Affiliations of authors: B. M. Pützer, T. Stiewe, F. Rödicker, C. Scherer (Department of Molecular Biology [Cancer Research]), O. Schildgen, M. Fiedler, M. Roggendorf (Department of Virology), S. Rühm (Department of Diagnostic Radiology), O. Dirsch (Department of Pathology), U. Damen (Department of Experimental Surgery), University of Essen Medical School, Germany; B. Tennant, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY; F. L. Graham, Department of Biology and Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.
Correspondence to: Brigitte M. Pützer, M.D., Ph.D., Centre for Cancer Research and Cancer Therapy, Institute of Molecular Biology, University of Essen, Hufelandstr. 55, D-45122 Essen, Germany (e-mail: brigitte.puetzer{at}uni-essen.de)
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ABSTRACT |
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INTRODUCTION |
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Of these cytokines, IL-12 appears to possess the strongest antitumor activity. The mechanism for IL-12-mediated tumor killing includes the activation of natural killer cells, T cells, lymphokine-activated killer cells, and macrophages (13). IL-12 stimulates IFN production from natural killer and T cells, promotes the cellular immune response by facilitating the proliferation and activation of Th1 T-helper cells (14), and inhibits angiogenesis in vivo (15). IL-12 interacts synergistically with the costimulatory molecule B7.1/CD80, normally expressed on professional antigen-presenting cells, to enhance protective antitumor immunity (9,1619). B7.1-mediated antitumor activity in these studies was largely attributed to the stimulation of natural killer and CD8+ T cells, whereas the requirement for CD4+ cells in tumor rejection is highly dependent on the tumor model (20).
Previously, we constructed an adenovirus vector carrying genes for both IL-12 and B7.1, termed "AdIL-12/B7.1," and demonstrated their synergistic effect on tumor growth by injecting the vector directly into breast adenocarcinomas transplanted from a polyoma middle-T-antigen (PyMT)-transgenic mouse model (9). In tumors derived from the transgenic model, one injection of the vector resulted in complete tumor regression in 70%90% of the treated animals and induced long-term systemic immunity (9). However, the animal model in this study, as in the majority of cancer immunotherapy trials, is based on transplantation of established tumor cell lines into immunocompetent animals. In contrast, during carcinogenesis, a primary tumor develops from a single cell that becomes malignant in its natural environment (21), thus allowing the development of immune escape mechanisms that may negatively affect immunotherapeutic approaches in humans (21).
To address this problem, we tested the ability of the AdIL-12/B7.1 vector to reduce the volume of primary, nontransplanted woodchuck hepatitis virus (WHV)induced hepatocellular carcinomas (HCCs) in woodchucks. WHV is closely related to the human hepatitis B virus in its structure, genomic organization, mechanism of replication, and the course of infection. Persistent WHV infection is associated frequently with the development of hepatic tumors (22). This experimental model has been applied extensively in the study of underlying mechanisms of human hepatitis B virus-induced hepatocarcinogenesis and in the development of prophylactic and therapeutic strategies of disease control (23,24).
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MATERIALS AND METHODS |
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The woodchuck liver cell line WH12/6 (25) was maintained in Ham's F-12 medium supplemented with 10% fetal calf serum (FCS). Human MRC5 fibroblasts (cell line CCL 171; American Type Culture Collection, Manassas, VA) were grown in modified Eagle medium plus 10% FCS. All viruses were grown in 293 cells [adenovirus 5 E1-transformed human embryonic kidney cells (26)] maintained in modified Eagle medium/F-11 with 10% FCS. Media were supplemented with 2 mM L-glutamine, penicillin at 100 µg/mL, and streptomycin at 100 U/mL.
Animals
Adult American woodchucks (Marmota monax) were chronically infected with WHV, which is closely related to human hepatitis B virus. All chronic WHV carriers were provided by Marmotech, Inc., Ithaca, NY, and had one or more hepatic tumors that were 2 cm or larger in diameter, characterized by ultrasound and magnetic resonance imaging (MRI). In this study, a total of five animals have been used. Four animals were treated by intratumoral administration of Ad vectors after laparotomy, and one was given an injection under MRI guidance.
Recombinant Adenovirus Vectors
Construction of the recombinant adenovirus vector expressing both murine IL-12 subunits in early region 1 (E1) and B7.1 in early region 3 has been described previously (9). The control adenovirus AdEGFP encoding the enhanced green fluorescent protein was described (27). Adenovirus vectors were propagated, purified, and titrated as described previously (28,29).
