Using adenovirus armed short hairpin RNA targeting transforming growth factor ß1 inhibits melanoma growth and metastasis in an ex vivo animal model.

The transforming growth factor ß (TGF-ß) is the key molecule implicated in impaired immune function in human patients with malignant melanoma. TGF-ß can promote tumor growth, invasion, and metastasis in advanced stages of melanoma. Blocking these tumor-promoting effects of TGF-ß provides a potentially important therapeutic strategy for the treatment of melanoma. In this study, we used an adenovirus-based shRNA expression system and successfully constructed Ad/TGF-ß1-RNA interference (RNAi) which mediated the RNAi for TGF-ß1 gene silencing. We examined the effects of TGF-ß1 protein knockdown by RNAi on the growth and metastasis of melanoma in C57BL/6 mice induced by the B16F0 cell line. The TGF-ß1 hairpin oligonucleotide was cloned into adenoviral vector. The resulting recombinant adenoviruses infected murine melanoma cell line, B16F0, and designated as B16F0/TGF-ß1-RNAi cells. The blank adenoviral vector also infected B16F0 cells and designed as B16F0/vector-control cells served as a control. TGF-ß1 expression was reduced in B16F0/TGF-ß1-RNAi cells compared with B16F0 cells and B16F0/vector-control cells. Three million wild-type B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-ß1-RNAi cells were injected subcutaneously into the right flanks of adult female syngeneic mice C57BL/6. The tumor sizes were 756.09 (65.35), 798.48 (78.77), and 203.55 (24.56) mm at the 14th day in the mice receiving B16F0 cells, B16F0/vector-control cells, and B16F0/TGFß1-RNAi cells, respectively. The P value was less than 0.01 by 1-way analysis of variance. TGF-ß1 knockdown in B16F0 cells enhanced the infiltration of CD4 and CD8 T cells in the tumor regions. C57BL/6 mice were evaluated for pulmonary metastasis after tail vein injection of 2 million B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-ß1-RNAi cells. The pulmonary metastasis also reduced significantly on days 14 day and 21 in mice injected with B16F0/TGF-ß1-RNAi tumors. The blood vessel density of the tumors markedly reduced in B16F0/TGF-ß1-RNAi tumors. Our results showed that Ad/TGF-ß1-RNAi could induce silencing of the TGF-ß1 gene effectively. Silencing of TGF-ß1 expression in B16F0 cells by RNAi technology can inhibit the growth and metastasis of this tumor after being transplanted to C57BL/6 mice. This kind of adenoviral vector based on RNAi might be a promising vector for cancer therapy.

Dual therapeutic effects of interferon-alpha gene therapy in a rat hepatocellular carcinoma model with liver cirrhosis.

Human hepatocellular carcinoma (HCC) often arises from a background of liver cirrhosis. Therefore, in order to develop therapeutic strategies for HCC, an animal model bearing multifocal liver tumors accompanied by liver cirrhosis is a preferred experimental setting. In this study, we developed a rapid and reproducible method for generating such a model in rats by weekly administration of diethylnitrosamine (DEN) at doses based on body weight (BW). By adjusting the duration of administration of DEN, the animals could be induced to develop HCC alone, or HCC and liver cirrhosis simultaneously. The latter model was used for evaluating the therapeutic effects of adenoviral delivery of interferon-alpha (IFN-alpha). Our results demonstrated that targeting of IFN-alpha expression to the liver significantly reduced liver tumor volume and ameliorated liver cirrhosis. Mechanistic studies revealed that IFN-alpha gene therapy induced immunomodulatory, antiproliferative, and proapoptotic activities that were effective in the control of tumor growth, and reduced the expressions of transforming growth factor-beta (TGF-beta) and tissue inhibitor of metalloproteinase-1 (TIMP-1), leading to amelioration of liver cirrhosis. These results suggest that IFN-alpha gene therapy is a promising strategy to treat HCC patients who have concomitant liver cirrhosis.

