In the past 5 years, the field of oncolytic virus research has matured significantly and is moving past the stage of being a laboratory novelty into a new era of preclinical and clinical trials. What have recent anticancer trials of oncolytic viruses taught us about this exciting new line of therapeutics?

Primary glial tumors of the central nervous system, most commonly glioblastoma multiforme (GBM), are aggressive lesions with a dismal prognosis. Despite identification and isolation of human brain tumor stem cells (BTSCs), characteristics that distinguish BTSCs from neural stem cells remain to be elucidated. We cultured cells isolated from gliomas, using the neurosphere culture system, to understand their growth requirements. Both CD133 1 and CD133 2 adult GBM BTSCs proliferated in the absence of exogenous mitogenic stimulation and gave rise to multipotent GBM spheres that were capable of self-renewal. Epidermal growth factor (EGF) and fibroblast growth factor-2 enhanced GBM BTSC survival, proliferation, and subsequent sphere size. Blockade of EGF receptor (EGFR) signaling reduced exogenous mitogen-independent GBM sphere growth. Implantation of as few as 10 exogenous mitogen-independent GBM BTSCs led to the formation of highly invasive intracranial tumors, which closely resembled human GBMs, in immunocompromised mice. These results demonstrate that exogenous mitogen independence, mediated in part through EGFR signaling, is one characteristic that distinguishes CD133 1 and CD133 2 GBM BTSCs from neural stem cells. This novel experimental system will permit the elucidation of additional constitutively activated mechanisms that promote GBM BTSC survival, self-renewal, and proliferation.

The invasive nature of cancers in general, and malignant gliomas in particular, is a major clinical problem rendering tumors incurable by conventional therapies. Using a novel invasive glioma mouse model established by serial in vivo selection, we identified the p75 neurotrophin receptor (p75NTR) as a critical regulator of glioma invasion. Through a series of functional, biochemical, and clinical studies, we found that p75NTR dramatically enhanced migration and invasion of genetically distinct glioma and frequently exhibited robust expression in highly invasive glioblastoma patient specimens. Moreover, we found that p75NTR-mediated invasion was neurotrophin dependent, resulting in the activation of downstream pathways and producing striking cytoskeletal changes of the invading cells. These results provide the first evidence for p75NTR as a major contributor to the highly invasive nature of malignant gliomas and identify a novel therapeutic target.

Myxoma virus, a poxvirus previously considered rabbit specific, can replicate productively in a variety of human tumor cells in culture. The purpose of this study was to determine if there was efficacy or toxicities of this oncolytic virus against experimental models of human malignant gliomas in vitro, in vivo, and ex vivo in malignant glioma specimens. In vitro, the majority of glioma cell lines tested (7 of 8, 87.5%) were fully permissive for myxoma virus replication and killed by infection. In vivo, intracerebral (i.c.) myxoma virus inoculation was well tolerated and produced only minimal focal inflammatory changes at the site of viral inoculation. U87 and U251 orthotopic xenograft models were used to assess myxoma virus efficacy in vivo. A single intratumoral injection of myxoma virus dramatically prolonged median survival compared with treatment with UVinactivated myxoma virus. Median survival was not reached in myxoma virus-treated groups versus 47.3 days (U87; P = 0.0002) and 50.7 days (U251; P = 0.0027) in UV-inactivated myxoma virus-treated groups. Most myxoma virus-treated animals (12 of 13, 92%) were alive and apparently ''cured'' when the experiment was finished (>130 days). Interestingly, we found a selective and long-lived myxoma virus infection in gliomas in vivo. This is the first demonstration of the oncolytic activity of myxoma virus in vivo. The nonpathogenic nature of myxoma virus outside of the rabbit host, its capacity to be genetically modified, its ability to produce a long-lived infection in human tumor cells, and the lack of preexisting antibodies in the human population suggest that myxoma virus may be an attractive oncolytic agent against human malignant glioma. (Cancer Res 2005; 65(21): 9982-90)

Reovirus has potent activity against human malignant gliomas in vitro, in vivo, and ex vivo. Oncolysis with reovirus may be a potentially useful treatment for a broad range of human cancers.

Purpose: The oncolytic effects of a systemically delivered, replicating, double-deleted vaccinia virus has been previously shown for the treatment of many cancers, including colon, ovarian, and others.The purpose of this study was to investigate the oncolytic potential of double-deleted vaccinia virus alone or in combination with rapamycin or cyclophosphamide to treat malignant gliomas in vitro and in vivo. Experimental Design: Rat (RG2, F98, C6) and human (A172, U87MG, U118) glioma cell lines were cultured in vitro and treated with live or UV-inactivated vaccinia virus. Viral gene [enhanced green fluorescent protein (EGFP)] expression by fluorescence-activated cell sorting, relative cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and assays for cytopathic effects were examined. S.c. murine tumor xenografts (U87MG, U118, C6) and i.c. (RG2, F98) tumor models in immunocompetent rats were treated with systemic administration of EGFP-expressing vaccinia virus (vvDD-EGFP), alone or in combination with rapamycin or cyclophosphamide, or controls. Tumor size, viral biodistribution, and animal survival were assessed. Lastly, the oncolytic effects of vvDD-EGFP on human malignant glioma explants were evaluated. Results: vvDD-EGFP was able to infect and kill glioma cells in vitro. A single systemic dose of vvDD-EGFP significantly inhibited the growth of xenografts in athymic mice. Systemic delivery of vvDD-EGFP alone was able to target solitary and multifocal i.c. tumors and prolong survival of immunocompetent rats, whereas combination therapy with rapamycin or cyclophosphamide enhanced viral replication and further prolonged survival. Finally, vvDD-EGFP was able to infect and kill ex vivo primary human malignant gliomas. Conclusions: These results suggest that vvDD-EGFP is a promising novel agent for human malignant glioma therapy, and in combination with immunosuppressive agents, may lead to prolonged survival from this disease.

Purpose: Glioblastoma is one of the most lethal cancers in humans, and with existing therapy, survival remains at 14.6 months. Current barriers to successful treatment include their infiltrative behavior, extensive tumor heterogeneity, and the presence of a stem-like population of cells, termed brain tumor-initiating cells (BTIC) that confer resistance to conventional therapies.Experimental Design: To develop therapeutic strategies that target BTICs, we focused on a repurposing approach that explored already-marketed (clinically approved) drugs for therapeutic potential against patient-derived BTICs that encompass the genetic and phenotypic heterogeneity of glioblastoma observed clinically.Results: Using a high-throughput in vitro drug screen, we found that montelukast, clioquinol, and disulfiram (DSF) were cytotoxic against a large panel of patient-derived BTICs. Of these compounds, disulfiram, an off-patent drug previously used to treat alcoholism, in the presence of a copper supplement, showed low nanomolar efficacy in BTICs including those resistant to temozolomide and the highly infiltrative quiescent stem-like population. Low dose DSF-Cu significantly augmented temozolomide activity in vitro, and importantly, prolonged in vivo survival in patient-derived BTIC models established from both newly diagnosed and recurrent tumors. Moreover, we found that in addition to acting as a potent proteasome inhibitor, DSF-Cu functionally impairs DNA repair pathways and enhances the effects of DNA alkylating agents and radiation. These observations suggest that DSF-Cu inhibits proteasome activity and augments the therapeutic effects of DNA-damaging agents (temozolomide and radiation).Conclusions: DSF-Cu should be considered as an adjuvant therapy for the treatment of patients with glioblastoma in both newly diagnosed and recurrent settings.

Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.