Experimental Glioma Model Research Articles

Endothelial Indoleamine-2,3-Dioxygenase-1 is not Critically Involved in Regulating Antitumor Immunity in the Central Nervous System.

The vascular niche of malignant gliomas is a key compartment that shapes the immunosuppressive brain tumor microenvironment (TME). The blood-brain-barrier (BBB) consisting of specialized endothelial cells (ECs) and perivascular cells forms a tight anatomical and functional barrier critically controlling transmigration and effector function of immune cells. During neuroinflammation and tumor progression, the metabolism of the essential amino acid tryptophan (Trp) to metabolites such as kynurenine has long been identified as an important metabolic pathway suppressing immune responses. Previous studies have demonstrated that indoleamine-2,3-dioxygenase-1 (IDO1), a key rate-limiting enzyme in tryptophan catabolism, is expressed within the TME of high-grade gliomas. Here, we investigate the role of endothelial IDO1 (eIDO1) expression for brain tumor immunity. Single-cell RNA sequencing data revealed that in human glioma tissue, IDO1 is predominantly expressed by activated ECs showing a JAK/STAT signaling pathway-related CXCL11+ gene expression signature. In a syngeneic experimental glioma model, eIDO1 is induced by low-dose tumor irradiation. However, cell type-specific ablation of eIDO1 in experimental gliomas did not alter frequency and phenotype of tumor-infiltrating T cells nor tumor growth. Taken together these data argue against a dominant role of eIDO1 for brain tumor immunity.

Differential roles of type I interferon signaling in tumor versus host cells in experimental glioma models

Despite multimodal treatment approaches including surgery, radiotherapy and chemotherapy, the median survival for patients with glioblastoma remains in the range of one year and thus poor. Type I interferons (IFN) are involved in immune responses to viral infection and exhibit anti-tumor activity in certain cancers.Here we explored the biological relevance of constitutive type I IFN signaling in murine glioma models in vitro and in vivo. CT-2A, GL-261, SMA-497, SMA-540 and SMA-560 murine glioma cells expressed IFN type I receptors IFNAR1 and IFNAR2 and were responsive to exogenous IFN stimulation. CRISPR/Cas9-mediated deletion of IFNAR1 decreased the baseline expression of type I IFN response genes in GL-261 cells, but neither in CT-2A nor in SMA-560 cells. IFNAR1 deletion slowed growth in GL-261 and SMA-560, but not in CT-2A cells. However, only the growth of IFNAR1-depleted GL-261 tumors and not that of SMA-560 tumors was delayed in vivo upon orthotopic tumor cell implantation into syngeneic mice. This survival gain was no longer detected when the IFNAR1-depleted GL-261 cells were inoculated into IFNAR1-deficient mice. Altogether these data suggest that constitutive type I IFN signaling in gliomas may be pro-tumorigenic, but only in a microenvironment that is proficient for type I IFN signaling in the host.

EXTH-33. RETROVIRAL REPLICATING VECTORS PSEUDOTYPED WITH GIBBON APE LEUKEMIA VIRUS ENVELOPE FOR PRODRUG ACTIVATOR GENE THERAPY IN PRECLINICAL GLIOMA MODELS

