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Glioblastoma is the most common primary malignant brain tumor. The tissue microenvironment adjacent to vasculature, termed the perivascular niche, has been implicated in promoting biological processes involved in glioblastoma progression such as invasion, proliferation, and therapeutic resistance. However, the exact nature of the cues that support tumor cell aggression in this niche are largely unknown. Soluble angiocrine factors secreted by tumor-associated vasculature have been shown to support such behaviors in other cancer types. Here, we exploit macroscopic and microfluidic gelatin hydrogel platforms to profile angiocrine factors secreted by self-assembled endothelial networks and evaluate their relevance to glioblastoma biology. Aggregate angiocrine factors support increases in U87-MG cell number, migration, and therapeutic resistance to temozolomide. We also identify a novel role for TIMP1 in facilitating glioblastoma tumor cell migration. Overall, this work highlights the use of multidimensional hydrogel models to evaluate the role of angiocrine signals in glioblastoma progression.Insight, Innovation, and IntegrationGlioblastoma progression is linked to interactions between tumor and vascular cells, which can influence invasion and therapeutic response. In co-culture studies to investigate tumor-vascular crosstalk, endothelial cells often are not presented in three-dimensional structures mimicking vasculature and the exact identity of secreted factors is not explored. Here, we use tissue engineering strategies to generate three-dimensional endothelial networks from which to collect soluble angiocrine signals and assess the impact of these signals on glioblastoma behavior. Furthermore, we use secretomic analysis to identify specific factors influencing glioblastoma invasion. We identify a novel role for TIMP1 in supporting glioblastoma migration and demonstrate that soluble angiocrine signals support chemoresistance to temozolomide.

Overexpression of NG2 in human glioblastoma cells is associated with an elevated drug resistance and thereby worsens clinical outcome. However, the regulatory mechanisms of NG2 expression are largely unknown. In this study, we identified miR-29b-3p as a posttranscriptional factor of NG2 expression. The basal mRNA levels of miR-29b-3p and NG2 were detected in the NG2-positive glioblastoma cell lines A1207 and U87 by qRT-PCR. The cells were transfected with miR-29b-3p-mimic or scrambled-miR (control) and the expression of NG2 was analyzed by qRT-PCR, flow cytometry and Western blot. Reporter gene analyses of the NG2 promotor region and 3’UTR were performed to study the effect of miR-29b-3p on NG2 expression. Finally, we analyzed the mRNA levels of NG2 and miR-29b-3p in samples from glioblastoma patients. We found that the two NG2-positive glioblastoma cell lines A1207 and U87 are positive for miR-29b-3p. Transfection with miR-29b-3p-mimic reduced NG2 mRNA levels in A1207 (29%±9.9; Mean±SD) and U87 (6%±2.8), resulting in a significantly decreased NG2 protein expression in A1207 (67%±6.4) and U87 (75%±4) when compared to controls. The analysis of the 3’UTR revealed that miR-29b-3p is a posttranscriptional regulator of NG2 expression. Moreover, miR-29b-3p affects the pretranscriptional NG2 expression by diminishing SP-1-dependent NG2 promotor activity. These results were confirmed by the analysis of glioblastoma patient-derived samples, demonstrating that a high NG2 expression is associated with low levels of miR-29b-3p. In conclusion, we identified miR-29b-3p as a crucial regulator of NG2 expression in glioblastoma. Hence, targeting NG2 expression by miR-29b-3p may provide a novel therapeutic strategy to overcome drug resistance in NG2-positive glioblastoma cells.

