Mouse Model Of High-grade Glioma Research Articles

IMMU-16. INTRA-TUMOURAL IL-12 DELIVERY ENABLES CAR T-CELL IMMUNOTHERAPY FOR HIGH-GRADE GLIOMA

Treatment with T-cells redirected to tumour specificity with a chimeric antigen receptor (CAR) may be well suited to treat intracranial tumours due to the ability of T-cells to access the central nervous system and migrate to infiltrative sites of disease. In adult glioblastoma, a case report of local and distant eradication of intracranial and spinal tumour deposits following intraventricular infusion of IL13Ra2-CAR T-cells indicates the potential of this approach. However, in contrast to the sustained complete remissions observed in haematological malignancies, in the majority of patients with glioblastoma CAR T-cell therapy has not resulted in clinical benefit. Tumour heterogeneity and the highly immune inhibitory tumour microenvironment (TME) are likely key barriers to achieving durable anti-tumour immunity. Here use intra-tumoural administration of IL-12 to enable CAR T-cell immunity. We employed CAR-T cells targeting the tumour-specific epidermal growth factor variant III (EGFRvIII). In an immunocompetent orthotopic mouse model of high-grade glioma, we show that CAR-T cells alone failed to control fully established tumour, but when combined with a single, locally delivered dose of IL-12, durable antitumor responses were achieved. IL-12 not only boosted cytotoxicity of CAR T-cells, but also reshaped the TME driving increased infiltration of proinflammatory CD4+ T-cells, decreased numbers of regulatory T-cells (Tregs) and activation of the myeloid compartment. Critically, immunotherapy enabling benefits of IL-12 were achieved with minimal systemic effects. Our findings show that local delivery of IL-12 is an effective adjuvant for CAR-T cell therapy for high-grade glioma. Assessment of application in high-risk childhood brain tumours is ongoing.

Everolimus improves the efficacy of dasatinib in PDGFRα-driven glioma.

Pediatric and adult high-grade gliomas (HGGs) frequently harbor PDGFRA alterations. We hypothesized that cotreatment with everolimus may improve the efficacy of dasatinib in PDGFRα-driven glioma through combinatorial synergism and increased tumor accumulation of dasatinib. We performed dose-response, synergism, P-glycoprotein inhibition, and pharmacokinetic studies in in vitro and in vivo human and mouse models of HGG. Six patients with recurrent PDGFRα-driven glioma were treated with dasatinib and everolimus. We found that dasatinib effectively inhibited the proliferation of mouse and human primary HGG cells with a variety of PDGFRA alterations. Dasatinib exhibited synergy with everolimus in the treatment of HGG cells at low nanomolar concentrations of both agents, with a reduction in mTOR signaling that persisted after dasatinib treatment alone. Prolonged exposure to everolimus significantly improved the CNS retention of dasatinib and extended the survival of PPK tumor-bearing mice (mutant TP53, mutant PDGFRA, H3K27M). Six pediatric patients with glioma tolerated this combination without significant adverse events, and 4 patients with recurrent disease (n = 4) had a median overall survival of 8.5 months. Our results show that the efficacy of dasatinib treatment of PDGFRα-driven HGG was enhanced with everolimus and suggest a promising route for improving targeted therapy for this patient population.

Voluntary Physical Exercise Reduces Motor Dysfunction and Hampers Tumor Cell Proliferation in a Mouse Model of Glioma.

Currently, high-grade gliomas are the most difficult brain cancers to treat and all the approved experimental treatments do not offer long-term benefits regarding symptom improvement. Epidemiological studies indicate that exercise decreases the risk of brain cancer mortality, but a direct relationship between physical exercise and glioma progression has not been established so far. Here, we exploited a mouse model of high-grade glioma to directly test the impact of voluntary physical exercise on the tumor proliferation and motor capabilities of affected animals. We report that exposing symptomatic, glioma-bearing mice to running wheels (i) reduced the proliferation rate of tumors implanted in the motor cortex and (ii) delayed glioma-induced motor dysfunction. Thus, voluntary physical exercise might represent a supportive intervention that complements existing neuro-oncologic therapies, contributing to the preservation of functional motor ability and counteracting the detrimental effects of glioma on behavioral output.