Abstract Amphotropic retroviral replicating vector (RRV) Toca 511, expressing the yeast cytosine deaminase (CD) prodrug activator gene, showed promising evidence of therapeutic benefit and increased survival in early-phase trials for recurrent high-grade glioma. While a multi-center Phase 3 trial did not meet its overall endpoints, highly statistically significant survival was observed within predetermined patient subgroups compared to matched randomized control patients, and clinical investigation is on-going. Hence it is worthwhile to consider strategies aimed at enhancing therapeutic efficacy, such as delivering combinations of multiple transgenes. However, RRVs encoated with the same envelope compete for the same cancer cell surface receptors. We have now developed novel RRV encoated (‘pseudotyped’) with a heterologous envelope derived from Gibbon ape leukemia virus (GALV), which utilizes a different cell surface receptor from the native amphotropic retrovirus envelope for cellular entry. RRV(GALV) vectors expressing either GFP or HSV thymidine kinase (TK) were constructed, and efficient replication and transgene expression was observed in > 90% of both established and primary human glioblastoma cells within 14 days after initial infection at 0.01 (1%) multiplicity of infection (MOI). Genomic stability of RRV(GALV) vectors was also confirmed over prolonged propagation. Established and primary human glioblastoma cells infected with RRV(GALV)-TK vector showed ≥ 50%-90% reduction in cell viability after exposure to Ganciclovir prodrug in the range of 1µM-100µM for 5 days, as compared to uninfected control cells or cells infected with RRV(GALV)-GFP control vector. Furthermore, dual infection with RRV(GALV)-TK and amphotropic RRV-CD (Toca 511) resulted in synergistic cytotoxicity upon simultaneous exposure to their respective prodrugs. Further data will be presented from on-going studies evaluating these vectors in intracerebral glioblastoma models. These results indicate that GALV envelope-pseudotyped RRV can efficiently deliver prodrug activator gene therapy in experimental glioma models, and open the door to combinatorial gene therapy regimens with this vector platform.

Comparative evaluation of T-cell receptors in experimental glioma-draining lymph nodes.

BackgroundGlioblastomas, the most common primary malignant brain tumors, are considered immunologically cold malignancies due to growth in an immune sanctuary site. While peptide vaccines have shown to generate intra-tumoral antigen-specific T cells, the identification of these tumor-specific T cells is challenging and requires detailed analyses of tumor tissue. Several studies have shown that CNS antigens may be transported via lymphatic drainage to cervical lymph nodes, where antigen-specific T-cell responses can be generated. Therefore, we investigated whether glioma-draining lymph nodes (TDLN) may constitute a reservoir of tumor-reactive T cells.MethodsWe addressed our hypothesis by flow cytometric analyses of chicken ovalbumin (OVA)-specific CD8+ T cells as well as T-cell receptor beta (TCRβ) next-generation-sequencing (TCRβ-NGS) of T cells from tumor tissue, TDLN, spleen, and inguinal lymph nodes harvested from experimental mouse GL261 glioma models.ResultsLongitudinal dextramer-based assessment of specific CD8+ T cells from TDLN did not show tumor model antigen reactivity. Unbiased immunogenomic analysis revealed a low overlap of TCRβ sequences from glioma-infiltrating CD8+ T cells between mice. Enrichment scores, calculated by the ratio of productive frequencies of the different TCRβ-CDR3 amino-acid (aa) rearrangements of CD8+ T cells derived from tumor, TDLN, inguinal lymph nodes, and spleen demonstrated a higher proportion of tumor-associated TCR in the spleen compared to TDLN.ConclusionsIn experimental glioblastoma, our data did not provide evidence that glioma-draining cervical lymph nodes are a robust reservoir for spontaneous glioma-specific T cells highlighting the requirement for detailed analyses of glioma-infiltrating T cells for the discovery of tumor-specific TCR.

EXTH-44. DEVELOPMENT OF RETROVIRAL REPLICATING VECTORS ENCODING SEQUENCE-OPTIMIZED NITROREDUCTASE FOR PRODRUG ACTIVATOR GENE THERAPY IN HUMAN GLIOMA MODELS

