Adenovirus-mediated lymphotactin gene transfer improves therapeutic efficacy of cytosine deaminase suicide gene therapy in established murine colon carcinoma

Abstract

Lymphotactin (Ltn) is the sole member of C chemokines which attracts T cells and NK cells specially. Ltn gene was transferred in vivo to improve the antitumor efficacy of cytosine deaminase (CD) gene therapy. Upregulation of CD80 and CD54 on murine CT26 colon carcinoma cells was observed after combined transfection with adenovirus encoding CD (AdCD) and adenovirus encoding murine Ltn (AdLtn) followed by administration of 5-fluorocytosine (5FC) in vitro. AdCD/5FC treatment also increased the expression of CD95 and induced obvious apoptosis of CT26 cells. After combined treatment with AdLtn and AdCD/5FC, the pre-established murine model with subcutaneous CT26 colon carcinoma exhibited most significant tumor growth inhibition, and four of eight tumor-bearing mice were tumor free, while tumors in other mice grew more progressively. Examination of lymphocyte infiltration and cytokine gene expression in tumor tissue revealed that tumors from AdLtn/AdCD/5FC-or AdLtn-treated mice were heavily infiltrated with CD4+, CD8+ T cells and NK cells, and IL-2 and IFN-γ mRNA expression were present in parallel with T cell and NK cell infiltration. Splenic NK and CTL activities increased significantly after the combination therapy. In vivo depletion analysis showed that NK cells, CD4+ T cells and CD8+T cells participated in the antitumor effect of the host with CD8+T cells being the main T cell subset responsible for the enhanced antitumor immune response. These findings suggested that increased immunogenicity and induction of apoptosis of the tumor cells, and efficient induction of local and systemic antitumor immunity of the host might contribute to the enhanced antitumor effects of the combined Ltn and CD suicide therapy.

Introduction

Colon carcinoma is one of the most common malignancies and also one of the most difficult tumors to cure because of its distant metastasis and easy recurrence. Chemotherapy with 5-fluorouracil (5FU) systemically is effective for this cancer. However, the toxicity associated with a high dose of 5FU, together with its limited capacity to eliminate tumor growth thoroughly, has inspired investigators to seek novel approaches to produce 5FU regionally at tumor sites without obvious adverse effects. Gene therapy, as a promising treatment for many human modalities, encompasses efficient potentials for treating cancers that have failed conventional therapy.12 Suicide gene therapy for the treatment of cancer has been studied intensively.34 The basic principle underlying metabolic suicide gene therapy is the conversion of a relatively nontoxic prodrug to its toxic metabolite by an enzyme expressed by the suicide gene, which is not normally present in the eukaryotes. 5-Fluorocytosine (5FC), being frequently used for the treatment of fungi and bacteria infections, could be metabolized to 5-FU and thus utilized for the selective killing of tumor cells in vivo. The growth of tumor cells modified to express CD could be inhibited significantly by administration of 5FC both in vitro and in vivo.5678910 One of the obstacles limiting the efficacy of suicide gene therapy is its insufficiency to induce potent antitumor immunity of the host, which is very important for the prevention of tumor recurrence and metastasis.11 In our previous study, we have shown that more potent antitumor effects could be achieved in established tumors by cotransfer of CD suicide gene and cytokine gene.121314 Recent studies have illustrated that tumor cells transduced with cytokine or chemokine genes showed increased immunogenicity and decreased tumorigenicty.1516171819

The recently discovered chemokine lymphotactin (Ltn) is the sole member of the third subfamily of chemokines, C chemokines. Ltn has been shown to be a chemoattractant specific for T lymphocytes and/or natural killer (NK) cells.2021222324 We hypothesized that in vivo Ltn gene transfer into tumor might recruit more lymphocytes which might be activated by the tumor antigens released from the killed tumor cells after CD gene therapy, and thus might improve the therapeutic efficacy of CD suicide gene therapy through the efficient induction of immune response in tumor-bearing animals. Adenovirus has been demonstrated to be an efficient vector for in vivo transfer of foreign genes. So in the present investigation, antitumor effect of adenovirus-mediated Ltn gene and CD suicide gene therapy was studied, and the results demonstrated that combination of Ltn gene and CD gene transfer followed by 5FC administration in vivo elicited potent antitumor effects in pre-established subcutaneous colon carcinoma. Efficient induction of nonspecific and specific antitumor immunity of the tumor bearing mice, both locally and systemically, was speculated to be responsible for the improved antitumor efficacy.

Results

Secretion of Ltn from CT26 cells infected with AdLtn in vitro

First, the microchemotaxis assay was performed to determine the secretion of Ltn by AdLtn-transfected CT26 colon carcinoma cells using Boyden chamber migration assay as we reported previously.13 One million CT26 cells were infected with 107 p.f.u. AdLtn or 107 p.f.u. AdLacZ in 1 ml of RPMI-1640 for 24 h in vitro. Then the supernatants were collected for Ltn bioactivity assay. The results demonstrated that supernatants from CT26 cells alone or AdLacZ-infected CT26 cells had no chemotactic activity on CD4+ T cells and CD8+ T cells. The supernatants (with dilution of 1:2) could attract both CD4+ T cells and CD8+ T cells with a chemotactic index of 2–3, which were equivalent to the chemotactic index induced by standard human Ltn 500 ng/ml (PharMingen, San Diego, CA, USA).