Ex vivo multi-electrode analysis reveals spatiotemporal dynamics of ictal behavior at the infiltrated margin of glioma

The purpose of this study is to develop a platform in which the cellular and molecular underpinnings of chronic focal neocortical lesional epilepsy can be explored and use it to characterize seizure-like events (SLEs) in an ex vivo model of infiltrating high-grade glioma. Microelectrode arrays were used to study electrophysiologic changes in ex vivo acute brain slices from a PTEN/p53 deleted, PDGF-B driven mouse model of high-grade glioma. Electrode locations were co-registered to the underlying histology to ascertain the influence of the varying histologic landscape on the observed electrophysiologic changes. Peritumoral, infiltrated, and tumor sites were sampled in tumor-bearing slices. Following the addition of zero Mg2+ solution, all three histologic regions in tumor-bearing slices showed significantly greater increases in firing rates when compared to the control sites. Tumor-bearing slices demonstrated increased proclivity for SLEs, with 40 events in tumor-bearing slices and 5 events in control slices (p-value = .0105). Observed SLEs were characterized by either low voltage fast (LVF) onset patterns or short bursts of repetitive widespread, high amplitude low frequency discharges. Seizure foci comprised areas from all three histologic regions. The onset electrode was found to be at the infiltrated margin in 50% of cases and in the peritumoral region in 36.9% of cases. These findings reveal a landscape of histopathologic and electrophysiologic alterations associated with ictogenesis and spread of tumor-associated seizures.

CADD-17. CT-179: AN INHIBITOR OF THE OLIG2 TRANSCRIPTION FACTOR WITH POTENT ANTI-TUMOUR ACTIVITY IN BRAIN CANCER

High Grade Glioma (HGG) is incurable and has a median survival of less than 5% at five years, highlighting a desperate need for new therapeutic strategies. OLIG2 is a basic helix-loop-helix (bHLH) transcription factor that is expressed in neural progenitor cells during embryonic development where it sustains their replication-competent state and regulates their oligodendrocyte and motor neuron multi-lineage potential. In HGG, OLIG2 is re-expressed at high levels and drives an oncogenic program that leads to dysregulation of the cell cycle and subsequent gliomagenesis. This central role for OLIG2 in HGG initiation and growth, along with its low expression in normal tissues, identifies OLIG2 as a target for HGG therapy. We report the characterisation of an orally bioavailable small-molecule OLIG2 inhibitor, CT-179, the first bHLH transcription factor targeting drug developed for the treatment of cancer. The drug is well tolerated and easily penetrates the blood brain barrier, where it reduces brain tumour burden in orthotopic mouse and zebrafish avatar models. Mechanistically, CT-179 displayed nanomolar anti-proliferative activity and induced significant apoptosis mediated through disruption of the cell cycle that resulted in mitotic catastrophe at prometaphase. CT-179 showed enhanced anti-tumour activity in mouse models of HGG when used in combination with standard of care radiotherapy and temozolomide. These studies demonstrate that the pharmacological inhibition of OLIG2 is an effective treatment strategy for HGG that warrants rapid translation into the clinic.

Ultra-High Mass Resolution MALDI Imaging Mass Spectrometry of Proteins and Metabolites in a Mouse Model of Glioblastoma

MALDI mass spectrometry imaging is able to simultaneously determine the spatial distribution of hundreds of molecules directly from tissue sections, without labeling and without prior knowledge. Ultra-high mass resolution measurements based on Fourier-transform mass spectrometry have been utilized to resolve isobaric lipids, metabolites and tryptic peptides. Here we demonstrate the potential of 15T MALDI-FTICR MSI for molecular pathology in a mouse model of high-grade glioma. The high mass accuracy and resolving power of high field FTICR MSI enabled tumor specific proteoforms, and tumor-specific proteins with overlapping and isobaric isotopic distributions to be clearly resolved. The protein ions detected by MALDI MSI were assigned to proteins identified by region-specific microproteomics (0.8 mm2 regions isolated using laser capture microdissection) on the basis of exact mass and isotopic distribution. These label free quantitative experiments also confirmed the protein expression changes observed by MALDI MSI and revealed changes in key metabolic proteins, which were supported by in-situ metabolite MALDI MSI.

Abstract 1367: Intra-cerebral pharmacokinetic monitoring of a tyrosine kinase inhibitor (theranostic 18[F]-PET/NIRF labeled dasatinib) delivered via convection-enhanced delivery

