Tumor Vessel Normalization, Immunostimulatory Reprogramming, and Improved Survival in Glioblastoma with Combined Inhibition of PD-1, Angiopoietin-2, and VEGF.

Glioblastoma (GBM) is a non-T-cell-inflamed cancer characterized by an immunosuppressive microenvironment that impedes dendritic cell maturation and T-cell cytotoxicity. Proangiogenic cytokines such as VEGF and angiopoietin-2 (Ang-2) have high expression in glioblastoma in a cell-specific manner and not only drive tumor angiogenesis and vascular permeability but also negatively regulate T-lymphocyte and innate immune cell responses. Consequently, the alleviation of immunosuppression might be a prerequisite for successful immune checkpoint therapy in GBM. We here combined antiangiogenic and immune checkpoint therapy and demonstrated improved therapeutic efficacy in syngeneic, orthotopic GBM models. We observed that blockade of VEGF, Ang-2, and programmed cell death protein-1 (PD-1) significantly extended survival compared with vascular targeting alone. In the GBM microenvironment, triple therapy increased the numbers of CTLs, which inversely correlated with myeloid-derived suppressor cells and regulatory T cells. Transcriptome analysis of GBM microvessels indicated a global vascular normalization that was highest after triple therapy. Our results propose a rationale to overcome tumor immunosuppression and the current limitations of VEGF monotherapy by integrating the synergistic effects of VEGF/Ang-2 and PD-1 blockade to reinforce antitumor immunity through a normalized vasculature.

Intratumoral Concentrations and Effects of Orally Administered Micellar Curcuminoids in Glioblastoma Patients.

BACKGROUND: The oral bioavailability of curcuminoids is low, but can be enhanced by incorporation into micelles. The major curcuminoid curcumin has antitumor effects on glioblastoma cells in vitro and in vivo. We therefore aimed to determine intratumoral concentrations and the clinical tolerance of highly bioavailable micellar curcuminoids in glioblastoma patients. METHODS: Thirteen glioblastoma patients ingested 70 mg micellar curcuminoids [57.4 mg curcumin, 11.2 mg demethoxycurcumin (DMC), and 1.4 mg bis-demethoxycurcumin (BDMC)] three times per day for 4 days (total amount of 689 mg curcumin, 134 mg DMC, and 17 mg BDMC) prior to planned resection of their respective brain tumors. Tumor and blood samples were taken during the surgery and analyzed for total curcuminoid concentrations. (31)P magnetic resonance spectroscopic imaging was performed before and after curcuminoid consumption. RESULTS: Ten patients completed the study. The mean intratumoral concentration of curcumin was 56 pg/mg of tissue (range 9-151), and the mean serum concentration was 253 ng/ml (range 129-364). Inorganic phosphate was significantly increased within the tumor (P = 0.034). The mean ratio of phosphocreatine to inorganic phosphate decreased, and the mean intratumoral pH increased (P = 0.08) after curcuminoid intervention. CONCLUSION: Oral treatment with micellar curcuminoids led to quantifiable concentrations of total curcuminoids in glioblastomas and may alter intratumoral energy metabolism.

MIF Receptor CD74 is Restricted to Microglia/Macrophages, Associated with a M1-Polarized Immune Milieu and Prolonged Patient Survival in Gliomas.

The macrophage migration inhibitory factor (MIF) receptor CD74 is overexpressed in various neoplasms, mainly in hematologic tumors, and currently investigated in clinical studies. CD74 is quickly internalized and recycles after antibody binding, therefore it constitutes an attractive target for antibody-based treatment strategies. CD74 has been further described as one of the most up-regulated molecules in human glioblastomas. To assess the potential relevance for anti-CD74 treatment, we determined the cellular source and clinicopathologic relevance of CD74 expression in human gliomas by immunohistochemistry, immunofluorescence, immunoblotting, cell sorting analysis and quantitative polymerase chain reaction (qPCR). Furthermore, we fractionated glioblastoma cells and glioma-associated microglia/macrophages (GAMs) from primary tumors and compared CD74 expression in cellular fractions with whole tumor lysates. Our results show that CD74 is restricted to GAMs in vivo, while being absent in tumor cells, the latter strongly expressing its ligand MIF. Most interestingly, a higher amount of CD74-positive GAMs was associated with beneficial patient survival constituting an independent prognostic parameter and with an anti-tumoral M1 polarization. In summary, CD74 expression in human gliomas is restricted to GAMs and positively associated with patient survival. In conclusion, CD74 represents a positive prognostic marker most probably because of its association with an M1-polarized immune milieu in high-grade gliomas.

