here we report the development of an iron oxide nanoparticle (NP) system for targeted delivery of siRNAs to suppress the TMZ-resistance gene (MGMT). We show that our NP is able to overcome biological barriers, bind specifically to tumor cells, and reduce MGMT expression in tumors of mice bearing orthotopic GBM serially-passaged patient-derived xenografts. The treatment with sequential administration of this NP and TMZ resulted in increased apoptosis of GBM stem-like cells, reduced tumor growth, and significantly-prolonged survival as compared to mice treated with TMZ alone. This study introduces an approach that holds great promise to improve the outcomes of GBM patients.

Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2 , is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research.

Abstract Recent work has uncovered oncogenic TRK fusions in a wide range of cancer types, including adult and pediatric gliomas. With some exceptions, many of these fusions tend to occur at very low frequencies below 1–2%, with unclear clinical significance, yet they highlight a potentially important and rapidly evolving role for NTRK1, NTRK2, NTRK3 in glioma biology. Basic scientific and clinical investigation surrounding TRKs’ role in cancer has often been hindered due to the nonspecific nature of antibodies and kinase inhibitors, combined with a lack of precise exon-specific expression data from patient populations. Tropomyosin receptor B (TrkB), encoded by the NTRK2 gene, is most known for its established roles in neuronal survival, proliferation, differentiation, apoptosis, learning, and memory. TrkB exerts diverse effects on cellular outcomes through interactions with downstream signaling cascades and has been shown to exhibit complex alternative splicing patterns. Here we show a novel role for a TrkB splice variant in human gliomas via NTRK2 transcript analyses in normal human brain and gliomas using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx). Using a novel antibody designed against this splice variant, immunostaining shows altered receptor localization within human gliomas compared to normal human brain. This NTRK2 splice variant enhances PDGF-driven gliomas in vivo in an RCAS-TVA mouse model and augments PDGF-induced signaling in vitro. Through the lens of NTRK2, these results highlight the importance of expanding upon whole gene-level and kinase-fusion analyses to explore TRK splicing in basic and translational research.

Most known for their essential roles in the development and maintenance of the nervous system, the neurotrophins consist of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), NT-3, NT-4 and their respective tropomyosin receptor kinases (TRKs) TrkA, TrkB, and TrkC along with the low affinity nerve growth factor receptor, p75. In addition to known roles in neuronal survival, proliferation, differentiation, and apoptosis, TRKs exert diverse effects on cellular outcomes through their interactions with downstream signaling cascades. Prior to TRKs’ established roles in neurobiology, oncogenic TRK was discovered as tropomysoin 3 (TPM3)-TrkA fusion and as a result, oncological studies of the neurotrophin family have identified additional TRK fusions. Putative oncogenic TRK fusions have been observed in various cancer types, but their clinical significance remains unclear and these fusions tend to occur at very low frequencies below 1–2%. The low incidence of these fusions combined with significant overexpression of various TRKs in a multitude of cancers raises the possibility that another aspect of TRK biology, in addition to kinase-domain fusions, may be relevant. Basic scientific and clinical investigation surrounding TRKs’ role in cancer has often been hindered due to the nonspecific nature of antibodies and kinase inhibitors, combined with a lack of precise exon-specific expression data from patient populations. Tropomyosin receptor B (TrkB), encoded by the NTRK2 gene, exhibits complex alternative splicing patterns. Here we show a novel role for a TrkB splice variant in gliomas via NTRK2 transcript analyses and immunostaining using a novel antibody engineered specifically to this variant. This NTRK2 splice variant enhances PDGF-driven gliomas in vivo and augments PDGF-induced signaling in vitro. Through the lens of NTRK2, these results highlight the importance of expanding upon whole gene-level and kinase-fusion analyses to explore TRK splicing in basic and translational research.