In vitro induction of apoptosis and increased immunogenicity of CT26 colon carcinoma cells after AdCD and AdLtn transfection followed by 5FC incubation

To investigate the mechanism of death of the CT26 cells after AdCD/5FC treatment, we analyzed the expression of CD95 and the apoptosis of CT26 colon carcinoma cells after AdCD and AdLtn transfection followed by 5FC incubation in vitro. The results in Figure 1a and b demonstrated that CD95 expression and apoptosis of the CT26 cells were induced obviously after AdCD/5FC treatment as compared with AdCD or AdLacZ/5FC treatment. The apoptosis induced by AdCD/5FC was almost completely blocked by anti-CD95 antagonistic antibodies, indicating that the apoptosis of CT26 cells induced by AdCD/5FC was through induction of CD95. AdLtn induced neither CD95 expression nor apoptosis of CT26 cells, and the inducing effects of AdCD/5FC were not affected by cotransfection with AdLtn (data not shown). These data suggested that AdCD/5FC could kill CT26 cells via induction of apoptosis, and CD95 up-regulation after AdCD/5FC treatment might contribute to the apoptosis of the CT26 cells.

Figure 1
figure1

CD95 expression (a) and apoptosis (b) of CT26 carcinoma cells treated with AdCD/5FC (––––), AdCD (–––), or AdLacZ (· · ·) or AdCD/5FC in the presence of antagonistic anti-CD95 antibody (·–·–·–) in vitro. CT26 cells were infected with recombinant adenovirus for 6 h and treated with 5FC 25 μg/ml for 6 h, and then the cells were incubated with PE-conjugated monoclonal antibody against CD95 or Annexin V for flow cytometry, and histograms were overlaid. Expression of CD80 (c) and CD54 (d) on CT26 carcinoma cells treated with AdLtn/AdCD/5FC (––––), AdCD/5FC (· · ·), or Ad Ltn (–––) in vitro.CT26 cells were infected with recombinant adenovirus for 6 h and treated with 5FC 25 μg/ml for 6 h and then the cells were incubated with monoclonal antibodies against CD80 and CD54 for 30 min followed by incubation with FITC-conjugated goat anti rat IgG. Becton Dickinson FACScalibur flow cytometer was for flow cytometer, and histograms were overlaid.

CT26 colon carcinoma cells grown under subconfluent conditions were treated with AdCD/5FC and/or AdLtn, and then CD80 and CD54 expressions on the CT26 cells were analyzed with flow cytometry. The results showed that the expressions of CD80 and CD54 did not increase significantly after treatment with either AdCD/5FC or AdLacZ when compared with those after PBS treatment (data not shown). The data in Figure 1c and d illustrated that the expressions of CD80 and CD54 on CT26 cells increased slightly after transfection with AdLtn. Combination treatment of the CT26 cells with AdLtn and AdCD/5FC increased the expressions of CD80 and CD54 significantly, thus indicating that the immunogenicity of the tumor cells might increase after combined treatment of AdLtn and AdCD/5FC.

Improved anti-tumor effects of AdCD/5FC suicide gene therapy when combined with AdLtn transfection in vivo

Then, we investigated the anti-tumor effect of AdLtn/AdCD/5FC combination treatment in a pre-established murine model with CT26 colon carcinoma. The data in Figure 2a showed that treatment of the tumor-bearing mice with intratumoral Ltn gene transfer suppressed the growth of the established tumors significantly as compared with treatments with AdLacZ/5FC or PBS (P < 0.01). AdCD/5FC gene therapy alone showed similar anti-tumor effects with AdLtn gene therapy. Combination treatment of the tumor-bearing mice with AdLtn and AdCD/5FC inhibited the growth of the subcutaneous CT26 tumors more significantly as compared with AdLtn alone or AdCD/5FC (P < 0.01).

Figure 2
figure2

Antitumor effects of the AdLtn and AdCD/5FC combined therapy on established murine CT26 colon carcinoma (n = 8 per group). BALB/c mice bearing the CT26 colon carcinoma were treated with various treatments as described in Materials and methods. The tumor growth inhibition (a) and survival rate (b) were shown. Tumor volume was expressed as (length × width2)/2.

The survival periods of the tumor-bearing mice after various treatments were observed. Eight mice in each group were observed for their survival time and the results (Figure 2b) showed that tumor-bearing mice treated with either AdLtn or AdCD/5FC survived much longer than those mice treated with PBS or AdLacZ/5FC (P < 0.05). The survival time of the tumor-bearing mice was prolonged most significantly after AdLtn/ AdCD/5FC administration as compared with that of mice receiving AdLtn alone or AdCD/5FC (P < 0.01), and four of eight mice were free of tumor 90 days after tumor inoculation.

In order to explore if systemic antitumor was induced in vivo in tumor-bearing mice after combined AdCD/5FC and AdLtn treatment, the four survival mice in AdCD/5FC/ADLtn-treated group, one survival mouse in AdCD/5FC-treated group and two survival mice in AdLtn-treated mice were re-challenged s.c. with 5 × 104 CT26 cells on the opposite flank region of the treated mice. All four mice following AdCD/5FC/AdLtn treatment and one mouse following AdLtn treatment remained tumor free 3 months after tumor inoculation. The survival mouse after AdCD/5FC treatment, the mouse after AdLtn treatment and all five mice without gene therapy but challenged with CT26 cells died within 50 days after CT26 tumor inoculation. These data suggested that strong systemic antitumor immunity was induced after combined AdCD/5FC and AdLtn gene therapy.

Efficient induction of local antitumor immunity after AdLtn/AdCD/5FC combined gene therapy

To conform our hypothesis that transfer of Ltn gene into tumor might recruit and activate T cells and NK cells to combat the established tumors, we analyzed the CD4+ T cell, CD8+ T cell and NK cell infiltration in the tumor mass of the tumor-bearing mice undergoing Ltn gene therapy in combination with AdCD/5FC suicide gene therapy. The tumor-infiltrating mononuclear cells were isolated from the killed mice and stained with monoclonal antibodies to identify their phenotypes by flow cytometry. As shown in Figure 3, only minor T cells and NK cells infiltrated in the tumor mass derived from AdLacZ/5FC- or AdCD/5FC-treated tumor-bearing mice. Administration of AdLtn, alone or in combination with AdCD/5FC, led to heavy infiltration of CD4+ T cells, CD8+ T cells and NK1.1+ cells in the tumor mass. No obvious damage of normal tissue was found around the tumors after injection of adenoviruses and 5FC.