Lovastatin and perillyl alcohol inhibit glioma cell invasion, migration, and proliferation--impact of Ras-/Rho-prenylation.

Alterations in small GTPase mediated signal transduction pathways have emerged as a central step in the molecular pathogenesis of glioblastoma (GBM), the most common malignant brain tumor in adults. Farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) are derived from mevalonate, whose production is catalyzed by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Prenylation by FPP and GGPP is required for membrane insertion and oncogenic function of Ras- and Rho-proteins, within the stimulation of the Ras-Raf-MEK-ERK pathway. A straightforward prediction from HMG-CoA reductase inhibitor studies is that statins decrease FPP and GGPP levels and diminish ERK signaling ensuring less proliferation and migration of cancer cells. Perillyl alcohol (POH), a naturally occurring monoterpene inhibits prenyltransferases and is able to inhibit cancer cell growth, but the underlying mechanism is still unclear. We here report that lovastatin (LOV) and POH impair the regulation of the mevalonate- and the Ras-Raf-MEK-ERK pathway in U87 and U343 glioblastoma cells. Both compounds affected the post-translational modification of H-Ras and Rac1. While LOV diminished the substrates of the transferase reaction that catalyze prenylation, POH inhibited the enzymes itself. Our data highlight the impact of isoprenoids for post-translational modification of small GTPases promoting proliferation, migration and invasion capabilities in glioma cells.

STAT3 silencing inhibits glioma single cell infiltration and tumor growth.

BACKGROUND: Diffuse infiltration remains the fulcrum of glioblastoma's incurability, leading inevitably to recurrence. Therefore, uncovering the pathological mechanism is imperative. Because signal transducer and activator of transcription 3 (STAT3) correlates with glioma malignancy and predicts poor clinical outcome, we determined its role in glioma single cell infiltration and tumor growth. METHODS: STAT3 was silenced in Tu-2449 glioma cells via lentiviral gene transfer. Target gene expression was measured by real-time reverse transcription PCR, Western blotting, and immunohistochemistry. Microvilli were visualized by staining with wheat germ agglutinin. Migration and invasion were measured by Scratch and Matrigel chamber assays. Diffuse infiltration was studied in 350-µm-thick organotypic tissue cultures over 14 days using cells tagged with enhanced green fluorescent protein and live confocal laser scanning microscopy. Survival of tumor-bearing syngeneic, immunocompetent B6C3F1 mice was analyzed by Kaplan-Meier plots. RESULTS: STAT3 silencing reduced cell migration and invasion in vitro and stopped single cell infiltration ex vivo, while STAT3-expressing cells disseminated through the neuropil at ∼100 µm/day. STAT3 silencing reduced transcription of several tumor progression genes. Mice with intracranial STAT3 knockdown tumors had a significant (P< .0007) survival advantage over controls, yielding 27% long-term survival. STAT3 knockdown reduced podoplanin expression 50-fold and inhibited concurrent microvilli formation. STAT3 knockdown tumors exhibited a weaker podoplanin immunoreactivity compared with controls. Podoplanin staining was diffuse, preferentially at tumor margins, and absent in normal brain. CONCLUSIONS: Our results show compelling evidence that STAT3 is a key driver of diffuse infiltration and glioma growth and might therefore represent a promising target for an anti-invasive therapy.

Comparative evaluation of preclinical in vivo models for the assessment of replicating retroviral vectors for the treatment of glioblastoma.

Despite impressive improvements in neurosurgical techniques, radiation and chemotherapy during the past few years, little progress has been made in the treatment of malignant gliomas. Recently, the efficacy of suicide gene therapy based on replication-competent retroviral (RCR) vectors as delivery vehicles for the therapeutic gene has been described in the treatment of experimental cancer, including gliomas. In this study, we have thus critically evaluated a panel of human and rodent glioma/glioblastoma cell lines (U-87MG, U-118MG, LN-18, LN-229, 8-MG-BA, 42-MG-BA, A-172, T-98G, UVW, C6, 9L, G-26, GL-261, Tu-2449, Tu-9648) with respect to RCR virus vector spread, sensitivity towards the cytosine deaminase (CD)/5-flurocytosine (5-FC)/5-flurouracil (5-FU) suicide system, and orthotopic growth characteristics in mice to identify suitable preclinical animal models for the development of a glioblastoma gene therapy. Rapid virus spread was observed in eight out of nine human cell lines tested in vitro. As expected, only CD-expressing cells became sensitive to 5-FC, due to their ability to convert the prodrug in its toxic form, 5-FU. All LD(50) values were within the range of concentrations obtained in human body fluids after conventional antifungal 5-FC administration. In addition, a significant bystander effect was observed in all human glioma cell lines tested. Injection of the RCR vector into pre-established orthotopic mouse tumor xenografts revealed substantial infection and virus spread of tumor tissue from most cell types.