ABSTRACT Efforts to reduce immunosuppression in the solid tumor microenvironment by blocking the recruitment or polarization of tumor associated macrophages (TAM), or myeloid derived suppressor cells (MDSCs), have gained momentum in recent years. Expanding our knowledge of the immune cell types, cytokines, or recruitment factors that are associated with high-grade disease, both within the tumor and in circulation, is critical to identifying novel targets for immunotherapy. Furthermore, a better understanding of how therapeutic regimens, such as Dexamethasone (Dex), chemotherapy, and radiation, impact these factors will facilitate the design of therapies that can be targeted to the appropriate populations and retain efficacy when administered in combination with standard of care regimens. Here we perform quantitative analysis of tissue microarrays made of samples taken from grades I-III astrocytoma and glioblastoma (GBM, grade IV astrocytoma) to evaluate infiltration of myeloid markers CD163, CD68, CD33, and S100A9. Serum, flow cytometric, and Nanostring analysis allowed us to further elucidate the impact of Dex treatment on systemic biomarkers, circulating cells, and functional markers within tumor tissue. We found that common myeloid markers were elevated in Dex-treated grade I astrocytoma and GBM compared to non-neoplastic brain tissue and grade II-III astrocytomas. Cell frequencies in these samples differed significantly from those in Dex-naïve patients in a pattern that depended on tumor grade. In contrast, observed changes in serum chemokines or circulating monocytes were independent of disease state and were due to Dex treatment alone. Furthermore, these changes seen in blood were often not reflected within the tumor tissue. Conclusions: Our findings highlight the importance of considering perioperative treatment as well as disease grade when assessing novel therapeutic targets or biomarkers of disease.

Glioblastoma (GBM) remains incurable, and recurrent tumors rarely respond to standard-of-care radiation and chemo-therapies. Therefore, strategies that enhance the effects of these therapies should provide significant benefits to GBM patients. We have developed a nanoparticle delivery vehicle that can stably bind and protect nucleic acids for specific delivery into brain tumor cells. These nanoparticles can deliver therapeutic siRNAs to sensitize GBM cells to radiotherapy and improve GBM treatment via systemic administration. We show that nanoparticle-mediated knockdown of the DNA repair protein apurinic endonuclease 1 (Ape1) sensitizes GBM cells to radiotherapy and extend survival in a genetic mouse model of GBM. Specific knockdown of Ape1 activity by 30% in brain tumor tissue doubled the extended survival achieved with radiotherapy alone. Ape1 is a promising target for increasing the effectiveness of radiotherapy, and nanoparticle-mediated delivery of siRNA is a promising strategy for tumor specific knockdown of Ape1.

Radiotherapy is an integral component of the treatment for pediatric and adult brain tumors. However, survival is frequently accompanied by one or more radiation-induced adverse sequelae, especially in children. Therefore, strategies that enhance radiotherapy in brain tumors while sparing adjacent normal brain are expected to improve life-long outcomes. We have developed a nanoparticle delivery vehicle that can stably bind, protect, and deliver nucleic acids to brain cancer cells and tumors. We found that nanoparticle-mediated siRNA delivery reduced expression and activity of the multifunctional DNA repair protein Ape1 specifically in glioblastoma cells and tumors, which diminished their response to radiotherapy. Importantly, nanoparticle treatment (intravenous delivery) combined with 10 Gy fractionated radiotherapy doubled the extension in survival compared to 10 Gy fractionated radiotherapy alone in a PDGFB-driven genetic mouse model of glioblastoma. Therefore, nanoparticle-mediated inhibition of Ape1 may be a useful strategy to enhance the therapeutic effect of radiotherapy in brain tumor patients and reduce treatment-induced morbidity.

