Microglia and astrocytes attenuate the replication of the oncolytic vaccinia virus LIVP 1.1.1 in murine GL261 gliomas by acting as vaccinia virus traps.

BACKGROUND: Oncolytic virotherapy is a novel approach for the treatment of glioblastoma multiforme (GBM) which is still a fatal disease. Pathologic features of GBM are characterized by the infiltration with microglia/macrophages and a strong interaction between immune- and glioma cells. The aim of this study was to determine the role of microglia and astrocytes for oncolytic vaccinia virus (VACV) therapy of GBM. METHODS: VACV LIVP 1.1.1 replication in C57BL/6 and Foxn1(nu/nu) mice with and without GL261 gliomas was analyzed. Furthermore, immunohistochemical analysis of microglia and astrocytes was investigated in non-, mock-, and LIVP 1.1.1-infected orthotopic GL261 gliomas in C57BL/6 mice. In cell culture studies virus replication and virus-mediated cell death of GL261 glioma cells was examined, as well as in BV-2 microglia and IMA2.1 astrocytes with M1 or M2 phenotypes. Co-culture experiments between BV-2 and GL261 cells and apoptosis/necrosis studies were performed. Organotypic slice cultures with implanted GL261 tumor spheres were used as additional cell culture system. RESULTS: We discovered that orthotopic GL261 gliomas upon intracranial virus delivery did not support replication of LIVP 1.1.1, similar to VACV-infected brains without gliomas. In addition, recruitment of Iba1(+) microglia and GFAP(+) astrocytes to orthotopically implanted GL261 glioma sites occurred already without virus injection. GL261 cells in culture showed high virus replication, while replication in BV-2 and IMA2.1 cells was barely detectable. The reduced viral replication in BV-2 cells might be due to rapid VACV-induced apoptotic cell death. In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected. Application of BV-2 microglial cells with M1 phenotype onto organotypic slice cultures with implanted GL261 gliomas resulted in reduced infection of BV-2 cells, whereas GL261 cells were well infected. CONCLUSION: Our results indicate that microglia and astrocytes, dependent on their activation state, may preferentially clear viral particles by immediate uptake after delivery. By acting as VACV traps they further reduce efficient virus infection of the tumor cells. These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.

Use of GLV-1h68 for Vaccinia Virotherapy and Monitoring.

Herein we describe the use of the vaccinia virus strain GLV-1h68 as a theragnostic agent in cancer models. To date, GLV-1h68 has been used successfully in more than 50 xenograft tumor models. The recombinant vaccinia virus strain has been equipped with heterologous expression cassettes for a luciferase-fluorescent protein fusion gene, bacterial beta-galactosidase, as well as a bacterial glucuronidase. The methods to investigate and monitor GLV-1h68 mediated virotherapy, including optical imaging and biomarker analysis, will be presented in detail.

Inducible gene expression in tumors colonized by modified oncolytic vaccinia virus strains.

UNLABELLED: Exogenous gene induction of therapeutic, diagnostic, and safety mechanisms could be a considerable improvement in oncolytic virotherapy. Here, we introduced a doxycycline-inducible promoter system (comprised of a tetracycline repressor, several promoter constructs, and a tet operator sequence) into oncolytic recombinant vaccinia viruses (rVACV), which were further characterized in detail. Experiments in cell cultures as well as in tumor-bearing mice were analyzed to determine the role of the inducible-system components. To accomplish this, we took advantage of the optical reporter construct, which resulted in the production of click-beetle luciferase as well as a red fluorescent protein. The results indicated that each of the system components could be used to optimize the induction rates and had an influence on the background expression levels. Depending on the given gene to be induced in rVACV-colonized tumors of patients, we discuss the doxycycline-inducible promoter system adjustment and further optimization. IMPORTANCE: Oncolytic virotherapy of cancer can greatly benefit from the expression of heterologous genes. It is reasonable that some of those heterologous gene products could have detrimental effects either on the cancer patient or on the oncolytic virus itself if they are expressed at the wrong time or if the expression levels are too high. Therefore, exogenous control of gene expression levels by administration of a nontoxic inducer will have positive effects on the safety as well as the therapeutic outcome of oncolytic virotherapy. In addition, it paves the way for the introduction of new therapeutic genes into the genome of oncolytic viruses that could not have been tested otherwise.

Vaccinia virus expressing bone morphogenetic protein-4 in novel glioblastoma orthotopic models facilitates enhanced tumor regression and long-term survival.