Dual Therapeutic Effects of Interferon-α Gene Therapy in a Rat Hepatocellular Carcinoma Model With Liver Cirrhosis.

Human hepatocellular carcinoma (HCC) often arises from a background of liver cirrhosis. Therefore, in order to develop therapeutic strategies for HCC, an animal model bearing multifocal liver tumors accompanied by liver cirrhosis is a preferred experimental setting. In this study, we developed a rapid and reproducible method for generating such a model in rats by weekly administration of diethylnitrosamine (DEN) at doses based on body weight (BW). By adjusting the duration of administration of DEN, the animals could be induced to develop HCC alone, or HCC and liver cirrhosis simultaneously. The latter model was used for evaluating the therapeutic effects of adenoviral delivery of interferon-α (IFN-α). Our results demonstrated that targeting of IFN-α expression to the liver significantly reduced liver tumor volume and ameliorated liver cirrhosis. Mechanistic studies revealed that IFN-α gene therapy induced immunomodulatory, antiproliferative, and proapoptotic activities that were effective in the control of tumor growth, and reduced the expressions of transforming growth factor-ß (TGF-ß) and tissue inhibitor of metalloproteinase-1 (TIMP-1), leading to amelioration of liver cirrhosis. These results suggest that IFN-α gene therapy is a promising strategy to treat HCC patients who have concomitant liver cirrhosis.

Combined GM-CSF and IL-12 gene therapy synergistically suppresses the growth of orthotopic liver tumors.

UNLABELLED: Immunotherapy is often effective only for small tumor burdens and, in many cases, is restricted to subcutaneous tumors. Here, we investigated the antitumor effects of combination therapy with GM-CSF and IL-12 on orthotopic liver tumors with intermediate or large tumor volumes, or on chemically-induced multifocal liver tumors in animals. Adenoviruses encoding GM-CSF or IL-12 were injected intratumorally to animals bearing transplanted tumors, or injected via intrahepatic artery in animals with primary multifocal liver tumors induced by diethylnitrosamine. Our results demonstrated that IL-12, but not GM-CSF, monotherapy displayed significant therapeutic effects, whereas combination therapy with both cytokines displayed synergistic antitumor effects not only on transplanted tumor models with intermediate or large tumor loads, but also on carcinogen-induced multifocal liver tumors. Effector cell analyses, revealed by in vivo cell subset depletion, flow cytometry analysis, and immunohistochemical staining of tumor infiltrates, indicated that NK cells were the prominent antitumor effectors for the IL-12-mediated antitumor activity, whereas CD8+ T cells, NKT cells, and macrophages were more important than NK cells in the combination therapy-mediated antitumor effects. Both IL-12 monotherapy and combination therapy could induce various types of effectors and high levels of IFN-gamma; however, the latter induced much higher levels than the former, which may explain why combination therapy is superior to IL-12 monotherapy. CONCLUSION: Combination therapy with GM-CSF and IL-12 represents a promising immunotherapy strategy for treating orthotopic, widespread liver tumors.

Effects of irradiated tumor vaccine and infusion of granulocyte-macrophage colony-stimulating factor and interleukin-12 on established gliomas in rats.