Nanoparticle-mediated delivery of chemotherapeutics has demonstrated potential in improving anticancer efficacy by increasing serum half-life and providing tissue specificity and controlled drug release to improve biodistribution of hydrophobic chemotherapeutics. However, suboptimal drug loading, particularly for solid core nanoparticles (NPs), remains a challenge that limits their clinical application. In this study we formulated a NP coated with a pH-sensitive polymer of O6-methylguanine-DNA methyltransferase (MGMT) inhibitor analog, dialdehyde modified O6-benzylguanosine (DABGS) to achieve high drug loading, and polyethylene glycol (PEG) to ameliorate water solubility and maintain NP stability. The base nanovector consists of an iron oxide core (9 nm) coated with hydrazide functionalized PEG (IOPH). DABGS and PEG-dihydrazide were polymerized on the iron oxide nanoparticle surface (IOPH-pBGS) through acid-labile hydrazone bonds utilizing a rapid, freeze-thaw catalysis approach. DABGS polymerization was confirmed by FTIR and quantitated by UV-vis spectroscopy. IOPH-pBGS demonstrated excellent drug loading of 33.4 ± 5.1% by weight while maintaining small size (36.5 ± 1.8 nm). Drug release was monitored at biologically relevant pHs and demonstrated pH dependent release with maximum release at pH 5.5 (intracellular conditions), and minimal release at physiological pH (7.4). IOPH-pBGS significantly suppressed activity of MGMT and potentiated Temozolomide (TMZ) toxicity in vitro, demonstrating potential as a new treatment option for glioblastomas (GBMs).

The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient‐derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self‐adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial–mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold‐grown cells also display higher expression of genes that promote resistance to hypoxia‐induced oxidative stress. In accord, scaffold‐grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments.

Cerebral vasospasm is a major cause of morbidity and mortality in patients with subarachnoid hemorrhage (SAH), causing delayed neurological deficits in as many as one third of cases. Existing therapy targets induction of cerebral vasodilation through use of various drugs and mechanical means, with a range of observed efficacy. Here, we perform a literature review supporting our hypothesis that transcranially delivered ultrasound may have the ability to induce therapeutic cerebral vasodilation and, thus, may one day be used therapeutically in the context of SAH. Prior studies demonstrate that ultrasound can induce vasodilation in both normal and vasoconstricted blood vessels in peripheral tissues, leading to reduced ischemia and cell damage. Among the proposed mechanisms is alteration of several nitric oxide (NO) pathways, where NO is a known vasodilator. While in vivo studies do not point to a specific physical mechanism, results of in vitro studies favor cavitation induction by ultrasound, where the associated shear stresses likely induce NO production. Two papers discussed the effects of ultrasound on the cerebral vasculature. One study applied clinical transcranial Doppler ultrasound to a rodent complete middle cerebral artery occlusion model and found reduced infarct size. A second involved the application of pulsed ultrasound in vitro to murine brain endothelial cells and showed production of a variety of vasodilatory chemicals, including by-products of arachidonic acid metabolism. In sum, nine reviewed studies demonstrated evidence of either cerebrovascular dilation or elaboration of vasodilatory compounds. Of particular interest, all of the reviewed studies used ultrasound capable of transcranial application: pulsed ultrasound, with carrier frequencies ranging between 0.5 and 2.0 MHz, and intensities not substantially above FDA-approved intensity values. We close by discussing potential specific treatment paradigms of SAH and other cerebral ischemic disorders based on MRI-guided transcranial ultrasound.

Human tumor necrosis factor α‐related apoptosis‐inducing ligand (TRAIL) is an attractive cancer therapeutic because of its ability to induce apoptosis in tumor cells while having a negligible effect on normal cells. However, the short serum half‐life of TRAIL and lack of efficient in vivo administration approaches have largely hindered its clinical use. Using nanoparticles (NPs) as carriers in gene therapy is considered as an alternative approach to increase TRAIL delivery to tumors as transfected cells would be induced to secrete TRAIL into the tumor microenvironment. To enable effective delivery of plasmid DNA encoding TRAIL into glioblastoma (GBM), we developed a targeted iron oxide NP coated with chitosan–polyethylene glycol–polyethyleneimine copolymer and chlorotoxin (CTX) and evaluated its effect in delivering TRAIL in vitro and in vivo. NP–TRAIL successfully delivers TRAIL into human T98G GBM cells and induces secretion of 40 pg mL −1 of TRAIL in vitro. Transfected cells show threefold increased apoptosis as compared to the control DNA bound NPs. Systemic administration of NP–TRAIL–CTX to mice bearing T98G‐derived flank xenografts results in near‐zero tumor growth and induces apoptosis in tumor tissue. Our results suggest that NP–TRAIL–CTX can potentially serve as a targeted anticancer therapeutic for more efficient TRAIL delivery to GBM.