Figure 3
figure3

NK (a), CD4+ (b) and CD8+ (c) T cell infiltration in tumor tissue of tumor-bearing mice after adenovirus-mediated Ltn gene and/or CD suicide gene transfer followed by 5FC administration. The tumors were taken out of the killed tumor-bearing mice 3 days after the last administration of 5FC, and the tumor tissue was minced to prepare single cell suspensions. The infiltrated mononuclear cells were isolated from the cell suspensions utilitzing discontinuous Ficoll–Hypaque gradient centrifugation. Monoclonal antibodies against NK1.1, CD4 and CD8 were used for the determination of correspondent cell populations by flow cytometry.

To confirm if the tumor-infiltrating T cells and NK cells were actually activated and participated in the antitumor immune response, we detected the mRNA expression of two cytokines, IL-2 and IFN-γ, in the tumor tissue utilizing RT-PCR. The results in Table 1 showed that neither IL-2 nor IFN-γ mRNA was detected in the tumor tissue derived from mice treated with PBS, AdLacZ/5FC or AdCD/5FC. Treatment of the mice with AdLtn, alone or in combination with AdCD/5FC, led to the expression of IL-2, IFN-γ and Ltn mRNA in tumor mass. IL-2 and IFN-γ, produced by T cells and NK cells, are important cytokines involved in the induction of antitumor immunity. These data suggested that the antitumor immune response in the tumor microenvironment was induced efficiently after Ltn gene transfer into tumor, and the activated local immunity might contribute to the antitumor effect of the combination therapy.

Table 1 mRNA analysis of IL-2, IFN-γ and Ltn in tumor masses of tumor-bearing mice after AdCD/5FC suicide gene therapy in combination with AdLtn gene therapy.

Potent activation of systemic antitumor immunity after AdLtn/AdCD/5FC combined gene therapy

We wanted to know whether the systemic antitumor immunity was activated after the combined therapy because of its crucial role in the prevention of tumor recurrence and metastasis from residual tumor cells after suicide gene therapy. The splenic lymphocytes were isolated from the killed tumor-bearing mice and then used in cytotoxic assays with effector:target (E:T) of 100:1, 50:1 and 25:1. The results showed that the NK and CTL activities in mice received AdLtn administration alone increased markedly when compared with those in mice treated with AdCD/5FC, AdLacZ/5FC or PBS (P < 0.01). AdCD/5FC treatment increased the NK and CTL activities slightly when compared with AdLacZ/5FC or PBS treatment (P > 0.05). The splenic NK and CTL activities increased most significantly after combination therapy with AdLtn/AdCD/5FC as compared with that after either AdCD/5FC or AdLtn therapy alone (P < 0.05, Figure 4). In determining CTL activity, no cytotoxicity on syngeneic Renca renal cell carcinoma cells was found with these induced lymphocytes (data not shown).

Figure 4
figure4

Cytotoxicity of splenic NK (a) and CTL (b) in the tumor-bearing mice after combined treatment with AdCD/5FC and/or AdLtn (n = 7 per group). The splenocytes were derived from the tumor-bearing mice 3 days after various therapies and then used as NK effector cells. The splenocytes were cocultured with inactivated CT26 cells for 7 days in the presence of IL-2 in vitro to induce CTL effector cells. The cytotoxicity was determined by a standard 4-h 51Cr release assay by utilizing YAC-1 cells and CT26 cells as target cells, respectively.

These data suggested that the non-specific and specific anti-tumor immunity were activated potently and might be involved in the anti-tumor response of the combined gene therapy. To confirm this conclusion, we also determined the production of IL-2 and IFN-γ by splenocytes derived from tumor-bearing mice 3 days after various treatments. The data in Figure 5 showed that splenocytes from mice treated with AdLtn produced significantly higher levels of IL-2 and IFN-γ than those treated with AdCD/5FC, AdLacZ/5FC or PBS (P < 0.01). The splenocytes from tumor-bearing mice after combined treatment with AdLtn/AdCD/5FC produced the highest levels of IL-2 and IFN-γ as compared with those from mice after other treatments, thus confirming the potent activation of systemic immunity after combined CD suicide gene and Ltn gene therapy.

Figure 5
figure5

Secretion of IL-2 (a) and IFN-γ (b) from splenocytes isolated from AdLtn- and/or AdCD/5FC-treated tumor-bearing mice (n = 7 per group). The splenocytes derived from the tumor-bearing mice 3 days after various therapies were cultured at 5 × 106 cells/ml in RPMI-1640 medium supplemented with 10% FCS at 37°C, 5% CO2 for 34 h. The IL-2 and IFN-γ in the supernatants were determined using EILSA kits (Endogen, Woburn, USA).

