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*Substance via MeSH
Medline Plus Health Information
*Genes and Gene Therapy
*Liver Cancer
The Journal of Immunology, 2007, 178: 574-583.
Copyright © 2007 by The American Association of Immunologists, Inc.

Prolonged, NK Cell-Mediated Antitumor Effects of Suicide Gene Therapy Combined with Monocyte Chemoattractant Protein-1 against Hepatocellular Carcinoma

Tomoya Tsuchiyama*, Yasunari Nakamoto*, Yoshio Sakai*, Yohei Marukawa*, Masaaki Kitahara*, Naofumi Mukaida{dagger} and Shuichi Kaneko1,*

* Department of Gastroenterology, Graduate School of Medical Science and {dagger} Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tumor recurrence rates remain high after curative treatments for hepatocellular carcinoma (HCC). Immunomodulatory agents, including chemokines, are believed to enhance the antitumor effects of tumor cell apoptosis induced by suicide gene therapy. We therefore evaluated the immunomodulatory effects of a bicistronic recombinant adenovirus vector (rAd) expressing both HSV thymidine kinase and MCP-1 on HCC cells. Using an athymic nude mouse model (BALB/c-nu/nu), primary s.c. tumors (HuH7; human HCC cells) were completely eradicated by rAd followed by treatment with ganciclovir. The same animals were subsequently rechallenged with HCC cells, tumor development was monitored, and the recruitment or activation of NK cells was analyzed immunohistochemically or by measuring IFN-{gamma} mRNA expression. Tumor growth was markedly suppressed as compared with that in mice treated with a rAd expressing the HSV thymidine kinase gene alone (p < 0.001). Suppression of tumor growth was associated with the elevation of serum IL-12 and IL-18. During suppression, NK cells were recruited exclusively, and Th1 cytokine gene expression was enhanced in tumor tissues. The antitumor activity, however, was abolished either when the NK cells were inactivated with anti-asialo GM1 Ab or when anti-IL-12 and anti-IL-18 Abs were administered. These results indicate that suicide gene therapy, together with delivery of MCP-1, eradicates HCC cells and exerts prolonged NK cell-mediated antitumor effects in a model of HCC, suggesting a plausible strategy to prevent tumor recurrence.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Despite curative treatments including surgical resection and liver transplantation for hepatocellular carcinoma (HCC),2 tumor recurrence rates remain high, probably because of insufficient therapeutic effects and the multicentric development of HCC in cirrhotic liver (1, 2, 3). Although nonsurgical treatments of HCC such as radiofrequency ablation, transcatheter arterial embolizations, and transcatheter arterial chemotherapy induce apoptosis of HCC cells, these treatments do not enhance antitumoral immunity sufficiently. Therefore, gene therapy aimed at enhancing antitumor immune responses may be a promising approach to induce sufficient inhibitory effects for the prevention of tumor recurrence.

Although killing tumor cells with cytotoxic genes such as suicide gene/prodrug systems consisting of HSV thymidine kinase (HSV-tk) and ganciclovir (GCV) may lead to the generation of effective immunity (4, 5), cell killing alone is insufficient to increase many antitumor responses (6, 7, 8). Recently, however, coexpression of HSV-tk and chemokines was found to increase tumor immunity in animal models in which neither HSV-tk nor chemokine expression alone was sufficient (9). In addition, we previously demonstrated that, at the local treatment site, the antitumor effects of the HSV-tk/GCV system were enhanced by codelivery of MCP-1, a member of the CC chemokine family (8, 10). MCP-1 has been shown to stimulate the cytotoxic activity of monocytes, enhance the expression of adhesion molecules such as CD11b and CD11c, and induce the cytotoxic and migratory activities of NK cells (11, 12, 13, 14). Moreover, transfection of the MCP-1 into human lung adenocarcinoma cells inhibited the formation of metastases, presumably via the activation of NK cells (15). It was recently reported that NK cells can mediate long-lived, Ag-specific adaptive recall responses independently of B cells and T cells (16). These observations suggest that MCP-1 can induce specific tumor immunity by enhancing NK cell functions even in this system.