Radiotherapy (RT) is an integral component of the treatment for medulloblastoma (MB) and the only effective adjuvant therapy for ependymoma (EP), two of the most common pediatric brain tumors. However, survival is frequently accompanied by one or more radiation-induced adverse developmental and psychosocial sequelae. Therefore, strategies that enhance the tumoricidal action of RT while sparing adjacent normal brain are expected to reduce the amount of radiation required to generate a therapeutic effect in these tumors. The multifunctional DNA repair protein Ape1/Ref-1 has been implicated in conferring radiation resistance in pediatric brain tumors. However, inhibiting Ape1 activity in the clinic has been hindered by the lack of safe and effective drugs and siRNA delivery vehicles. Here, we used our nanoparticle delivery vehicles to deliver siRNA against Ape1 (siApe1) and improve cell kill after RT. Nanoparticles were loaded with siApe1, or siGFP as a control, and used to treat UW228 (MB) and Res196 (EP) cells. Ape1 expression and activity were measured using PCR, Western blot, and an abasic endonuclease activity assay. DNA repair and clonogenic survival were assessed after exposure to 137Cs-γ-rays. We found that nanoparticle-mediated siApe1 delivery reduced Ape1 expression and activity by greater than 80%, which was accompanied by a greater abundance of DNA damage. This diminished the shoulder of resistance in survival curves decreasing survival to ∼50% at 1 Gy. Sensitization was specific to abasic site generating treatments as response to paclitaxel was not affected. Therefore, nanoparticle-mediated inhibition of Ape1 may help enhance the therapeutic effect of RT in pediatric brain tumor patients and reduce treatment-induced morbidity.

Medulloblastoma (MB) and ependymoma (EP) are the most common pediatric brain tumors, afflicting 3000 children annually. Radiotherapy (RT) is an integral component in the treatment of these tumors; however, the improvement in survival is often accompanied by radiation-induced adverse developmental and psychosocial sequelae. Therefore, there is an urgent need to develop strategies that can increase the sensitivity of brain tumors cells to RT while sparing adjacent healthy brain tissue. Apurinic endonuclease 1 (Ape1), an enzyme in the base excision repair pathway, has been implicated in radiation resistance in cancer. Pharmacological and specificity limitations inherent to small molecule inhibitors of Ape1 have hindered their clinical development. Here we report on a nanoparticle (NP) based siRNA delivery vehicle for knocking down Ape1 expression and sensitizing pediatric brain tumor cells to RT. The NP comprises a superparamagnetic iron oxide core coated with a biocompatible, biodegradable coating of chitosan, polyethylene glycol (PEG), and polyethyleneimine (PEI) that is able to bind and protect siRNA from degradation and to deliver siRNA to the perinuclear region of target cells. NPs loaded with siRNA against Ape1 (NP:siApe1) knocked down Ape1 expression over 75% in MB and EP cells, and reduced Ape1 activity by 80%. This reduction in Ape1 activity correlated with increased DNA damage post-irradiation, which resulted in decreased cell survival in clonogenic assays. The sensitization was specific to therapies generating abasic lesions as evidenced by NP:siRNA not increasing sensitivity to paclitaxel, a microtubule disrupting agent. Our results indicate NP-mediated delivery of siApe1 is a promising strategy for circumventing pediatric brain tumor resistance to RT.

Background The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors. Results We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations. Conclusions Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.

Background: CpG methylation in the O-6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies.Methods: We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP).Results: Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P <= 0.001) and continuous (P <= 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P <= 0.005) and longer PFS (P <= 0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P <= 0.005), and among unmethylated tumors with less than median activity (P <= 0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs.Conclusions: Our results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas.General significance: MGMT activity is an attractive target for anti-resistance therapy regardless of methylation status. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND 4.0 license (http://creativecommons.org/licenses/by/4.0/).