Depletion analysis with anti-CD4 and/or anti-CD8 monoclonal antbodies

In vivo depletion analysis with anti-NK1.1, anti-CD4 and/or anti-CD8 monoclonal antibodies was performed to elucidate the roles of NK cells and different T cell subsets in mediating the anti-tumor immune response of the AdLtn/AdCD/5FC combined gene therapy. The results illustrated that the tumor-bearing mice received neither AdLtn/AdCD/5FC therapy nor antibody treatment (‘No therapy’ group) developed tumors most progressively, and AdLtn/AdCD/5FC treatment suppressed the growth of the tumors significantly. The mice injected with anti-NK1.1 antibody showed larger tumors when compared with AdLtn/AdCD/5FC-treated mice which were injected with control IgG, indicating that NK cells might participate in the anti-tumor response. The mice treated with anti-CD4 antibody also showed larger tumors when compared with the mice treated with control IgG, suggesting that CD4+ T cells participated in the anti-tumor response of the host. The mice treated with anti-CD4 in combination with anti-CD8 antibodies developed tumors with almost the same sizes as the mice treated the anti-CD8 antibodies alone, which abolished most of the anti-tumor effects of AdLtn/AdCD/5FC combined gene therapy (Figure 6). Two months after tumor inoculation, the survival rates of the tumor-bearing mice were assessed, and the results in Table 2 also showed that CD8+ T cells might be the main T cell subset responsible for the anti-tumor immune response of the AdLtn/AdCD/5FC combined gene therapy, while both NK cells and CD4+ T cells participated in this process.

Figure 6
figure6

Effect of host immunosuppression on the inhibition of subcutaneous CT26 colon carinoma treated with AdLtn and/or AdCD/5FC. For selective depletion analysis, 10 mice in each group were injected i.p. with 100 μg of antiNK1.1, anti-CD4 and/or anti-CD8 antibodies 2 days before tumor inoculation and 4 h, 3, 7 and 13 days after tumor inoculation. AdLtn and/or AdCD/5FC therapy began 3 days after tumor inoculation. The tumor volume were determined as (length × width2)/2.

Table 2 Survival rates of tumor-bearing mice undergoing AdLtn/AdCD/5FC combination therapy and in vivo immunosuppression with anti-CD4 and/or anti CD8 antibodies.

Discussion

Many approaches have been explored to address the obstacles limiting the efficacy of suicide gene therapy. Some of them, including fusion of CD and HSVtk genes, combination with radiation, or utilization of tumor- or tissue-specific promoter, are effective in increasing the killing of tumor cells and reducing its toxicity.25262728 Among these approaches, co-transfection of suicide gene and cytokine gene has been found to elicit potent antitumor effects and induce potent antitumor immunity efficiently. Various cytokine genes, including GM-CSF, IL-2, IL-4, IFN-α and IFN-γ have been transferred into tumor cells in combination with HSVtk or CD suicide gene therapy, and more potent antitumor effects and more efficient induction of antitumor immune response have been observed with these combination thera- pies.1213142930313233 In this investigation, we first explored the antitumor efficacy of chemokine gene transfer in combination with CD/5FC gene therapy. Chemokines are pivotal in the trafficking of leukocytes. Recently, chemokine gene transfer has been utilized for the modification of tumor cells. Human RANTES chemokine was found to abolish tumorigenicity of an immunogenic fibrosarcoma in an in vivo murine model, and the process was mediated by various subpopulations of immune effector cells including CD8+/Thy-1+ tumor-infiltrating lymphocytes (TIL) and macrophages.34 The tumor cells transfected with macrophage inflammatory protein 1α (hu-MIP-1α), monocyte chemotactant protein-1 (MCP-1/MCAF/IE), or monocyte chemotactic protein-3 (MCP-3) gene showed reduced tumorigenicity and the animals that had rejected tumor cells transfected with these chemokine genes were resistant to a subsequent challenge with the parental cells. Infiltration of lymphocytes, macrophages and neutrophils was observed at the site of inoculation with chemokine gene transfected tumor cells.171835 Ltn is a new protein belonging to the C or γ subfamily of chemokines with only two of the four cysteine residues. Ltn has been shown to be a chemoattractant specific for CD8+ T cells and/or NK cells, and to be produced by CD8+ T cells, NK cells, and mast cells.36 Ltn was reported to act specifically on T lymphocytes and not on monocytes and neutrophils.24 We have transferred Ltn gene into dendritic cells, and the vaccine prepared with peptide-pulsed, Ltn gene-modified dendritic cells could stimulate antitumor immune response more potently.13 When IL-2 gene was coexpressed with Ltn gene, expanded T lymphocytes were attracted by Ltn and significant growth suppression of established tumor was observed in a CD4+ T cell- and CD8+ T cell-dependent manner.37 Cheon et al16 reported that chemogene therapy could achieve better antitumor effects with reduced toxicity than the conventional chemotherapy or gene therapy protocols alone. They chose low-dose methotrexate to increase the chemosensitization of the tumor cells in combination with suicide gene therapy with an overall reduced toxicity and enhanced therapeutic efficacy.

This is the first report to illustrate that cotransfection of a chemokine gene could augment the antitumor effects of CD suicide gene therapy through induction of apoptosis and increased immunogenicity of the tumor cells, and efficient induction of local and systemic anti-tumor immune response after combined Ltn and CD gene therapy. The supernatants from Ltn gene transfected CT26 cells were capable of attracting T cells in a chemotaxis assay using Boyden chamber, and Ltn mRNA expression could be detected in tumor tissue using RT-PCR, indicating that adenovirus could deliver Ltn gene into CT26 colon carcinoma cells efficiently. The mechanisms involved in the improved antitumor efficacy of the combination gene therapy might be related to the following respects.

First, the therapeutic effect of CD gene therapy is based on a ‘bystander effect’ whereby CD gene-modified tumor cells are toxic to nearby unmodified tumor cells when exposed to 5FC.3839 Apoptosis of B16F10 cells both in vitro and in vivo was reported in AdCD/5FC system.12 In this investigation, we used Annexin V to evaluate the apoptosis of the tumor cells, and obvious apoptosis of the CT26 cells was observed when the cells were treated with AdCD/5FC in vitro. To elucidate the mechanism underlying the apoptosis of the tumor cells, we found that the expression of CD95, also known as fas or Apo-1, upregulated significantly when the CT26 cells were incubated with AdCD and treated with 5FC, indicating that the CT26 cells were killed by induction of fas-mediated apoptosis after AdCD/5FC treatment.