Abstract Early-phase clinical trials of Toca 511, a retroviral replicating vector (RRV) expressing an optimized form of yeast cytosine deaminase for prodrug activator gene therapy, showed highly promising evidence of therapeutic benefit and increased survival in recurrent high-grade glioma, but an international Phase 3 trial failed to meet primary and secondary endpoints. Nonetheless, promising results observed within predetermined subgroups, combined with the highly favorable safety profile of this approach, make it worthwhile to consider alternative prodrug-activating enzymes in order to increase therapeutic efficacy. In this study, we employed RRV to deliver modified versions of another prodrug activator gene, E.coli nitroreductase (NTR), which activates the prodrug CB1954 to generate a potent bifunctional alkylating agent. We constructed RRV encoding two different isoforms of wild-type E.coli NTR genes (NfsA, NfsB) as well as variants of both isoforms extensively modified to optimize human codon usage and vector stability. Further efforts to improve the potency of these prodrug activator enzymes included the construction of additional new vectors incorporating point mutations at the active site which have previously been demonstrated to improve prodrug conversion efficiency. NTR transgene insertion did not affect RRV replication, which resulted in increasing NTR expression over time in human glioma cells in culture. Sequence optimization resulted in enhanced genomic stability of the vectors upon serial passage and also in significantly reduced cell viability in vitro on treatment of fully transduced U87 and U87-EGFRviii glioma cells with CB1954 prodrug. In orthotopic U87-EGFRviii intracerebral tumor models, stereotactic intratumoral injection of sequence-optimized RRV-NTR and repeated cycles of prodrug treatment resulted in rapid and significant reduction of tumor burden as monitored by bioluminescence imaging, leading to prolonged survival benefit. These data indicate that improved therapeutic efficacy can be achieved with optimized NTR prodrug activator gene therapy delivered by RRV in experimental glioma models.

Synergistic Toll-like Receptor 3/9 Signaling Affects Properties and Impairs Glioma-Promoting Activity of Microglia.

In murine experimental glioma models, TLR3 or TLR9 activation of microglial/macrophages has been shown to impair glioma growth, which could, however, not been verified in recent clinical trials. We therefore tested whether combined TLR3 and TLR9 activation of microglia/macrophages would have a synergistic effect. Indeed, combined TLR3/TLR9 activation augmented the suppression of glioma growth in organotypic brain slices from male mice in a microglia-dependent fashion, and this synergistic suppression depended on interferon β release and phagocytic tumor clearance. Combined TLR3/TLR9 stimulation also augmented several functional features of microglia, such as the release of proinflammatory factors, motility, and phagocytosis activity. TLR3/TLR9 stimulation combined with CD47 blockade further augmented glioma clearance. Finally, we confirmed that the coactivation of TLR3/TLR9 also augments the impairment of glioma growth in vivo Our results show that combined activation of TLR3/TLR9 in microglia/macrophages results in a more efficient glioma suppression, which may provide a potential strategy for glioma treatment.SIGNIFICANCE STATEMENT Glioma-associated microglia/macrophages (GAMs) are the predominant immune cells in glioma growth and are recently considered as antitumor targets. TLRs are involved in glioma growth, but the TLR3 or TLR9 ligands were not successful in clinical trials in treating glioma. We therefore combined TLR3 and TLR9 activation of GAMs, resulting in a strong synergistic effect of tumor clearance in vitro, ex vivo, and in vivo Mechanisms of this GAM-glioma interaction involve IFNβ signaling and increased tumor clearance by GAMs. Interfering with CD47 signaling had an additional impact on tumor clearance. We propose that these signaling pathways could be exploited as anti-glioma targets.

Blockade of Cell Volume Regulatory Protein NKCC1 Increases TMZ-Induced Glioma Apoptosis and Reduces Astrogliosis.

Glioma is one of the most common primary malignant tumors of the central nervous system accounting for approximately 40% of all intracranial tumors. Temozolomide is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk gliomas, including grade II to grade IV. Our bioinformatic analysis of The Cancer Genome Atlas and Chinese Glioma Genome Atlas datasets and immunoblotting assay show that SLC12A2 gene and its encoded Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) protein are abundantly expressed in grade II-IV gliomas. NKCC1 regulates cell volume and intracellular Cl- concentration, which promotes glioma cell migration, resistance to temozolomide, and tumor-related epilepsy in experimental glioma models. Using mouse syngeneic glioma models with intracranial transplantation of two different glioma cell lines (GL26 and SB28), we show that NKCC1 protein in glioma tumor cells as well as in tumor-associated reactive astrocytes was significantly upregulated in response to temozolomide monotherapy. Combination therapy of temozolomide with the potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated the cytotoxic effects of temozolomide, decreased tumor-associated reactive astrogliosis, and restored astrocytic GLT-1 and GLAST glutamate transporter expression. The combinatorial therapy also led to suppressed tumor growth and prolonged survival of mice bearing GL26 glioma cells. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in the pathogenesis of glioma tumors and presents as a therapeutic target for reducing temozolomide-mediated resistance and tumor-associated astrogliosis.

