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Glioblastoma multiforme (GBM) represents the most aggressive form of malignant brain tumors and remains a therapeutically challenge. Intense research in the field has lead to the testing of oncolytic viruses to improve tumor control. Currently, a variety of different oncolytic viruses are being evaluated for their ability to be used in anti-cancer therapy and a few have entered clinical trials. Vaccinia virus, is one of the viruses being studied. GLV-1h68, an oncolytic vaccinia virus engineered by Genelux Corporation, was constructed by insertion of three gene cassettes, RUC-GFP fusion, β-galactosidase and β- glucuronidase into the genome of the LIVP strain. Since focal tumor radiotherapy is a mainstay for cancer treatment, including glioma therapy, it is of clinical relevance to assess how systemically administered oncolytic vaccinia virus could be combined with targeted ionizing radiation for therapeutic gain. In this work we show how focal ionizing radiation (IR) can be combined with multiple systemically delivered oncolytic vaccinia virus strains in murine models of human U-87 glioma. After initial experiments which confirmed that ionizing radiation does not damage viral DNA or alter viral tropism, animal studies were carried out to analyze the interaction of vaccinia virus and ionizing radiation in the in vivo setting. We found that irradiation of the tumor target, prior to systemic administration of oncolytic vaccinia virus GLV-1h68, increased viral replication within the U-87 xenografts as measured by viral reporter gene expression and viral titers. Importantly, while GLV-1h68 alone had minimal effect on U-87 tumor growth delay, IR enhanced GLV-1h68 replication, which translated to increased tumor growth delay and mouse survival in subcutaneous and orthotopic U-87 glioma murine models compared to monotherapy with IR or GLV-1h68. The ability of IR to enhance vaccinia replication was not restricted to the multi-mutated GLV-1h68, but was also seen with the less attenuated oncolytic vaccinia, LIVP 1.1.1. We have demonstrated that in animals treated with combination of ionizing radiation and LIVP 1.1.1 a strong pro-inflammatory tissue response was induced. When IR was given in a more clinically relevant fractionated scheme, we found oncolytic vaccinia virus replication also increased. This indicates that vaccinia virus could be incorporated into either larger hypo-fraction or more conventionally fractionated radiotherapy schemes. The ability of focal IR to mediate selective replication of systemically injected oncolytic vaccinia was demonstrated in a bilateral glioma model. In mice with bilateral U-87 tumors in both hindlimbs, systemically administered oncolytic vaccinia replicated preferentially in the focally irradiated tumor compared to the shielded non- irradiated tumor in the same mouse We demonstrated that tumor control could be further improved when fractionated focal ionizing radiation was combined with a vaccinia virus caring an anti-angiogenic payload targeting vascular endothelial growth factor (VEGF). Our studies showed that following ionizing radiation expression of VEGF is upregulated in U-87 glioma cells in culture. We further showed a concentration dependent increase in radioresistance of human endothelial cells in presence of VEGF. Interestingly, we found effects of vascular endothelial growth factor on endothelial cells were reversible by adding purified GLAF-1 to the cells. GLAF-1 is a single- chain antibody targeting human and murine VEGF and is expressed by oncolytic vaccinia virus GLV-109. In U-87 glioma xenograft murine models the combination of fractionated ionizing radiation with GLV-1h164, a vaccinia virus also targeting VEGF, resulted in the best volumetric tumor response and a drastic decrease in vascular endothelial growth factor. Histological analysis of embedded tumor sections 14 days after viral administration confirmed that blocking VEGF translated into a decrease in vessel number to 30% of vessel number found in control tumors in animals treated with GLV-164 and fractionated IR which was lower than for all other treatment groups. Our experiments with GLV-1h164 and fractionated radiotherapy have shown that in addition to ionizing radiation and viral induced tumor cell destruction we were able to effectively target the tumor vasculature. This was achieved by enhanced viral replication translating in increased levels of GLAF-2 disrupting tumor vessels as well as the radiosensitization of tumor vasculature to IR by blocking VEGF. Our preclinical results have important clinical implications of how focal radiotherapy can be combined with systemic oncolytic viral administration for highly aggressive, locally advanced tumors with the potential, by using a vaccinia virus targeting human vascular endothelial growth factor, to further increase tumor radiation sensitivity by engaging the vascular component in addition to cancer cells.
Effects of stem cell transcription factor-expressing vaccinia viruses in oncolytic virotherapy
(2012)
Cancer remains the second leading cause of death in the industrialized. The data from many different studies investigating the nature of cancer-initiating cells coined the description ‘cancer stem cells’ and has major implications on conventional cancer therapy. Thus, to improve the outcome of cancer treatment and to lower negative side effects, the development of novel therapeutic regimens is indispensable. It has been demonstrated in many preclinical studies that oncolytic virotherapy using vaccinia virus may provide a powerful and well-tolerable new tool in cancer therapy which is currently investigated in several clinical trials (Phase I & II) as stand-alone treatment or in combination with conventional cancer therapy. Cancer-initiating cells and stem cells share a variety of characteristics like the ability to self-renew, differentiation potential, quiescence, drug and radiation resistance, activation and inhibition of similar signaling pathways as well as expression of cell surface markers and stem cell-related genes. In this work, two new recombinant vaccinia viruses expressing the transcription factors Nanog (GLV-1h205) and Oct4 (GLV-1h208) were engineered to provide deeper insight of these stem cell master regulators in their significance of cancer-initiation and their impact on oncolytic virotherapy. Both viruses were analyzed for their replication potential in A549 and PC-3 human cancer cells. Marker gene expression was assessed by RT-PCR, SDS-PAGE and Western blotting, ELISA or immunocytochemistry.Furthermore, the effect of GLV-1h205 infection on the cell cycle in A549 cells was analyzed. Next, the effects of virus-mediated expression of stem cell transcription factors on therapeutic efficacy and survival rates in A549 xenograft mouse models was analyzed. A non-functional Nanog mutant-expressing virus strain (GLV-1h321) was engineered to analyze whether the observed therapeutic benefits were promoter- or payload-driven. Furthermore, this study analyzed the potential of GLV-1h68 to infect, replicate in, and lyse colorectal cancer cell lines to study whether oncolytic vaccinia viruses can be potential new and less invasive treatment regimens for late stage colorectal cancer. Marker gene expression was assessed by fluorescence microscopy and FACS. The transcription factor Klf4 is highly expressed in quiescent, terminally differentiated cells in the colonic epithelium whereas it is dramatically downregulated in colon cancers. Klf4 expression leads to cell growth arrest and inhibits Wnt signaling by binding to beta-catenin. To further improve the treatment of colorectal cancers, new recombinant vaccinia viruses (GLV-1h290-292) mediating the expression of differing amounts of the tumor suppressor Klf4 by using different promoter strengths were engineered. Initial characterization of recombinant vaccinia viruses expressing Klf4 by replication assay, cell viability assay, SDS-PAGE and Western blotting, immuncytochemistry and analysis of protein functionality by qPCR and ELISA analysis for cellular beta-catenin expression, demonstrated promoter strength-dependent expression of and impact of Klf4. To further boost the effects of tumor suppressor Klf4, a vaccinia virus strain expressing Klf4 with a C-terminal fusion of the TAT transduction domain (GLV-1h391) was engineered. Treatment of HT-29 non-responder tumors in vivo with GLV-1h291 and GLV-1h391 led to significant tumor growth inhibition and improved overall survival compared to GLV-1h68. This makes the Klf4-TAT expressing GLV-1h391 a promising candidate for the treatment of colorectal cancer in man.