Inhibition of the JAK-2/STAT3 signaling pathway impedes the migratory and invasive potential of human glioblastoma cells.

The objective of current treatment strategies for glioblastoma (GBM) is cytoreduction. Unfortunately, the deleterious migratory and invasive behavior of glial tumors remains largely unattended. The transcription factor signal transducer and activator of transcription (STAT) 3 is known to be involved in the development and progression of many different tumor types, including malignant gliomas. Beside other biological effects, STAT3 controls cell proliferation and tissue remodeling, processes common to both wound healing and tumor dissemination. Here, we report on impeded migratory and invasive potential of five different glioblastoma cell lines after treatment with AG490, a pharmacological inhibitor of the upstream STAT3 activator Janus kinase (JAK) 2. STAT3 was constitutively activated in all the cell lines tested, and treatment with AG490 eliminated the biologically active, tyrosine705-phosphorylated form of STAT3 in a dose-dependent fashion, as determined by Western blot analysis. Inhibition of activated STAT3 was paralleled by a decrease in transcriptional expression of the STAT3 target genes MMP-2 and MMP-9, and led to reduced proteolytic activity, as determined by zymography. Accordingly, the migratory behavior of all five GBM cell lines was impeded in monolayer wound-healing assays; invasive capacity in matrigel-coated trans-well assays was also hampered by treatment with AG490. The proliferative activity of the cell lines was also significantly reduced after treatment with AG490. The effects elicited by STAT3 inhibition were observed in both PTEN-expressing and PTEN-deficient cells. Because pharmacological inhibition of the JAK-2/STAT3 signaling pathway affects not only tumor cell proliferation but also the characteristic features of malignant gliomas, i.e. migration and invasion pertinent to invariable tumor recurrence and high morbidity, our findings support the idea that STAT3 is a suitable target in the treatment of brain tumors.

Dietary curcumin attenuates glioma growth in a syngeneic mouse model by inhibition of the JAK1,2/STAT3 signaling pathway.

PURPOSE: Glioblastomas are the most common and most deadly primary brain tumors. Here, we evaluated the chemotherapeutic effect of the natural polyphenol curcumin on glioma cells in vitro and in vivo using an immunocompetent orthotopic mouse model. EXPERIMENTAL DESIGN: Curcumin's effects on proliferation, cell cycle, migration, invasion, JAK/STAT3 signaling, STAT3 target gene expression, and STAT3C rescue experiments were determined in murine glioma cell lines in vitro. Therapeutic effects of curcumin in vivo were evaluated in tumor-bearing mice fed a Western-type diet fortified with curcumin (0.05%, w/w) and in control animals. Tumor growth patterns and survival were evaluated by immunohistochemistry, morphometric analyses, and Kaplan-Meier plots. RESULTS: In vitro, curcumin inhibited JAK1,2/STAT3 tyrosine-phosphorylation in a dose-dependent fashion in murine glioma cell lines. Real-time RT-PCR revealed that curcumin downregulated transcription of the STAT3 target genes c-Myc, MMP-9, Snail, and Twist, and of the proliferation marker Ki67. Curcumin dose-dependently suppressed cell proliferation by inducing a G2/M phase arrest. In wound healing and Matrigel invasion assays, curcumin treatment resulted in a dose-dependent attenuation of the glioma cells' migratory and invasive behavior, which could be rescued by constitutively active STAT3C. In vivo, curcumin intake reduced the growth and midline crossing of intracranially implanted tumors and proliferation of tumor cells ensuing in significant long-term survival compared with control diet. CONCLUSION: This preclinical study shows that curcumin is capable of suppressing malignant glioma growth in vitro and in vivo. Our data suggest that the pharmacologically safe agent curcumin holds promise for clinical application in glioma therapy.

Cardiac oxidative stress and inflammation are similar in SAMP8 and SAMR1 mice and unaltered by curcumin and Ginkgo biloba extract intake.