TMIC-30. MICROGLIA/BRAIN MACROPHAGES PROMOTE GLIOMA GROWTH BY EXPRESSING GLYCOPROTEIN NMB

Abstract BACKGROUND Glioma-associated microglia and blood-derived macrophages (GAMs) promote tumor growth in experimental mouse glioma models. Using microarray and RNA sequencing, we have previously shown that GAMs upregulate the expression of Glycoprotein NMB/Osteoactivin (GPNMB) when compared to naïve microglia. GPNMB is a type 1 transmembrane glycoprotein expressed intracellularly under healthy conditions. Malignancies such as glioma induce a translocation into the plasma membrane where the extracellular domain can be cleaved and released. METHODS We used qRT-PCR, immunocytochemistry, Western Blot and flow cytometry to determine the cellular localization of GPNMB expression in human and mouse glioblastoma. To test the impact of microenvironment-derived GPNMB on glioma growth, we inoculated GL261 and RCAS-PDGFb glioblastoma cells into organotypic brain slices obtained from wildtype and GPNMB-/- mice. In addition, we quantified glioma growth after injection of RCAS-PDGFb cells into wildtype and GPNMB-/- mice. The soluble extracellular domain of GPNMB was used to stimulate primary human glioblastoma and RCAS-PDGFb cells in vitro. SRB assays were performed to assess proliferation. RESULTS Our data indicate that GAMs are the predominant source of GPNMB in both human and mouse glioblastoma and that the levels of expression in GAMs in the tumor microenvironment is higher than in naïve microglia. In the organotypic brain slice model we found that tumors were significantly smaller in slices derived from GPNMB-/- mice as compared to wildtype. The tumor growth in vivo was nearly completely blocked in the absence of GPNMB. Stimulation of glioma cells with the extracellular domain of GPNMB did not increase proliferation. CONCLUSION Our results show that GPNMB is predominantly expressed in GAMs of human and murine samples. Loss of GPNMB impaired tumor growth ex vivo and glioblastoma progression in vivo. GPNMB seems to play a crucial role in the pro-tumorigenic activity of microglia and blood-derived macrophages in the tumor microenvironment.

Irradiation enhances the therapeutic effect of the oncolytic adenovirus XVir-N-31 in brain tumor initiating cells.

Virotherapy using oncolytic viruses is an upcoming therapy strategy for cancer treatment. A variety of preclinical and clinical trials have indicated that adenoviruses may be used as potent agents in the treatment of a variety of cancers, and also for the treatment of brain tumors. In these studies, it has also been shown that oncovirotherapy is safe in terms of toxicity and side effects. In addition, previous studies have presented evidence for a significant role of oncovirotherapy in the activation of anti-tumor immune responses. With regard to oncolytic adenoviruses, we have demonstrated previously that the multifunctional protein Y-box binding protein-1 (YB-1) is a potent factor that was used to develop an YB-1-dependent oncolytic adenovirus (XVir-N-31). XVir-N-31 provides the opportunity for tumor-selective replication and exhibited marked oncolytic properties in a mouse glioma tumor model using therapy-resistant brain tumor initiating cells (BTICs). In a number of, but not all, patients with glioma, YB-1 is primarily located in the nucleus; this promotes XVir-N-31-replication and subsequently tumor cell lysis. However, in certain BTICs, only a small amount of YB-1 has been identified to be nuclear, and therefore virus replication is suboptimal. YB-1 in BTICs was demonstrated to be translocated into the nucleus following irradiation, which was accompanied by an enhancement in XVir-N-31 production. R28 glioma spheres implanted in living organotypic human brain slices exhibited a significantly delayed growth rate when pre-irradiated prior to XVir-N-31-infection as compared with single treatment methods. Consistent with the in vitro data, R28 glioma-bearing mice exhibited a prolonged mean and median survival following single tumor irradiation prior to intratumoral XVir-N-31 injection, compared with the single treatment methods. In conclusion, the present study demonstrated that in an experimental glioma model, tumor irradiation strengthened the effect of an XVir-N-31-based oncovirotherapy.

