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 (125–4000 μg/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.

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 α-smooth muscle actin (α-SMA) in those large HCC, which were resistant to adenoviral infection. Intra-arterial infusion of vasoactive compounds (histamine, angiotensin II or nitric oxide donor nitroglycerin) before vector administration enhanced gene transfer to tumor nodules that were poorly transduced without pre-treatment. Nitroglycerin was active to enhance transduction of large tumors with trabecular or pseudoglandular histological pattern, which were impermeable to adenoviral vectors even after histamine or angiotensin treatments. Our data indicate the presence of a physical barrier between blood and neoplastic cells, which prevents transduction of the tumor by vectors given by the intravascular route. The thickness and impermeability of the barrier increases as the tumor nodule grows. Vasoactive compounds may be of value in gene therapy of liver cancer by increasing transduction efficiency by intravascularly administered vectors.

Background: Bioluminescent imaging (BLI) is based on the detection of light emitted by living cells expressing a luciferase gene. Stable transfection of luciferase in cancer cells and their inoculation into permissive animals allows the noninvasive monitorization of tumor progression inside internal organs. We have applied this technology for the development of a murine model of colorectal cancer involving the liver, with the aim of improving the pre-clinical evaluation of new anticancer therapies.Results: A murine colon cancer cell line stably transfected with the luciferase gene (MC38Luc1) retains tumorigenicity in immunocompetent C57BL/6 animals. Intrahepatic inoculation of MC38Luc1 causes progressive liver infiltration that can be monitored by BLI. Compared with ultrasonography (US), BLI is more sensitive, but accurate estimation of tumor mass is impaired in advanced stages. We applied BLI to evaluate the efficacy of an immunogene therapy approach based on the liver-specific expression of the proinflammatory cytokine interleukin-12 (IL-12). Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival.Conclusion: We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver. Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.

The preclinical evaluation of toxicity and antitumor effect of conditionally replicative (oncolytic) adenoviruses is hampered by the inability of human adenoviruses to replicate efficiently in murine cells. The Syrian golden hamster (Mesocricetus auratus) has been suggested as a permissive animal for adenoviral replication, and cancer cell lines derived from various hamster tumors are available. We provide evidence that wild-type adenovirus type 5 is able to infect and replicate in the pancreatic cancer cell lines HaP-T1 and H2T both in vitro and in vivo. Determination of cytopathic effect, viral spread, progeny production, and the expression of late viral proteins indicates that the complete viral cycle of adenovirus takes place, albeit less efficiently than in highly permissive human cancer cell lines A549 and HuH7. Intrahepatic inoculation of HaP-T1 and H2T cells gave rise to tumors in the liver of hamsters that resemble metastases of pancreatic cancer. The growth of HaP-T1-induced nodules was faster compared with those derived from H2T, but both caused progressive liver infiltration and peritoneal dissemination. When adenovirus was inoculated in these lesions, productive replication took place and newly formed infective virions could be recovered 4 days after administration. In conclusion, the Syrian hamster models described here offer the opportunity to evaluate the effect of oncolytic adenoviruses in an immunocompetent animal and may be a valuable tool in the preclinical evaluation of these agents.