Trail armed oncolytic poxvirus suppresses lung cancer cell by inducing apoptosis.

Lung cancer has a high morbidity rate worldwide and is often resistant to therapy. Oncolytic virus therapy is a developing trend for cancer treatment. Thus, we constructed an oncolytic poxvirus carrying human trail gene that expresses a membrane-binding tumor necrosis factor and associated apoptosis-inducing ligand (TRAIL, Oncopox-trail). We hypothesized that the expression of trail would increase the efficacy of the oncolytic poxvirus. The effect of the TRAIL protein depends on the death receptors on the surface of different cancer cells. The expression of death receptors in lung cancer cell lines was analyzed by western blot analysis. In vitro, the oncolytic poxvirus carrying the trail gene displayed a better cytotoxicity at the cell level in the lung cancer cell line than that carrying the Oncopox-empty. TRAIL protein mainly induced apoptosis and inhibited necrosis. In vivo, two transplanted tumor models of human A549 lung cancer cells and mouse Lewis lung cancer cells were used to verify the anti-cancer effect of the oncolytic poxvirus carrying the trail gene. TUNEL staining results of the tumor histological sections also verified the anti-cancer effect. Similarly, through systemic administration of Oncopox-trail, the oncolytic poxvirus also exhibited anti-cancer effect.

Bacterial-mediated knockdown of tumor resistance to an oncolytic virus enhances therapy.

Oncolytic viruses (OVs) and bacteria share the property of tumor-selective replication following systemic administration. In the case of nonpathogenic bacteria, tumor selectivity relates to their ability to grow extracellularly within tumor stroma and is therefore ideally suited to restricting the production of bacterially produced therapeutic agents to tumors. We have previously shown the ability of the type 1 interferon antagonist B18R to enhance the replication and spread of vesicular stomatitis virus (VSV) by overcoming related cellular innate immunity. In this study, we utilized nonpathogenic bacteria (E. coli) expressing B18R to facilitate tumor-specific production of B18R, resulting in a microenvironment depleted of bioactive antiviral cytokine, thus "preconditioning" the tumor to enhance subsequent tumor destruction by the OV. Both in vitro and in vivo infection by VSVΔ51 was greatly enhanced by B18R produced from E. coli. Moreover, a significant increase in therapeutic efficacy resulted from intravenous (i.v.) injection of bacteria to tumor-bearing mice 5 days prior to i.v. VSVΔ51 administration, as evidenced by a significant reduction in tumor growth and increased survival in mice. Our strategy is the first example where two such diverse microorganisms are rationally combined and demonstrates the feasibility of combining complementary microorganisms to improve therapeutic outcome.

Live attenuated measles virus vaccine induces apoptosis and promotes tumor regression in lung cancer.

Although the treatment of lung carcinoma has improved, at least 65% of patients with this tumor succumb to progressive disease. Measles virus oncolytic therapy has been reported to be effective in reducing tumor burden in immunocompetent or nude mice; however, its potential to reduce tumor burden in lung carcinoma remains to be determined. Herein, we report the potent antitumor effects of a live attenuated measles vaccine virus Hu-191 strain (MV) against lung carcinoma. Immunocompetent C57BL/6 mice bearing Lewis lung carcinoma (LLC) cells were treated with MV (1x104 to 1x106 CCID50/ml) once every other day for 10 days. Our results showed that treatment with MV effectively suppressed tumor growth and significantly prolonged the survival time of tumor-bearing animals. Histological examination revealed that the antitumor effects of MV therapy may result from increased induction of apoptosis, tumor necrosis and elevated lymphocyte infiltration. Our data suggest that MV, one of the widely used vaccines in China, has the ability to inhibit the growth of mouse lung carcinoma and may prove useful in the further exploration of the application of this approach in the treatment of human advanced lung cancer.

Adoptively transferred tumor-specific T cells stimulated ex vivo using herpes simplex virus amplicons encoding 4-1BBL persist in the host and show antitumor activity in vivo.

