Lentiviral-induced high-grade gliomas in rats: the effects of PDGFB, HRAS-G12V, AKT, and IDH1-R132H.

In human gliomas, the RTK/RAS/PI(3)K signaling pathway is nearly always altered. We present a model of experimental gliomagenesis that elucidates the contributions of genes involved in this pathway (PDGF-B ligand, HRAS-G12V, and AKT). We also examine the effect on gliomagenesis by the potential modifier gene, IDH1-R132H. Injections of lentiviral-encoded oncogenes induce de novo gliomas of varying penetrance, tumor progression, and histological grade depending on the specific oncogenes used. Our model mimics hallmark histological structures of high-grade glioma, such as pseudopalisades, glomeruloid microvascular proliferation, and diffuse tumor invasion. We use our model of gliomagenesis to test the efficacy of an experimental brain tumor gene therapy. Our model allowed us to test the contributions of oncogenes in the RTK/RAS/PI(3)K pathway, and their potential modification by over-expression of mutated IDH1, in glioma development and progression in rats. Our model constitutes a clinically relevant system to study gliomagenesis, the effects of modifier genes, and the efficacy of experimental therapeutics.

Temozolomide does not impair gene therapy-mediated antitumor immunity in syngeneic brain tumor models.

PURPOSE: Glioblastoma multiforme is the most common primary brain cancer in adults. Chemotherapy with temozolomide (TMZ) significantly prolongs the survival of patients with glioblastoma multiforme. However, the three-year survival is still approximately 5%. Herein, we combined intratumoral administration of an adenoviral vector expressing Flt3L (Ad-Flt3L) with systemic temozolomide to assess its impact on therapeutic efficacy. EXPERIMENTAL DESIGN: Wild-type or immunodeficient mice bearing intracranial glioblastoma multiforme or metastatic melanoma were treated with an intratumoral injection of Ad-Flt3L alone or in combination with the conditionally cytotoxic enzyme thymidine kinase (Ad-TK), followed by systemic administration of ganciclovir and temozolomide. We monitored survival and measured the tumor-infiltrating immune cells. RESULTS: Although treatment with temozolomide alone led to a small improvement in median survival, when used in combination with gene therapy-mediated immunotherapy, it significantly increased the survival of tumor-bearing mice. The antitumor effect was further enhanced by concomitant intratumoral administration of Ad-TK, leading to 50% to 70% long-term survival in all tumor models. Although temozolomide reduced the content of T cells in the tumor, this did not affect the therapeutic efficacy. The antitumor effect of Ad-Flt3L+Ad-TK+TMZ required an intact immune system because the treatment failed when administered to knock out mice that lacked lymphocytes or dendritic cells. CONCLUSIONS: Our results challenge the notion that chemotherapy leads to a state of immune-suppression which impairs the ability of the immune system to mount an effective antitumor response. Our work indicates that temozolomide does not inhibit antitumor immunity and supports its clinical implementation in combination with immune-mediated therapies.

Immune-mediated loss of transgene expression from virally transduced brain cells is irreversible, mediated by IFNγ, perforin, and TNFα, and due to the elimination of transduced cells.

The adaptive immune response to viral vectors reduces vector-mediated transgene expression from the brain. It is unknown, however, whether this loss is caused by functional downregulation of transgene expression or death of transduced cells. Herein, we demonstrate that during the elimination of transgene expression, the brain becomes infiltrated with CD4(+) and CD8(+) T cells and that these T cells are necessary for transgene elimination. Further, the loss of transgene-expressing brain cells fails to occur in the absence of IFNγ, perforin, and TNFα receptor. Two methods to induce severe immune suppression in immunized animals also fail to restitute transgene expression, demonstrating the irreversibility of this process. The need for cytotoxic molecules and the irreversibility of the reduction in transgene expression suggested to us that elimination of transduced cells is responsible for the loss of transgene expression. A new experimental paradigm that discriminates between downregulation of transgene expression and the elimination of transduced cells demonstrates that transduced cells are lost from the brain upon the induction of a specific antiviral immune response. We conclude that the anti-adenoviral immune response reduces transgene expression in the brain through loss of transduced cells.

B cells are critical to T-cell-mediated antitumor immunity induced by a combined immune-stimulatory/conditionally cytotoxic therapy for glioblastoma.

We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6(-/-) mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6(-/-) mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression.

Identification and visualization of CD8+ T cell mediated IFN-γ signaling in target cells during an antiviral immune response in the brain.

CD8(+) T cells infiltrate the brain during an anti-viral immune response. Within the brain CD8(+) T cells recognize cells expressing target antigens, become activated, and secrete IFNγ. However, there are no methods to recognize individual cells that respond to IFNγ. Using a model that studies the effects of the systemic anti-adenoviral immune response upon brain cells infected with an adenoviral vector in mice, we describe a method that identifies individual cells that respond to IFNγ. To identify individual mouse brain cells that respond to IFNγ we constructed a series of adenoviral vectors that contain a transcriptional response element that is selectively activated by IFNγ signaling, the gamma-activated site (GAS) promoter element; the GAS element drives expression of a transgene, Cre recombinase (Ad-GAS-Cre). Upon binding of IFNγ to its receptor, the intracellular signaling cascade activates the GAS promoter, which drives expression of the transgene Cre recombinase. We demonstrate that upon activation of a systemic immune response against adenovirus, CD8(+) T cells infiltrate the brain, interact with target cells, and cause an increase in the number of cells expressing Cre recombinase. This method can be used to identify, study, and eventually determine the long term fate of infected brain cells that are specifically targeted by IFNγ. The significance of this method is that it will allow to characterize the networks in the brain that respond to the specific secretion of IFNγ by anti-viral CD8(+) T cells that infiltrate the brain. This will allow novel insights into the cellular and molecular responses underlying brain immune responses.