Glioblastoma (GBM) is the most common type of adult brain tumour, representing 45.2% of all malignant brain and central nervous system tumours. It is also the most lethal, with a median post-therapy survival of 14 months, due to the high likelihood of tumour recurrence. Intratumour heterogeneity is thought to be a key contributor to GBM tumour recurrence, because treatments such as radiotherapy and chemotherapy typically only successfully target the core of the tumour. Due to its high visibility, this core is excised during surgery, but there is another more subtle region of the tumour, the invasive margin, which generally remains behind. This invasive margin is a mixture between normal brain and highly invasive tumour cells. If left over from surgery, these remaining tumour cells infiltrate the normal brain, and cause recurrence within 2cm of the original site. However, if surgeons are too vigorous in removing the invasive margin, they may cause unnecessary removal of healthy brain tissue, thereby causing other complications. This make developing specific methods to target the cancer cells in the invasive margin a priority area in GBM research. In order to specifically target the invasive margin, we need to have a way of differentiating it reliably from the other regions. In this thesis, I investigated the role that micro RNAs (miRNAs), a type of small non-coding RNA, play in this intratumour heterogeneity. miRNAs act as post-transcriptional regulators of gene expression and can simultaneously suppress multiple genes within the same pathway, which makes them potentially valuable routes for therapy development. I performed a miRNA microarray on tissue samples from three tumour regions (tumour core, rim, and invasive margin) and validated the results with qRT-PCR. I then performed computational predictions of gene targets of the differentially expressed miRNAs and validated them in cell lines and patient samples at both the mRNA (qRT-PCR) and protein (Western blot and immunohistochemistry) levels. I then performed a bi-phasic extraction of polar and non-polar metabolites and employed liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectroscopy techniques to profile the intracellular metabolome and lipidome of U87 cells overexpressing miR-619-5p and miR-4793-3p. Transwell invasion assays were performed to investigate the effect of each miRNA and its target on GBM cell invasion. In this work, I found that two miRNAs, miR-619-5p and miR-4793-3p, are significantly downregulated in the invasive margin. Moreover, these miRNAs control the expression of genes that are involved in regulating lipid metabolism pathways. A second key finding was that the knockdown or pharmacological inhibition of the miR-619-5p target, CPT2, also significantly reduced the invasion of the cells in the invasive margin. Because CPT2 lies in the lipid metabolism pathway, my findings indicate that lipid metabolism may present a possible vulnerability of the GBM invasive cell population, and the understanding the function of miRNAs in regulating lipid homeostasis in GBM may provide novel avenues for GBM therapy.

BACKGROUND: Among the new approaches to treat glioma, lies the idea to heat locally the tumor cells using nanoparticles exposed to an alternating magnetic field (AMF). Until now, chemically synthesized nanoparticles were tested, leading to partial antitumor activity due to their sub-optimal magnetic properties. We had the idea to use a new type of nanoparticles synthesized by magnetotactic bacteria called magnetosomes with a series of advantageous properties: i), a chain arrangement that leads to uniform nanoparticle and heat distribution in the tumor, ii), ferrimagnetic properties that enhance nanoparticle heating power and iii), a controllable release of endotoxins that can attract immune cells possibly involved in tumor destruction. MATERIAL AND METHODS: We administered a suspension of magnetosomes in intracranial U87-MG and subcutaneous GL-261 glioma tumors followed by several AMF applications and we monitored the evolution of the tumor size after treatment using either an IVIS spectrum for the intracranial luminescent tumors or a caliper for the subcutaneous ones. Mice were kept during 6 months after treatment to verify that GBM tumor does not regrow and a few mice were euthanized 6 months after treatment to examine if the brain had been damaged by the treatment. RESULTS: we show that we can induce the disappearance of GBM tumors using the magnetosomes, a behavior that was not observed using chemically synthesized nanoparticles administered at equivalent quantity. Mice treated with the magnetosomes and AMF applications are alive 6 months following treatment and histological analysis of their brain tissue does not show any sign of brain damage. Interestingly, it also appears that full glioma destruction is achieved when magnetosomes occupy only 10% of the whole tumor volume suggesting involvement of an indirect anti-tumor mechanism which is valuable in treatment of infiltrating tumor, such as glioma, that can hardly be fully covered by nanoparticles. CONCLUSION: In this study, we present a method that yields the full disappearance of GBM tumors in two different GBM mouse models using biological nanoparticles heated by an AMF. The treatment appears to be efficient in mice without resulting in significant observable side effects.