PURPOSE: We investigated granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12) infused into the injection site of irradiated tumor vaccine (TV) as therapy for gliomas. METHODS: Rats with subcutaneous RT-2 gliomas were treated with irradiated TV and/or subcutaneous infusion of GM-CSF and/or IL-12 via osmotic minipump 5 days after tumor-cell inoculation. Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell activity were analyzed to investigate immune responses. Rats with intracerebral gliomas were treated with irradiated TV and infused GM-CSF/IL-12 3 days after tumor-cell inoculation. Tumor growth rates and animal survival were followed. Survivors were re-challenged with wild-type RT-2 cells subcutaneously or intracerebrally to study long-term anti-tumor immunity. RESULTS: Rats with subcutaneous gliomas treated with GM-CSF and IL-12 or TV plus GM-CSF or IL-12 did not have increased survival rate (P>0.2), but did have prolonged survival time (P<0.05); in contrast, rats treated with TV plus GM-CSF/IL-12 had increased survival rate (P<0.05) and prolonged survival time (P<0.05) compared with controls. These treatment strategies showed enhanced CTL and NK cell activities. Rats with intra-cerebral gliomas treated with TV plus GM-CSF/IL-12 did not have increased survival rate (P=0.11), but did have prolonged survival time (P<0.0001). Survivors in each group were re-challenged with wild-type RT-2 cells, and all had long-term survival. CONCLUSIONS: Irradiated TV plus continuous localized infusion of GM-CSF/IL-12 may induce a tumor-specific anti-tumor immune response on established subcutaneous or intra-cerebral gliomas, and such a treatment strategy deserves consideration as adjuvant treatment for glioma.

Induction of T-cell apoptosis in rats by genetically engineered glioma cells expressing granulocyte-macrophage colony-stimulating factor and B7.1.

PURPOSE: To evaluate antitumor effects on intracerebral gliomas of genetically engineered tumor vaccines expressing granulocyte-macrophage colony-timulating factor (GM-CSF), B7.1, or both (combination). EXPERIMENTAL DESIGN: A rat glioma cell line, RT-2, was engineered with a retroviral vector to express GM-CSF, B7.1, or combination. Tumorigenicity of engineered cells and therapeutic effects of s.c. given irradiated or live tumor vaccines on parental intracerebral gliomas were studied. Immune cell infiltration induced at vaccine and tumor sites was examined by histologic and immunohistochemical staining. Apoptosis of T cells from vaccine sites was analyzed with fluorescence-activated cell sorting. RESULTS: Engineered RT-2 cells exhibited reduced s.c. tumorigenicity in rats with reduced tumor growth and prolonged animal survival time compared with control rats. Rats with intracerebral gliomas s.c. treated with irradiated or live GM-CSF-expressing vaccines had 60% and 100% survival rates, respectively, significantly better than the control groups (P < 0.05). In contrast, rats treated with vaccines expressing B7.1 or the combination had no or mild therapeutic effects. Studies revealed less T-cell infiltration at both vaccine and tumor sites in rats treated with vaccines expressing B7.1 or the combination than in rats treated with a vaccine expressing GM-CSF. Cell sorting analyses revealed higher proportions of apoptotic T cells at vaccine sites of rats treated with the combination than those treated with vaccine expressing GM-CSF. CONCLUSIONS: Combination of GM-CSF- and B7.1-expressing tumor vaccines exerted no synergistic, or even worse, therapeutic effects on gliomas compared with single GM-CSF-secreting tumor vaccine. The worse therapeutic effects of the GM-B7.1-expressing tumor vaccine than the GM-CSF-expressing tumor vaccine were related to the reduced T-cell amount and increased T-cell apoptosis in the former.

The immunization site of cytokine-secreting tumor cell vaccines influences the trafficking of tumor-specific T lymphocytes and antitumor efficacy against regional tumors.

Tumor cells engineered to secrete cytokines, referred to as tumor cell vaccines, can often generate systemic antitumor immunity and, in many cases, cause tumor regression. We compared the efficacy of s.c. immunization or intrahepatic immunization of GM-CSF-expressing tumor cell vaccines on the growth of s.c. or orthotopic liver tumors. A chemically transformed hepatic epithelial cell line, GP7TB, derived from Fischer 344 rats, was used to generate tumor models and tumor cell vaccines. Our results demonstrated that two s.c. injections of an irradiated tumor cell vaccine significantly controlled the growth of s.c. tumors, but was completely ineffective against orthotopic liver tumors. Effector cell infiltration in liver tumors was markedly reduced compared with s.c. tumors. Enhanced apoptosis of some effector cells was observed in the liver tumors compared with the s.c. tumors. Furthermore, the T cells induced by s.c. immunization preferentially migrated to s.c. tumor sites, as demonstrated by adoptive transfer experiments. In contrast, intrahepatic immunization, using parental tumor cells admixed with adenoviruses carrying the GM-CSF gene, yielded significantly better therapeutic effects on the liver tumors than on the s.c. tumors. Adoptive transfer experiments further confirmed that the T cells induced by liver immunization preferentially migrated to the liver tumor sites. Our results demonstrate that distinct T cell populations are induced by different immunization routes. Thus, the homing behavior of T cells depends on the route of immunization and is an important factor determining the efficacy of immunotherapy for regional tumors.