Enzyme-Linked Immunosorbent Assay and Flow Cytometry Analysis
Hepatoma cells infected at a multiplicity of infection of 10 plaque-forming units (pfu) per cell were incubated in growth medium as indicated. Expression levels of secreted murine IL-12 were quantitated from infected cell supernatant by an enzyme-linked immunosorbent assay (ELISA) with the use of the mouse IL-12p70 DuoSet ELISA development system (R&D Systems Europe, Oxon, U.K.), according to the manufacturer's protocol. For B7.1 detection, cells were trypsinized, resuspended in ice-cold phosphate-buffered saline (PBS) containing 1% fetal bovine serum, incubated with the fluorescein isothiocyanate-conjugated anti-mouse B7.1 antibody (PharMingen, Hamburg, Germany) for 60 minutes on ice, washed twice in PBS, and analyzed for fluorescence with a FACSVantage flow cytometer (Becton Dickinson Biosciences, Heidelberg, Germany). Human MRC5 fibroblasts were used as a negative control for B7.1 expression because these cells do not react with the mouse B7.1 antibody.
Ribonuclease Protection Assay
Total RNA was extracted from frozen liver biopsy samples with the RNeasy Mini kit (Qiagen, Hilden, Germany) by following the manufacturer's directions. Ribonuclease protection assays (RPAs) for the analysis of cytokine and T-lymphocyte transcripts were performed with the RiboQuantTM RPA kit (PharMingen, San Diego, CA) according to the instruction manual. The protected RNA bands were quantitated on a Cyclone storage phospho screen imager (Packard, Dreiech, Germany).
Intratumoral Administration of Adenovirus Vectors
Hepatic tumors were selected preoperatively by ultrasound. Four woodchucks were anesthetized with 1 mL/kg of ketamine (Dr. Graeub, AG, Bern, Switzerland) and 0.3 mL/kg of xylazine (Bayer, Leverkusen, Germany). Median laparotomies were performed, and grossly identifiable tumors were located and measured with calipers before injection of adenovirus vectors and at various times after treatment. The tumor volume was calculated from the longest diameter and average width by assuming a prolate spheroid shape (6). Four woodchucks, each with two or more hepatic tumors that were 2.0 cm or larger in diameter, were treated with a single type of vector as follows: One woodchuck (animal 1) received multiple intratumoral injections (a total of 1 x 109 pfu of AdEGFP in 350 µL of PBS) in separate sites of one tumor nodule. One woodchuck (animal 2) received multiple intratumoral injections (a total of 1 x 109 pfu of AdIL-12/B7.1 in 350 µL of PBS) in separate sites of one tumor nodule. In two woodchucks (animals 3 and 4), two tumor nodules of equal size were treated with multiple intratumoral injections of either AdEGFP or AdIL-12/B7.1; each tumor nodule received a total of 1 x 109 pfu in 350 µL of PBS. Animals were killed on day 7 (animal 3), day 10 (animal 4), or day 14 (animals 1 and 2) after injection; the liver was exposed; and hepatic tumors were measured. Tumor and normal liver tissue samples were fixed in phosphate-buffered formalin, embedded in paraffin, and sectioned. Sections were stained with hematoxylineosin for histologic examination. Samples of hepatic tumors and non-neoplastic liver were frozen in liquid nitrogen and stored at -70 °C for RPAs.
Selective Adenovirus Vector Administration and Tumor Size Assessment by MRI Guidance
For each MRI session, the woodchuck was fully anesthetized with ketamine (1 mL/kg) and xylazine (0.3 mL/kg). The tumor was selected, and the injection needle was placed directly into the tumor nodule under guidance by an MRI by use of a 1.5-tesla magnet (Magnetom Sonata; Siemens, Erlangen, Germany). The animal received a single intratumoral injection of 1 x 109 pfu of the AdIL-12/B7.1 vector in 350 µL of PBS. Other tumors identified served as controls. To maximize the signal-to-noise ratio, a standard head coil was used for data reception. The animal underwent axial MRI examinations of the liver with the following imaging parameters: 1) T1-weighted fast low-angle-shot (FLASH) sequence (162-ms/4.0-ms repetition time/echo time, 256 x 256 matrix, 5-mm slice thickness, 24-cm2 x 14-cm2 field of view, one signal acquired, 12-second acquisition time); 2) T2-weighted HASTE (half-Fourier-acquisition single-shot turbo-spin-echo) sequence (1000-ms/60-ms repetition time/echo time, 256 x 256 matrix, 5-mm slice thickness, 24-cm2 x 14-cm2 field of view, one signal acquired, 20-second acquisition time); and 3) T1-weighted FLASH sequence (162-ms/4.0-ms repetition time/echo time, 256 x 256 matrix, 5-mm slice thickness, 24-cm2 x 14-cm2 field of view, one signal acquired, 12-second acquisition time) with fat-saturation postintravenous application of 1 mL of Gd-diethylenetriaminepentaacetate; Magnevist; Schering, Berlin, Germany). Because tumors were most conspicuous on T1-weighted postcontrast images, this sequence was used to assess tumor volume and thus to monitor the response of tumor treatment. Therefore, region-of-interest measurements were performed on a computer workstation with the region-of-interest size adapted to encompass the tumor contours on the individual image sections. The calculated area was then multiplied by the craniocaudal extension of the tumor.