Glioblastoma (WHO grade IV, GBM) is a malignant, very aggressive, primary brain tumor which due to tumor cell heterogeneity, genetic alterations and therapy resistance remains incurable. GBM is heavily infiltrated with microglia and macrophages which play an important role in proliferation, diffusive growth and responses to treatment. Microglia and peripheral macrophages accumulate in malignant gliomas and constitute 30-50% of the tumor mass. Glioma cells overexpress and secrete proteins that reprogram microglia and peripheral macrophages into cells which potentiate tumor invasion and growth, at the same suppressing antitumor immunity. We have previously demonstrated that glioma-derived granulocyte macrophage colony-stimulating factor - GM-CSF (encoded by a CSF-2 gene) induces accumulation and protumorigenic activation of microglia/macrophages. We hypothesized that a humanized peptide that selectively binds to GM-CSF, would block its binding to respective receptors on myeloid cells, and inhibit activation of the receptors and downstream signaling pathways, which should result in inhibition of glioma invasiveness. First, we designed a peptide library containing 27 peptides, each 14-residues long. Next, we identified the peptide binding to GM-CSF using peptide microarrays, enzyme-linked immunosorbent assay (ELISA) and a technique based on surface plasmon resonance (SPR). Subsequently, using the Matrigel Matrix cell invasion assay we selected the peptide (G7) with most potent capacity for inhibition of human U87 MG and LN18 glioma cell invasiveness in the presence of glioma-microglia co-cultures. We further confirmed that this peptide blocks binding of GM-CSF to its receptor using SPR and its relatively good stability using mass spectrometry. Anti-tumor activity of G7 peptide in vivo was confirmed in orthotopic U87 xenograft mouse model. Intracranial delivery of this peptide inhibitor omits blood-brain barrier and minimizes nonspecific toxicity.

Introduction: Despite that Temozolomide (TMZ) is the standard of care recognized for its effectiveness in the treatment of glioblastoma (GBM), its prognosis remains poor. Therefore, combination chemotherapy using TMZ with other antitumor agents is now under investigation. We evaluated antitumor efficacy, tolerability and long-term response of the combination of TMZ with Aldoxorubicin (Aldoxo), a novel drug-conjugate of Doxorubicin, in a growing GBM xenograft mouse model. Materials and Methods: A human GBM cell line that expresses a luciferase reporter (U87-luc) was injected in the right lobe of the brain of Foxn1 mice. Following initial tumor growth for 7 days, mice were treated with Aldoxo (16 mg/kg, weekly intravenous administrations), TMZ (0.9 mg/kg, daily oral administrations) or their combination up to 5 weeks. Bioluminescence IVIS acquisitions have been performed at day 0, +3, +7 and then weekly until the end of experiment in order to quantify tumor growth and long-term response to therapy (until day +90). Results: In the first 28 days, all animals had developed tumors of varying size without significant difference between treated and untreated mice. At day +42, (end of treatment), there was a significant decrease in the tumors with TMZ and Aldoxo-TMZ therapy (P

Migration and invasion of malignant glioma play a major role in tumor progression and can be increased by low doses of gamma or X-ray irradiation, especially when the migrated tumor cells are located at a distance from the main tumor mass or postoperative cavity and are irradiated in fractions. We studied the influence of proton beam irradiation on migration and invasion of human U87 malignant glioma (U87MG) cells. Irradiation at 4 and 8 Gy increased cell migration by 9.8% (±4, P = 0.032) and 11.6% (±6.6, P = 0.031) and invasion by 45.1% (±16.5, P = 0.04) and 40.5% (±12.7, P = 0.041), respectively. After irradiation at 2 and 16 Gy, cell motility did not differ from that at 0 Gy. We determined that an increase in proton beam irradiation dose to over 16 Gy might provide tumor growth control, although additional specific treatment might be necessary to prevent the potentially increased motility of glioma cells during proton beam therapy.

BACKGROUND: Grade IV astrocytomas, formerly known as glioblastoma multiforme (GBM), are the most common primary brain tumors and have the highest mortality. The therapeutic standard for managing this malignancy remains a combination of surgery, chemotherapy, and radiotherapy; however, there is no cure, nor has there been any significant advancement in the clinical approach to GBM since this protocol was established in 2005. This study aims to better understand the molecular and metabolic characteristics of GBM to better define treatment groups and potentially identify new avenues for therapy. This study utilized continuous, commercially-available GBM cell lines U87, U118, A172, and H4, and examined the concentrations of Bcl-2 family proteins on mitochondria and metabolic activity for each of the cell lines. The measures were correlated to temozolamide (TMZ) sensitivity. RESULTS: Western blot analysis of pro-survival and pro-apoptotic Bcl-2 proteins revealed that U118 and U87 expressed high levels of Bcl-2, while A172 had increased Bcl-xL expression. Interestingly, Bcl-2 and Bcl-xL were not detected in H4 cells. Incidentally, pro-apoptotic BH3-only protein levels were increased in H4 and A172 cells. Metabolic analysis of the cell lines revealed that U118 cells were a glutaminolytic, while U87, A172, and H4 were classified as glycolytic. We found that U118 cells were the most resistant followed by U87, A172, and H4, respectively. CONCLUSIONS: We found that Bcl-2 protein profiling was a useful means to determine therapeutic response and resistance. This was enhanced by addingmetabolic stratification, wherein glycolytic cells were shown to be more sensitive TMZ than glutaminolytic cells. We will expand the number of cell lines in our study and increase the cellular profiling to include mitochondrial dynamics. Using this approach, we will produce a method to define GBM responses and outcomes based on the mitochondrial and metabolic context of individual tumors.

PURPOSE: Despite aggressive treatment with radiation therapy plus concurrent and adjuvant temozolomide (TMZ), the prognosis for glioblastoma remain poor. We investigated the potential of targeting signal transducer and activator of transcription-3 (STAT3) to improve the therapeutic outcome of combined radiotherapy and TMZ in glioblastoma. METHODS AND MATERIALS: We evaluated the preclinical potential of a STAT3 inhibitor, Cpd188 combined with temozolomide and radiation by in vitro clonogenic assays using two established glioblastoma cell lines (U251, U87) and two patients-derived glioblastoma cell lines (GBL12, GBL28) and in vivo studies using nude mice bearing intracranial U251 xenografts. RESULTS: Cpd188 potentiated the radiosensitizing effect of TMZ in U251 glioblastoma cell line which has high levels of p-STAT3 expression and in vitro. Increased radiosensitizing effects of TMZ were associated with the induction of apoptosis and the reversion of epithelial-mesenchymal transition (EMT). Cpd188 delayed in vivo tumor growth both alone and in combination with fractionated radiation and TMZ with a trend toward improved survival rates. Immunohistochemical staining of tumor sections showed that Cpd188 decreased the expression of CD31 (a marker of endothelial proliferation), vascular endothelial growth factor, and hypoxia-inducible factor-1α, suggesting that Cpd188 also has anti-angiogenic effects. We also confirmed the radiosensitizing effect of Cpd188 of GBL28 cell line which was originated from a patient who had a glioblastoma and also was confirmed high level of STAT3 expression and unmethylated MGMT. CONCLUSION: These data indicate that Cpd188 has the potential to improve the therapeutic outcome of combined radiotherapy and TMZ in human glioblastoma, especially in patients whose tumor has a high level of STAT3 expression regardless of MGMT methylation status. Work supported by grant (#2012-0004867 & #2013R1A1A2074531) from National Research Foundation, Korean Ministry of Future Creative Science to In Ah Kim.