The increased expression of CD80 and CD54 on the tumor cells after combined Ltn and CD gene transfection followed by 5FC treatment might be related to the increased immunogenicity of the tumor cells. CD80, also known as B7–1, a well identified costimulatory molecule, is very important for the activation of T cells during the presentation of tumor antigen.40 CD54, also named intercellular adhesion molecule-1 (ICAM-1), is known to associate with cellular migration into inflammatory sites and with facilitating interactions between lymphocytes and tumor cells in the pathway of cell-mediated cytotoxicity. CD54 is also reported to be implicated in the costimulation of T cells.41 Ramesh et al42 have found that expression of costimulatory molecules B7 and ICAM could be up-regulated after the tumor cells were transfected with HSVtk suicide gene. In this study, we observed significant up-regulation of CD80 and CD54 on CT26 cells after combined AdLtn/AdCD/5FC treatment in vitro, which was speculated to be responsible for the increased immunogenicity of the tumor cells.

Secondly, after we observed the induction of apoptosis and increased immunogenicity of the CT26 cells, we expected that these dead tumor cells might release various known and unknown tumor antigens or present these antigens to lymphocytes. Therefore, we cotransferred Ltn gene intratumorally to recruit more lymphocytes for the activation of an antitumor immune response. Local immune suppression at the tumor site is one of the important reasons for the escape of tumor growth from the immuno-surveillance of the host. So reversal of tumor suppression at the tumor site is necessary and efficient for the eradication of tumors. We found that administration of AdLtn, alone or in combination with AdCD/5FC, led to heavy infiltration of CD4+ T cells, CD8+ T cells and NK cells in the tumor mass. IL-2 and IFN-γ mRNA expression was found in parallel with T cell and NK cell infiltration in the tumor tissues. IL-2 and IFN-γ, produced by T cells and NK cells, are key cytokines in the induction and amplification of antitumor immunity. We also analyzed the histological changes of tumor tissues in tumor-bearing mice and large areas of tumor necrosis and a considerable amount of lymphocyte infiltration were found inside the tumor mass after the combined therapy (data not shown). Recent work by Hall et al43 also found that antitumor NK cell activity could be induced in established tumor models when treated with HSVtk gene therapy. These results illustrated that the local immune response in tumor mass was activated and might contribute to the enhanced antitumor efficacy after combined AdLtn and AdCD/5FC gene therapy.

Lastly, induction of systemic antitumor immunity after combination therapy might play an important role in the augmented antitumor effects of AdCD/5FC in combination with AdLtn. The splenic NK and CTL activities increased significantly after the combination therapy. The production of IL-2 and IFN-γ also increased markedly after the cotransfer of Ltn and CD gene followed by 5FC administration. Depletion experiments with ant-NK1.1, anti-CD4 and/or anti-CD8 monoclonal antibodies illustrated that the main T cell subsets involved in the antitumor response of the host are CD8+ T cells. The antitumor effects of AdLtn/AdCD/5FC in tumor-bearing mice could not be blocked completely as compared with the mice receiving no therapy. AdCD/5FC and NK cytotoxicity might be responsible for the remaining antitumor effects. Our data indicated a crucial role for T cells and NK cells in mediating the antitumor activity of this therapy. Transduction of tumor cells with both GM-CSF and IFN-γ resulted in a powerful synergistic antitumor effect in CT26-bearing mice, and both T cells and NK cells were found to be responsible for the antitumor response.32 These data indicated that the efficient induction of specific and non-specific cellular immunity of the host might be an important reason for the antitumor effects of the combined therapy.

To have a conclusion, adenovirus-mediated CD suicide gene therapy and Ltn gene therapy could elicit antitumor efficacy in pre-established murine colon carcinoma more potently. Efficient induction of nonspecific and specific antitumor immunity, both locally and systemically, might be responsible for the enhanced antitumor efficacy of the combined therapy.

Materials and methods

Animals and cell lines

Male or female BALB/c mice, 6 weeks of age, provided by Joint Venture SIPPR BK Experimental Animal Co, Shanghai, China, were housed in a specific pathogen-free state for all experiments. CT26, a murine colon carcinoma cell line derived from BALB/c mice, NK sensitive YAC-1 cells (TIB-160, American Type Culture Collection, ATCC, Manassas, USA) and 293 (CRL-1573, ATCC), a continuous cell line derived from human embryonic kidney, were maintained in RPMI-1640 medium supplemented with penicillin 100 U/ml, streptomycin 100 μg/ml, 2-mercaptoethanol 50 mmol/l and 10% fetal calf serum (FCS). All culture media and fetal calf serum (FCS) were purchased from Gibco-BRL (Gaithersburg, MD, USA).

Recombinant adenovirus preparation

Replication-defective recombinant adenovirus AdLacZ encoding β-galactosidase, AdCD encoding E. coli CD, AdLtn encoding murine lymphotactin, were constructed from human adenovirus serotype 5 using homologous recombination.1344 The expression of these genes was driven by CAG promoter.13 These adenoviruses were propagated in 293 cells and the titers of the adenovirus were determined with a plaque forming unit (p.f.u.) assay.

