Intracranial Glioma Model Research Articles

Inflammatory activation of microglia by Staphylococcus aureus caused phenotypic alterations and affected glioblastoma growth.

As the most common type of tumour in brain, glioma has a high rate of morbidity and mortality and easily penetrates the surrounding normal brain parenchyma. The immunosuppressive microenvironment, which is similar to that in other neoplasms, is believed to participate in the tumorigenesis of glioma. Thus, many experts are seeking to exploit microenvironment as a therapeutic target. In the present study, we focused on microglia polarization to investigate the anti-glioma response of microglia inflammatory activation by Staphylococcus aureus in vitro and in vivo. First, we found that intratumor injection of S. aureus delayed glioma growth in C57/BL6 mice. Then, we showed that inflammatory microglia activated by S. aureus inhibited glioma cell proliferation, migration, and invasion. This inhibition was likely related to the phenotypic switch observed in microglia. In intracranial tumour models, the microglia activated by S. aureus exerted antitumour efficacy and prolonged animal survival. Taken together, our results suggest that microglia activated by S. aureus have antitumour efficacy, which may be a potential therapeutic agent for glioma. SIGNIFICANCE OF THE STUDY: In this study, we mainly demonstrated the antitumour efficacy of microglia after activated by S. aureus. Firstly, we found that intratumor injection of S. aureus inhibited the tumour growth in intracranial orthotopic glioma model. In addition, we found that the microglia around the glioma may exert antitumour efficacy, and its phenotype may be altered by stimulation of S. aureus. Our data manifested that S. aureus did not directly suppress cell proliferative, migration, and invasion capacity, but by activating microglia. And in mice GL261 GBMs, injection of microglia after cocultured with S. aureus inhibited tumour growth and prolonged animal survival.

Targeted Magnetic Resonance Imaging and Modulation of Hypoxia with Multifunctional Hyaluronic Acid-MnO2 Nanoparticles in Glioma.

Manganese dioxide (MnO2 )-based nanoparticles are a promising tumor microenvironment-responsive nanotheranostic carrier for targeted magnetic resonance imaging (MRI) and for alleviating tumor hypoxia. However, the complexity and potential toxicity of the present common synthesis methods limit their clinical application. Herein, multifunctional hyaluronic acid-MnO2 nanoparticles (HA-MnO2 NPs) are synthesized in a simple way by directly mixing sodium permanganate with HA aqueous solutions, which serve as both a reducing agent and a surface-coating material. The obtained HA-MnO2 NPs show an improved water-dispersibility, fine colloidal stability, low toxicity, and responsiveness to the tumor microenvironment (high H2 O2 and high glutathione, low pH). After intravenous injection, HA-MnO2 NPs exhibit a high imaging sensitivity for detecting rat intracranial glioma with MRI for a prolonged period of up to 3 d. These nanoparticles also effectively alleviate the tumor hypoxia in a rat model of intracranial glioma. The downregulation of VEGF and HIF-1α expression in intracranial glioma validates the sustained attenuation effect of HA-MnO2 NPs on tumor hypoxia. These results show that HA-MnO2 NPs can be used for sensitive, targeted MRI detection of gliomas and simultaneous attenuation of tumor hypoxia.

Identification of long non-coding RNA HERC2P2 as a tumor suppressor in glioma.

Long non-coding RNAs (lncRNAs) have been reported to play important roles in glioma; however, most of them promote glioma progression. We constructed a competing endogenous (ceRNA) network based on the Chinese Glioma Genome Atlas dataset, and lncRNA hect domain and RLD 2 pseudogene 2 (HERC2P2) is the core of this network. Highly connected genes in the ceRNA network classified the glioma patients into three clusters with significantly different survival rates. The expression of HERC2P2 is positively correlated with survival and negatively correlated with clinical grade. Cell colony formation, Transwell and cell scratch tests were performed to evaluate the role of HERC2P2 in glioblastoma growth. Furthermore, we overexpressed HERC2P2 in U87 cells and established a mouse intracranial glioma model to examine the function of HERC2P2 in vivo. In conclusion, we identified a lncRNA with tumor suppressor functions in glioma that could be a potential biomarker for glioma patients.

Modulation of temozolomide dose differentially affects T-cell response to immune checkpoint inhibition.