INTRODUCTION: Locally administered agents against malignant gliomas demand sophisticated metabolic imaging to monitor local and systemic treatment effects in animal models. The purpose of this feasibility study was to create an animal model suitable to represent the effect of locally delivered, soluble temozolomide in orthotopically implanted gliomas using micro-[18F] FLT-PET for high quality metabolic information with a distinct resolution for observation of treatment effects. METHODS: U87 glioma cells were stereotactically implanted in 48 female T-cell deficient athymic nude rats (RNU). Tumor existence and proliferation was proven by [18F] FLT-PET and microMRI. Rats were allocated to four treatment groups. Group one and two received a one-time intratumoral stereotactic injection of soluble temozolomide (TMZ) (group 1: 0.03mg; group 2: 0.015mg). Group three was administered systemic TMZ for 2 days (5mg). Group four served as untreated control group. Treatment was performed on day 15 post implantation. FLT-PET and MRI were performed 7 and 14 days after treatment. Animals showing signs of neurological impairment or reduced general health were immediately examined by MRI and sacrificed according to the animal ethical guidelines. RESULTS: The tumor take rate was 100 percent. Characteristically, U87 tumor cell formation was rather solid. Tumor size (MRI) and proliferation rate (PET) were congruent in all groups prior to treatment. Post treatment, FLT-PET was more specific for tumor growth compared to MRI. Tumor proliferation rates were significantly lower in the local treatment groups. MRI confirmed intratumoral TMZ fluid distribution which decreased within 7 days. CONCLUSION: Our orthotopic rat glioma model proved to be feasible and efficient for the performance of local therapy studies. Sophisticated Micro-PET offers distinct monitoring of tumor proliferation and supplements MR tumor imaging very well. The FLT-PET tracer is most suitable for the monitoring of local treatment effects of TMZ.

Overexpression of the Axl receptor tyrosine kinase is associated with a negative outcome for glioblastoma patients. The aim of the study was to evaluate the effect of the specific Axl-inhibitor BGB 324 on glioma cell proliferation and migration in vitro as well as on orthotopic tumor growth in vivo. Axl expression was analyzed in adherent glioblastoma cell lines (N = 32) as well as in spherical stem-like glioblastoma cell lines (N = 9) by microarray and qPCR analyses, Western blot and flow cytometry. Axl was strongly expressed in glioblastoma cells with a proliferative, differentiated phenotype, such as G55 and U87, while its expression was weak in stem-like GBM cells. Full length Axl protein was present at the cell surface, while its soluble form sAxl was detectable in conditioned media and was released from the cells in a metalloproteinase-dependent fashion. The Axl ligand Gas6 was also present in conditioned medium, indicating an autocrine stimulation loop. In addition, unprocessed full length Axl was found in conditioned medium, suggesting an association of Axl with glioblastoma-derived exosomes. Erk1/2 and Akt phosphorylation induced by recombinant Gas6 was abrogated by interfering with Axl tyrosine kinase function using BGB324, resulting in a dose-dependent decrease in proliferation and cell migration. Most importantly, oral administration of 25 mg/kg BGB324 to tumor-bearing mice twice daily significantly increased the median survival of treated animals compared to vehicle controls for both G55 (23 vs 19 days, p < 0.0001) and U87 (26.5 vs 24days, p < 0.001) xenograft models. In summary, targeting Axl by oral administration of BGB324 appears a promising strategy for Axl-overexpressing glioblastoma cells that have undergone proliferative differentiation. The impact of Axl inhibition glioblastoma stem-like cells remains to be elucidated.