Thus, we evaluated the long-term systemic immunomodulatory effects of a bicistronic recombinant adenovirus vector (rAd) expressing both HSV-tk and MCP-1 (Ad-tk-MCP1). After the primary s.c. HCC tumors in athymic nude mice were eradiated by using Ad-tk-MCP1, the same HCC cells were injected into an another site of the same mice to prove the presence of NK cell-mediated, long-term immunity. Moreover, we explored whether innate immune responses induced by NK cells were involved in these procedures. In this study, we provide definitive evidence to indicate that codelivery of a suicide gene and MCP-1 exerts prolonged NK cell-mediated antitumor effects in this model, suggesting a plausible strategy to prevent HCC recurrence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Recombinant adenoviruses

The bicistronic Ad-tk-MCP1 (10), which harbors the HSV-tk gene and the human MCP-1 gene in sequence and is driven by a CAG promoter constructed from a cytomegalovirus enhancer, a chicken beta-actin promoter and part of rabbit beta-globin, was prepared, purified, and titrated according to the protocols supplied by the manufacturer (Takara Bio) as described (17, 18). Briefly, using the internal ribosomal entry site (IRES) fragment of the encephalomyocarditis virus, the plasmid ptk-IRES-MCP1 (tk-MCP1) was constructed and the fragment was inserted into the cosmid vector (pAd-tk-MCP1). Ad-tk-MCP1 was subsequently generated by transfecting 293 cells with pAd-tk-MCP1 and EcoT22I-digested adenovirus 5-dIX DNA-terminal protein complex. The rAd expressing HSV-tk (Ad-tk), lacZ (Ad-lacZ) and MCP-1 (Ad-MCP1) were constructed in the same way (8). The rAds were purified on cesium gradients and their titers were determined by the 50% tissue culture infectious dose (TCID50) method (19).

Cell lines and culture

The human HCC cell line HuH7 (20) and the mouse HCC cell line BNL 1ME A.7R.1 (BNL) were cultured in DMEM (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies). When infected with Ad-tk-MCP1 or Ad-MCP1, BNL cells produced MCP-1 protein at similar levels as HuH7 cells (data not shown), suggesting that human MCP-1 protein was efficiently expressed in the infected human and mouse HCC cell lines.

Preparation of dendritic cells (DCs) and monocytes

Murine DCs were generated using the method of Lutz et al. (21). Briefly, bone marrow cells were harvested from 6-wk-old male BALB/c-nu/nu mice (CLEA Japan). Erythrocytes were lysed with ammonium chloride potassium buffer (BioWhittaker), and the nucleated cells were plated in plastic bacteriologic dishes in 10 ml of RPMI 1640 supplemented with 10% heat-inactivated FBS and 20 ng/ml murine GM-CSF (PeproTec), with the culture medium refreshed every 3 days. On day 8, the nonadherent DCs were collected. Purity was routinely >95% CD11c+ DC as determined by FACS analysis.

Thioglycollate-elicited murine peritoneal exudate cells were collected as described (22). Briefly, nude mice were i.p. injected with 2 ml of 3% fluid thioglycollate medium (Wako Pure Chemical) and sacrificed 4 days later, followed by peritoneal lavage with 10 ml of cold PBS. Approximately 90% of the collected peritoneal cells were positive for both Mac-1 (CD11b) and I-Ad MHC class II when stained with PE-conjugated anti-Mac-1 Ab (clone M1/70; BD Pharmingen) and FITC-conjugated I-Ad MHC class II (clone AMS-32.1; BD Pharmingen).

Human monocytes and DCs were isolated from healthy blood donors (23). Briefly, PBMCs were isolated by centrifugation in Lymphoprep tubes (Nycomed). PBLs were then incubated in 6-well cell culture plates and the resultant adherent cells were collected as a monocyte population consisting of ~70% CD14+ (clone M{phi}P9; BD Pharmingen) cells, as determined by flow cytometric analysis. The monocyte population was further grown into differentiated DCs by culturing them for 1 wk in CellGro DC medium (Good Manufacturing Practice grade; Cell Genix) supplemented with 100 ng/ml GM-CSF (Cell Genix) and 50 ng/ml IL-4 (Cell Genix). The cells were collected with viability of >80%, and >60% of cells were identified as CD14–HLA-DR+ (clone L243; BD Pharmingen) DCs.

Assays for IL-12 production in vitro

HuH7 cells were infected with Ad-tk-MCP1, Ad-tk, or Ad-lacZ at a multiplicity of infection (MOI) of 5 for 24 h. Aliquots of 105 DCs or monocytes were cocultured with 105 rAd- or mitomycin C (MMC)-treated HuH7 cells in 1.0 ml of culture medium in a 24-well tissue culture plate and treated with or without GCV for two days at 37°C. The concentrations of IL-12 in the medium were quantitated using an immunoassay kit (BioSource International).