The changes induced in host immunity and the tumor microenvironment by chemotherapy have been shown to impact immunotherapy response in both a positive and a negative fashion. Temozolomide is the most common chemotherapy used to treat glioblastoma (GBM) and has been shown to have variable effects on immune response to immunotherapy. Therefore, we aimed to determine the immune modulatory effects of temozolomide that would impact response to immune checkpoint inhibition in the treatment of experimental GBM. Immune function and antitumor efficacy of immune checkpoint inhibition were tested after treatment with metronomic dose (MD) temozolomide (25 mg/kg × 10 days) or standard dose (SD) temozolomide (50 mg/kg × 5 days) in the GL261 and KR158 murine glioma models. SD temozolomide treatment resulted in an upregulation of markers of T-cell exhaustion such as LAG-3 and TIM-3 in lymphocytes which was not seen with MD temozolomide. When temozolomide treatment was combined with programmed cell death 1 (PD-1) antibody therapy, the MD temozolomide/PD-1 antibody group demonstrated a decrease in exhaustion markers in tumor infiltrating lymphocytes that was not observed in the SD temozolomide/PD-1 antibody group. Also, the survival advantage of PD-1 antibody therapy in a murine syngeneic intracranial glioma model was abrogated by adding SD temozolomide to treatment. However, when MD temozolomide was added to PD-1 inhibition, it preserved the survival benefit that was seen by PD-1 antibody therapy alone. The peripheral and intratumoral immune microenvironments are distinctively affected by dose modulation of temozolomide.

Elevated Na/H exchanger 1 (SLC9A1) emerges as a marker for tumorigenesis and prognosis in gliomas

BackgroundSodium/hydrogen exchanger 1 (NHE1), encoded by the SLC9A1 gene (SoLute Carrier family 9A1) in humans, is the main H+ efflux mechanism in maintaining alkaline intracellular pH (pHi) and Warburg effects in glioma. However, to date, there are no clinical studies exploring pharmacological inhibition of NHE1 protein in cancer treatment. In this study, we investigated NHE1 expression in gliomas and its relationship with glioma clinical outcome.MethodsThe Chinese Glioma Genome Atlas (CGGA) dataset containing transcriptome sequencing data of 325 glioma samples and the Cancer Genome Atlas (TCGA) with 698 glioma mRNAseq data were analyzed in this study. Mouse SB28 and GL26 intracranial syngeneic glioma models in C57BL/6 J mice were established to investigate NHE1 expression and impact of NHE1 protein inhibition with its inhibitor HOE642 on tumorigenesis and anti-PD1 therapy. Tumor angiogenesis, immunogenicity, and progression were assessed by immunofluorescence staining and flow cytometric profiling.ResultsAnalysis of SLC9A1 mRNA expression in two data sets, CGGA and TCGA, reveals significantly higher SLC9A1 mRNA levels in higher grade gliomas. The SLC9A1 mRNA expression was especially enriched in isocitrate dehydrogenase (IDH)1/2 wild-type glioblastoma (GBM) and in mesenchymal glioma subtypes. Worsened survival probabilities were correlated with the elevated SLC9A1 mRNA levels in gliomas. The underlying mechanisms include promoting angiogenesis, and extracellular matrix remodeling. Increased SLC9A1 mRNA expression was also associated with tumor-associated macrophage accumulation. NHE1 inhibitor HOE642 reduced glioma volume, invasion, and prolonged overall survival in mouse glioma models. Blockade of NHE1 protein also stimulated immunogenic tumor microenvironment via activating CD8 T-cell accumulation, increasing expression of interferon-gamma (Ifng), and sensitized animals to anti-PD-1 therapy.ConclusionOur findings strongly suggest that NHE1 protein emerges as a marker for tumorigenesis and prognosis in glioma. Blocking NHE1 protein is a novel strategy for adjuvant anti-cancer therapies.

Blockade of Na/H exchanger stimulates glioma tumor immunogenicity and enhances combinatorial TMZ and anti-PD-1 therapy

The weak immunogenicity of gliomas presents a barrier for effective immunotherapy. Na/H exchanger isoform 1 (NHE1) maintains alkaline intracellular pH (pHi) of glioma cells and acidic microenvironment. In addition, NHE1 is expressed in tumor-associated microglia and tumor-associated macrophages (TAMs) and involved in protumoral communications between glioma and TAMs. Therefore, we hypothesize that NHE1 plays a role in developing tumor resistance and immunosuppressive tumor microenvironment. In this study, we investigated the efficacy of pharmacological inhibition of NHE1 on combinatorial therapies. Here we show that temozolomide (TMZ) treatment stimulates NHE1 protein expression in two intracranial syngeneic mouse glioma models (SB28, GL26). Pharmacological inhibition of NHE1 potentiated the cytotoxic effects of TMZ, leading to reduced tumor growth and increased median survival of mice. Blockade of NHE1 stimulated proinflammatory activation of TAM and increased cytotoxic T cell infiltration into tumors. Combining TMZ, anti-PD-1 antibody treatment with NHE1 blockade significantly prolonged the median survival in the mouse glioma model. These results demonstrate that pharmacological inhibition of NHE1 protein presents a new strategy for potentiating TMZ-induced cytotoxicity and increasing tumor immunogenicity for immunotherapy to improve glioma therapy.

Rapid dynamic contrast-enhanced MRI for small animals at 7T using 3D ultra-short echo time and golden-angle radial sparse parallel MRI.

To develop a rapid dynamic contrast-enhanced MRI method with high spatial and temporal resolution for small-animal imaging at 7 Tesla. An ultra-short echo time (UTE) pulse sequence using a 3D golden-angle radial sampling was implemented to achieve isotropic spatial resolution with flexible temporal resolution. Continuously acquired radial spokes were grouped into subsets for image reconstruction using a multicoil compressed sensing approach (Golden-angle RAdial Sparse Parallel; GRASP). The proposed 3D-UTE-GRASP method with high temporal and spatial resolutions was tested using 7 mice with GL261 intracranial glioma models. Iterative reconstruction with different temporal resolutions and regularization factors λ showed that, in all cases, the cost function decreased to less than 2.5% of its starting value within 20 iterations. The difference between the time-intensity curves of 3D-UTE-GRASP and nonuniform fast Fourier transform (NUFFT) images was minimal when λ was 1% of the maximum signal intensity of the initial NUFFT images. The 3D isotropic images were used to generate pharmacokinetic parameter maps to show the detailed images of the tumor characteristics in 3D and also to show longitudinal changes during tumor growth. This feasibility study demonstrated that the proposed 3D-UTE-GRASP method can be used for effective measurement of the 3D spatial heterogeneity of tumor pharmacokinetic parameters.

Hyperbaric oxygen inhibits production of CD3+ T cells in the thymus and facilitates malignant glioma cell growth.

ObjectiveHyperbaric oxygen (HBO) is an emerging complementary alternative medical approach in glioma treatment. However, its mode of action is unknown, so this was investigated in the present study.MethodsWe constructed an intracranial glioma model of congenic C57BL/6J mice. Glioma growth under HBO stimulation was assessed by bioluminescent imaging and magnetic resonance imaging. Flow cytometry assessed direct effects of HBO on reactive oxygen species (ROS) signaling of transplanted glioma cells and organs, and quantified mature T cells and subgroups in tumors, the brain, and blood.ResultsHBO promoted the growth of transplanted GL261-Luc glioma in the intracranial glioma mouse model. ROS signaling of glioma cells and brain cells was significantly downregulated under HBO stimulation, but thymus ROS levels were significantly upregulated. CD3+ T cells were significantly downregulated, while both Ti/Th cells (CD3+CD4+) and Ts/Tc cells (CD3+CD8+) were inhibited in tumors of the HBO group. The percentage of regulatory T cells in Ti/Th (CD3+CD4+) cells was elevated in the tumors and thymuses of the HBO group.ConclusionHBO induced ROS signaling in the thymus, inhibited CD3+ T cell generation, and facilitated malignant glioma cell growth in vivo in the intracranial glioma mouse model.

Targeted therapy of intracranial glioma model mice with curcumin nanoliposomes.

BackgroundGlioma is the most aggressive and lethal brain tumor in humans, it comprises about 30 per cent of all brain tumors and central nervous system tumors.PurposeThe objective of this study was to create novel brain-targeting nanoliposomes to encapsulate curcumin as a promising option for glioma therapy.Patients and methodsHuman glioma cells (U251MG) were used to determine cell uptake efficiency and possible internalization mechanism of the curcumin-loaded nanoliposomes modified by a brain-targeting peptide RDP. In addition, intracranial glioma mice model was prepared by transplantation of U251MG cells into the mice striatum, and then the liposomes were intravenously administered into the glioma-bearing mice to evaluate the anti-glioma activity.ResultsRDP-modified liposomes (RCL) could enter the brain and glioma region, and were internalized by the glioma cells perhaps through acetylcholine receptor-mediated endocytosis pathway. Furthermore, the RCL prolonged the survival time of the glioma-bearing mice from 23 to 33 days, and the inhibition mechanism of the RCL on glioma cell was partly due to cell cycle arrest at the S phase and induction of cell apoptosis.ConclusionThis study would provide a potential approach for targeted delivery of drug-loaded liposomes for glioma treatment.

Intracranial rat glioma model for tumor resection and local treatment

BackgroundAlthough tumor resection is among the most important prognostic factors, high grade gliomas regrow in most cases. Also, resection of glial tumors in eloquent brain regions is not or only partially possible. Despite these severe restraints, however, only a few in-vivo models have been established to investigate tumor recurrence and local treatment. Here we characterize the intracranial BT4Ca rat glioma as a model for these aspects. New methodBT4Ca cells were stereotaxically implanted into the frontal cortex of BDIX rats. Rats were than allocated to (1) a control group, which received no further treatment; (2) a catheter group, where a catheter was implanted for repeated microinjection of vehicle every 3rd day as catheter-control; (3) a resection group, where the tumor was microsurgically removed eight days after cell injection. Postoperatively, survival time, weight and general health condition were scored and the tumor size was histologically assessed. ResultsInjection of BT4Ca cells induced fast-growing tumors with a mean survival time of 16 days in the control and catheter groups. Resection significantly prolonged survival time whereby the tumor regrew in all rats. Tumor size was similar between all groups. Comparison with existing method(s)We here present a robust and reliable intracranial rat glioma model, which is suitable to simulate tumor recurrence after surgical resection and local treatment. Importantly, this model does not require advanced imaging or elaborate surgical techniques. ConclusionsThe intracranial BT4Ca glioma model appears to be a feasible tool to investigate tumor recurrence after resection and to test local treatment.

\u03b2-catenin-mediated YAP signaling promotes human glioma growth

BackgroundHippo/YAP pathway is known to be important for development, growth and organogenesis, and dysregulation of this pathway leads to tumor progression.We and others find that YAP is up-regulated in human gliomas and associated with worse prognosis of patients. However, the role and mechanism of YAP in glioma progression is largely unknown.MethodsThe expression of YAP in glioma tissues was detected by quantitative polymerase chain reaction (qPCR) and immunoblotting. The effect of YAP on glioma progression was examined using cell growth assays and intracranial glioma model. The effect of YAP on β-catenin protein level, subcellular location and transcription activity was examined by immunoblotting, immunofluorescence and RT-PCR.ResultsFirstly, knockdown of YAP inhibited glioma cell proliferation in vitro and tumor growth in vivo. In addition, YAP modulated the protein level, subcellular location and transcription activity of β-catenin via regulating the activity of GSK3β. Lastly, β-catenin partially mediated the effect of YAP on glioma cell proliferation.ConclusionOur findings identify that YAP promotes human glioma growth through enhancing Wnt/β-catenin signaling. In addition, this study provides a new crosstalk mechanism between Hippo/YAP and Wnt/β-catenin pathways, which suggests a new strategy for human glioma treatment.

Abstract 2454: Genetic landscape of glioma reveals defective neuroactive ligand receptor interaction pathway as a poor prognosticator in glioblastoma patients