All animal experiments were approved by and carried out according to guidelines set forth by the Animal Research Ethics Board of the University of Essen.
Statistical Analysis
The statistical significance of the relative increase in tumor size was calculated by paired Student's t test. All statistical tests were two-sided.
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RESULTS |
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In vivo IL-12 gene delivery by adenovirus vectors into established transplanted tumors can inhibit tumor growth and induce tumor regression in various mouse tumor models (8,3034). We have demonstrated previously (9) that the antitumor activity of IL-12 in transplanted tumors can be increased substantially by also inserting B7.1. When we injected this vector into the transplanted tumors at 2.5 x 107 pfu, a low virus dose, we observed complete tumor regression and induction of systemic immunity in the majority of animals. To determine whether AdIL-12/B7.1 could be used to treat established nontransplanted (orthotopic) tumors, we used the woodchuck animal model. Before the vector was given to woodchucks, however, we infected WH12/6 cells in vitro and determined that murine IL-12 and B7.1 were expressed 3 and 5 days after infection (Fig. 1). Next, we selected large tumors in four woodchucks that were chronic WHV carriers and injected the tumors with AdIL-12/B7.1 or the control vector AdEGFP. The absolute tumor volumes of six tumor nodules, three injected with the AdIL-12/B7.1 virus and three treated with AdEGFP virus, are presented in Table 1
before and 714 days after treatment; the absolute tumor volumes of two untreated tumors are also presented after 14 days. On average, over a 2-week period, tumors given an injection intratumorally with AdIL-12/B7.1 showed an 80% reduction in volume from their original volume (Fig. 2
, A), whereas untreated and AdEGFP-injected tumors showed an approximately twofold increase in volume (Fig. 2
, A). Fig. 2
, B, presents representative MRI images from one woodchuck at three representative time points (before treatment and 4 and 7 weeks after treatment), showing the long-term antitumor response after direct intratumoral injection of 1 x 109 pfu of AdIL-12/ B7.1 into a selected liver tumor nodule under MRI guidance. In this experiment, AdIL-12/B7.1 treatment had a pronounced effect on tumor growth over a 7-week period, leading to an approximately 95% reduction in tumor size from 8.6 cm3 at the time of injection to 0.5 cm3 by day 50 (Fig. 2
, B-I to B-III). In contrast, no reduction in tumor size was noted in the surrounding untreated tumors. Thus, large established nontransplanted liver tumors can be treated efficiently by injection of an IL-12/B7.1-expressing adenovirus vector.
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Gene transfer studies (5,35,36) in several mouse models have indicated that rejection of transplanted tumors expressing IL-12 and/or B7.1 is dependent on CD4+ and CD8+ T lymphocytes and natural killer cells. To analyze the immune response elicited by injection of AdIL-12/B7.1 into established nontransplanted liver tumors, we next examined liver and tumor biopsy samples from woodchucks 14 before and after treatment (Fig. 3, A). Histologic sections from HCCs showed the typical features of neoplastic hepatic cells forming pseudotubulary structures with striking changes in the nuclear morphology, characterized by condensed chromatin and macronucleoli (Fig. 3
, A-III). In contrast to nodules injected with the control vector AdEGFP (Fig. 3
, A-IV), necrosis and massive inflammatory infiltration were detected only in AdIL-12/B7.1-treated tumors (Fig. 3
, A-V). In the nontumoral liver parenchyma, we found typical morphologic patterns of chronic hepatitis after persistent WHV infection, characterized by moderate fibrosis and mild mononuclear infiltrate of portal or periportal tracts with occasional necrosis [(37) and Fig. 3
, A-I]. However, no evidence of adenovirus- induced hepatic toxicity was observed in normal liver tissue 10 days after intratumoral vector administration (Fig. 3
, A-II).