Animal studies

The following investigations were conducted in accordance with the Institutional Animal Care and Use Committee guidelines of Kanazawa University. Six-week-old male athymic nude mice were s.c. injected with 5 x 106 HuH7 cells on day 0. On days 3 and 4, 5 x 107 TCID50 (100 µl) of Ad-tk-MCP1, Ad-tk, or Ad-MCP1 was injected into the s.c. tumors, and the mice were treated with 75 mg/kg GCV injected into the peritoneal cavity every day for the next 5 days (days 5–9). Following complete eradication of the primary tumors, the mice were s.c. rechallenged on day 14 with 3 x 106 HuH7 cells or injected with 1 x 103 BNL cells at a distance of >3 cm from the primary challenge site. Nine of 80 (11.3%) mice treated with Ad-tk-MCP1 and 9 of 44 (20.4%) treated with Ad-tk did not show a complete eradication of the primary tumor by the final measurement and therefore were excluded from a rechallenge experiment. In some experiments, Ad-tk-MCP1-treated animals were i.p. administered 200 µl of 1 mg/ml polyclonal rabbit anti-asialo GM1 (AGM1) Ab (Wako Pure Chemical), an Ab against NK cells (24, 25), or 200 µl of rabbit serum (Sigma-Aldrich), 1 ml of 2 mg/ml carrageenan (Sigma-Aldrich), which inactivates macrophages in vivo (26, 27, 28), or 1 ml of PBS on days 11, 12, 13, 20, 27, 34, 41, and 48. In another series of experiments, Ad-tk-MCP1-treated animals were i.p. administered 250 µg of neutralizing goat anti-mouse IL-12 Ab (Sigma-Aldrich), 225 µg of anti-IL-12 Ab plus 25 µg of anti-mouse IL-18 Ab (93-10C; Medical & Biological Laboratories), or 250 µg of control IgG Ab (goat and/or rat; Sigma-Aldrich) on days 14 and 17. Tumor sizes were measured every 4 days after the second tumor injection, and tumor volumes were calculated according to the formula (longest diameter) x (shortest diameter)2/2.

In another series of experiments, immunocompetent BALB/c-jcl mice (CLEA Japan) were s.c. injected with 1 x 105 BNL cells infected with each rAd at an in vitro MOI of 100 on day 0, GCV was administered i.p. for the next 5 days (days 1–5), and the primary tumors were completely eradicated. These mice were s.c. injected with 1 x 104 BNL cells in other sites on day 14, and the tumor sizes were measured every 7 days.

ELISA for serum IL-12 and IL-18

Mouse sera were collected before the injection of s.c. primary tumors and after the rechallenge with tumors, and IL-12 and IL-18 concentrations were measured using immunoassay kits (IL-12 from BioSource International and IL-18 from Medical & Biological Laboratories).

Immunohistochemical analysis

Tumor tissues and spleens were resected on day 16 (2 days after tumor rechallenge). The tissue samples, except those used for F4/80 (A3-1; Serotec) staining, were embedded in OCT compound (Sakura Finetek) and snap frozen in liquid nitrogen. Cryostat sections of frozen tissues were fixed in cold acetone for 10 min, followed by rinsing three times in PBS. The tissue samples used for F4/80 staining were fixed in 10% phosphate-buffered formalin and embedded in paraffin. To avoid nonspecific staining, avidin and biotin in the tissues were blocked using a blocking kit (Vector Laboratories). The slides were subsequently incubated with Abs against AGM1, F4/80, Mac-1, CD11c (HL3; BD Pharmingen), or CD45R (RA3-6B2; BD Pharmingen) for 30 min at room temperature. Negative controls included staining with the corresponding isotype for each Ab and subsequent staining with the secondary Ab. The reactions were visualized using a VECTASTAIN ABC Standard kit (Vector Laboratories), followed by counterstaining with hematoxylin.

RT-PCR for IFN-{gamma} gene expression

Total RNA was extracted from tumor tissues resected on day 10 using a total cellular RNA isolation kit (Ambion) according to the manufacturer’s protocol. Each RT-PCR was performed using 1 µg of total RNA and an oligo(dT) adaptor primer and an RNA PCR kit (avian myeloblastosis virus), version.2.1 (Takara Bio). The amplification protocol consisted of an initial denaturation at 94°C for 2 min followed by 30 or 40 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and an extension at 72°C for 1.5 min. The PCR primers for the mouse IFN-{gamma} and GAPDH genes were purchased from R&D Systems.

Flow cytometry

Single cell suspensions of splenocytes were resuspended in PBS containing 1% BSA and 0.1% sodium azide and incubated for 30 min on ice with FITC-conjugated rat anti-mouse-F4/80 and PE-conjugated rat anti-mouse pan NK cells (DX5; BD Pharmingen) or with FITC-conjugated rat anti-mouse-CD4 (BD Pharmingen) and PE-conjugated rat anti-mouse CD8 (BD Pharmingen). The cells were washed, resuspended in PBS, and analyzed in a FACScan with CellQuest software.

Statistical analysis

All results were expressed as means ± SE. The statistical significance of differences between groups was evaluated by repeated measures ANOVA for the duration of the serum levels of IL-12 or the Mann-Whitney U test for the other results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Apoptotic HCC cells expressing MCP-1 augment IL-12 production by monocytes and DCs in vitro

IL-12, which was originally identified as an NK-stimulatory factor and a cytotoxic lymphocyte maturation factor, is one of the most promising cytokines in cancer treatment because of its multiple effects. IL-12 is produced by APCs such as macrophages, DCs, and B cells following the appropriate stimuli (29, 30, 31). To evaluate the immunomodulatory effects of rAds expressing HSV-tk with or without MCP-1 (Fig. 1), we measured IL-12 production by monocytes and DCs, both of which had been cocultured with HCC cells that had been infected with rAds (Fig. 1). Murine peritoneal exudate cells, consisting mostly of macrophages, and human monocytes cocultured with apoptotic HCC cells induced by the HSV-tk/GCV system plus MCP-1 produced greater amounts of IL-12 than did those cocultured with apoptotic HCC cells induced by the HSV-tk/GCV system alone (Fig. 1, A and C). Murine bone marrow DCs tended to produce IL-12 when cocultured with HCC cells infected with rAds expressing MCP-1 without regard to HSV-tk-induced apoptosis (Fig. 1B). Human DCs produced large amounts of IL-12 when cocultured with HSV-tk/GCV-induced apoptotic tumor cells, which expressed MCP-1, as did human monocytes (Fig. 1D). Thus, the phenomena observed in this xenograft model may also be observed under human allogeneic conditions.


Figure 1
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  		FIGURE 1. IL-12 production by monocytes and DCs cocultured with apoptotic or nonapoptotic HCC cells infected with rAds in vitro. HuH7 cells were infected with Ad-tk-MCP1, Ad-tk, and Ad-lacZ at an MOI of 5 for 24 h. Aliquots of 105 mouse (A) and human (C) monocytes or 105 mouse (B) and human (D) DCs were cocultured with 105 rAd- or MMC-treated HuH7 cells and treated with or without GCV for two days, and the concentrations of IL-12 in the medium were evaluated using an immunoassay. Each value is the mean ± SE of triplicate experiments. *, p < 0.05 compared to Ad-tk with PBS (Ad-tk, PBS); **, p < 0.05 compared to Ad-tk with GCV (Ad-tk, GCV) by the Mann-Whitney U test.

 
When we measured DC maturation markers we found that their expression levels did not change when these cells were cocultured with tk/MCP-1 transduced HCC cells, whereas CD86 expression was elevated when the DCs were incubated with apoptotic HCC cells (data not shown).

Prolongation of the antitumor effects of the HSV-tk/GCV system by codelivery of the MCP-1 gene in an athymic nude mouse model of HCC

To determine the effects of HSV-tk/GCV plus MCP-1 in a murine model of HCC, HuH7 cells were s.c. transplanted into athymic nude mice and eradicated with rAds harboring HSV-tk with or without MCP-1, and the mice were rechallenged with HuH7 cells (Fig. 2A). We found that tumor regrowth was significantly lower when the primary tumor cells had been eradicated with Ad-tk-MCP1 as compared with Ad-tk (tumor volume 40 days after rechallenge, 59.2 ± 24.9 mm3 (n = 22) vs 471.2 ± 118.6 mm3 (n = 20), p < 0.01) (Fig. 2B). No growth inhibition was observed when Ad-tk-MCP1 or Ad-MCP1 was administered in the absence of HuH7 cell transplantation (tumor volume, 339.6 ± 124.3 mm3, n = 18, and 575.3 ± 179.1 mm3, n = 12, respectively) or when Ad-lacZ was administered along with MMC-treated HuH7 cells (tumor volume, 554.8 ± 125.6 mm3, n = 18). The results demonstrate that, when the primary tumors were eradicated with the HSV-tk/GCV system plus MCP-1, the antitumor effects were maintained.


Figure 2
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  		FIGURE 2. Prolonged antitumor effects of rAds expressing HSV-tk with or without MCP-1 in an athymic nude mouse model of HCC. A, Mice were s.c. injected with 5 x 106 HuH7 cells on day 0. On days 3 and 4, 5 x 107 TCID50 of Ad-tk-MCP1, Ad-tk, Ad-lacZ, or Ad-MCP1 were injected into the tumors, and the mice were i.p. injected with 75 mg/kg GCV every day for the next 5 days (days 5–9). Following complete eradication of the primary tumors, the mice were s.c. rechallenged with 3 x 106 HuH7 cells at other sites on day 14. B, Tumor sizes were measured every 4 days. The results are the means of three independent experiments. *, p < 0.001 compared to Ad-tk with HuH7 (Ad-tk, HuH7) by the Mann-Whitney’s U test.

