Giulia Fulci

Cyclophosphamide enhances glioma virotherapy by inhibiting innate immune responses.

Clinical trials are testing oncolytic viruses (OVs) as therapies for cancer. We have shown that animals that have brain tumors and are treated with a herpes simplex virus (HSV)-derived OV live significantly longer when cyclophosphamide (CPA) is preadministered. Here, we explore the mechanisms behind this finding. In a syngeneic rat glioma model, intratumoral HSV administration is associated with rapid increase of natural killer cells, microglia/macrophages (CD68+ and CD163+), and IFN-γ. Pretreatment with CPA enhances HSV replication and oncolysis and reduces an HSV-mediated increase in CD68+ and CD163+ cells and intratumoral IFN-γ. Molecular imaging shows CPA pretreatment to inhibit HSV-induced infiltration of tumor-associated phagocytic cells. Our results reveal molecular and cellular mechanisms that inhibit intratumoral spread of HSV and suggest a therapeutic path for improving the efficacy of virotherapy as a treatment for cancer.

p53 gene mutation and ink4a-arf deletion appear to be two mutually exclusive events in human glioblastoma.

P16 and P14ARF are two tumor suppressors encoded by the locus ink4a-arf which is frequently deleted in human tumors. Recent experiments performed with mouse embryonic fibroblasts have shown that P14ARF is an upstream regulator of the P53 pathway. This raises the question as to whether in human tumors the loss of p14arf and mutation of p53 are mutually exclusive events which segregate with genetic alterations at other loci. To examine this question we performed a multigenic analysis on 29 gliomas. We analysed p53 and p14arf in relation with five other genetic loci encoding the most frequently mutated genes in human gliomas: cdkn2a, mdm2, egfr, pten and the chromosomal regions 10q23.3 and 10q25-26. Our study shows for the first time that p53 mutations and p14arf deletions appear mutually exclusive in human glioblastoma, suggesting that they may be functionally redundant in glioma tumorigenesis. The P53 pathway is, therefore, disrupted in 81.8% of malignant gliomas (WHO grades III and IV), either by mutation of the p53 gene (31.8%) or by p14arf deletion (54.5%). These tumors further showed MDM2 overexpression (9.1%), egfr oncogene amplification/egfr overexpression (50%), pten mutations (27.3%) and loss of heterozygosity (LOH) at the chromosomal regions 10q23.3 (86.4%) and 10q25-26 (100%). These alterations did not segregate with p53 mutations or p14arf deletions, while p14arf and cdkn2a were always deleted.

Glioma Virotherapy: Effects of Innate Immune Suppression and Increased Viral Replication Capacity

Oncolytic viruses are genetically altered replication-competent viruses that infect, and reproduce in, cancer cells but do not harm normal cells. On lysis of the infected cells, the newly formed viruses burst out and infect other tumor cells. Experiments with injecting mutant herpes simplex virus 1 (hrR3) into glioma implanted in brains of rats show lack of efficacy in eradicating the cancer. This failure is attributed to interference by the immune system. Initial pretreatment with immunosuppressive agent cyclophosphamide reduces the percentage of immune cells. We introduce a mathematical model and use it to determine how different protocols of cyclophosphamide treatment and how increased burst size of the mutated virus will affect the growth of the cancer. One of our conclusions is that the diameter of the cancer will decrease from 4 mm to eventually 1 mm if the burst size of the virus is triple that which is currently available. The effect of repeated cyclophosphamide treatment is to maintain a low density of uninfected cells in the tumor, thus reducing the probability of migration of tumor cells to other locations in the brain.

Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma.

Altered expression of antiviral cytokine mRNAs associated with cyclophosphamide's enhancement of viral oncolysis

Oncolytic viruses (OVs) are being used as anticancer agents in preclinical and clinical trials. Propagation of OVs inside infected tumors is critical to their efficacy and is mediated by the productive generation of progeny OVs within infected tumor cells. In turn, this progeny can spread the infection to other tumor cells in successive rounds of oncolysis. Previously, we had found that, in rats, cyclophosphamide (CPA) pretreatment increased infection of brain tumors by an intra-arterially administered herpes simplex virus type 1 OV, because it inhibited activation of complement responses, mediated by innate IgM. We also have previously shown that other pharmacologic inhibitors of complement, such as cobra venom factor (CVF), allowed for increased infection. However, in these studies, further inhibition of complement responses by CVF did not result in additional infection of brain tumor cells or in propagation of OV to surrounding tumor cells. In this study, we sought to determine if CPA did lead to increased infection/propagation from initially infected tumor cells. Unlike our results with CVF, we find that CPA administration does result in a time-dependent increase in infection of tumor cells, suggestive of increased propagation, in both syngeneic and athymic models of brain tumors. This increase was due to increased survival of OV within infected tumors and brain surrounding tumors. CPAs effect was not due to a direct enhancement of viral replication in tumor cells, rather was associated with its immunosuppressive effects. RT-PCR analysis revealed that CPA administration resulted in impaired mRNA production by peripheral blood mononuclear cells (PBMCs) of several cytokines (interferons α/β, interferon γ, TNFα, IL-15, and IL-18) with anti-HSV function. These findings suggest that the CPA-mediated facilitation of OV intraneoplastic propagation is associated with a general decrease of antiviral cytokines mRNAs in PBMCs. These findings not only suggest a potential benefit for the addition of transient immunosuppression in clinical applications of oncolytic HSV therapy, but also suggest that innate immunomodulatory pathways may be amenable to manipulation, in order to increase OV propagation and survival within infected tumors.

p53 and brain tumors: from gene mutations to gene therapy.

