Cancer DNA vaccines encoding tumor associated antigen represent a novel form of cancer treatment. These vaccines have been found to be an effective means of cancer therapy by several researchers.1, 2, 3, 4 It is relatively easy to devise a cancer DNA vaccine to insert antigen into mammalian expression vector containing CMV promoter, and thus to deliver the required DNA via various routes in living organisms.5, 6 Following the administration of tumor associated antigen encoding cancer DNA vaccine into a living organism, antigen peptide of the cancer DNA vaccine is delivered to immune related cells, and thus immune response against the antigen peptide is induced in the host. Immune systems stimulated by cancer DNA vaccines may protect host from cancer occurrence or recurrence.7 However, cancer DNA vaccines are not always effective because of their limited potencies, regulatory T cells hindrance, cytokine imbalances in the host or others.8, 9, 10, 11
Sodium iodide symporter (NIS) is a specialized active iodide transporter that cotransports sodium and an iodide ion.12, 13, 14 Many researchers have tried to apply this system to cancer gene therapy using viral or nonviral gene systems by directly transferring the NIS gene into living organisms. Moreover, NIS gene expression in cancer cells allows effective radioiodine (131I, 188Re) therapy, which has led to good prognoses in preclinical and clinical models.15, 16, 17 Moreover, some investigators have reported that NIS-specific radioiodine accumulation within NIS-transfected cells can induce significant tumor reduction in athymic nude mice.18, 19, 20, 21
Cancer DNA vaccine researchers have added various genes to cancer DNA vaccines to increase therapeutic effects, and these various gene supplements have resulted in significant tumor regression in living subjects.22, 23, 24, 25 Since radioiodine gene therapy has been shown to be a powerful tool for cancer gene therapy, we attempted to develop a new combination therapy to enhance the preventive effects of cancer DNA vaccines by utilizing a NIS approach. In this study, we attempted to enhance the tumor growth inhibition induced by hMUC1 DNA vaccination by using radioiodine gene therapy of human NIS.
Material and methods
Specific pathogen-free 6-week-old female BALB/c mice were obtained from SLC (Hamamatsu, Japan).26 All experimental animals were housed under specific pathogen-free conditions and were handled in accord with the guidelines issued by the Seoul National University Animal Research Committee.
Generation of cDNA constructs and plasmid preparation
The human pancreatic mucin1 gene, hMUC1 (accession no. J05582), was cloned into the BamHI site of pcDNA3 vector (Invitrogen, Carlsbad, CA). Plasmid DNA was amplified in E. coli DH5α and purified by large-scale plasmid preparation using endotoxin-free Giga Prep columns (QIAGEN, Chatsworth, CA). DNA was dissolved in endotoxin-free TE buffer for storage.
Generation of Lentivirus
To construct hNIS expressing lentiviral vector under the control of ubiquitin C promoter, the hNIS gene was cloned into pLenti6/UbC/V5-DEST (Invitrogen, Carlsbad, CA). Replication-incompetent lentivirus was produced by cotransfecting lentiviral vector carrying hNIS and a ViraPower™ Packaging Mix (Invitrogen, Carlsbad, CA) into 293FT producer cell line. 293FT cells (6 × 106) were transient transfected using 36 μl Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in 10 cm tissue culture plates. Cells were cotransfected with 5 μg of lentiviral vector and 10 μg of packaging plasmids (gag, pol, vsv-g, rev). Growth medium (DMEM containing 10% FBS, and 1% penicillin/streptomycin) was changed at 24 hr post-transfection and lentivirus-containing supernatant was harvested at 48 hr post-transfection. Harvested supernatants were centrifuged at 3,000 rpm for 15 min at 4°C to pellet cell debris and stored at −80°C for later use. To assess the activity of recombinant virus encoding hNIS reporter gene, HT1080 human fibrosarcoma cells were infected by adding thawed lentivirus-supernatant containing 10 μg/ml of Polybrene. Human sodium-iodide symporter (hNIS) expression was confirmed by examining 125I uptake.