Experimental groups and therapeutic regimens

CT26 colon carcinoma cells were taken from continuous culture and resuspended in PBS without FCS for inoculation into mice. BALB/c mice were inoculated s.c. with 5 × 104 CT26 cells to prepare an in vivo tumor model. Three days after tumor inoculation, the tumor-bearing mice were divided into five groups with 15 mice in each group. Then the mice in each group were injected intratumorally with any of the following preparations: PBS, AdLacZ, AdLtn, AdCD, AdCD plus AdLtn. A booster of the same injections (109 viruses in 0.1 ml PBS) was given after 7 days. Twenty-four hours after the first injection of adenoviruses, 300 mg/kg of 5FC (Sigma Chemical, St Louis, MO, USA, 5-FC was dissolved in PBS at a concentration of 12 mg/ml) was injected i.p. into AdLacZ-, AdCD-, or AdCD/AdLtn-treated mice, and 5FC administration was performed daily for 10 consecutive days. The length and width of the tumors were measured with caliber every 3 days after tumor inoculation and the tumor volume was expressed as (length × width2)/2.

In vivo depletion experiments

For selective immunosuppression, 10 tumor-bearing mice in each group undergoing CD/5FC/Ltn therapy were injected i.p. with 0.5 ml of PBS containing 100 μg of anti-NK1.1 puritied from ascites of hybridoma PK136 (anti-NK1.1, ATCC HB-191), 100 μg of anti-CD4 puritied from ascites of hybridoma GK1.5 (TIB-207, ATCC) or 100 μg of anti-CD8 from hybridoma 2.43 (TIB-210, ATCC) 2 days before tumor inoculation and 4 h and 3, 7, 10, and 13 days after tumor inoculation. Flow cytometry was utilized for the analysis of residual blood and spleen cells from these mice, and the targeted leukocytes were decreased selectively to <1/5000 blood leukocytes during the treatment. Control mice were given 100 μg of rat IgG. The tumor volumes and survival periods of the tumor-bearing mice were observed after in vivo depletion of CD4/CD8 T cells.4546

Assay of mRNA expression

Tumor mass was taken from the killed mice 3 days after AdCD/5FC and/or AdLtn gene therapy. The tumor mass was minced and total RNA isolated. RT-PCR method was used for IL-2, IFN-γ and Ltn mRNA assay in the tumor microenvironment. The primers for the analysis of PCR were as follows: IL-2: sense, TCC ACT TCA AGC TCT ACA G; antisense, GAG TCA AAT CCA GAA CAT DCC; IFN-γ: sense, CAT GAA AAT CCT GCA GAG CC; antisense, GGA CAA TCT CTT CCC CAC CC; Ltn, sense, TGG GGA CTG AAG TCC TAG AAG; antisense, TTA CCC AGT CAG GGT TAC TGC TGT G; murine β-actin: sense, TGG AAT CCT GTG GCA TCC ATG AAA C; antisense, TAA AAC GCA GCT CAG TAA CAG TCC G.13 First strand cDNA was synthesized from total RNA by reverse transcription and PCR reactions were carried out according to the method by Ramesh et al.43 Amplification was carried out for 35 cycles (95°C for 20 s, 54–57°C for 30 s and 72°C for 30 s) depending on the template to be amplified, followed by a final extension for 15 min. The amplified PCR products were detected by agarose gel electrophoresis.

Cytotoxic assay

Splenic lymphocytes were isolated from killed tumor-bearing mice 3 days after the last injection of 5FC and used as NK effector cells. The lymphocytes were cocultured with inactivated CT26 cells for 7 days in the presence of mIL-2 20 IU/ml (Genzyme, Cambridge, MA, USA), and then collected as CTL effector cells. The NK and CTL activities were determined by a standard 4-h 51Cr release assay utilizing YAC-1 and CT26 as targets, respectively.15 Two million YAC-1 cells or CT26 cells in 0.5 ml RPMI-1640 with 20% FCS were labeled with 200 μCi Na51CrO4 (Amersham, Arlington Heights, IL, USA) for 2 h. The labeled cells were washed three times in serum-free medium. Ten thousand target cells were then mixed with effector cells for 4 h at 37°C at the ratio indicated. For the maximal 51Cr release control, 0.1 ml of 0.1 N HCl was added to the target cells, and for the spontaneous 51Cr control, 0.1 ml of medium was added to the labeled cells. The amount of 51Cr released was determined by γ counting on a 1275 Minigamma Counter (LKB-Wallac, Turku, Finland), and the percentage of specific lysis was calculated as follows: NK or CTL activity (%) = (experimental c.p.m. − spontaneous c.p.m.)/ (maximal c.p.m. − spontaneous c.p.m.) × 100.

Flow cytometry analysis

For in vitro analysis of CD95 expression and apoptosis, CT26 cells were infected with AdCD at MOI of 20:1 for 6 h and treated with 5FC 25 μg/ml for 6 h, and then the cells were incubated with PE-conjugated monoclonal antibody against CD95 or Annexin V (PharMingen, San Diego, CA, USA) for flow cytometry. CT26 cells were incubated with antagonistic anti-CD95 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 2 h before infection with AdCD and treatment with 5FC to confirm the role of CD95 in AdCD/5FC induced apoptosis of CT26 cells. For analysis of CD80 and CD54 expression, CT26 cells were infected with AdLtn and AdCD for 6 h and treated with 5FC 25 μg/ml for 6 h, and then the cells were incubated with monoclonal antibodies against CD80 and CD54 (PharMingen) for 30 min at 4°C followed by washing with PBS three times. The cells were then incubated with FITC-conjugated goat anti-rat IgG for another 30 min at 4°C followed by washing with PBS. The cells were resuspended in PBS containing 1% formaldehyde for flow cytometry. The control cells were incubated without the primary antibody. The cell-surface fluorescence was determined by Becton Dickinson FACScalibur. A scatter window was set to eliminate dead cells and cell debris, and histograms were overlaid.

The tumors were taken out of the killed tumor-bearing mice 3 days after the last administration of 5FC and the tumor mass was minced to prepare single cell suspensions. The infiltrated mononuclear cells were isolated from the cell suspensions utilizing discontinuous Ficoll–Hypaque gradient centrifugation. Rat anti-mouse CD4, CD8 or NK1.1 monoclonal antibodies (PharMingen) were utilized for the determination of correspondent cell populations by flow cytometry.12

Cytokine assays

Splenic cells were derived from killed tumor-bearing mice 3 days after the last administration of 5FC and the cells were washed with PBS for three times. The splenocytes (5 × 106 cells/ml) were then resuspended in RPMI-1640 medium supplemented with 10% FCS and cultured in 24-well plates at 37°C, 5% CO2 for 24 h. The supernatants were collected and the contents of IL-2 and IFN-γ were determined using ELISA kits purchased from Endogen (Woburn, USA).47

Statistics

All experiments were run in triplicate and the results are means ± s.d. of triplicate determinations or representative data of three independent experiments. The differences in tumor mean volume between treatment groups at each time point were compared using the independent t test. The differences in survival periods of the tumor-bearing mice after various treatments were compared using log-rank tests. Other statistical analyses were performed using the Student's t test. A P < 0.05 was considered to be statistically significant.

References

  1. 1

    Mullen CA . Metabolic suicide genes in gene therapy Pharm Ther 1994 63: 199–207

  2. 2

    Tiberghien P . Use of suicide genes in gene therapy J Leuk Biol 1994 56: 203–209

  3. 3

    Deonarain MP, Spooner RA, Epenetos AA . Genetic delivery of enzymes for cancer therapy Gene Therapy 1995 2: 235–244

  4. 4

    Dilber MS et al. Suicide gene therapy for plasma cell tumors Blood 1996 88: 2192–2200

  5. 5

    Crystal RG et al. Phase I study of direct administration of a replication deficient adenovirus vector containing the E. coli cytosine deaminase gene to metastatic colon carcinoma of the liver in association with the oral administration of the pro-drug 5-fluorocytosine Hum Gene Ther 1997 8: 985–1001

  6. 6

    Dong Y et al. In vivo replication-deficient adenovirus vector-mediated transduction of the cytosine deaminase gene sensitizes glioma cells to 5-fluorocytosine Hum Gene Ther 1996 7: 713–720

  7. 7

    Li Z et al. Enzyme/prodrug gene therapy approach for breast cancer using a recombinant adenovirus expressing Escherichia coli cytosine deaminase Cancer Gene Ther 1997 4: 113–117

  8. 8

    Topf N, Worgall S, Hackett NR, Crystal RG . Regional ‘pro-drug’ gene therapy: intravenous administration of an adenoviral vector expressing the E. coli cytosine deaminase gene and systemic administration of 5-fluorocytosine suppresses growth of hepatic metastasis of colon carcinoma Gene Therapy 1998 5: 507–513

  9. 9

    Wolff G et al. Ex vivo breast cancer cell purging by adenovirus-mediated cytosine deaminase gene transfer and short-term incubation with 5-fluorocytosine completely prevents tumor growth after transplantation Hum Gene Ther 1998 9: 2277–2284

  10. 10

    Pederson LC et al. Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma cells in vitro and in vivo Cancer Res 1997 57: 4325–4332

  11. 11

    Freeman SM, Ramesh R, Marrogi AJ . Immune system in suicide-gene therapy Lancet 1997 349: 2–3

  12. 12

    Cao X et al. Adenovirus-mediated GM-CSF gene and cytosine deaminase gene transfer followed by 5-fluorocytosine administration elicit more potent antitumor tesponse in tumor-bearing mice Gene Therapy 1998 5: 1130–1136

  13. 13

    Cao X et al. Lymphotactin gene-modified bone marrow dendritic cells act as more potent adjuvants for peptide delivery to induce specific antitumor immunity J Immunol 1998 161: 6238–6244

  14. 14

    Ju DW, Wang B, Cao X . Combined suicide gene and interleukin 2 gene therapy for the treatment of established tumor and induction of antitumor immunity in leukemia-bearing mice J Cancer Res Clin Oncol 1998 124: 683–689

  15. 15

    Ju DW, Cao X, Acres B . Intratumoral injection of GM-CSF gene encoded recombinant vaccinia virus elicits potent antitumor response in a murine melanoma model Cancer Gene Ther 1997 4: 139–144

  16. 16

    Cheon J et al. Chemogene therapy: osteocalcin promoter-based suicide gene therapy in combination with methotrexate in a murine osteosarcoma model Cancer Gene Ther 1997 4: 359–365

  17. 17

    Fioretti F et al. Reduced tumorigenicity and augmented leukocyte infiltration after monocyte chemotactic protein-3 (MCP-3) gene transfer: perivascular accumulation of dendritic cells in peritumoral tissue and neutrophil recruitment within the tumor J Immunol 1998 161: 342–346

  18. 18

    Bottazzi B et al. Monocyte chemotactic cytokine gene transfer modulates macrophage infiltration, growth, and susceptibility to IL-2 therapy of a murine melanoma J Immunol 1992 148: 1280–1285

  19. 19

    Nakashima E et al. A candidate for cancer gene therapy: MIP-1 alpha gene transfer to an adenocarcinoma cell line reduced tumorigenicity and induced protective immunity in immunocompetent mice Pharm Res 1996 13: 1896–1901

  20. 20

    Kelner GS et al. Lymphotactin: a cytokine that represents a new class of chemokine Science 1994 226: 1395–1399

  21. 21

    Hedrick JA et al. Lymphotactin is produced by NK cells and attracts both NK cells and T cells in vivo J Immunol 1997 158: 1533–1540

  22. 22

    Hedrick JA, Zlotnik A . Lymphotactin Clin Immunol Immunopathol 1998 87: 218–222

  23. 23

    Hedrick JA, Zlotnik A . Lymphotactin: a new class of chemokine Meth Enzymol 1997 287: 206–215

  24. 24

    Giancarlo B et al. Migratory response of human natural killer cells to lymphotactin Eur J Immunol 1996 26: 3238–4241

  25. 25

    Aghi M et al. Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine deaminase gene therapies J Natl Cancer Inst 1998 90: 370–380

  26. 26

    Hanna NN et al. Virally directed cytosine deaminase/5-fluorocytosine gene therapy enhances radiation response in human cancer xenografts Cancer Res 1997 57: 4205–4209

  27. 27

    Freytag SO et al. A novel three-pronged approach to kill cancer cells selectively: concomitant viral, doule suicide gene, and radiotherapy Hum Gene Ther 1998 9: 1323–1333

  28. 28

    Rogulski KR, Kim JH, Kim SH, Freytag SO . Glioma cells transduced with an Escherichia coli CD/HSV 1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity Hum Gene Ther 1997 8: 73–85

  29. 29

    Kwong YL et al. Combination therapy with suicide and cytokine genes for hepatic metastases of lung cancer Chest 1997 112: 1332–1337

  30. 30

    Santodonato L et al. Cure of mice with established metastatic friend leukemia cell tumors by a combined therapy with tumor cells expressing both interferon-α1 and herpes simplex thymidine kinase followed by ganciclovir Gene Therapy 1996 7: 1–10

  31. 31

    Nanni P et al. The immune response elicited by mammary adenocarcinoma cells transduced with interferon-γ and cytosine deaminase genes cures lung metastases by parental cells Hum Gene Ther 1998 9: 217–224

  32. 32

    Yoon SJ et al. Synergistic antitumor effects with co-expression of GM-CSF and IFN-γ in murine tumors Int J Cancer 1998 77: 907–912

  33. 33

    Benedetti S et al. Limited efficacy of the HSV-TK/GCV system for gene therapy of malignant gliomas and perspectives for the combined transduction of the interleukin-4 gene Hum Gene Ther 1997 8: 1345–1353

  34. 34

    Mule JJ et al. RANTES secretion by gene-modified tumor cells results in loss of tumorigenicity in vivo: role of immune cell subpopulations Hum Gene Ther 1996 7: 1545–1553

  35. 35

    Nakashima E et al. Synergistic antitumor interaction of human monocyte chemotactant protein-1 gene transfer and modulator for tumor-infiltrating macrophages Pharm Res 1998 15: 685–689

  36. 36

    Wang J et al. Lymphotactin: a key regulator of lymphocyte trafficking during acute graft rejection Immunology 1998 95: 56–61

  37. 37

    Dilloo D et al. Combined chemokine and cytokine gene transfer enhances antitumor immunity Nature Med 1996 2: 1090–1095

  38. 38

    Bi W et al. An HSVtk-mediated local and distant antitumor bystander effect in tumors of head and neck origin in athymic mice Cancer Gene Ther 1997 4: 246–252

  39. 39

    Denning C, Pitts JD . Bystander effects of different enzyme-prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes Hum Gene Ther 1997 8: 1825–1835

  40. 40

    Chong H et al. Tumour cell expression of B7 costimulatory molecules and interleukin-12 or granulocyte–macrophage colony-stimulating factor induces a local antitumour response and may generate systemic protective immunity Gene Therapy 1998 5: 223–232

  41. 41

    Uzendoski K et al. Construction and characterization of a recombinant vaccinia virus expressing murine intercellular adhesion molecule-1: induction and potentiation of antitumor responses Hum Gene Ther 1997 8: 851–860

  42. 42

    Ramesh R et al. Expression of costimulatory molecules: B7 and ICAM up-regulation after treatment with a suicide gene Cancer Gene Ther 1996 3: 373–384

  43. 43

    Hall SJ, Sanford MA, Atkinson G, Chen SH . Induction of potent antitumor natural killer activity by herpes simplex virus-thymidine kinase and ganciclovir therapy in an mouse model of prostate cancer Cancer Res 1998 58: 3221–3225

  44. 44

    Kanai F et al. In vivo gene therapy for alpha fetoprotein producing hepatocellular carcinoma by adenovirus mediated transfer of cytosine deaminase gene Cancer Res 1997 57: 461–465

  45. 45

    Cavallo F et al. Role of neutrophils and CD4+ T lymphocytes in the primary and memory response of nonimmunogenic murine mammary adenocarcinoma made immunogenic by IL-2 gene J Immunol 1992 149: 3627–3635

  46. 46

    Consalvo M et al. 5-Fluorocytosine induced eradication of murine adenocarcinomas engineered to express the cytosine deaminase suicide gene requires host immune competence and leaves an efficient memory J Immunol 1995 154: 5302–5312

  47. 47

    Toda M, Martuza RL, Kojima H, Rabkin SD . In situ cancer vaccination: an IL-12 defective vector/replication-competent herpes simplex virus combination induces local and systemic antitumor activity J Immunol 1998 160: 4457–4464

Download references

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 39600181) and National High Biotechnology Project of China (Z20–01–03).

Author information

Correspondence to X Cao.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ju, D., Tao, Q., Cheng, D. et al. Adenovirus-mediated lymphotactin gene transfer improves therapeutic efficacy of cytosine deaminase suicide gene therapy in established murine colon carcinoma. Gene Ther 7, 329–338 (2000). https://doi.org/10.1038/sj.gt.3301082

Download citation

Keywords

  • lymphotactin
  • cytosine deaminase
  • gene therapy
  • adenovirus
  • antitumor immunity

Further reading