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DISCUSSION |
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Because our previous data indicated that AdIL-12/B7.1-injected murine tumors were effectively rejected within approximately 2 weeks, the immune response against nontransplanted woodchuck liver tumors was first analyzed shortly after treatment. Consistent with the results obtained from the PyMT model (9), much of the tumor mass was rejected by day 14, suggesting that AdIL-12/B7.1 had a similar inhibitory effect on growth of transplanted and nontransplanted tumors at this early stage of treatment. The use of MRI guidance allowed us to locate and select large tumor nodules into which we could directly inject the vector, thereby avoiding the negative and stressful effects of surgery. MRI monitoring of a selected tumor revealed long-term tumor regression, resulting in nearly complete tumor elimination. However, there was a clear difference in the time course of tumor reduction between the tumor injected at one site under MRI guidance and the tumors injected at multiple sites during laparotomy.
In murine models, intratumoral injection of adenovirus vectors carrying IL-12 and/or B7.1 can stimulate systemic immunity that can act effectively on distal tumors (79). No clear systemic effect, however, has been observed in the woodchuck model, which may reflect a variation in the immunogenicity of different tumor nodules (Roggendorf M: unpublished data). Because these tumors are not of clonal origin and might express different tumor antigens, an effect on surrounding tumors might be obtained when vascular anastomoses allow the virus to spread between the tumors. Therefore, a lack of anastomoses between the tumor and the normal liver in this model might contribute to the lack of systemic response. In addition, higher doses of cytokine vectors were more efficient in inhibiting tumor growth than lower doses (9,30,40). If the body weight of a woodchuck and the tumor size of 25 cm in diameter are considered, the virus dose of 1 x 109 pfu, used for woodchucks, is low compared with doses used in mice. Therefore, we cannot exclude the possibility that the IL-12/B7.1 virus might induce systemic immunity in primary cancer at higher vector doses.
Our results suggest that regression of WHV-induced primary liver tumors is associated with massive intratumoral lymphocyte infiltration. We also show that intratumoral adenoviral IL-12/B7.1 expression in primary HCCs leads to increased intratumoral levels of IFN , consistent with results from a number of models indicating that the antitumor activity of IL-12 is IFN
dependent (8,41,42). From recent observations, expression of major histocompatibility complex (MHC) class I molecules is altered in liver cells of animals chronically infected with WHV (43) and also commonly reduced on neoplastic cells of HCC nodules (Roggendorf M: unpublished data). Consequently, this reduction in MHC molecules may diminish the susceptibility of WHV-infected hepatocytes to virus-specific T cells, WHV-infected hepatocytes may evade the antiviral immunologic surveillance system, and thus liver damage may be perpetuated. The current model emphasizes the role of IFN
for the modulation of peptide processing and stimulation of genes involved in antigen presentation by the MHC class I molecules (44). Thus, the activation of MHC class I-associated genes by local induction of IFN
through tumor-infiltrating T lymphocytes may be a possible mechanism for efficient tumor rejection after IL-12/B7.1 immunotherapy in this model.
However, our observation varies from the observations made in established nontransplanted 3-MC-induced mouse tumors after IL-7/B7.1 gene delivery (45). In that model, failure to induce tumor regression depends on the inability of T cells to infiltrate nontransplanted tumors. The observed difference might result from a difference in T-cell infiltration of tumors induced by viruses or by carcinogens rather than a general difference between transplanted and primary, nontransplanted malignant tumors.
The animal model used in this study is expensive and difficult to use, and only a limited number of tumor-bearing WHV-infected woodchucks were available. Nevertheless, our results demonstrate that adenovirus vector-mediated immunotherapy is certainly not limited to artificial mouse tumor models and provide a powerful approach for the in vivo treatment of large, primary tumors in a model of hepadnavirus-induced carcinogenesis, reflecting the real-world scenario of HCC in humans. Direct injection into selected HCC nodules under MRI guidance appears to be a relatively nontoxic and feasible method for administrating viral vectors in human clinical trials. However, our data emphasize the need to evaluate the mechanisms involved in effective intratumoral penetration by immune effector cells as a prerequisite for successful cytokine treatment of natural tumors.
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NOTES |
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Supported in part by grant PU188/21 from the Deutsche Forschungsgemeinschaft and by the Internal Research Support Program from the University of Essen, Germany (to B. M. Pützer).
We thank Silke Bosk for excellent technical assistance in performing the magnetic resonance tomography, Li Jun for surgical assistance, and Michael Lowak for help in animal handling.
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Manuscript received September 22, 2000; revised December 28, 2000; accepted January 9, 2001.
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