Changes in tissue composition caused by brain tumor growth involve a series of complex biochemical alterations which can be imaged on unstained native tissue using multiphoton microscopy: We used coherent anti-Stokes Raman scattering (CARS) imaging that resonantly excites the symmetric stretching vibration of CH2 groups at 2850 cm−1 and visualizes lipid content in combination with imaging of endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) to discern different types of tumors from normal tissue in unstained, native brain samples. Experimental brain tumors were induced in nude mice NMRI nu/nu (n = 25) by stereotactic implantation of glioblastoma (U87), melanoma (A375) and breast cancer (MCF-7) cell lines. Label-free multiphoton microscopy of brain cryosections provided exhaustive information of the tumor morphochemistry. The tumor border was defined with cellular resolution by a strong reduction of CARS signal intensity to 61% (glioblastoma), 71% (melanoma) and 68% (breast cancer). This reduction of lipid content within the tumor was confirmed by Raman spectroscopy. Micrometastases infiltrating normal tissue (size 50 - 200 µm) were identified in glioblastoma and melanoma. Additionally, multiphoton microscopy proved a reduction of CARS signal intensity in all human glioblastoma samples analyzed (to 72%, n = 6). Additionally, relevant SHG and TPEF signals were detected in human primary and secondary brain tumor samples and enabled to image variations in tumor associated vasculature, fibrosis, necrosis and nuclear size and density. All primary or secondary brain tumors investigated were characterized by a lower intensity of the CARS signal, therefore offering a simple tool for objective tumor detection and delineation. The combination of techniques allows retrieving a quantity of information on native unstained tissue which is comparable to H&E staining. Therefore, label-free multiphoton microscopy has the potential to become a technology routinely used intraoperatively for inspection and by neuropathology for frozen section equivalent assessment of brain tumors.

Despite post-operative radio-chemotherapy, the prognosis of the patients with a glioblastoma (GBM) remains poor because of a local recurrence, mainly due to resistance of the GBM cells to radiotherapy. We have previously demonstrated that FGF-2 controls cell radioresistance and that expression of FGFR1 in the tumor of patients with GBM was an independent prognostic factor of overall survival and of time to progression after radio-chemotherapy. These results led to the hypothesis that FGFR1 could be involved in the radioresistance of GBM and that inhibiting FGFR1 in tumor cells may increase the efficiency of radiotherapy. To investigate this, we used in vitro and in vivo silencing strategies in GBM radioresistant U87 cells mainly expressing FGFR1. We first showed that the surviving fraction after a 5Gy irradiation (SF5) of U87 cells was significantly reduced when FGFR-1 has been silenced compared to U87 control cells (SF5=0.58%±0.06 to 0.33%±0.03; p < 0.001). Moreover, inhibition of FGFR1 led to a significant increase of the mitotic cell death after irradiation. We then showed that inhibiting FGFR1 led to an inhibition of PLCγ phosphorylation which radiosensitizes U87 cells in vitro. We then performed an in vivo approach by generating subcutaneous limb xenografts with two different FGFR-1 silenced U87 clones, locally or sham irradiated with two 3 Gy fractions when tumor size reached 300 mm3. We showed a significant growth delay in the two silenced FGFR1 xenografts compared to control cell xenografts. In addition, a dramatic decrease in expression of hypoxia inducible factor, HIF-1α has been found by immunohistochemistry in silenced FGFR-1 xenografts compared to control cell xenografts and confirmed in vitro by western blot. Our data establishes the proof-of-concept of FGFR1 inhibition as a new treatment for radiosensitizing these aggressive tumors. Considering that we showed that FGFR-1 expression in GBM patients is an independent prognostic factor of survival and time to progression after radio-chemotherapy, our present study highly suggests the interest to associating a FGFR1 inhibitor with radiotherapy in clinical trials for patients with glioblastoma.