BACKGROUND: Glioblastoma multiforme (GBM) is one of the most aggressive forms of cancer with a high rate of recurrence. We propose a novel oncolytic vaccinia virus (VACV)-based therapy using expression of the bone morphogenetic protein (BMP)-4 for treating GBM and preventing recurrence. METHODS: We have utilized clinically relevant, orthotopic xenograft models of GBM based on tumor-biopsy derived, primary cancer stem cell (CSC) lines. One of the cell lines, after being transduced with a cDNA encoding firefly luciferase, could be used for real time tumor imaging. A VACV that expresses BMP-4 was constructed and utilized for infecting several primary glioma cultures besides conventional serum-grown glioma cell lines. This virus was also delivered intracranially upon implantation of the GBM CSCs in mice to determine effects on tumor growth. RESULTS: We found that the VACV that overexpresses BMP-4 demonstrated heightened replication and cytotoxic activity in GBM CSC cultures with a broad spectrum of activity across several different patient-biopsy cultures. Intracranial inoculation of mice with this virus resulted in a tumor size equal to or below that at the time of injection. This resulted in survival of 100% of the treated mice up to 84 days post inoculation, significantly superior to that of a VACV lacking BMP-4 expression. When mice with a higher tumor burden were injected with the VACV lacking BMP-4, 80% of the mice showed tumor recurrence. In contrast, no recurrence was seen when mice were injected with the VACV expressing BMP-4, possibly due to induction of differentiation in the CSC population and subsequently serving as a better host for VACV infection and oncolysis. This lack of recurrence resulted in superior survival in the BMP-4 VACV treated group. CONCLUSIONS: Based on these findings we propose a novel VACV therapy for treating GBM, which would allow tumor specific production of drugs in the future in combination with BMPs which would simultaneously control tumor maintenance and facilitate CSC differentiation, respectively, thereby causing sustained tumor regression without recurrence.

Characterization and evaluation of a new oncolytic vaccinia virus strain LIVP6.1.1 for canine cancer therapy.

Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is one novel approach for canine cancer therapy. In this study we described for the first time the characterization and the use of new VACV strain LIVP6.1.1 as an oncolytic agent against canine cancer in a panel of four canine cancer cell lines including: soft tissue sarcoma (STSA-1), melanoma (CHAS), osteosarcoma (D-17) and prostate carcinoma (DT08/40). Cell culture data demonstrated that LIVP6.1.1 efficiently infected and destroyed all four tested canine cancer cell lines. In two different xenograft models on the basis of the canine soft tissue sarcoma STSA-1 and the prostate carcinoma DT08/40 cell lines, a systemic administration of the LIVP6.1.1 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. In summary, the pre-clinical evaluation has demonstrated the efficacy of LIVP6.1.1 for canine cancer therapy. Furthermore, a clinical trial with canine cancer patients has already been started.

Biotherapeutic approaches to target cancer stem cells.

With the cementing of the cancer stem cell (CSC) concept, cancer biology and cancer drug discovery have attained a new avenue to target cancer. Studying the hierarchy of tumor tissue organization and how to inhibit the cell that resides at the very top of this hierarchy has opened up a new branch of tumor biology and given the opportunity to develop novel cancer-targeting strategies. With the discovery of CSCs in majority of cancer indications there seems to be a universal applicability of the concept. However, the CSC field is still at an early fledgling state and a lot more needs to be done in terms of understanding their emergence, maintenance, role in metastasis and their function in shaping the tumor architecture. CSCs are considered to be responsible for tumor initiation, metastasis and resistance to conventional radio and chemotherapy. Therefore, different approaches to targeting these tumorigenic and rare cells are urgently needed in order to improve the efficacy of anti-cancer therapy. We outline here the cancer stem cell concept and its relevance as well as biotherapeutic approaches to CSC targeting, including oncolytic viruses, monoclonal antibodies, cytokines and cytotoxic T lymphocytes.

Preferential replication of systemically delivered oncolytic vaccinia virus in focally irradiated glioma xenografts.

PURPOSE: Radiotherapy is part of the standard of care in high-grade gliomas but its outcomes remain poor. Integrating oncolytic viruses with standard anticancer therapies is an area of active investigation. The aim of this study was to determine how tumor-targeted ionizing radiation (IR) could be combined with systemically delivered oncolytic vaccinia virus. EXPERIMENTAL DESIGN: U-87 glioma xenografts were grown subcutaneously or orthotopically. Oncolytic vaccinia viruses GLV-1h68 and LIVP 1.1.1 were injected systemically and IR was given focally to glioma xenografts. In a bilateral tumor model, glioma xenografts were grown in both flanks, oncolytic vaccinia was injected systemically and radiation was delivered specifically to the right flank tumor, whereas the left flank tumor was shielded. Viral replication and tumor regression, after systemic injection, was analyzed and compared in irradiated and nonirradiated glioma xenografts. RESULTS: Systemically administered oncolytic vaccinia virus replicated to higher titers in preirradiated U-87 xenografts than in nonirradiated glioma xenografts. This increased oncolytic viral replication correlated with increased tumor xenograft regression and mouse survival in subcutaneous and orthotopic U-87 glioma models compared with monotherapies. The ability of focal IR to mediate selective replication of oncolytic vaccinia was shown in a bilateral glioma model in which systemically administered oncolytic vaccinia replicated preferentially in the irradiated tumor compared with the nonirradiated tumor in the same mouse. CONCLUSION: These findings show a potential clinical role of focal IR in sensitizing irradiated tumor sites for preferential vaccinia virus-mediated oncolysis.

Magnetic resonance imaging of tumors colonized with bacterial ferritin-expressing Escherichia coli.

BACKGROUND: Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties. METHODS AND FINDINGS: Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors. CONCLUSIONS: Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes.