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.
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.
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.
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).
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.
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.
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).
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.
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.
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.
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
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
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.
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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).
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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
- cytosine deaminase
- gene therapy
- antitumor immunity
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