BACKGROUND: Neural stem cells (NSCs) have an inherent brain tumor tropism that can be exploited for targeted delivery of therapeutic genes to invasive gliomas. We have recently introduced the concept of intranasal application of NSCs as a noninvasive and direct alternative delivery method for glioma-targeting NSCs. Here, we demonstrate that the repeated non-invasive intranasal administration of tumor-targeting NSC is able to deliver a novel suicide gene (TK.007) to intracerebrally growing human glioblastoma xenografts. METHODS: Murine NSC were genetically modified to stably express a novel, codon-optimized HSVtk(A168H) mutant (TK.007). The biological activity of the NSC-mediated TK.007/ganciclovir (GCV) system was assessed in cell survival and bystander assays using various human glioma cell lines. Therapeutic effects of intratumoral and intranasal NSC-TK007 applications alone and the sequential combinations of both was tested using an intracranial U87 human glioblastoma model in nude mice. All animals received 50 mg/kg GCV i.p. for five consecutive days. In all experiments two control groups received either NaCl instead of GCV or NSC containing the control vector instead of NSC-TK.007. Therapeutic efficiency was determined by assessment of tumor size by 7T MR-imaging and by establishment of Kaplan-Meier survival curves. RESULTS: Cell survival and bystander assays confirmed the GCV catalytic activity of NSC-expressed TK007 in a GCV dose dependent manner and a significant bystander effect at 12.5% of cells. Tumor targeted migration was demonstrated by intravital imaging of luciferase-expressing NSC. In glioma-bearing mice a single intratumoral application of NSC-TK007 followed by systemic prodrug application of GCV lead to a significant tumor growth inhibition of 72% versus the control groups at day 22 and translated in a prolonged survival time. Mice recieving a cyclic intranasal application of NSC-TK.007 alone displayed a signifcant longer survival than the controls. This effect was significantly enhanced when cyclic intranasal NSC-TK.007 therapy was preceded by a single intratumoral application of NSC-TK007 and led to a significant tumor growth inhibition of >95% and improved survival time. CONCLUSIONS: Our data demonstrates that the NSC-mediated TK.007/GCV therapy, which is based on the clinically most widely used suicide system to date, is safe, nontoxic, and effective in glioblastoma-bearing mice. Most importantly, our findings establish that the intranasal application of therapeutically-modified NSC is a safe and non-invasive application method which allows a chronic treatment during the disease course. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Glioblastoma multiforme, a World Health Organization grade IV glioma, has a poor prognosis in humans despite current treatment options. Here, we present magnetic resonance imaging (MRI) data regarding the regression of aggressive rat F98 gliomas and human U87 glioma xenografts after treatment with the nitrone compound OKN-007, a disulfonyl derivative of α-phenyl-tert-butyl nitrone.MRI was used to assess tumor volumes in F98 and U87 gliomas, and bioluminescence imaging was used to measure tumor volumes in F98 gliomas encoded with the luciferase gene (F98(luc)). Immunohistochemistry was used to assess angiogenesis (vascular endothelial growth factor [VEGF] and microvessel density [MVD]), cell differentiation (carbonic anhydrase IX [CA-IX]), hypoxia (hypoxia-inducible factor-1α [HIF-1α]), cell proliferation (glucose transporter 1 [Glut-1] and MIB-1), proliferation index, and apoptosis (cleaved caspase 3) markers in F98 gliomas. VEGF, CA-IX, Glut-1, HIF-1α, and cleaved caspase 3 were assessed in U87 gliomas.Animal survival was found to be significantly increased (P.001 for F98, P.01 for U87) in the group that received OKN-007 treatment compared with the untreated groups. After MRI detection of F98 gliomas, OKN-007, administered orally, was found to decrease tumor growth (P.05). U87 glioma volumes were found to significantly decrease (P.05) after OKN-007 treatment, compared with untreated animals. OKN-007 administration resulted in significant decreases in tumor hypoxia (HIF-1α [P.05] in both F98 and U87), angiogenesis (MVD [P.05], but not VEGF, in F98 or U87), and cell proliferation (Glut-1 [P.05 in F98, P.01 in U87] and MIB-1 [P.01] in F98) and caused a significant increase in apoptosis (cleaved caspase 3 [P.001 in F98, P.05 in U87]), compared with untreated animals.OKN-007 may be considered as a promising therapeutic addition or alternative for the treatment of aggressive human gliomas.