The effect of locally delivered cisplatin is dependent on an intact immune function in an experimental glioma model

Several chemotherapeutic drugs are now considered to exert anti-tumour effects, by inducing an immune-promoting inflammatory response. Cisplatin is a potent chemotherapeutic agent used in standard medulloblastoma but not glioblastoma protocols. There is no clear explanation for the differences in clinical efficacy of cisplatin between medulloblastomas and glioblastomas, despite the fact that cisplatin is effective in vitro against the latter. Systemic toxicity is often dose limiting but could tentatively be reduced by intratumoral administration. We found that intratumoral cisplatin can cure GL261 glioma-bearing C57BL/6 mice and this effect was abolished in GL261-bearing NOD-scid IL2rγnull (NSG) mice. Contrary to previous results with intratumoral temozolomide cisplatin had no additive or synergistic effect with whole cell either GL261 wild-type or GM-CSF-transfected GL261 cells whole cell vaccine-based immunotherapy. While whole tumour cell immunizations increased CD8+ T-cells and decreased F4/80+ macrophages intratumorally, cisplatin had no effect on these cell populations. Taken together, our results demonstrate that intratumoral cisplatin treatment was effective with a narrow therapeutic window and may be an efficient approach for glioma or other brain tumour treatment.

Abstract B075: Modeling response and resistance to immune checkpoint blockade in syngeneic mouse glioma

Abstract Immune checkpoint inhibitors are now implemented into the standard therapy of an increasing number of tumor entities and elicit remarkable durable therapy responses. However, gliomas seem resistant to checkpoint inhibition as recent evidence from a randomized clinical trial did not show a therapeutic benefit of PD-1 blockade in an unselected population of patients with recurrent glioblastoma. The blood-brain barrier per se does not seem to be a hurdle in transducing an effective peripheral immune response into tumors as evidenced by responses seen in selected glioma patients and patients with brain metastases treated with checkpoint inhibitors. This project investigates the mechanisms of response and resistance to checkpoint blockade targeting CTLA-4 and PD-1 in an experimental syngeneic Gl261 glioma model, where we found a clear and unanticipated dichotomy between responders and non-responders. We demonstrate that response to PD-1 and CTLA-4 blockade is driven by increased numbers and effector function of cytotoxic tumor-infiltrating T-cells as well as an enhanced TCRβ repertoire clonality of tumor infiltrating CD8 T-cells. Surprisingly, little overlap of the TCRβ repertoire between responder CD8 TILs was detected with only one shared TCRβ sequence motif, suggestive of a common tumor-antigen driving the expansion of reactive clones in responding mice. Moreover, we identified putative tumor neoepitopes that were predominantly abundant in non-responding tumors and thus might have undergone effective targeting by tumor-reactive T-cell in responding tumors. Resistance to PD-1 and CTLA-4 blockade was associated with increased frequencies of intratumoral macrophages (TAMs) expressing high levels of immunosuppressive markers such as PD-L1, CD38 and CD73. TAMs of nonresponding mice induced enhanced suppression of CD4 T-cell proliferation which was partially restored by PD-L1 blockade. Strikingly, additional PD-L1 blockade enhanced response rates to PD-1 and CTLA-4 blockade in Gl261-bearing mice, potentially by inhibiting the ligation of PD-L1 on TAMs to its alternative interaction partner CD80 on TILs. Collectively, we suggest a syngeneic mouse model for assessing mechanisms of response and resistance to checkpoint blockade in gliomas demonstrating a surprising heterogeneity of the TCRβ repertoire of tumor-infiltrating CD8 T-cells despite strict syngeneicity. We also provide evidence for a suppressive TAM subset associated with resistance to immune checkpoint inhibition in glioma, providing a rationale for combinatorial therapy strategies to overcome resistance to checkpoint blockade. Citation Format: Katrin Deumelandt, Jens Blobner, Jana K. Sonner, Mirco Friedrich, Edward Green, Michael O. Breckwoldt, Manuel Fischer, Jochen Meyer, Felix Sahm, Daniel Schrimpf, Andreas von Deimling, Michael Platten. Modeling response and resistance to immune checkpoint blockade in syngeneic mouse glioma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B075.