Pediatric brain tumors are the leading cause of death in children, and survival is frequently accompanied by one or more radiation-induced adverse developmental and psychosocial sequelae. Radiotherapy (RT) is an integral component of the treatment for medulloblastoma (MB) and the only effective adjuvant therapy for ependymoma (EP). Therefore, there is an urgent need to develop strategies to enhance the tumoricidal action of RT while sparing adjacent normal tissue. The multifunctional DNA repair protein Ape1/Ref-1 has been implicated in conferring radiation resistance in pediatric brain tumors. However, inhibiting Ape1 activity in the clinic has been hindered by the lack of safe and effective drugs and siRNA delivery vehicles. We have previously developed a nanoparticle that can deliver siRNA specifically to brain tumors for efficient knockdown of GFP. Here, we aimed to deliver siRNA against Ape1 to improve tumor cell kill after RT. Nanoparticles were loaded with siApe1, or siGFP as a control, and used to treat UW228 (MB) and Res196 (EP) cells. Ape1 expression levels were measured using PCR and Western blot, and the abasic endonuclease activity of Ape1 was determined using an Ape activity assay. Cells were then treated with 137Cs-γ-rays and cell survival monitored using clonogenic assays. DNA repair in cells was assessed though quantification of abasic sites, and resulting double-strand breaks were imaged by γH2AX foci immunostaining. We found that nanoparticle-mediated siApe1 delivery reduced Ape1 expression and activity by greater than 80%. This diminished the shoulder of resistance in survival curves decreasing survival to ∼50% at 1 Gy, and was accompanied by inhibition of DNA repair. Sensitization was specific to abasic site generating treatments as response to paclitaxel was not affected. Therefore, siApe1 loaded nanoparticles may help enhance the therapeutic effect of RT in pediatric brain tumor patients by inhibiting DNA repair.

Emerging evidence implicates cancer stem cells (CSCs) as primary determinants of the clinical behavior of human cancers, representing an ideal target for next-generation anti-cancer therapies. However CSCs are difficult to propagate in vitro, severely limiting the study of CSC biology and drug development. Here we report that growing cells from glioblastoma (GBM) cell lines on three dimensional (3D) porous chitosan-alginate (CA) scaffolds dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. CA scaffold-grown cells were found more tumorigenic in nude mouse xenografts than cells grown from monolayers. Growing in CA scaffolds rapidly promoted expression of genes involved in the epithelial-to-mesenchymal transition that has been implicated in the genesis of CSCs. Our results indicate that CA scaffolds have utility as a simple and inexpensive means to cultivate CSCs in vitro in support of studies to understand CSC biology and develop more effective anti-cancer therapies.

Resistance to temozolomide (TMZ) based chemotherapy in glioblastoma multiforme (GBM) has been attributed to the upregulation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Inhibition of MGMT using O6-benzylguanine (BG) has shown promise in these patients, but its clinical use is hindered by poor pharmacokinetics that leads to unacceptable toxicity. To improve BG biodistribution and efficacy, we developed superparamagnetic iron oxide nanoparticles (NP) for targeted convection-enhanced delivery (CED) of BG to GBM. The nanoparticles (NPCP-BG-CTX) consist of a magnetic core coated with a redox-responsive, cross-linked, biocompatible chitosan-PEG copolymer surface coating (NPCP). NPCP was modified through covalent attachment of BG and tumor targeting peptide chlorotoxin (CTX). Controlled, localized BG release was achieved under reductive intracellular conditions and NPCP-BG-CTX demonstrated proper trafficking of BG in human GBM cells in vitro. NPCP-BG-CTX treated cells showed a significant reduction in MGMT activity and the potentiation of TMZ toxicity. In vivo, CED of NPCP-BG-CTX produced an excellent volume of distribution (Vd) within the brain of mice bearing orthotopic human primary GBM xenografts. Significantly, concurrent treatment with NPCP-BG-CTX and TMZ showed a 3-fold increase in median overall survival in comparison to NPCP-CTX/TMZ treated and untreated animals. Furthermore, NPCP-BG-CTX mitigated the myelosuppression observed with free BG in wild-type mice when administered concurrently with TMZ. The combination of favorable physicochemical properties, tumor cell specific BG delivery, controlled BG release, and improved in vivo efficacy demonstrates the great potential of these NPs as a treatment option that could lead to improved clinical outcomes.