Abstract Glioblastoma (GBM; grade IV), is highly proliferative, infiltrative and treatment refractory. Hence, understanding the complete genetic alteration profile of GBM would help us in identifying molecules or pathways that have strong implications in GBM pathogenesis, thus opening up avenues for targeted therapy. Recent large scale studies suggest that three pathways - receptor-tyrosine kinase, TP53 and RB, are significantly altered in GBM. However, even with the tremendous increase in our understanding of the tumor, advancement in therapeutics is minimal and the median survival still remains at 15 months. Hence, we need to elucidate novel altered molecules and pathways in GBM progression such that more effective therapeutic options can be explored. Here, we have carried out whole exome sequencing of grade II, grade III and GBM samples which revealed the mutation spectrum of glioma from our patient set. Further, we performed integrative analysis of mutated genes from our patient cohort as well as TCGA cohort (The Cancer Genome Atlas) to find out mutated pathways that predict survival in GBM patients. The most significant pathway - neuroactive ligand-receptor interaction pathway was explored further. Patients with mutations in one or more genes of this pathway had poor survival. The pathway comprises of G-protein coupled receptors, ion channels and ligands which functions in modulation of neural plasticity, memory processes, behavior etc. Of the enriched genes belonging to this pathway, Calcitonin Receptor (CALCR), which was highest mutated in GBM (2.75%), was taken up for further investigation. CALCR was found to be downregulated in GBM and mutation or downregulation of the gene was found to predict poor survival in patients. Functional studies through cell-line based experiments revealed CALCR is a tumor suppressor in GBM. The peptide hormone calcitonin (CT), a high affinity CALCR ligand, inhibited proliferation, migration and anchorage-independent growth of glioma cells expressing CALCR with a concomitant decrease in the phosphorylation levels of ERK, AKT and JNK signaling molecules. However, CT failed to do these functions in CALCR silenced glioma cells. Exogenous overexpression of CALCR in glioma cells expressing low levels of the receptor was found to inhibit proliferation, migration and anchorage independent growth and this effect was further augmented when CT was added. Further, introduction of tumor-derived mutations in CALCR led to the abrogation of its tumor suppressor function. Studies are ongoing to demonstrate the tumor suppressive nature of CALCR using in vitro astrocyte transformation and intracranial orthotopic mouse glioma model. Thus, our study finds CT-CALCR signaling axis is an important tumor suppressor pathway in glioma development and underscores the importance of using CT as a novel therapeutic molecule for GBM treatment. Citation Format: Jagriti Pal, Vikas Patil, Anupam Kumar, Kavneet Kaur, Chitra Sarkar, Kumaravel Somasundaram. Genetic landscape of glioma reveals defective neuroactive ligand receptor interaction pathway as a poor prognosticator in glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2454. doi:10.1158/1538-7445.AM2017-2454

Abstract 3771: Concurrent GVAX and anti-OX40 therapy in murine intracranial glioma model

Abstract Introduction: Malignant glioma is the most aggressive brain tumor with a median survival time of less than 1.5 years post diagnosis despite standard treatments. However, recent advancements in immunotherapy hold significant promise. Irradiated whole tumor cell vaccination stimulates antitumor immunity and prolongs survival in mice with intracranial glioma tumors. We have shown that vaccination and immune checkpoint blockade is synergistic and these efforts are underway in the clinic. In contrast to immune checkpoint blockade, agonist stimulation of OX40, a member of the TNFR/TNF superfamily that is expressed on activated CD4 and CD8 T lymphocytes increases T cell proliferation, clonal memory differentiation, and cytokine release. We hypothesized that subcutaneous vaccination with irradiated GM-CSF expressing glioma cells (GVAX) would, therefore, synergize with systemic delivery of stimulating anti-OX40 antibody and provide effective immunity against intracranial gliomas. Methods: Gl261 cells were implanted in the right frontal lobes of syngeneic c57/bl6 mice using a stereotactic implantation technique. Mice received concurrent subcutaneous injection of irradiated GM-CSF expressing glioma cells and intraperitoneal injection of OX40 antibody, either monotherapy, or control treatment on days 3, 6 and 9 following tumor implantation. Results: Combination immunotherapy with irradiated GM-CSF expressing glioma cells and intraperitoneal delivery of agonist monoclonal antibody against OX40 was more effective than either therapy alone and prolonged survival in mice with established intracranial GL261 tumors. Elispot analyses of peripheral lymphocytes revealed that combination immunotherapy reverses the Th2 cytokine skew that is induced by the presence of intracranial glioma. OX40 signaling alone and in combination with vaccination drove high levels of interferon gamma expression by splenocytes harvested from glioma-bearing animals, while IL-10 expression was little changed. In the tumor microenvironment, vaccination improves the CD8+ /FoxP3+ T lymphocyte ratio, while OX40 ligation overcomes tumor-driven lymphocyte exhaustion, demonstrated by reduced expression of PD1 and LAG3, measured by flow cytometery 2 weeks after initiation of treatment. Conclusions: Combination treatment with GVAX and OX-40 agonist antibody increased survival in mice bearing intracranial gliomas and are mechanistically complementary. Citation Format: Nusrat Jahan, Hammad Talat, William T. Curry. Concurrent GVAX and anti-OX40 therapy in murine intracranial glioma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3771. doi:10.1158/1538-7445.AM2017-3771

Golgi phosphoprotein 3 promotes glioma progression via inhibiting Rab5-mediated endocytosis and degradation of epidermal growth factor receptor.