EGFL7 enhances surface expression of integrin α5β1 to promote angiogenesis in malignant brain tumors

Glioblastoma (GBM) is a typically lethal type of brain tumor with a median survival of 15 months postdiagnosis. This negative prognosis prompted the exploration of alternative treatment options. In particular, the reliance of GBM on angiogenesis triggered the development of anti‐VEGF (vascular endothelial growth factor) blocking antibodies such as bevacizumab. Although its application in human GBM only increased progression‐free periods but did not improve overall survival, physicians and researchers still utilize this treatment option due to the lack of adequate alternatives. In an attempt to improve the efficacy of anti‐VEGF treatment, we explored the role of the egfl7 gene in malignant glioma. We found that the encoded extracellular matrix protein epidermal growth factor‐like protein 7 (EGFL7) was secreted by glioma blood vessels but not glioma cells themselves, while no major role could be assigned to the parasitic miRNAs miR‐126/126*. EGFL7 expression promoted glioma growth in experimental glioma models in vivo and stimulated tumor vascularization. Mechanistically, this was mediated by an upregulation of integrin α5β1 on the cellular surface of endothelial cells, which enhanced fibronectin‐induced angiogenic sprouting. Glioma blood vessels that formed in vivo were more mature as determined by pericyte and smooth muscle cell coverage. Furthermore, these vessels were less leaky as measured by magnetic resonance imaging of extravasating contrast agent. EGFL7‐inhibition using a specific blocking antibody reduced the vascularization of experimental gliomas and increased the life span of treated animals, in particular in combination with anti‐VEGF and the chemotherapeutic agent temozolomide. Data allow for the conclusion that this combinatorial regimen may serve as a novel treatment option for GBM.

Modeling phenotypes of malignant gliomas.

Malignant gliomas are primary tumors of the central nervous system characterized by diffuse infiltration into the brain and a high recurrence rate. Advances in comprehensive genomic studies have provided unprecedented insight into the genetic and molecular heterogeneity of these tumors and refined our understanding of their evolution from low to high grade. However, similar levels of phenotypic characterization are indispensable to understanding the complexity of malignant gliomas. Experimental glioma models have also achieved great progress in recent years. Advances in transgenic technologies and cell culture have allowed the establishment of mouse models that mirror the human disease with increasing fidelity and which support single‐cell resolution for phenotypic analyses. Here we review the major types of preclinical glioma models, with an emphasis on how recent developments in experimental modeling have shed new light on two fundamental aspects of glioma phenotype, their cell of origin and their invasive potential.

Magnetic targeting with superparamagnetic iron oxide nanoparticles for in vivo glioma

AbstractThe purpose of this study was to review the use of the magnetic targeting technique, characterized by magnetic driving compounds based on superparamagnetic iron oxide nanoparticles (SPIONs), as drug delivery for a specific brain locus in gliomas. We reviewed a process mediated by the application of an external static magnetic field for targeting SPIONs in gliomas. A search of PubMed, Cochrane Library, Scopus, and Web of Science databases identified 228 studies, 23 of which were selected based on inclusion criteria and predetermined exclusion criteria. The articles were analyzed by physicochemical characteristics of SPIONs used, cell types used for tumor induction, characteristics of experimental glioma models, magnetic targeting technical parameters, and analysis method of process efficiency. The study shows the highlights and importance of magnetic targeting to optimize the magnetic targeting process as a therapeutic strategy for gliomas. Regardless of the intensity of the patterned magnetic field, the time of application of the field, and nanoparticle used (commercial or synthesized), all studies showed a vast advantage in the use of magnetic targeting, either alone or in combination with other techniques, for optimized glioma therapy. Therefore, this review elucidates the preclinical and therapeutic applications of magnetic targeting in glioma, an innovative nanobiotechnological method.