 
Recruitment and activation of NK cells in rechallenged tumors

Serum MCP-1 concentration was below the detection limit of the ELISA used when the s.c. tumors were injected with rAds, whereas the tumor produced MCP-1 in vitro upon infection with Ad-tk-MCP-1 (data not shown). Moreover, we could not detect adenovirus DNA in these rechallenged tumors by using PCR (data not shown), negating the possibility that adenovirus infection contributed to the rejection of the rechallenged tumor. These results indicate that the injected human MCP-1 gene functioned locally in the primary s.c. tumors, thereby modulating the subsequent response to the rechallenged tumor. Because athymic nude mice possess NK cells and macrophages but not T lymphocytes, we determined the migration of these cells by an immunohistochemical analysis. The number of AGM1+ NK cells was significantly higher upon tumor rechallenge in mice whose primary tumors had been eradicated with Ad-tk-MCP1 plus GCV than in those whose primary tumors had been eradicated with Ad-tk plus GCV (p < 0.05) (Fig. 3, A and B). Similarly, the numbers of F4/80 or Mac-1 positive cells (32, 33) tended to be higher upon tumor rechallenge in mice whose primary tumors had been eradicated with Ad-tk-MCP1. Moreover, the mRNA of IFN-{gamma} secreted by NK cells (34) became detectable after 30 PCR cycles in the rechallenged tumors of animals whose primary tumors had been eradicated with Ad-tk-MCP1 and was greatly amplified after 40 PCR cycles (Fig. 3C). These results demonstrate that NK cells were recruited and activated into rechallenged tumor tissues, presumably inhibiting tumor cell growth in mice whose primary tumors had been eradicated with HSV-tk/GCV plus MCP-1.


Figure 3
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  		FIGURE 3. Expression of AGM1, F4/80, and Mac-1 Ags and IFN-{gamma} mRNA in rechallenged tumor tissues. In the experiment described in the legend to Fig. 2, tumor tissues were resected 2 days after tumor rechallenge and analyzed immunohistochemically and estimated for IFN-{gamma} mRNA expression by RT-PCR. A, Tumor tissues obtained from mice whose primary tumors were treated with Ad-tk-MCP1, Ad-tk, and Ad-LacZ were stained with anti-AGM1, F4/80, and Mac-1 Abs. Original magnification, x100. B, Quantitative morphometric analysis showing the proportions of positive cells in areas of 100 tumor cells. Values are the means ± SE of triplicate experiments. *, p < 0.05 compared to Ad-tk by the Mann-Whitney’s U test. C, RT-PCR were conducted in accordance with the manufacture’s protocol as described in Materials and Methods. Bands corresponding to IFN-{gamma} (384 bp) and GAPDH (265 bp) were detected. Splenocytes treated with 1 µg/ml LPS were used as a positive marker and tumor tissues treated with PBS were used as a negative control. carr., Carrageenan.

 
To monitor the activation state of innate immunity in extrahepatic lymphoid organs, we determined immunohistochemically the numbers of immune cells in the spleen after tumor rechallenge using anti-AGM1, F4/80, Mac-1, CD11c, and CD45R Abs (Fig. 4, A and B). The numbers of F4/80+ and Mac-1+ cells were significantly increased in the spleens of mice treated with Ad-tk-MCP1 compared with mice treated with Ad-tk (p < 0.05). In contrast, the numbers of AGM1+ and CD45R+ cells tended to be higher in the spleens of mice treated with Ad-tk-MCP1, but there was little difference in the numbers of CD11c+ cells. A flow cytometrical analysis of splenocyte single cell suspensions demonstrated that the numbers of DX5+ and F4/80+ cells tended to be higher in the spleens of mice treated with Ad-tk-MCP1 (Fig. 4C). In contrast, treatment with carrageenan decreased the number of macrophages in the spleen and at rechallenge sites and slightly increased the number of NK cells in the spleen. Collectively, these results suggest that alterations in the proportions of cell subsets in splenocytes may reflect the activation status of the innate immune system following the eradication of primary tumors by HSV-tk/GCV plus MCP-1. Finally, an anti-AGM1 Ab (35, 36) significantly inhibited the antitumor immunity conferred by Ad-tk-MCP1 (tumor volume 40 days after rechallenge, 385.4 ± 106.3 mm3 (n = 22) vs 64.2 ± 43.6 mm3 (n = 16), p < 0.05), and carrageenan partially inhibited the antitumor immunity of Ad-tk-MCP1 (tumor volume, 242.6 ± 100.8 mm3 (n = 14) vs 53.8 ± 22.9 mm3 (n = 22), p = 0.22) (Fig. 4D). The results indicate that antitumor effects were mainly mediated by NK cells.


Figure 4
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  		FIGURE 4. A–C, Immunohistochemical detection of AGM1, F4/80, Mac-1, CD11c, and CD45R in spleens. In the experiment described in the legend to Fig. 2, spleens were resected 2 days after the tumor rechallenge. A, The numbers of immune cells in the spleens were analyzed immunohistochemically using Abs against AGM1, F4/80, Mac-1, CD11c, and CD45R. Original magnification, x100. B, Quantitative morphometric analysis showing the percentage of positive cells in 50 x400 power fields. Each value is the mean ± SE of triplicate experiments. *, p < 0.05 compared with Ad-tk by the Mann-Whitney U test. C, Surface expression of DX5 and F4/80 in cell populations obtained from spleens was assessed by FACS. The results are representative of two independent experiments. carr., Carrageenan. D, The effects of anti-AGM1 Ab or carrageenan on the growth of rechallenged tumors. At the rechallenge with HuH7 cells, Ad-tk-MCP1-treated animals were i.p. administered with 200 µl of 1 mg/ml anti-AGM1 Ab (Ad-tk-MCP1, AGM1 Ab), 200 µl of rabbit serum (Ad-tk-MCP1, serum) or 1 ml of 2 mg/ml carrageenan (Ad-tk-MCP1, carrageenan) as described in Materials and Methods. Tumor sizes were measured every 4 days. The results are the means of two independent experiments. *, p < 0.05 compared to Ad-tk-MCP1 with PBS or serum (Ad-tk-MCP1, serum) by the Mann-Whitney U test.

 
Involvement of IL-12 and IL-18 in sustained antitumor effects

IL-18 is a proinflammatory cytokine produced by activated macrophages that has been shown to augment both innate and acquired immunity (37) and, in combination with IL-12, induce Th 1 cell development and NK cell activation (38). We therefore assayed IL-12 and IL-18 production after tumor rechallenge. Serum concentrations of IL-12 and IL-18 were significantly higher after tumor rechallenge in mice whose primary tumors had been eradicated with Ad-tk-MCP1 compared with mice whose tumors had been eradicated with Ad-tk (p < 0.05) (Fig. 5A). Moreover, serum concentrations of IL-12 peaked after primary tumors were eradicated (day 9) and were sustained thereafter (p < 0.05) (Fig. 5B). Furthermore, the administration of anti-IL-12 significantly inhibited the antitumor effects conferred by Ad-tk-MCP1 (Fig. 5C) and reduced the serum concentrations of IL-12 to an undetectable level (data not shown). The combined treatment of anti-IL-12 and anti-IL-18 Ab further diminished antitumor effects (Fig. 5C) and reduced both serum IL-12 and IL-18 levels to undetectable levels (data not shown). The results suggest the critical involvement of IL-12 and IL-18 in the antitumor effects induced by Ad-tk-MCP1 on tumor regrowth.


Figure 5
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  		FIGURE 5. Roles of IL-12 and IL-18 in growth suppression of rechallenged HuH7 cells. A, Mouse sera were collected before s.c. injection of primary tumor cells (untreated) and 2 days after rechallenge with HuH7 cells, and IL-12 and IL-18 concentrations were measured using immunoassay kits. Each value is the mean ± SE of triplicate experiments. *, p < 0.01; **, p < 0.05 compared to Ad-tk by the Mann-Whitney U test. B, Serum concentrations of IL-12 were monitored every 9 days after the injection of primary tumors. Each value is the mean ± SE of triplicate experiments. *, p < 0.05 compared to Ad-tk with HuH7 (Ad-tk, HuH7) by repeated measures ANOVA. C, At rechallenge with HuH7 cells, Ad-tk-MCP1-treated animals were i.p. administered with 250 µg of anti-IL-12 Ab (Ad-tk-MCP1, IL-12Ab), 225 µg of anti-IL-12 Ab plus 25 µg of anti-IL-18 Ab (Ad-tk-MCP1, IL-12Ab+IL-18Ab), or 250 µg of control IgG Ab (Ad-tk-MCP1, goat IgG or Ad-tk MCP1, goat IgG+rat IgG). Tumor sizes were measured every 4 days. The results are representative of two independent experiments. *, p < 0.05 compared to Ad-tk-MCP1 with goat IgG (Ad-tk-MCP, goat IgG); **, p < 0.01 compared to Ad-tk-MCP1 with goat IgG plus rat IgG (Ad-tk-MCP, goat IgG+rat IgG) by the Mann-Whitney U test.

 
Innate immune responses to heterologous tumor injection in an athymic nude mouse

To estimate the involvement of innate immune responses in the antitumor effects observed with HSV-tk/GCV plus MCP-1, we rechallenged mice with heterologous tumor administration. The growth of a second unprimed cell line (BNL; transformed liver cells derived from BALB/c mice) was significantly suppressed when HuH7 cells had been eradicated with Ad-tk-MCP1 as compared with Ad-tk (tumor volume, 1059.5 ± 110.6 mm3 (n = 12) vs 1825.4 ± 197.9 mm3 (n = 12), p < 0.01) when Ad-lacZ was administered with MMC-treated HuH7 cells (tumor volume, 1960.8 ± 183.8 mm3, n = 12) (Fig. 6). These results indicate that the innate immune responses contributed to the prolonged antitumor effects of HSV-tk/GCV plus MCP-1 gene transfer.