Golgi phosphoprotein 3 (GOLPH3) is associated with worse prognosis of gliomas, but its role and mechanism in glioma progression remain largely unknown. This study aimed to explore the role and mechanism of GOLPH3 in glioma progression. The expression of GOLPH3 in glioma tissues was detected by quantitative PCR, immunoblotting, and immunohistochemistry. GOLPH3's effect on glioma progression was examined using cell growth assays and an intracranial glioma model. The effect of GOLPH3 on epidermal growth factor receptor (EGFR) stability, endocytosis, and degradation was examined by immunoblotting and immunofluorescence. The activity of Rab5 was checked by glutathione S-transferase pulldown assay. GOLPH3 was upregulated in gliomas, and its downregulation inhibited glioma cell proliferation both in vitro and in vivo. Furthermore, GOLPH3 depletion dampened EGFR signaling by enhancing EGFR endocytosis, driving EGFR into late endosome and promoting lysosome-mediated degradation. Interestingly, GOLPH3 bound to Rab5 and GOLPH3 downregulation promoted the activation of Rab5. In addition, Rab5 depletion abolished the effect of GOLPH3 on EGFR endocytosis and degradation. Our results imply that GOLPH3 promotes glioma cell proliferation via inhibiting Rab5-mediated endocytosis and degradation of EGFR, thereby activating the phosphatidylinositol-3 kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway. We find a new mechanism by which GOLPH3 promotes tumor progression through regulating cell surface receptor trafficking. Extensive and intensive understanding of the role of GOLPH3 in glioma progression may provide an opportunity to develop a novel molecular therapeutic target for gliomas.

Intratumoral delivery of bortezomib: impact on survival in an intracranial glioma tumor model.

OBJECTIVE Glioblastoma (GBM) is the most prevalent and the most aggressive of primary brain tumors. There is currently no effective treatment for this tumor. The proteasome inhibitor bortezomib is effective for a variety of tumors, but not for GBM. The authors' goal was to demonstrate that bortezomib can be effective in the orthotopic GBM murine model if the appropriate method of drug delivery is used. In this study the Alzet mini-osmotic pump was used to bring the drug directly to the tumor in the brain, circumventing the blood-brain barrier; thus making bortezomib an effective treatment for GBM. METHODS The 2 human glioma cell lines, U87 and U251, were labeled with luciferase and used in the subcutaneous and intracranial in vivo tumor models. Glioma cells were implanted subcutaneously into the right flank, or intracranially into the frontal cortex of athymic nude mice. Mice bearing intracranial glioma tumors were implanted with an Alzet mini-osmotic pump containing different doses of bortezomib. The Alzet pumps were introduced directly into the tumor bed in the brain. Survival was documented for mice with intracranial tumors. RESULTS Glioma cells were sensitive to bortezomib at nanomolar quantities in vitro. In the subcutaneous in vivo xenograft tumor model, bortezomib given intravenously was effective in reducing tumor progression. However, in the intracranial glioma model, bortezomib given systemically did not affect survival. By sharp contrast, animals treated with bortezomib intracranially at the tumor site exhibited significantly increased survival. CONCLUSIONS Bypassing the blood-brain barrier by using the osmotic pump resulted in an increase in the efficacy of bortezomib for the treatment of intracranial tumors. Thus, the intratumoral administration of bortezomib into the cranial cavity is an effective approach for glioma therapy.

Blood-Brain-Barrier-Penetrating Albumin Nanoparticles for Biomimetic Drug Delivery via Albumin-Binding Protein Pathways for Antiglioma Therapy.