Generation of Retrovirus
Firefly luciferase gene under EF-1α promoter was cloned into pMSCVneo (BD Bioscience Clontech, CA). Retrovirus was produced by cotransfection into a human 293FT producer cell line using retroviral vector carrying the luciferase gene and packaging plasmids (gag, pol, vsv-g) (BD Bioscience Clontech, CA). Transient transfection of 293FT cells (6 × 106) was performed using 36 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in 10 cm tissue culture plates. Cells were cotransfected with 5 μg of retroviral vector and 10 μg of packaging plasmids. Growth media (DMEM containing 10% FBS and 1% penicillin/streptomycin) were changed at 24 hr post-transfection and retrovirus-containing supernatants were harvested at 48 hr post-transfection. Harvested supernatants were centrifuged at 3,000 rpm for 15 min at 4°C to pellet cell debris and stored at −80°C for later use. To assess the activity of recombinant virus encoding firefly-luciferase reporter gene, HT1080 human fibrosarcoma cells were infected by adding thawed retrovirus-containing supernatant and 10 μg/ml of Polybrene. Cell luciferase expression was confirmed by checking luciferase activity using a luminometer (Applied Biosystems, Forster, CA).
Murine tumor cell line expressing hMUC1, hNIS and firefly luciferase
hMUC1 expressing CT26 (CT26/hMUC1) was kindly provided by Dr. Jung-Ah Cho.26 Viral supernatants were transduced into CT26/hMUC1 of the H-2d MHC type. Stable clones (CT26/hMUC1-hNIS-Fluc referred to as CMNF) were also subjected to luciferase assays and 125I uptake using a microplate luminometer (Applied Biosystems, Forster, CA) and gamma counter (GMI, Ramsey, MN).
To examine hMUC1 expressions on CMNF cell surfaces, cells were harvested and suspended in 0.1% BSA-containing PBS. Primary antibodies were added to these suspensions, and anti-MUC1 (Biomeda, CA) mouse antibody was used as the primary antibody.27 After 1 hr incubation on ice, cells were washed and pelleted to remove unbound antibodies. FITC-tagged anti-mouse antibody was used as a secondary antibody and incubated for 30 min on ice. Cold 2% PFA-containing PBS was used to fix the cells, and finally, fluorescence intensities were measured using a Coulter FACScan. FACS analysis showed that CMNF cells highly expressed hMUC1 tumor antigen. In addition, we observed that bioluminescent signals increased with cell numbers.
In vitro clonogenic assay
The procedure used, with minor modifications, has been described previously.28 Briefly, cells were grown in a 75-cm2 flask and incubated for 7 hr at 37°C in 5 ml HBSS containing 37 MBq/10 ml (1 mCi/10 ml) Na131I. The reaction was terminated by removing the radioisotope-containing medium and washing the cells twice with HBSS. The cells were then trypsinized, counted and plated at densities of 250 or 1,000 cells per well in DMEM in 6-well plates. Cells were grown for 10 days, fixed with 3:1 methanol/acetic acid, stained with crystal violet and macroscopic colonies numbers were counted. Survival rates were defined as colony numbers in radionuclide treated plates expressed as percentage of numbers in plates treated with HBSS only.
Monitoring of tumor growth inhibition in living mice
The IVIS200 imaging system (Xenogen, Alameda, CA), which includes an optical CCD camera mounted on a light-tight specimen chamber, was used for data acquisition and analysis. Firefly D-luciferin potassium salt (Fluc substrate), was diluted to 3 mg/100 ul in PBS before use, and mice were injected i.p. with 100 μl of this D-luciferin solution. Mice were placed individually in the specimen chamber containing the CCD camera, and then cooled to −105°C. Light emitted by luciferase in mice was then measured. Gray scale photographic images and bioluminescent color images were superimposed using LIVINGIMAGE V. 2.12 (Xenogen, Alameda, CA) and IGOR image analysis software (WaveMetrics, Lake Oswego, OR). Bioluminescent signals were expressed in units of photons per cm3 per second per steradian (P/cm2/s/sr).
pcDNA3.1 (50 μg/100 μl) or pcDNA3-hMUC1 (50 μg/100 μl) was injected i.m. into quadriceps muscles of right hind legs once a week for 2 weeks. One week after the final immunization, each group was inoculated s.c. with 1 × 105 CMNF cells in right thighs. Mice had received a low-iodine diet and T4 supplementation in their drinking water for 2 weeks post CMNF challenge to maximize radioiodine uptake in tumors and to reduce iodide uptake by thyroid glands. Each animal was administered 111 MBq (3 mCi) of 131I or saline i.p. Mice were repeatedly imaged at 7, 13, 21, 28, 35 days post CMNF challenge using an optical CCD camera to acquire photons, 10 min after injecting D-luciferin. To quantify emitted light, regions of interest were drawn over the tumor region and total photon effluxes over an exposure time of 10 sec were determined. For scintigraphic imaging, 99mTc-pertechnetate was injected i.p and mice were imaged using a γ-ray camera (ON-410) at 21 days postchallenge. Tumor size was measured using a caliper at 17, 21, 28, 32, 39 days and tumor weights were measured at 39 days.
Statistical significances were determined using an unpaired Student's t test, and Kaplan Meier curves were generated for survival analysis. The survival curves of 2 groups were compared using the log rank test. P values of <0.05 were considered significant.
Establishment of a cancer cell line expressing high levels of hMUC1, hNIS and firefly luciferase.
To determine hMUC1, hNIS and Fluc gene expressions in selected stable cell lines (CT26/hMUC-hNIS-Fluc, referred to a CMNF), we performed FACS analysis, and luciferase and 125I uptake assays, as shown in Figure 1. FACS analysis showed that stable transfectants highly expressed the hMUC1 gene (Fig. 1a), and bioluminescence signals and 125I uptakes were found to correlate well with cell numbers (Figs. 1b and 1c).
In vitro clonogenic assay
As shown in Figure 2, the survival rates of CT26/hMUC-hNIS-Fluc cells were markedly reduced to (14.6 ± 1.5)% in response to 131I vs. CT26 cells (p < 0.001).
Tumor growth inhibition induced by hMUC1 vaccination plus radioiodine gene therapy
We performed following experimental procedures for radioiodine gene therapy and immunotherapy (Fig. 3). NIS gene expression was observed in all 4 mouse groups (Fig. 4a). Also, we observed tumor growth inhibition induced by hMUC1 vaccination in the two pcDNA3-hMUC1 vaccination groups (pcDNA3-hMUC1+PBS, pcDNA3-hMUC1+131I groups) compared with the two pcDNA3.1 vaccination groups (pcDNA3.1, pcDNA3.1 + 131I groups) by bioluminescent imaging (Fig. 4b). Following 131I treatment to all groups, tumor progression was monitored by bioluminescent imaging and caliper measurements until 39 days postchallenge (Figs. 4b–4d), and a difference in tumor growth inhibition was observed between the pcDNA3-hMUC1 + PBS and pcDNA3-hMUC1 +131I groups at 28 days postchallenge. Significant tumor growth inhibition was observed in the pcDNA3-hMUC1 +131I group vs. the pcDNA3.1 + PBS, pcDNA3.1+131I and pcDNA3-hMUC1+PBS groups (p < 0.05). At 39 days postchallenge, animals were euthanized and tumor masses were removed and weighted. Significant tumor growth inhibition was observed in the pcDNA3-hMUC1 + 131I group vs. the pcDNA3.1, pcDNA3.1+131I and pcDNA3-hMUC1+PBS groups (p < 0.05) (Fig. 4e). As shown in the Kaplan-Meier plots in Figure 4f, 80% of tumor bearing mice survived until 50 days postchallenge in the combination therapy group(pcDNA3-hMUC1 + 131I group) using hMUC1 vaccination + 131I treatment, whereas almost all animals in the other groups died (pcDNA3.1 + PBS, pcDNA3.1+131I and pcDNA3-hMUC1+PBS group: 0, 10, 20 %, respectively).
Immunotherapy using cancer DNA vaccines is now viewed as a means of inhibiting tumor growth in living organisms. Various strategies have been applied to increase the potency of DNA vaccines; for example, targeting tumor-associated antigens to facilitate rapid cellular degradation, directing tumor-associated antigens to chemokines, coinjecting cytokines or coadministering CpG motif.29, 30, 31, 32, 33, 34, 35, 36, 37 However, immunotherapies based on cancer DNA vaccines in combination with these strategies have been found to be ineffective at inhibiting tumor growth. Thus, immunotherapy researchers have tried to utilize other modalities to overcome the preventive (or therapeutic) limitations of cancer DNA vaccines.
