Induction of immunosuppressive molecules and regulatory T cells counteracts the antitumor effect of interleukin-12-based gene therapy in a transgenic mouse model of liver cancer☆
Introduction
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, with an estimated 600,000 new cases per year worldwide [1]. HCC responds poorly responsive to conventional chemotherapy and hormonal treatments [2]. The incidence of HCC is increasing considerably worldwide and the development of novel and efficient therapies for this malignancy has become an urgent need.
Gene therapy represents a promising modality to treat HCC since it allows to concentrate the expression of therapeutic gene products within or around the tumor lesions. IL-12 is a cytokine naturally secreted by activated dendritic cells and monocytes/macrophages [3], [4], this triggers proliferation of activated natural killer (NK) and T cells, and induces the secretion of IFN-γ, the major mediator of IL-12 biological activity [3]. IL-12 promotes a Th1 type of response and enhances tumor infiltration by effector CD4+ and CD8+ T lymphocytes, NK cells and macrophages, which are capable of mediating tumor cell death [5], [6]. In addition, IL-12 displays important antiangiogenic activities [3], [7], [8]. Gene therapy-based immunotherapy using IL-12 targeted to tumor nodules or the liver has been proposed to increase the tumor-blood gradient of the cytokine within the tumor area [9]. This would enhance the antineoplastic effect of IL-12 whilst reducing its systemic toxicity.
Intratumor injection of a first generation adenoviral vector encoding IL-12 results in remission of transplantable orthotopic HCC in rats [7]. However, only a limited and transient antitumor effect was evident using a similar approach in a phase I clinical trial, most probably due to low and short-term expression of the transgene [10], [11]. In this work, we aimed to treat HCC by inducing the expression of IL-12 in the liver for long periods of time using a system that enables modulation of transgene production [12].
Results obtained in syngeneic orthotopic rodent models have influenced the design of gene therapy strategies against liver cancer [13], [14]. However, transplantable tumors often lack the stromal organization and adaptation to host immune defences that occur in tumors that arise as a result of a slow process of progressive genetic and epigenetic changes [15], [16], [17]. A recent study demonstrated a similar molecular pattern between HCCs that spontaneously arise in c-myc, E2f1, and c-myc/E2f1 transgenic mice and a group of human HCC with good prognosis [18]. The L-PK/c-myc transgenic mice over-express the c-myc oncogene in the liver and after 6–8 months these animals develop well-differentiated HCCs that closely resemble human HCC in terms of biology, histology and relevant gene expression and protein functions [19], [20]. Thus, transgenic animal models of liver cancer can provide the most valuable data to predict the efficacy of a given therapeutic modality to treat HCC in patients.
In this study, we have used L-PK/c-myc transgenic mice to evaluate the antitumor activity of long-term and inducible IL-12 production in the liver. Our results show that, although induction of IL-12 for a prolonged period of time can improve the prognosis of murine HCC, the efficacy of the treatment may be limited by the upsurge of immunosuppressive mechanisms in the tumor lesions.
Section snippets
L-PK/c-myc mice
We established a transgenic mice colony by crossing L-PK/c-myc (+/−) males [19] with 129S2/SvHsd wt females (Harlam, Barcelona, Spain). The littermates were genotyped as described earlier [19]. Heterozygous L-PK/c-myc mice (28% of littermates) were fed with a 75% carbohydrate-rich diet (Safe, Augy, France) to enhance c-myc expression [20]. About 60% of these animals developed single and multifocal tumors in the liver by 7–8 months. A total of 182 animals were bred, 56 were transgenic for c-myc,
Antitumor efficacy of long-term intrahepatic expression of mIL-12 in L-PK/c-myc mice
L-PK/c-myc transgenic mice slowly develop HCCs due to sustained over-expression of the c-myc oncogene in the liver [19], [20]. Two groups of L-PK/c-myc mice with tumors of 6.24 ± 3.53 mm in diameter were established with a similar distribution of males and females. The treated group received pTonL2(T)-mIL12 plasmid via hydrodynamic injection and the control group received the same volume of saline. Both groups were given 3 cycles of 2 mg/ml of Dox in drinking water separated by 3–5 weeks rest (Fig.
Discussion
In this work, we have evaluated the antitumor efficacy of repeated induction of IL-12 expression over an extended period of time in the L-PK/c-myc transgenic mice model of HCC. These mice develop liver tumors with similar pathobiology to human HCC [20]. We used a plasmid encoding IL-12 under the control of a liver-specific inducible system, the plasmid was injected into the liver by the hydrodynamics procedure. Such a procedure was recently shown to be efficient in transferring genes to the
Acknowledgements
We thank Pablo Sarobe, Ignacio Melero, Rubén Hernández-Alcoceba, and Gloria González-Aseguinolaza for helpful discussions, and technical help obtained from Jose Ignacio Riezu, Carmen Berasain, Mercedes Reboredo, Laura Guembe, Marta Garcı´a-Granero, January Salas, and Paula Garcés. We are grateful to Paul Neeson (Peter Mac Callum Cancer Institute, Australia) for help with English. This work was financed by UTE project CIMA and Grants from Fondo de Investigacion Sanitaria (FIS), Ministerio de
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The authors declare that they do not have anything to disclose regarding conflict of interest with respect to this manuscript.