Convection-enhanced delivery of a hydrophilic nitrosourea ameliorates deficits and suppresses tumor growth in experimental spinal cord glioma models.

Convection-enhanced delivery (CED) is a technique allowing local infusion of therapeutic agents into the central nervous system, circumventing the blood-brain or spinal cord barrier. To evaluate the utility of nimustine hydrochloride (ACNU) CED in controlling tumor progression in an experimental spinal cord glioma model. Toxicity studies were performed in 42 rats following the administration of 4μl of ACNU CED into the mid-thoracic spinal cord at concentrations ranging from 0.1 to 10mg/ml. Behavioral analyses and histological evaluations were performed to assess ACNU toxicity in the spinal cord. A survival study was performed in 32 rats following the implantation of 9L cells into the T8 spinal cord. Seven days after the implantation, rats were assigned to four groups: ACNU CED (0.25mg/ml; n = 8); ACNU intravenous (i.v.) (0.4mg; n = 8); saline CED (n = 8); saline i.v. (n = 8). Hind limb movements were evaluated daily in all rats for 21days. Tumor sizes were measured histologically. The maximum tolerated ACNU concentration was 0.25mg/ml. Preservation of hind limb motor function and tumor growth suppression was observed in the ACNU CED (0.25mg/ml) and ACNU i.v. groups. Antitumor effects were more prominent in the ACNU CED group especially in behavioral analyses (P < 0.05; log-rank test). ACNU CED had efficacy in controlling tumor growth and preserving neurological function in an experimental spinal cord tumor model. ACNU CED can be a viable treatment option for spinal cord high-grade glioma.

Bioluminescence and MR Imaging of the Safety and Efficacy of Vascular Disruption in Gliomas.

The goal of the present study was to examine the safety and efficacy of the vascular disrupting agent (VDA) EPC2407 (Crolibulin™) in experimental glioma models using bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). Experimental imaging studies were performed in subcutaneous human U87 glioma xenografts and orthotopic murine gliomas established by intracranial implantation of luciferase-transfected glioma cells (GL261-luc). Correlative histopathology and long-term survival analysis was also performed. Treatment with EPC2407 decreased tumor perfusion and increased necrosis and tumor doubling times in subcutaneous U87 xenografts. Dynamic BLI and T1-weighted contrast-enhanced MRI showed reduction in blood flow of intracranial GL261-luc gliomas within a few hours of VDA treatment. T2-weighted MRI did not show any evidence of hemorrhaging or edema in uninvolved brain tissue of EPC2407-treated animals. A significant increase in median survival (p < 0.05) was observed in the orthotopic GL261-luc model following VDA treatment compared to untreated controls. We demonstrate, for the first time, the biological activity of EPC2407 in experimental gliomas. Further investigation into the potential of VDAs in combination with chemoradiation therapy against gliomas is warranted.

Advantages and disadvantages of experimental glioma models in animals

Advantages and disadvantages of experimental glioma models in animals

Decrease of VEGF-A in myeloid cells attenuates glioma progression and prolongs survival in an experimental glioma model.

Glioblastomas are highly vascularized tumors with a prominent infiltration of macrophages/microglia whose role in promoting glioma growth, invasion, and angiogenesis has not been fully elucidated. The contribution of myeloid-derived vascular endothelial growth factor (VEGF) to glioma growth was analyzed in vivo in a syngeneic intracranial GL261 glioma model using a Cre/loxP system to knock out the expression of VEGF-A in CD11b + myeloid cells. Changes in angiogenesis-related gene expression profile were analyzed in mutant bone marrow-derived (BMD) macrophages in vitro. Furthermore, we studied the influence of macrophages on GL261 growth, invasiveness, and protein expression profile of angiogenic molecules as well as the paracrine effect of mutant macrophages on angiogenesis in vitro. Myeloid cell-restricted VEGF-A deficiency leads to a growth delay of intracranial tumors and prolonged survival. The tumor vasculature in mutant mice was more regular, with increased pericyte coverage. Expression analysis revealed significant downregulation of VEGF-A and slight upregulation of TGFβ-1 in BMD macrophages from mutant mice. Endothelial tube formation was significantly decreased by conditioned media from mutant macrophages. The expression of angiogenesis-related proteins in GL261 glioma cells in co-culture experiments either with wild-type or mutant macrophages remained unchanged, indicating that effects observed in vivo are due to myeloid-derived VEGF-A deficiency. Our results highlight the importance of VEGF derived from tumor-infiltrating myeloid cells for initiating vascularization in gliomas. The combination of antiangiogenic agents with myeloid cell-targeting strategies might provide a new therapeutic approach for glioblastoma patients.

