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We have recently demonstrated that a 4-in-1 gene therapy strategy that contains two anti-angiogenic genes [endostatin and pigment epithelium-derived factor] and two cytokine genes [granulocyte macrophage colony-stimulating factor and interleukin 12] has a considerable antitumor effect on large tumors in a woodchuck hepatoma model. The current study further investigates the underlying mechanisms for the antitumor effect observed by using small rodent models. We found that immunotherapy alone increased immunosuppressive cells in large tumors over time, whereas the anti-angiogenic therapy contained in the 4-in-1 strategy alleviated immunosuppression and made tumors vulnerable to immunotherapy, thus resulting in a synergistic antitumor effect., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Cytokine and antiangiogenic gene therapies have proved effective in implanted hepatocellular carcinoma (HCC) models in which small tumor burdens were established in small rodents. These models, however, may not reflect human HCCs, which are frequently detected at a stage when tumors are large and multifocal. In addition, HCC in patients is often associated with viral hepatitis. To investigate the effectiveness of a mixture type of gene therapy strategy on large tumor burdens, we used the woodchuck model in which woodchuck hepatitis virus-induced HCCs are large and multifocal, simulating the conditions in humans. Adenoviruses encoding antiangiogenic factors (pigment epithelium-derived factor and endostatin) or cytokines (GM-CSF and IL-12) were delivered via the hepatic artery separately or in combination into woodchuck livers bearing HCCs. Our results showed that the mixture type of strategy, which contained two cytokines and two antiangiogenic factors, had better antitumor effects on large tumors as compared with monotherapy either with antiangiogenic or cytokine genes. The immunotherapy recruited significant levels of CD3(+) T cells that infiltrated the tumors, whereas the antiangiogenesis-based therapy significantly reduced tumor vasculature. The mixture type of gene therapy achieved both effects. In addition, it induced high levels of natural killer cells and apoptotic cells and reduced the levels of immunosuppressive effectors in the tumor regions. Hence, antiangiogenic therapy may provide the advantage of reducing immune tolerance in large tumors, making them more vulnerable to the immune reactions. Our study implies that in the future, the combination therapy may prove effective for the treatment of patients with advanced HCC.

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.

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.

Hepatocellular carcinoma (HCC) is one of the most frequent cancers worldwide. Effective therapy to this cancer is currently lacking, creating an urgent need for new therapeutic strategies for HCC. Gene therapy approach that relies on the transduction of cells with genetic materials, such as apoptotic genes, suicide genes, genes coding for antiangiogenic factors or immunomodulatory molecules, small interfering RNA (siRNA), or oncolytic viral vectors, may provide a promising strategy. The aforementioned strategies have been largely evaluated in the animal models with HCC or liver metastasis. Due to the diversity of vectors and therapeutic genes, being used alone or in combination, gene therapy approach may generate great beneficial effects to control the growth of tumors within the liver.

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., (Copyright 2003 John Wiley & Sons, Ltd.)

Background and Aims: We established an orthotopic animal model of rectal cancer in mice and applied this model to the study of the antitumor effects of cytokine-assisted tumor vaccine., Materials and Methods: The CT-26 murine colon adenocarcinoma cells were inoculated into the submucosa of the rectum of the mice to induce the rectal tumor. The tumor growth rate and the survival time of the mice were observed. The cDNA of granulocyte-macrophage colony-stimulating factor (GM-CSF) was transduced to the CT-26 cell line via a retroviral vector, and the therapeutic effects of irradiated GM-CSF secreting tumor vaccine on the rectal tumor were investigated., Results: All the mice implanted with the wild-type tumor cells had tumor growth in the rectum and died. The mean survival time of the mice was 28.9 days. Two doses of irradiated GM-CSF secreting tumor vaccine administered on days 0 and 3 after tumor cell implantation significantly prolonged the survival of the mice with rectal tumor compared with that of the control groups ( P<0.0001). In contrast, no antitumor effect was observed when the treatment with GM-CSF secreting tumor vaccine was delayed to 3 days after tumor cell implantation ( P>0.17)., Conclusion: The results suggest that cytokine gene therapy exerts an antitumor effect on small tumors and may be considered as an adjuvant immunotherapy of rectal cancers and prevention of reimplantation of tumor cells disseminated during or following surgery. The orthotopic animal model of the rectal cancer in mice could be applied to the in vivo experimental studies of rectal cancer.