4-1BB is a T-cell costimulatory receptor which binds its ligand 4-1BBL, resulting in prolonged T cell survival. We studied the antitumor effects of adoptively transferred tumor-specific T cells expanded ex vivo using tumors transduced with herpes simplex virus (HSV) amplicons expressing 4-1BBL as a direct source of antigen and costimulation. We constructed HSV amplicons encoding either the 4-1BBL (HSV.4-1BBL) or B7.1 (HSV.B7.1) costimulatory ligands. Lewis lung carcinoma cells expressing ovalbumin (LLC/OVA) were transduced with HSV.4-1BBL, HSV.B7.1, or control HSV amplicons and used to stimulate GFP+ OVA-specific CD8+ T cells (OT-1/GFP) ex vivo. Naive or ex vivo stimulated OT-1/GFP cells were adoptively transferred into LLC/OVA tumor-bearing mice. Higher percentages of OT-1/GFP cells were seen in the peripheral blood, spleen, and tumor bed of the HSV.4-1BBL-stimulated OT-1/GFP group compared with all other experimental groups. OT-1 cells identified within the tumor bed and draining lymph nodes of the HSV.4-1BBL-stimulated OT-1 group showed enhanced bromodeoxyuridine (BrdUrd) incorporation, suggesting ongoing expansion in vivo. Mice receiving HSV.4-1BBL-stimulated OT-1/GFP had significantly decreased tumor volumes compared with untreated mice (P<0.001) or to mice receiving naive OT-1/GFP (P<0.001). Transfer of HSV.B7.1-stimulated OT-1/GFP did not protect mice from tumor. Mice that received HSV.4-1BBL-stimulated OT-1/GFP exhibited increased cytolytic activity against LLC/OVA and higher percentages of Ly-6C+ OT-1/GFP in the spleen and tumor bed compared with controls. Tumor-specific T cells stimulated ex vivo using tumor transduced with HSV.4-1BBL expand in vivo following adoptive transfer, resulting in tumor eradication and the generation of tumor-specific CD44+Ly-6C+CD62L- effector memory T cells.

Coadministration of a herpes simplex virus-2 based oncolytic virus and cyclophosphamide produces a synergistic antitumor effect and enhances tumor-specific immune responses.

Despite their unique property of selective replication and propagation in tumor tissues, oncolytic viruses have had only limited antitumor effects in cancer patients. One of the major reasons is probably the host's immune defense mechanisms, which can restrict the ability of the virus to replicate and spread within tumors. The innate immune system, which can be rapidly activated during virus infection, likely plays a more pivotal antiviral role than does acquired immunity, as the antitumor effect of an oncolytic virus is mainly generated during the acute phase of virus replication. To exploit the potential of cyclophosphamide, a cancer chemotherapeutic drug that also inhibits innate immune responses, to enhance the activity of oncolytic viruses, we evaluated the effect of coadministration of this drug with a herpes simplex virus-2-based oncolytic virus (FusOn-H2) against Lewis lung carcinoma, which is only semipermissive to infection with FusOn-H2. This strategy synergistically enhanced the antitumor effect against lung carcinoma growing in mice. It also potentiated the ability of FusOn-H2 to induce tumor-specific immune responses. Together, our results suggest that coadministration of FusOn-H2 with cyclophosphamide would be a feasible way to enhance the antitumor effects of this oncolytic virus in future clinical trials.

Induction of apoptosis and tumor regression by vesicular stomatitis virus in the presence of gemcitabine in lung cancer.

Vesicular stomatitis virus (VSV) has been shown to replicate rapidly in vitro and kill selectively a variety of tumor cell lines. The present study was designed to determine whether gemcitabine potentiates the antitumor activity of VSV in vitro and in vivo. A549 human lung adenocarcinoma cells and LLC Lewis lung carcinoma cells were treated with VSV (0.1-10 plaque-forming units per cell) plus gemcitabine (20 nM to 20 microM). Mice bearing A549 or LLC were treated with VSV (5 x 10(4) to 1 x 10(8) plaque-forming units) daily for 5 days plus gemcitabine (5-125 mg/kg/day) once every 3 days for 4 times. Induction of apoptosis and effects on growth inhibition were assessed. The lung cancer cells treated with VSV plus gemcitabine displayed the apparently increased apoptotic cells compared with treatment with VSV or gemcitabine alone. The combined treatment with VSV plus gemcitabine induced the apparent antitumor activity with complete regression of the established lung cancer in both A549 and LLC lung cancer models and augmented the induction of apoptosis in lung cancer cells in vivo as well. This study suggests that the combined treatment with VSV plus gemcitabine may augment the induction of apoptosis in lung cancer cells in vitro and in vivo, and that the augmented antitumor activity in vivo may result from the increased induction of apoptosis in lung cancer cells. The present findings may be of importance to the further exploration of the potential application of this combined approach in the treatment of lung cancer.

Anticancer activity of bacteriophage T4 and its mutant HAP1 in mouse experimental tumour models.