Chronic inflammation and oxidative stress increase with advancing age and appear to be involved in the pathogenesis of coronary heart disease, the leading cause of death worldwide. There is a need for animal models that reflect the increases in pro-inflammatory cytokines and oxidative damage observed during aging in humans. We therefore aimed to investigate the suitability of the fast-aging senescence-accelerated mouse-prone 8 (SAMP8) strain and its normally aging control senescence-accelerated mouse-resistant 1 (SAMR1) to study the age-dependent changes in cytokines, oxidative damage and antioxidants in the heart. To this end, 2-months-old male SAMR1 and SAMP8 mice were fed a Western type diet (control groups) for 5 months. Two groups of SAMP8 mice were simultaneously fed identical diets fortified with 0.5 g curcumin or 1.0 g Ginkgo biloba extract EGb 761(®) per kg diet. Heart tissue homogenates were analysed for protein carbonyls, glutathione, glutathione disulfide, methionine, cysteine and uric acid as well as the cytokines tumor-necrosis factor-α, interleukin-1ß, interleukin-6, and monocyte chemoattractant protein 1. Neither the strain (SAMR1 or SAMP8) nor antioxidant intake (curcumin or EGb 761(®)) affected the concentrations of the measured parameters. In conclusion, our data do not support the suitability of the SAMP8 and SAMR1 strains as a model to study age-related changes in pro-inflammatory cytokines and oxidative stress parameters in the heart.

The nontoxic natural compound Curcumin exerts anti-proliferative, anti-migratory, and anti-invasive properties against malignant gliomas.

BACKGROUND: New drugs are constantly sought after to improve the survival of patients with malignant gliomas. The ideal substance would selectively target tumor cells without eliciting toxic side effects. Here, we report on the anti-proliferative, anti-migratory, and anti-invasive properties of the natural, nontoxic compound Curcumin observed in five human glioblastoma (GBM) cell lines in vitro. METHODS: We used monolayer wound healing assays, modified Boyden chamber trans-well assays, and cell growth assays to quantify cell migration, invasion, and proliferation in the absence or presence of Curcumin at various concentrations. Levels of the transcription factor phospho-STAT3, a potential target of Curcumin, were determined by sandwich-ELISA. Subsequent effects on transcription of genes regulating the cell cycle were analyzed by quantitative real-time PCR. Effects on apoptosis were determined by caspase assays. RESULTS: Curcumin potently inhibited GBM cell proliferation as well as migration and invasion in all cell lines contingent on dose. Simultaneously, levels of the biologically active phospho-STAT3 were decreased and correlated with reduced transcription of the cell cycle regulating gene c-Myc and proliferation marking Ki-67, pointing to a potential mechanism by which Curcumin slows tumor growth. CONCLUSIONS: Curcumin is part of the diet of millions of people every day and is without known toxic side effects. Our data show that Curcumin bears anti-proliferative, anti-migratory, and anti-invasive properties against GBM cells in vitro. These results warrant further in vivo analyses and indicate a potential role of Curcumin in the treatment of malignant gliomas.

Orthotopic transplantation of v-src-expressing glioma cell lines into immunocompetent mice: establishment of a new transplantable in vivo model for malignant glioma.

OBJECT: The aim of this study was to develop and characterize a new orthotopic, syngeneic, transplantable mouse brain tumor model by using the cell lines Tu-9648 and Tu-2449, which were previously isolated from tumors that arose spontaneously in glial fibrillary acidic protein (GFAP)-v-src transgenic mice. METHODS: Striatal implantation of a 1-microl suspension of 5000 to 10,000 cells from either clone into syngeneic B6C3F1 mice resulted in tumors that were histologically identified as malignant gliomas. Prior subcutaneous inoculations with irradiated autologous cells inhibited the otherwise robust development of a microscopically infiltrating malignant glioma. Untreated mice with implanted tumor cells were killed 12 days later, when the resultant gliomas were several millimeters in diameter. Immunohistochemically, the gliomas displayed both the astroglial marker GFAP and the oncogenic form of signal transducer and activator of transcription-3 (Stat3). This form is called tyrosine-705 phosphorylated Stat3, and is found in many malignant entities, including human gliomas. Phosphorylated Stat3 was particularly prominent, not only in the nucleus but also in the plasma membrane of peripherally infiltrating glioma cells, reflecting persistent overactivation of the Janus kinase/Stat3 signal transduction pathway. The Tu-2449 cells exhibited three non-random structural chromosomal aberrations, including a deletion of the long arm of chromosome 2 and an apparently balanced translocation between chromosomes 1 and 3. The GFAP-v-src transgene was mapped to the pericentromeric region of chromosome 18. CONCLUSIONS: The high rate of engraftment, the similarity to the high-grade malignant glioma of origin, and the rapid, locally invasive growth of these tumors should make this murine model useful in testing novel therapies for human malignant gliomas.