Nutrient transporters have been explored for biomimetic delivery targeting the brain. The albumin-binding proteins (e.g., SPARC and gp60) are overexpressed in many tumors for transport of albumin as an amino acid and an energy source for fast-growing cancer cells. However, their application in brain delivery has rarely been investigated. In this work, SPARC and gp60 overexpression was found on glioma and tumor vessel endothelium; therefore, such pathways were explored for use in brain-targeting biomimetic delivery. We developed a green method for blood-brain barrier (BBB)-penetrating albumin nanoparticle synthesis, with the capacity to coencapsulate different drugs and no need for cross-linkers. The hydrophobic drugs (i.e., paclitaxel and fenretinide) yield synergistic effects to induce albumin self-assembly, forming dual drug-loaded nanoparticles. The albumin nanoparticles can penetrate the BBB and target glioma cells via the mechanisms of SPARC- and gp60-mediated biomimetic transport. Importantly, by modification with the cell-penetrating peptide LMWP, the albumin nanoparticles display enhanced BBB penetration, intratumoral infiltration, and cellular uptake. The LMWP-modified nanoparticles exhibited improved treatment outcomes in both subcutaneous and intracranial glioma models, with reduced toxic side effects. The therapeutic mechanisms were associated with induction of apoptosis, antiangiogenesis, and tumor immune microenvironment regulation. It provides a facile method for dual drug-loaded albumin nanoparticle preparation and a promising avenue for biomimetic delivery targeting the brain tumor based on combination therapy.

A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy.

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

Inhibition of fatty acid synthase suppresses neovascularization via regulating the expression of VEGF-A in glioma.

Fatty acids (FAs) are essential for membrane lipids biosynthesis and energy consumption in cancer cells. De novo FAs synthesis is catalyzed by fatty acid synthase (FASN), which is overexpressed and correlates with histological grade in glioma. Herein, we focused on the role of FASN in glioma neovascularization. The expression levels of FASN, Ki67 and CD34 were determined using immunohistochemistry (IHC). FASN specific-targeted shRNA and C75 were applied to evaluate the influence of FASN on glioma stem cell proliferation, migration and tube formation ability in vitro. An intracranial glioma model was established to study the effects of FASN on tumor growth and neovascularization in vivo. IHC staining showed that the expression level of FASN correlated with tumor grade, Ki67 levels and microvessels density (MVD) in human gliomas. Inhibition of FASN using shRNAs or C75 decreased tumor growth, prolonged the overall survival of xenograft mice and decreased MVD in brain tumor sections. Moreover, inhibition of FASN blocked hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor A (VEGF-A) signaling and upregulated the anti-angiogenic isoform-VEGF165b. Our results suggest that FASN plays a pivotal role in glioma neovascularization, and inhibition of FASN may be a potential target for anti-angiogenic therapy for glioma.

Abstract A43: MAPK-activated protein kinase MK2 exerts immune regulatory functions in the microenvironment of orthotopic tumors

Abstract The MAPK signaling pathway represents a central player in inflammatory processes. As a downstream target of p38, MAPK-activated protein kinase 2 (MK2) contributes to signal transduction regulating the expression and translation of various targets, among them several cytokines such as tumor necrosis factor (TNF) α. While MK2 was shown to promote an inflammatory macrophage phenotype, we have reported its Th1-attenuating function in dendritic cells (DCs). These observations of differential regulatory functions prompted us to investigate MK2 in the context of DCs and other myeloid cells in the tumor microenvironment. Murine DCs lacking MK2 activity exhibit an enhanced potential to differentiate Th1 cells upon Toll-like receptor (TLR) ligation in vitro and in vivo. In line with this finding, we observe significantly reduced B16.F10 melanoma growth in CD11cCre-MK2fl/fl mice upon DC activation by administration of lipopolysaccharide (LPS) together with whole tumor cell lysate as compared to wildtype littermate controls. Interestingly, CD11cCre-MK2fl/fl mice show an overall reduced myeloid cell tumor infiltration. Looking at MK2 expression in DCs isolated from orthotopic B16.F10 tumor-bearing mice, we find elevated MK2 levels in tumor-resident as opposed to splenic DCs, which has also been confirmed in a model of intracranial glioma (GL-261). This MK2 up-regulation in glioma-infiltrating DCs is associated with an enhanced expression of IL-10. Furthermore, tumor-resident myeloid-derived suppressor cells (MDSCs) show elevated MK2 expression, further underlining its potential involvement in immunosuppressive mechanisms in the myeloid lineage. In DCs we have observed cross-regulation of MAPK signaling by MK2, which promotes ERK1/2 while attenuating p38 activation and is further implicated to enhance STAT3 phosphorylation – all together contributing to a tolerogenic DC phenotype. Therefore we are now looking to unravel mechanisms by which MK2 impacts on signaling networks involved in myeloid immunosuppression in the tumor microenvironment. Our data contrast the previously described role of MK2 in pro-inflammatory mechanisms of the p38 signaling route and suggest an additional immunosuppressive feedback function in myeloid cells, which might be exploited by tumor cells to escape immune recognition and elimination. These indications are particularly interesting with regard to glioma, since the glioma microenvironment has been shown to be rich in various cytokines promoting favourable conditions for tumor growth. Finally, MK2 represents a promising target for combination therapy, as it has been reported to mediate chemo- as well as radioresistance in different types of tumors. Nevertheless, its function in tumor-infiltrating immune cells remains to be elucidated. We therefore aim to contribute to a deeper understanding of the complex interplay between extra- and intracellular signaling molecules regulating tumor as well as immune cell phenotypes. Citation Format: Klara Soukup, Angela Halfmann, Fiona Poyer, Katharina Martin, Bernadette Blauensteiner, Bastien Huber, Mario Kuttke, Gernot Schabbauer, Lydia Zopf, Jelena Zinnanti, Alexander Michael Dohnal. MAPK-activated protein kinase MK2 exerts immune regulatory functions in the microenvironment of orthotopic tumors. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A43.