Curative potential of GM-CSF-secreting tumor cell vaccines on established orthotopic liver tumors: mechanisms for the superior antitumor activity of live tumor cell vaccines.

In preclinical studies, tumor cells genetically engineered to secrete cytokines, hereafter referred to as tumor cell vaccines, can often generate systemic antitumor immunity. This study investigated the therapeutic effects of live or irradiated tumor cell vaccines that secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) on established orthotopic liver tumors. Experimental results indicated that two doses (3 x 10(7) cells per dose) of irradiated tumor cell vaccines were therapeutically ineffective, whereas one dose (3 x 10(6) cells) of live tumor cell vaccines caused complete tumor regression. In vivo depletion of CD8+ T cells, but not natural killer cells, restored tumor formation in the live vaccine-treated animals. Additionally, the treatment of cells with live vaccine induced markedly higher levels of cytotoxic T lymphocyte activity than the irradiated vaccines in the draining lymph nodes. The higher levels of cytokine and antigen loads could partly explain the superior antitumor activity of live tumor cell vaccines, but other unidentified mechanisms could also play a role in the early T cell activation in the lymph nodes. A protocol using multiple and higher dosages of irradiated tumor cell vaccines also caused significant regression of liver tumors. These results suggest that the GM-CSF-secreting tumor cell vaccines are highly promising for orthotopic liver tumors if higher levels of immune responses are elicited during early tumor development.

Concurrent delivery of GM-CSF and endostatin genes by a single adenoviral vector provides a synergistic effect on the treatment of orthotopic liver tumors.

BACKGROUND: The immune resistance of large tumors represents a major problem for cancer immunotherapy, whereas the need for repeated injections of high doses of recombinant anti-angiogenic proteins represents a similar problem for anti-angiogenic therapy. To test whether antitumor activity could be increased by combining the above two mechanisms, this study examined the therapeutic effect of combination gene therapy using a murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) gene and a human endostatin (hED) gene on a rat orthotopic liver tumor model. METHODS: An adenoviral vector was constructed that simultaneously carried two transcriptional cassettes, for the expression of mGM-CSF and hED, respectively, or that carried a single cassette of either gene. The adenoviruses were intratumorally administered to 3-day-old or 7-day-old tumors. Moreover, the antitumor effects of the combination therapy and monotherapy were assessed and compared. RESULTS: The double-gene-containing adenoviral vector expressed transgenes as efficiently as the single-gene-containing vector. Moreover, the adenovirally expressed endostatin was biologically active, as demonstrated in vitro and in vivo. Results from animal experiments demonstrated a synergistic antitumor effect induced by the combined mGM-CSF and hED therapy. The combination of hED with mGM-CSF enhanced tumor-specific CTL activity, but did not interfere with the infiltration of cellular effectors in the tumor regions. The blood vessel density of the liver tumors markedly reduced as a result of hED expression in both monotherapy and combination therapy. Furthermore, combination therapy significantly increased the number of apoptotic cells in the tumor regions. CONCLUSIONS: The experimental results suggest that the combined gene therapy against tumor cells and the tumor vascular system using antitumor immune mechanisms and anti-angiogenic mechanisms holds promise as a strategy for treating cancers.