Interleukin-6 gene ablation in a transgenic mouse model of malignant skin melanoma.

Interleukin (IL)-6 is a pleiotropic cytokine that has been shown to inhibit the growth of early stage and to promote the proliferation of advanced stage melanoma cells in vitro. In patients with metastasizing melanomas, highly increased IL-6 blood levels correlate with a poor response to chemotherapy and a worse overall prognosis, suggesting that IL-6 promotes melanoma progression in vivo. Here, we analyzed the role of IL-6 in melanoma development and progression in a transgenic mouse model. We bred IL-6-deficient mice with MT-ret transgenic animals predisposed for melanomas. While MT-ret transgenic animals develop severe melanosis of the skin and subcutis and subsequent melanomas at an incidence of 80% during their first year of life, MT-ret mice devoid of IL-6 developed preneoplastic melanosis and consecutive melanomas significantly less frequently (47%; P < 0.05). Moreover, the tumors were significantly smaller in the groups of MT-ret mice lacking one (P < 0.05) or both (P < 0.01) copies of the IL-6 gene. Immunoblot analysis revealed that ret transgene expression was not reduced in the skin of mice lacking IL-6, indicating that the observed decrease of melanoma incidence and of tumor sizes was not because of a down-regulation of transgene expression. Taken together, these results indicate that IL-6 enhances both the development of melanoma precursor lesions and the subsequent growth of the resulting tumors in the MT-ret model of melanoma development.

Interleukin-6 induces transcriptional activation of vascular endothelial growth factor (VEGF) in astrocytes in vivo and regulates VEGF promoter activity in glioblastoma cells via direct interaction between STAT3 and Sp1.

Interleukin-6 (IL-6) expression is strongly correlated with the degree of human glioma malignancy and necessary for tumor formation in a mouse model of spontaneous astrocytomas. Yet, exactly how IL-6 contributes to malignant progression of these brain tumors is still unclear. We have scrutinized the mechanism of transcriptional activation of vascular endothelial growth factor (VEGF) expression by IL-6 in the mouse brain and in glioblastoma cells. We demonstrate here that IL-6 drives transcriptional upregulation of VEGF in astrocytes in vivo using glial fibrillary acidic protein (GFAP)-IL-6/VEGF-green fluorescent protein (GFP) double transgenic mice. We further show that IL-6-induced VEGF transcription and VEGF secretion by human glioblastoma cells is dependent on signal transducer and activator of transcription 3 (STAT3). By progressive 5'-deletion analysis we defined the minimal VEGF promoter region for IL-6-responsiveness to nucleotides -88/-50. Surprisingly, this promoter region is rich in GC-boxes and does not contain STAT3 binding elements. Electrophoretic mobility shift and supershift assays revealed binding of Sp1 and Sp3 to the -88/-50 element upon IL-6 stimulation. Interestingly, preincubation with STAT3 antibody prevented the binding of Sp1 and Sp3 to the -88/-50 element, indicating that STAT3 is involved in IL-6-driven Sp1/Sp3 protein-DNA complex formation. Physical interaction of STAT3 and Sp1 was demonstrated by coimmunoprecipitation. The functional relevance of the STAT3/Sp1 association was corroborated by transient transfection experiments, which showed that overexpression of constitutively active STAT3 increased the minimal VEGF promoter activity. Taken together, our study suggests that IL-6 promotes tumor angiogenesis in gliomas and describes a novel transcriptional activation mechanism for STAT3 in the context of a STAT3 binding element (SBE)-free promoter.

IL-6 is required for glioma development in a mouse model.

The pleiotropic cytokine interleukin-6 (IL-6) contributes to malignant progression and apoptosis resistance of various cancer types. Although IL-6 is elevated in malignant gliomas, and glioma cells respond to IL-6, its functional role in gliomagenesis is unclear. We have investigated this role of IL-6 in a mouse model of spontaneous astrocytoma by crossbreeding glial fibrillary acidic protein (GFAP)-viral src oncogene transgenic mice with IL-6-deficient mice. We show here that ablation of IL-6 prevents tumour formation in these predisposed animals, but did not affect preneoplastic astrogliosis. Moreover, we demonstrate phosphorylation and nuclear translocation of the transcription factor signal transducer and activator of transcription (STAT)3, a prerequisite for IL-6 signalling, in 51 human gliomas WHO grade II-IV and all experimental mouse tumours investigated. Together with the observation that STAT3 activation increases with malignancy, these findings indicate an important role for IL-6 in the development and malignant progression of astrocytomas.