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Background and aims New options are needed for the management and prevention of colorectal cancer liver metastases. Interleukin 12 (IL-12) is an immunostimulatory cytokine with proven antitumour effect in animal models. Despite evidence indicating its biological effect in humans, neither the recombinant protein nor gene therapy vectors expressing IL-12 have shown a relevant benefit in patients with cancer. Objective To develop a new approach to overcome the difficulties in obtaining a suitable expression pattern and the immunosuppressive milieu in the tumours which contribute to this poor performance. Methods A high-capacity ('gutless') adenoviral vector carrying a liver-specific, mifepristone (Mif)-inducible system for the expression of IL-12 (HC-Ad/RUmIL-12) was used in combination with chemotherapy. Tumours were established in the liver of C57BL/6 mice by inoculation of MC38 colon cancer cells. Results Intrahepatic injection of HC-Ad/RUmIL-12 and tailored induction regimens allowed the maintenance of safe and efficient levels of IL-12 in vivo. An individualised, stepwise increase in the dose of Mif (125e4000 mg/kg) was needed to compensate for the progressive but transient downregulation of the inducible system. Repeated cycles of Mif induction (every 24 h for 10 days) were needed for optimal tumour eradication. However, complete protection against tumour rechallenge was seen in <25% of the animals. The administration of oxaliplatin (5 mg/kg intraperitoneally) 3 days before starting the induction regimen achieved efficient elimination of liver metastases with a single cycle of IL-12 induction, and improved protection against tumour rechallenge. This was associated with a shift in the tumour microenvironment towards a more pro-immunogenic phenotype, with an increase in the CD8+/T regulatory cell ratio and a reduction in myeloid-derived suppressor cells. These effects were not seen with 5-fluorouracil, irinotecan or gemcitabine. Conclusions Long-term controlled expression of IL-12 using an HC-Ad vector in combination with oxaliplatin is effective and clinically applicable against hepatic colon cancer metastases.< Additional figures are published online only. To view these files please visit the journal online (http://gut.bmj. com).

We have evaluated gene transfer efficiency to tumor nodules in diethylnitrosoamine (DENA)-induced hepatocellular carcinoma (HCC) in rats using adenoviral vectors administered by three different routes: intraportal, intra-arterial and intratumoral injection. Our results showed that intraportal infusion could not transduce tumor nodules greater than 1 mm in diameter while the intra-arterial route allowed transduction of nodules up to 2-5 mm in diameter. Tumors greater than this size were resistant to transduction by intravascular route, but could be transduced by direct intratumoral injection, indicating that the obstacle preventing gene transfer to tumor cells was mainly at the level of tumor vasculature and not at the level of neoplastic cells. We have studied the extracellular matrix in tumoral lesions to assess whether nodules with different size and histological pattern have different profiles in relation to transduction efficacy. Immunohistochemical detection showed a high expression of fibronectin (FN), laminin (LN) and ␣-smooth muscle actin (␣-

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An improved viral vector for cancer gene therapy should be capable of infecting tumors with high efficiency, inducing specific and high-level expression of transgene in the tumor and selectively destroying tumor cells. In the design of such a vector to treat hepatocellular carcinoma, we took advantage of (a) the high infectivity of adenoviruses for hepatic cells, (b) the high level of protein expression and proapoptotic properties that characterize Semliki Forest virus (SFV) replicon, and (c) tumor selectivity provided by A-fetoprotein (AFP) promoter. We constructed a hybrid viral vector composed of a helper-dependent adenovirus containing an SFV replicon under the transcriptional control of AFP promoter and a transgene driven by SFV subgenomic promoter. Hybrid vectors containing murine interleukin-12 (mIL-12) genes or reporter gene LacZ showed very specific and high-level expression of transgenes in AFP-expressing hepatocellular carcinoma cells, both in vitro and in an in vivo hepatocellular carcinoma animal model. Infected hepatocellular carcinoma cells were selectively eliminated due to the induction of apoptosis by SFV replication. In a rat orthotopic liver tumor model, treatment of established tumors with a hybrid vector carrying mIL-12 gene resulted in strong antitumoral activity without accompanying toxicity. This new type of hybrid vectors may provide a potent and safe tool for cancer gene therapy.