Abstract Introduction: Convection enhanced delivery (CED) has been recently explored as an advantageous therapeutic strategy for central nervous system (CNS) tumors. One current limitation is the inability to quantitatively monitor distribution of chemotherapeutic agents. The use of surrogate tracers probably underestimates differences in distribution and clearance owing to discordant features between contrast molecules and therapeutic agents, including bioactivity, degradation, conductivity, and diffusivity. Ideally, direct labelling of a therapeutic compound would eliminate these concerns and afford a noninvasive method to monitor critical pharmacokinetic information. This ability would pave the way for designing infusion parameters and drug schedules that are expected to be unique for CED-based therapy. Methods: The small molecule kinase inhibitor dasatinib was modified with a dual-probe technology that utilizes a boronate trap to conjugate 18[F] with a near-infrared fluorophore into a single molecule, 1, allowing for visualization by positron emission tomography (PET) and near-infrared fluorescence (NIRF). The novel boronate trap allows for direct conjugation of 18[F] with minimal disruption of dasatinib's mechanism of action, resulting in a small, versatile probe with potential for other clinically relevant targets. Results: The modified drug was first tested in vitro in a PDGF-B driven p53 deficient DIPG tumor line from Nestin tv-a; p53 floxed mouse. 1 was shown to enter cells on fluorescence microscopy and inhibit cell proliferation. Antagonist drug potency of 1 as evaluated on pontine glioma in ATP-dependent luminescent cell viability, cell-permeant calcein AM, and an MTS cell proliferation assays show that the IC50, of 1 is ∼10nM. In addition, infusions of 1 were performed via CED and intravenous systemic delivery in a RCAS/tv-a PDGF-B driven with p53 deficiency Ntv-a mouse model of high-grade glioma. Compared to systemic delivery, CED was shown to be roughly 230 times more effective in delivery of the infusion to the tumor site. Maximal glioma delivery occurred at 115 min post a 20 min infusion, with clearance occurring with a half-life of 45 min after maximum delivery. Conclusion: Dasatinib was modified to give 1, an agent that can be imaged non-invasively by fluorescence and positron emission tomography (PET). The modified dasatinib showed little modification in biologic activity when compared to unmodified dasatinib and exhibited cytotoxic effects in vitro in glioma cell culture. CED shows a clear benefit to intravenous delivery when the target site is in the CNS. The dual-label probe technology's versatility and minimal biologic profile lends itself to various clinical uses, including the study of different therapeutic agents and delivery routes without the need to rely on surrogate tracers. Citation Format: Melinda Wang, Zhiping Zhou, Hari Krishna Kommidi, Melanie Schweitzer, Mark Chan, Yue Linda Wu, Ranjodh Singh, Richard Ting, Mark M. Souweidane. Intra-cerebral pharmacokinetic monitoring of a tyrosine kinase inhibitor (theranostic 18[F]-PET/NIRF labeled dasatinib) delivered via convection-enhanced delivery. [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 1367.

Dynamics of Circulating γδ T Cell Activity in an Immunocompetent Mouse Model of High-Grade Glioma.

Human γδ T cells are potent effectors against glioma cell lines in vitro and in human/mouse xenograft models of glioblastoma, however, this effect has not been investigated in an immunocompetent mouse model. In this report, we established GL261 intracranial gliomas in syngeneic WT C57BL/6 mice and measured circulating γδ T cell count, phenotype, Vγ/Vδ repertoire, tumor histopathology, NKG2D ligands expression, and T cell invasion at day 10–12 post-injection and at end stage. Circulating γδ T cells transiently increased and upregulated Annexin V expression at post-tumor day 10–12 followed by a dramatic decline in γδ T cell count at end stage. T cell receptor repertoire showed no changes in Vγ1, Vγ4, Vγ7 or Vδ1 subsets from controls at post-tumor day 10–12 or at end stage except for an end-stage increase in the Vδ4 population. Approximately 12% of γδ T cells produced IFN-γ. IL-17 and IL-4 producing γδ T cells were not detected. Tumor progression was the same in TCRδ-/- C57BL/6 mice as that observed in WT mice, suggesting that γδ T cells exerted neither a regulatory nor a sustainable cytotoxic effect on the tumor. WT mice that received an intracranial injection of γδ T cells 15m following tumor placement showed evidence of local tumor growth inhibition but this was insufficient to confer a survival advantage over untreated controls. Taken together, our findings suggest that an early nonspecific proliferation of γδ T cells followed by their depletion occurs in mice implanted with syngeneic GL261 gliomas. The mechanism by which γδ T cell expansion occurs remains a subject for further investigation of the mechanisms responsible for this immune response in the setting of high-grade glioma.

Irradiation of freeze-thaw lysate used to pulse dendritic cells further increases antitumor immunity in a mouse model of high-grade glioma

The prognosis of high-grade glioma is poor despite of advanced neurosurgery, radio-and chemotherapy. Dendritic cell (DC)-based immunotherapy has emerged as a promising and feasible treatment approach because of it's high degree of selectivity and the ability to induce a memory response. In our research group we are investigating the efficacy of vaccinating both primary (HGG-2006 trial) and relapsed HGG patients (HGG-IMMUNO-2003 trial) with DC pulsed with autologous whole tumor lysate. Both trials showed a better overall survival compared to historical controls. Currently we are running a placebo-controlled randomized phase IIb clinical trial (HGG-2010) in primary HGG patients.