The p53 tumor suppressor gene (TP53) is the most frequently altered gene in human cancer and is also found mutated in several types of brain tumors. Loss of p53 function plays a central role in the development of cancer. The characterization of the biochemical pathways by which p53 alteration triggers tumorigenesis is the foundation for the design of novel therapeutic approaches.

Replicative oncolytic herpes simplex viruses in combination cancer therapies.

Viruses that kill the host cell during their replication cycle have attracted much interest for the specific killing of tumor cells and this oncolytic virotherapy is being evaluated in clinical trials. The rationale for using replicative oncolytic viruses is that viral replication in infected tumor cells will permit in situ viral multiplication and spread of viral infection throughout the tumor mass thus overcoming the delivery problems of gene therapy. Improved understanding of the life cycle of viruses has evidenced multiple interactions between viral and cellular gene products, which have evolved to maximize the ability of viruses to infect and multiply within cells. Differences in viral-cell interactions between normal and tumor cells have emerged that have led to the design of a number of genetically engineered viral vectors that selectively kill tumor cells while sparing normal cells. These viruses have undergone further modifications to carry adjunct therapy genes to increase their anti-cancer abilities. Since these viruses kill cells by oncolytic mechanisms differing from standard anticancer therapies, there is an opportunity that synergistic interactions with other therapies might be found with the use of combination therapy. In this review, we focus on the oncolytic Herpes Simplex Virus-1 (HSV-1) vectors that have been examined in preclinical and clinical cancer models and their use in combination with chemo-, radio-, and gene therapies.

Viral therapy for glioblastoma.

Malignant gliomas remain amongst the most difficult cancer to treat. Viral-based gene therapies have been employed for the last decade in preclinical and clinical modes as a novel treatment modality. In this review, such therapies are summarized. The overwhelming majority of clinical studies point one to conclude that methodologies that will increase tumor infection/transduction will lead to enhanced therapeutic results.

Bevacizumab With Angiostatin-armed oHSV Increases Antiangiogenesis and Decreases Bevacizumab-induced Invasion in U87 Glioma

Bevacizumab (BEV) is an antiangiogenic drug approved for glioblastoma (GBM) treatment. However, it does not increase survival and is associated with glioma invasion. Angiostatin is an antiangiogenic polypeptide that also inhibits migration of cancer cells, but is difficult to deliver. Oncolytic viruses (OV) can potentially spread throughout the tumor, reach isolated infiltrating cells, kill them and deliver anticancer agents to uninfected cells. We have tested a combination treatment of BEV plus an OV expressing angiostatin (G47Δ-mAngio) in mice-bearing human GBM. Using a vascular intracranial human glioma model (U87) in athymic mice, we performed histopathological analysis of tumors treated with G47Δ-mAngio or BEV alone or in combination, followed tumor response by magnetic resonance imaging (MRI), and assessed animal survival. Our results indicate that injection of G47Δ-mAngio during BEV treatment allows increased virus spread, tumor lysis, and angiostatin-mediated inhibition of vascular endothelial growth factor (VEGF) expression and of BEV-induced invasion markers (matrix metalloproteinases-2 (MMP2), MMP9, and collagen). This leads to increased survival and antiangiogenesis and decreased invasive phenotypes. We show for the first time the possibility of improving the antiangiogenic effect of BEV while decreasing the tumor invasive-like phenotype induced by this drug, and demonstrate the therapeutic advantage of combining systemic and local antiangiogenic treatments with viral oncolytic therapy.

The status of gene therapy for brain tumors

The advent of gene therapy in the early 1990’s raised expectations for brain tumor therapies; however, whereas clinical trials in patients with malignant gliomas provided evidence of safety, therapeutic benefit was not convincing. These early forays resembled the historical introductions of other therapies that seemed promising, only to fail in human trials. Nevertheless, re-study in the laboratory and retesting in iterative laboratory–clinic processes enabled therapies with strong biological rationales to ultimately show evidence of success in humans and become accepted. Examples, such as organ transplantation, monoclonal antibody therapy and antiangiogenic therapy, provide solace that a strategy’s initial lack of success in humans provides an opportunity for its further refinement in the laboratory and development of solutions that will translate into patient success stories. The authors herein summarize results from clinical trials of gene therapy for malignant gliomas, and discuss the influence of these results on present thought in preclinical research.