Targeted toxins for glioblastoma multiforme: pre-clinical studies and clinical implementation.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. GBM is very aggressive due to its poor cellular differentiation and invasiveness, which makes complete surgical resection virtually impossible. Therefore, GBM's invasive nature as well as its intrinsic resistance to current treatment modalities makes it a unique therapeutic challenge. Extensive examination of human GBM specimens has uncovered that these tumors overexpress a variety of receptors that are virtually absent in the surrounding non-neoplastic brain. Human GBMs overexpress receptors for cytokines, growth factors, ephrins, urokinase-type plasminogen activator (uPA), and transferrin, which can be targeted with high specificity by linking their ligands with highly cytotoxic molecules, such as Diptheria toxin and Pseudomonas exotoxin A. We review the preclinical development and clinical translation of targeted toxins for GBM. In view of the clinical experience, we conclude that although these are very promising therapeutic modalities for GBM patients, efforts should be focused on improving the delivery systems utilized in order to achieve better distribution of the immuno-toxins in the tumor/resection cavity. Delivery of targeted toxins using viral vectors would also benefit enormously from improved strategies for local delivery.

Gene therapy and targeted toxins for glioma.

The most common primary brain tumor in adults is glioblastoma. These tumors are highly invasive and aggressive with a mean survival time of 15-18 months from diagnosis to death. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma. As such, glioma is an attractive target for developing novel therapeutic approaches utilizing gene therapy. This review will examine the available preclinical models for glioma including xenographs, syngeneic and genetic models. Several promising therapeutic targets are currently being pursued in pre-clinical investigations. These targets will be reviewed by mechanism of action, i.e., conditional cytotoxic, targeted toxins, oncolytic viruses, tumor suppressors/oncogenes, and immune stimulatory approaches. Preclinical gene therapy paradigms aim to determine which strategies will provide rapid tumor regression and long-term protection from recurrence. While a wide range of potential targets are being investigated preclinically, only the most efficacious are further transitioned into clinical trial paradigms. Clinical trials reported to date are summarized including results from conditionally cytotoxic, targeted toxins, oncolytic viruses and oncogene targeting approaches. Clinical trial results have not been as robust as preclinical models predicted; this could be due to the limitations of the GBM models employed. Once this is addressed, and we develop effective gene therapies in models that better replicate the clinical scenario, gene therapy will provide a powerful approach to treat and manage brain tumors.

Gene therapy and virotherapy: novel therapeutic approaches for brain tumors.

Glioblastoma multiforme (GBM) is a deadly primary brain tumor in adults, with a median survival of ~12-18 months post-diagnosis. Despite recent advances in conventional therapeutic approaches, only modest improvements in median survival have been achieved; GBM usually recurs within 12 months post-resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are desperately needed. Our group and others are pursuing virotherapy and gene therapy strategies for the treatment of GBM. In this review, we will discuss various virotherapy and gene therapy approaches for GBM currently under pre-clinical and clinical evaluation including direct or conditional cytotoxic, and/or immunostimulatory approaches. We also discuss cutting-edge technologies for drug/gene delivery and targeting brain tumors, including the use of stem cells as delivery platforms, the use of targeted immunotoxins, and the therapeutic potential of using GBM microvesicles to deliver therapeutic siRNAs or virotherapies. Finally, various animal models available to test novel GBM therapies are discussed.

Exogenous fms-like tyrosine kinase 3 ligand overrides brain immune privilege and facilitates recognition of a neo-antigen without causing autoimmune neuropathology.

Soluble antigens diffuse out of the brain and can thus stimulate a systemic immune response, whereas particulate antigens (from infectious agents or tumor cells) remain within brain tissue, thus failing to stimulate a systemic immune response. Immune privilege describes how the immune system responds to particulate antigens localized selectively within the brain parenchyma. We believe this immune privilege is caused by the absence of antigen presenting dendritic cells from the brain. We tested the prediction that expression of fms-like tyrosine kinase ligand 3 (Flt3L) in the brain will recruit dendritic cells and induce a systemic immune response against exogenous influenza hemagglutinin in BALB/c mice. Coexpression of Flt3L with HA in the brain parenchyma induced a robust systemic anti-HA immune response, and a small response against myelin basic protein and proteolipid protein epitopes. Depletion of CD4(+)CD25+ regulatory T cells (Tregs) enhanced both responses. To investigate the autoimmune impact of these immune responses, we characterized the neuropathological and behavioral consequences of intraparenchymal injections of Flt3L and HA in BALB/c and C57BL/6 mice. T cell infiltration in the forebrain was time and strain dependent, and increased in animals treated with Flt3L and depleted of Tregs; however, we failed to detect widespread defects in myelination throughout the forebrain or spinal cord. Results of behavioral tests were all normal. These results demonstrate that Flt3L overcomes the brain's immune privilege, and supports the clinical development of Flt3L as an adjuvant to stimulate clinically effective immune responses against brain neo-antigens, for example, those associated with brain tumors.