Evaluation of GX1 and RGD-GX1 peptides as new radiotracers for angiogenesis evaluation in experimental glioma models.

Gliomas are the most common type among all central nervous system tumors. The aggressiveness of gliomas is correlated with the level of angiogenesis and is often associated with prognosis. The aim of this study is to evaluate the novel GX1 peptide and the heterodimer RGD-GX1 radiolabeled with technetium-99m, for angiogenesis detection in glioma models. Radiolabeling and radiochemical controls were assessed for both radioconjugates. In vitro binding studies in glioma tumor cells were performed, as well as biodistribution in SCID mice bearing tumor cells, in order to evaluate the biological behavior and tumor uptake of the radiocomplexes. Blocking and imaging studies were also conducted. MicroSPECT/CT images were acquired in animals with experimentally implanted intracranial tumor. Open field activity was performed to evaluate behavior, as well as perfusion and histology analysis. The radiochemical purity of both radiotracers was greater than 96 %. In vitro binding studies revealed rather similar binding profi le for each molecule. The highest binding was for RGD-GX1 peptide at 120 min in U87MG cells (1.14 ± 0.35 %). Tumor uptake was also favorable for RGD-GX1 peptide in U87MG cells, reaching 2.96 ± 0.70 % at 1 h p.i. with 47 % of blocking. Imaging studies also indicated better visualization for RGD-GX1 peptide in U87MG cells. Behavior evaluation pointed brain damage and histology studies confirmed actual tumor in the uptake site. The results with the angiogenesis seeking molecule (99m)Tc-HYNIC-E-[c(RGDfk)-c(GX1)] were successful, and better than with (99m)Tc-HYNIC-PEG4-c(GX1). Future studies targeting angiogenesis in other glioma and nonglioma tumor models are recommended.

Blood vessels expressing CD90 in human and rat brain tumors.

Blood vessels in brain tumors, particularly glioblastomas, have been shown to express CD90. CD90(+) cells in and around blood vessels in cancers including brain tumors have been identified as endothelial cells, cancer stem cells, fibroblasts or pericytes. In this study, we aimed to determine the nature or type(s) of cells that express CD90 in human brain tumors as well as an experimental rat glioma model by double immunofluorescence staining. The majority of CD90(+) cells in human glioblastoma tissue expressed CD31, CD34 and von Willebrand factor, suggesting that they were endothelial cells. Vasculatures in a metastatic brain tumor and meningioma also expressed CD90. CD90(+) cells often formed glomeruloid structures, typical of angiogenesis in malignant tumors, not only in glioblastoma but also in metastatic tumors. Some cells in the middle and outer layers of the vasculatures expressed CD90. Similar results were obtained in the rat glioma model. There were cells expressing both α-smooth muscle actin and CD90 in the middle layer of blood vessels, indicating that smooth muscle cells and/or pericytes may express CD90. CD90(+) vasculatures were surrounded by tumor-associated macrophages (TAMs). Thus, in addition to endothelial cells, some other types of cells, such as smooth muscle cells, pericytes and fibroblasts constituting the vasculature walls in brain tumors expressed CD90. Because CD90 has been shown to interact with integrins expressed by circulating monocytes, CD90 might be involved in angiogenesis through recruitment and functional regulation of TAMs in tumors. CD90(+) vasculatures may also interact with tumor cells through interactions with integrins. Because CD90 was not expressed by vasculatures in normal brain tissue, it might be a possible therapeutic target to suppress angiogenesis and tumor growth.