Abstract 2527: MDA-9/Syntenin (SDCBP) promotes self renewal and malignancy in cancer stem cells

Abstract Cancer metastasis is a complex process that culminates in the majority of patient deaths. A key component of the cancer paradigm involves cancer stem cells (CSCs), the primary mediators of cancer progression, resistance to therapy and recurrence of cancer after remission/latency. Although the clinical significance of CSCs is well accepted, the key upstream regulatory mechanisms that define these unique subsets of cancer cells requires further clarification. Previous studies demonstrate that MDA-9/Syntenin or syndecan binding protein (SDCBP) levels are elevated in large spectrum of tumors, including melanoma, breast carcinoma, gastric carcinoma and glioblastoma multiforme (GBM), versus normal cellular counterparts. We now uncover a unique function of MDA-9 as a facilitator of cancer stemness and survival. Expression profiles of mda-9, Nanog, Oct4, Sox2 and c-myc were studied in different clinical samples and cell lines for correlations. Cells were sorted by FACS into stem and non-stem populations and the same genes were assayed for correlative expression. Stemness gene expression array profile analysis was performed on control and mda-9 silenced CSCs. Genetic (gain-of-function, loss-of-function) approaches were used to modify mda-9 expression in normal human astrocytes and prostate cells, non-stem cancer cells (NSCCs) and CSCs from breast prostate and GBM. Recovery-of-function approaches were also applied to elucidate the pathways responsible for MDA-9-mediated regulation of CSCs. The impact of MDA-9 on CSC-mediated tumorigenicity and progression was evaluated in vivo by intracranial glioma and subcutaneous xenograft mice models. Apoptosis was assayed by caspase activation and Annexin V staining. A positive and statistically significant correlation was observed between mda-9 and stemness regulating genes (Nanog, Oct4, Sox2 and c-myc), in 50 clinical samples and 3 cell culture systems, including breast, prostate and GBM. CSCs expressed elevated levels of mda-9 vs. non-stem cancer cells or normal stem cells from a total of 11 cell lines including breast, prostate and GBM and two clinical GBM samples. Over-expression of mda-9 in NSCCs increased populations of CSCs as well as expression of stemness regulating genes. Loss of MDA-9 affected 84 genes linked to CSC regulation. Control intracranial glioma xenograft mouse models had large tumors with spongioblastic morphology typical of human gliomas, whereas the loss of mda-9 caused a large decrease in tumor size. Mechanistically, MDA-9 regulated multiple stemness genes and knockdown of MDA-9 caused a loss of stemness and tumorigenicity with initiation of apoptosis. Our data uncover a previously unidentified relationship between MDA-9 and CSCs. CSCs appear more dependent on MDA-9 expression than normal stem cells. MDA-9 regulates CSCs on multiple levels of stemness and survival, reinforcing the relevance of this gene as a potential therapeutic target. Citation Format: Sarmistha Talukdar, Swadesh Das, Anjan K. Pradhan, Luni Emdad, Xue-Ning Shen, Jolene J. Windle, Devanand Sarkar, Paul B. Fisher. MDA-9/Syntenin (SDCBP) promotes self renewal and malignancy in cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2527.