Glioblastoma multiforme (GBM) is a devastating disease that is notoriously resistant to current therapies, leading to dismal patient outcomes and a median survival of just 14.6 months.1 As such, translation of our rapidly expanding knowledge of the molecular biology of GBM together with the growing repertoire of molecular-based chemotherapeutics now available has the potential to dramatically improve patient outcomes. Signal transducer and activator of transcription (STAT)3 signaling has recently come to the forefront of the glioma literature as a common pathway downstream of paracrine leukemia inhibitory factor, interleukin-6, or erythropoietin signaling in GBM.2–4 Recent transcriptional profiling and signaling network analyses have also identified STAT3 and CCAAT (cytidine-cytidine-adenosine-adenosine-thymidine)-enhancer-binding proteins β as 2 key master transcription factors underlying the mesenchymal subtype of GBM.5 Consequently, STAT3 has become a therapeutic target of significant interest in glioma, and a number of small-molecule inhibitors of the STAT3' upstream regulator Janus kinase (JAK)2 are currently in early preclinical investigation in GBM. Inhibition of JAK2 with AG490 or its more potent second-generation analog WP1066 has demonstrated efficacy in reducing survival and proliferation of U87 and U251 glioma cells in vitro and decreasing tumorigenicity of U87 in subcutaneous xenografts.6,7 JAK2/STAT3 blockade has also demonstrated promise as an adjuvant for other therapeutics. Concurrent inhibition of JAK2/STAT3 with cucurbitacin-I (JSI-124) and blockade of epidermal growth factor receptor (EGFR) with Iressa has been recently reported to potently induce apoptosis in GBM cell lines and sensitize them to temozolomide.8 Emerging evidence suggests that high levels of STAT3 activation are also present within brain tumor stem cells (BTSCs), and STAT3 inhibition has been reported to decrease the self-renewal survival and resistance to temozolomide of glioma stemlike cells in vitro.2–4,9,10 Thus, there is strong evidence in the glioma literature for the crucial role of STAT3 in human GBM, and JAK2-targeted therapeutics have yielded promising results against GBM cell lines in vitro. However, the true translational potential of JAK2/STAT3 therapeutics for GBM remained an unanswered question, as these drugs had yet to be thoroughly investigated in a model that accounted for the molecular heterogeneity of human GBM and assessed on-target efficacy in intracranial humanlike GBM tumors in vivo. BTSCs have been postulated to be at the root of the disease initiation, recurrence, and therapeutic resistance that is characteristic of GBM. BTSCs isolated from human GBM tumors capture the molecular heterogeneity and mutation spectrum that are characteristic of the disease.11,12 BTSCs also initiate orthotopic xenograft tumors in nonobese diabetic/severe combined immunodeficient (NOD SCID) mice that emulate the phenotypic and molecular characteristics of their parent human tumors, hence making them the most relevant model currently available for preclinical evaluation of novel GBM therapeutics. Our group has successfully established a large collection of BTSC lines from glioma patients that display the molecular heterogeneity of the disease,13 including rare lines endogenously expressing isocitrate dehydrogenase (IDH1)R132H, differential MGMT promoter methylation status, and mutations in common targets such as PTEN, TP53, and EGFR variant III (unpublished), amongst others.14–16 Here, we use a large panel of patient-derived GBM BTSCs to investigate whether JAK2/STAT3 is (i) a clinically relevant signaling pathway whose activity is crucial in molecularly diverse BTSCs and (ii) amenable to therapeutic targeting in vivo in orthotopic humanlike GBM xenografts. We demonstrate that JAK2/STAT3 signaling is a key driver for proliferation in GBM BTSCs and that on-target inhibition of this pathway results in decreased BTSC survival in vitro and slows disease progression in vivo.