Figure 6
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  		FIGURE 6. Antitumor effects of rAds expressing HSV-tk with or without MCP-1 against a second unprimed cell line (BNL) in an athymic nude mouse model of HCC. As described in the legend to Fig. 3, following complete eradication of the primary tumors the mice were s.c. injected with 1 x 103 BNL cells at other sites on day 14. Tumor sizes were measured every four days. The results are the means of two independent experiments. *, p < 0.01 compared to Ad-tk with HuH7 (Ad-tk, HuH7) by the Mann-Whitney’s U test.

 
Prolonged antitumor effects against mouse HCC of rAd expressing HSV-tk and MCP-1 in an immunocompetent mouse

Finally, we evaluated the antitumor responses in immune-competent mice using the same experimental procedures (Fig. 7A). The growth of rechallenged tumors was significantly lower when the primary tumor cells had been eradicated with Ad-tk-MCP1 as compared with Ad-tk (tumor volume 42 days after rechallenge, 170.3 ± 54.2 mm3 (n = 22) vs 488.9 ± 120.1 mm3 (n = 22), p < 0.01), similarly observed on athymic nude mice injected with human HCC. In addition, the growth of rechallenged tumors was significantly suppressed in mice whose primary tumors had been eradicated with Ad-tk as compared with those treated with Ad-lacZ and MMC (488.9 ± 120.1 mm3 (n = 22) vs 1666.4 ± 259.2 mm3 (n = 22), p < 0.01). Furthermore, when we isolated splenocytes 70 days after the injection of primary tumor cells we found that the numbers of CD4– and CD8+ cells were increased in mice treated with Ad-tk-MCP1 (Fig. 7B). Collectively, these results confirm that antitumor effects may be dependent not only on innate immunity but on acquired immune responses.


Figure 7
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  		FIGURE 7. Prolonged antitumor effects of rAds expressing HSV-tk with or without MCP-1 in an immunocompetent mouse model of HCC. A, On day 0, mice were s.c. injected with 1 x 105 BNL cells infected with Ad-tk-MCP1, Ad-tk, or Ad-lacZ at an in vitro MOI of 100. The mice were i.p. injected with 75 mg/kg GCV per day for the next 5 days (days 1–5). Following complete eradication of the primary tumors, the mice were s.c. rechallenged with 1 x 104 BNL cells at other sites on day 14. Tumor sizes were measured every 7 days. The results are the means of three independent experiments. **, p < 0.001 compared to Ad-lacZ with MMC-treated BNL (Ad-lacZ, MMC treated BNL); *, p < 0.01 compared to Ad-tk with BNL (Ad-tk, BNL) or Ad-lacZ with MMC-treated BNL (Ad-lacZ, MMC-treated BNL) by the Mann-Whitney U test. B, Spleens were resected 70 days after the injection of primary tumor cells, and surface expression of DX5, F4/80, CD4, and CD8 in cell populations obtained from spleens was assessed by FACS. The results are representative of two independent experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the current study, we observed that when monocytes were cocultured with apoptotic HCC cells infected with Ad-tk-MCP1, these immune cells produced large amounts of IL-12. Interestingly, in both nude and immunocompetent mice the growth of rechallenged HCC cells was markedly suppressed after the primary tumor cells had been eradicated with Ad-tk-MCP1 followed by GCV administration. Furthermore, these prolonged in vivo antitumor effects were associated with the production of IL-12 and IL-18 and mediated by NK cells.

Monocytes produced large amounts of IL-12 when cocultured with apoptotic HCC cells induced by the HSV-tk/GCV system plus MCP-1. APCs, such as macrophages, DCs, and B cells produce IL-12, which was originally identified as an NK-stimulatory factor and shown to exhibit considerable antineoplastic activity (39, 40). APCs were found to be activated upon the recognition of Ags from apoptotic target cells (41), and both macrophages and DCs secrete large amounts of IL-12 when treated with MCP-1 in vitro (33, 42, 43). These findings suggest that the recognition of apoptotic tumor cells together with MCP-1 may activate macrophages and DCs, thereby enhancing IL-12 secretion.