Irradiation of necrotic tumor cells used to pulse dendritic cells (DCs) potentiates DC vaccine-induced anti-tumor immunity in a mouse model of high-grade glioma

Meeting abstracts The prognosis of high-grade glioma (HGG) is poor despite advancements in neurosurgery, radio-and chemotherapy. DC-based immunotherapy has emerged as a promising and feasible treatment approach due to its high degree of selectivity and its ability to induce an antigen-specific

Abstract 2367A: Understanding the role of stem-like tumor cells in mouse models of high-grade gliomas

Abstract High-grade gliomas (WHO grade III-IV) are typified by poor therapeutic response and rapid lethality. Since the discovery of glioma cells with properties reminiscent of neural stem cells, there has been a great deal of interest in understanding their contributes to tumor growth and therapeutic resistance. Self-renewal is the defining characteristic of both normal and cancer stem cells, and it has been hypothesized that glioma cells with self-renewal capacity contribute disproportionately to tumor growth compared to other, more differentiated lineages. However, analyses are complicated by the lack of reliable markers to definitively identify and functionally characterize each distinct cell lineage. In the normal neurogenic niche, ID (Inhibitor of DNA-binding) genes regulate self-renewal and multipotency of adult neural stem cells. Here, using mouse models of high-grade glioma driven by PDGF- or mutKRAS, we show that high Id1 expression (Id1high) identifies self-renewing, multipotent tumor cells. By contrast, low Id1 expression (Id1low) identifies progenitor-like cells with proliferative potential but limited self-renewal capacity. While Id1high cells generate tumors upon orthotopic transplantation, Id1low cells do so with shorter latency and higher penetrance. Further, eliminating tumor cell self-renewal through targeted deletion of ID genes has modest effects on overall survival, while knockdown of the bHLH transcription factor, Olig2, within the Id1low population, results in a significant survival benefit. Together, these data highlight an important distinction between self-renewal capacity and tumor growth potential in two mouse models of gliomagenesis, underscoring the importance of non-stem cell lineages in disease progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2367A. doi:1538-7445.AM2012-2367A

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Self-Renewal Does Not Predict Tumor Growth Potential in Mouse Models of High-Grade Glioma

Within high-grade gliomas, the precise identities and functional roles of stem-like cells remain unclear. In the normal neurogenic niche, ID (Inhibitor of DNA-binding) genes maintain self-renewal and multipotency of adult neural stem cells. Using PDGF- and KRAS-driven murine models of gliomagenesis, we show that high Id1 expression (Id1(high)) identifies tumor cells with high self-renewal capacity, while low Id1 expression (Id1(low)) identifies tumor cells with proliferative potential but limited self-renewal capacity. Surprisingly, Id1(low) cells generate tumors more rapidly and with higher penetrance than Id1(high) cells. Further, eliminating tumor cell self-renewal through deletion of Id1 has modest effects on animal survival, while knockdown of Olig2 within Id1(low) cells has a significant survival benefit, underscoring the importance of non-self-renewing lineages in disease progression.

Abstract 4187: A rapid and robust transgenic high-grade glioma mouse model for therapy-intervention studies

Abstract High-grade glioma is a devastating and uniformly fatal disease for which better therapy is urgently needed. Glioma cells extensively infiltrate into surrounding pivotal brain structures, thereby rendering current treatments largely ineffective. Evaluation of novel therapies requires the availability of appropriate glioma mouse models. We here present a novel transgenic mouse model of high-grade glioma that can be conveniently used for testing therapy-intervention strategies. High-grade gliomas were induced by stereotactic intracranial injection of self-deleting lentiviral GFAP-Cre or CMV-Cre vectors into adult immune-competent compound LoxP-conditional mice, resulting in localized expression of K-Rasv12 in conjunction with loss of p16Ink4a/p19Arf with or without concomitant loss of p53 or Pten. Tumors have been characterized by histology, immune-histochemistry and magnetic resonance imaging and reproduced many of the features that are characteristic for human high-grade gliomas, including invasiveness and blood-brain barrier functionality. We are currently assessing which molecular alterations in cell signaling pathways have occurred. Especially CMV-Cre injection into p53;Ink4a/Arf;K-Rasv12 mice resulted in grade IV (glioblastoma multiforme) with a short tumor latency (2-3 weeks) and full penetrance. Early detection and follow-up of tumor growth can be accomplished conveniently by non-invasive bioluminescence imaging and the practical utility for therapy-intervention has been demonstrated in a study with temozolomide. Temozolomide delayed tumor progression modestly but significantly in line with what is observed in patients. Importantly, this novel model can be used with almost the same convenience as traditional xenograft models, thus allowing the implementation of realistic high grade glioma models at the forefront of preclinical evaluation. We expect that this model will be particularly useful to assess the potential clinical utility of agents that may augment temozolomide cytotoxicity, such as PARP-inhibitors or Wee1 kinase inhibitors, but also for agents targeting activated RAS and/or PI3K-Akt-mTor signaling pathways. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4187.