BACKGROUND: Previously, we have shown the ability of the bacteriophage T4 and its substrain HAP1 (selected for a higher affinity to melanoma cells) to reveal antimetastatic activity in a mouse melanoma model. Here, we investigated the potential phage anticancer activity in primary tumour models. MATERIALS AND METHODS: Mice were inoculated subcutaneously with B16 or LLC cells (collected from in vitro culture). Bacteriophages T4 and HAP1 were injected intraperitoneally daily (8 x 10(8)pfu/mouse, except the experiment concerning the dose-dependence). RESULTS: Treatment with purified preparations of bacteriophage T4 resulted in significant reduction of tumour size, the effect being dose-dependent. HAP1 was more effective than T4 and its activity was also dose-dependent. Parallel experiments with non-purified bacteriophage lysates resulted in significant stimulation of tumour growth. CONCLUSION: These data suggest that purified bacteriophages may inhibit tumour growth, a phenomenon with potentially important clinical implications in oncology.

Systemic reovirus therapy of metastatic cancer in immune-competent mice.

The human reovirus is an oncolytic virus that specifically targets cancer cells with an activated Ras pathway. Because it is replication competent and highly specific for cancer cells, this virus has the potential to be an effective antimetastatic cancer agent through remote site delivery. In this study, we exploited the ability of reovirus to replicate in murine cells to test the efficacy of this virus in eliminating distal and/or metastatic tumors in immune-competent mice. We found that i.v. therapy with reovirus not only inhibited metastatic tumor growth but also led to a significant improvement in animal survival. Combining i.v. reovirus treatment with immune suppression (cyclosporine A or anti-CD4/anti-CD8 antibodies) resulted in further reduction in tumor size and a considerable prolongation in survival, compared with viral therapy alone. Combined therapy was also effective in overcoming a preexisting immunity to reovirus (a common occurrence in humans and thus a potential impediment to oncolytic effectiveness) to induce metastatic tumor regression. This is the first study to use systemic delivery of an oncolytic agent in conjunction with immune-suppressive drugs to effectively prolong animal survival. Altogether, our results suggest that i.v. reovirus therapy may present a feasible, novel alternative in the treatment of metastatic cancer in humans.

CD8+ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity.

The fate of dendritic cells (DC) after they have initiated a T cell immune response is still undefined. We have monitored the migration of DC labeled with a fluorescent tracer and injected s.c. into naive mice or into mice with an ongoing immune response. DC not loaded with Ag were detected in the draining lymph node in excess of 7 days after injection with maximum numbers detectable approximately 40 h after transfer. In contrast, DC that had been loaded with an MHC class I-binding peptide disappeared from the lymph node with kinetics that parallel the known kinetics of activation of CD8+ T cells to effector function. In the presence of high numbers of specific CTL precursors, as in TCR transgenic mice, DC numbers were significantly decreased by 72 h after injection. The rate of DC disappearance was extremely rapid and efficient in recently immunized mice and was slower in "memory" mice in which memory CD8+ cells needed to reacquire effector function before mediating DC elimination. We also show that CTL-mediated clearance of Ag-loaded DC has a notable effect on immune responses in vivo. Ag-specific CD8+ T cells failed to divide in response to Ag presented on a DC if the DC were targets of a pre-existing CTL response. The induction of antitumor immunity by tumor Ag-loaded DC was also impaired. Therefore, CTL-mediated clearance of Ag-loaded DC may serve as a negative feedback mechanism to limit the activity of DC within the lymph node.

Oncolytic therapy using a mutant type-1 herpes simplex virus and the role of the immune system.

BACKGROUND: Herpes simplex virus (HSV)-1716, a replication-restricted herpes simplex virus type 1, has shown efficacy as an oncolytic treatment for central nervous system tumors, breast cancer, ovarian cancer, and malignant mesothelioma. We evaluated the efficacy of HSV-1716 in a murine lung cancer model, Lewis lung carcinoma. METHODS: Lewis lung carcinoma cells were infected with HSV-1716 and implanted in the flanks of mice at varying ratios of infected to uninfected cells. Tumor burden was assessed by measurement of the weight of the tumor nodule. The role of the immune system was examined by performing experiments in both immunocompetent and SCID mice. Tumors were implanted in the opposite flank to evaluate the vaccine effect. RESULTS: In immunocompetent and SCID animals, ratio of 1:10 (infected-to-uninfected) cells completely prevented tumor formation and ratio of 1:100 suppressed tumor growth. Established tumors at a distant site in the groups receiving HSV-1716 infected cells showed no difference in size versus control, suggesting absence of a vaccine effect. CONCLUSIONS: We conclude that HSV-1716 may provide a oncolytic therapy for lung cancer even in the absence of immune system induction and a "carrier" cell could potentially deliver this vector.