We demonstrated that the antitumor effects were maintained when the tumor cells had been eradicated with Ad-tk-MCP1, a vector that expresses both a suicide gene and a chemokine, but that either alone was not sufficient to prolong immunity in our models. We previously demonstrated that MCP-1 secreted by apoptotic HuH7 cells may recruit and activate macrophages efficiently, although these effects did not occur when the tumor cells were treated with the rAd expressing either HSV-tk or MCP-1 (8, 10). Moreover, we observed that the numbers of Mac-1+ and F4/80+ cells were increased in the spleens of mice after tumor rechallenge. Indeed, MCP-1 has been shown to activate murine peritoneal macrophages and enhance the expression of CD11b (Mac-1) in BALB/c mice (32, 33). Collectively, these results suggest that during eradication of the primary tumors, activated macrophages in the tumor tissues and the peripheral lymphoid organs can induce the secretion of cytokines, including IL-12 and IL-18, that can activate NK cells, thus exerting antitumor effects.

IL-12-stimulated NK cells exhibit potent cytotoxic activity against various tumor cells (31, 44, 45). NK cells are a part of the innate immune system, a first-line defense against tumor cells, and exert antitumor effects of NK cells rapidly without any prior sensitization (46). The depletion of NK cells has been shown to promote metastases or tumor growth after rechallenge with primary tumor cells (15, 44, 47). We demonstrated here that the growth of rechallenged parental tumor cells or newly challenged heterologous tumor cells was suppressed after eradication of the primary tumors. Therefore, augmentation of NK-mediated innate immune responses may be an attractive strategy for preventing HCC recurrence, including the growth of differentially transformed tumor cells.

We observed that NK cell-mediated antitumor effects were prolonged after primary tumor cells had been eradicated with Ad-tk-MCP1. Several lines of evidence indicate that the inhibitory effects of NK cells on tumor growth were maintained and were detectable at the site of the primary tumor even after treatment discontinuation (36, 48). Although the mechanisms involved in these responses are not yet known, a number of tumor model systems have demonstrated the important roles of NK cells in early tumor clearance, leading to the establishment of adaptive immunity. It was recently reported that NK cell-mediated immune responses featured hallmarks of adaptive immunity such as acquired immunity, long-lived memory, and Ag specificity (16). DCs expressing IL-12 have been shown to confer NK-mediated tumor protection in which NK activation is dependent on both DC-NK interaction and IL-12 secretion (49). Moreover, NK cell-derived IFN-{gamma} may provide early immune regulation that alters the outcome and quality of adaptive immunity (50). Furthermore, MCP-1 has been shown to induce DC migration to lesions where NK cytolytic responses are activated (51). Consistent with these observations, we demonstrated that the antitumor responses were abolished when NK cells were inactivated by treatment with the AGM1 Ab and that NK cells were recruited and IFN-{gamma} production enhanced in the rechallenged tumors.

We observed that the growth of rechallenged heterologous tumors was suppressed to a lesser extent than that of homologous tumors in our nude mice model. Athymic nude mice lack T lymphocyte-mediated immune responses, but the numbers and functions of macrophages and NK cells are preserved. Moreover, nude mice have limited populations of extrathymically matured T lymphocytes, including {gamma}{delta} T cells (52), and these may be reduced slightly by treatment with AGM1 Ab (53). Both NK cells and V{delta}1{gamma} {delta}T lymphocytes have been reported to prevent the growth of s.c. melanoma cells, with both cell types detected at the sites of the s.c. tumors (47). Therefore, we cannot exclude the possibility that the memory subset of {gamma} {delta}T cells affects antitumor immunity against homologous and heterologous cells, thus leading to differences in the magnitude of tumor suppression.

Although the results presented here are promising, a number of problems remain to be solved before this approach can be used clinically. First, s.c. tumor models using an HCC cell line may not be fully comparable to HCCs in patients. Second, problems using rAds need to be resolved before they can be applied clinically. However, in patients treated with nonsurgical procedures such as percutaneous radiofrequency ablation therapy and transcatheter arterial chemotherapy, the administration of rAd vectors may ensure tumor cell killing, thus enhancing the antitumor effects on residual tumor cells and recurrent HCC.


   Acknowledgments
 
We thank Akemi Nakano and Yuzu Hasebe for assistance with histology and immunohistochemistry. We are also grateful to Maki Kawamura and Chiharu Minami for animal care.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Shuichi Kaneko, Department of Gastroenterology, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan. E-mail address: skaneko{at}medf.m.kanazawa-u.ac.jp Back

2 Abbreviations used in this paper: HCC, hepatocellular carcinoma; AGM1, asialo GM1; BNL, BNL 1ME A.7R.1 HCC cell line; DC, dendritic cell; GCV, ganciclovir; HSV-tk, HSV thymidine kinase; MMC, mitomycin C; MOI, multiplicity of infection; rAd, recombinant adenovirus vector; Ad-tk, rAd expressing HSV-tk; Ad-tk-MCP1, rAd expressing both HSV-tk and MCP-1; Ad-MCP1, rAd expressing MCP-1; Ad-lacZ, rAd expressing lacZ; TCID50, 50% tissue culture infectious dose. Back

Received for publication October 27, 2005. Accepted for publication October 13, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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