Sodium iodide symporter (NIS) has been used for radionuclide gene therapy as an adjunct to treatment in various types of cancer. Some investigators have found that effective gene expression is induced in living subjects by infecting various cancer cells with NIS, and have achieved good therapeutic effects in preclinical models.15, 16 Other reports have shown that NIS gene transfer using tissue-specific promoter allows the targeting of NIS gene expression in specific cancer cells, thereby maximizing tissue-specific cytotoxicities and minimizing toxic side-effects in normal cells.18, 19, 20 NIS gene transfer into cancer cells causes therapeutic radionuclides (131I, 188Re) to concentrate in these cells, and thus facilitating the use of therapeutic radionuclides to induce cancer cell apoptosis. Our group has previously reported that NIS expressing human hepatocellular carcinoma can be selectively killed given 131I and 188Re accumulation via NIS gene expression,17 and demonstrated that therapy and imaging based on NIS gene transfer could be used to treat anaplastic thyroid carcinomas.38
It has also been shown that the human MUC1 mucin is over-expressed in an incompletely glycosylated form in various human cancers,39, 40 and because high expression of tumor associated antigen hMUC1 is related with rapid tumor progression and a poor prognosis in several types of human cancer, it is considered an attractive immunotherapeutic target. Moreover, cancer DNA vaccine encoding hMUC1 has been studied for the targeting of epithelial cancers expressing high levels of MUC1, and it has been shown that MUC1 DNA vaccination can inhibit MUC1 expressing tumor growth in a preclinical model.41, 42, 43, 44
In the present study, we developed a combination therapy based on an MUC1 vaccine and radionuclide gene therapy. To do this we established an adenocarcinoma colon cancer cell line stably expressing hMUC1, firefly luciferase and sodium iodide symporter by retro- or lentivirus infection, which we refer to as the CMNF cell line. Because CMNF cells express immunogenic target and therapeutic genes, we were able to perform combined cancer DNA immunotherapy and radionuclide gene therapy in living mice. In addition, bioluminescent signals emitted by CMNF allow the monitoring of therapeutic effects.
This combination therapy was found to have the desired effects in vitro and in vivo, and the survival of NIS expressing CMNF cells was effectively reduced to (14.6 ± 1.5)% of the control after 131I treatment. In addition, we observed effective in vivo tumor growth inhibition in the combination therapy group (the pcDNA3-hMUC1 + 131I treatment group) but not in mono therapy group (pcDNA3.1, pcDNA3.1+131I, pcDNA3-hMUC1+PBS treatment group). Tumor growth inhibition occurred in mice immunized with pcDNA3-hMUC1 but not in mice immunized with pcDNA3.1 at 28 days post-CMNF challenge based on caliper and bioluminescent imaging findings (Fig. 4b). However, tumor growth inhibition was not sustained in pcDNA3-hMUC1 + PBS treatment group from 28 days postchallenge.
Although we did not investigate immunologic aspects, we believe that this phenomenon (reduction in tumor growth inhibition) may have been due to an immunogenic effect of the DNA vaccine, the hindrance regulator T-cells, and disruption of cytokine balance in the host.8, 9, 10, 11 Furthermore, we could suggest following 2 reasons for the possible mechanisms of more significant tumor growth inhibition effect after I-131 treatment in MUC1 vaccinated group than other groups. The one is the immune booster response to the MUC1 antigen, which was released from the destroyed CMNF cells through the therapeutic effects by I-131 therapy. As a similar feature, T cell populations are significantly increased after radioiodine therapy in patients with Graves' disease.45 The other mechanism, which might be involved, is that the released hNIS itself from dead cells after radionuclide treatment could also raise immune response against viable CNMF cells with hNIS expression. We recently reported that hNIS vaccination with MIDGE plasmid vector could raise hNIS-associated cell mediated immune responses in mice tumor model.46
This study has a limitation that should be mentioned. We injected MUC1 vaccine before tumor inoculation, which is not accord with the normal situation in cancer patients, but which is consistent with cancer vaccine treatment in a postoperative setting after tumor resection. Thus, it would be more accurate to say that the described combination therapy helps prevent tumor recurrence. In terms of future studies, the repertoire of immune responses initiated by radioiodine gene therapy in living subjects requires investigation. Moreover, because recent sodium iodide symporter radioiodine gene therapies have concentrated on the adenoviral system, which is controlled by specific-promoter regulation, further efforts are required to broaden the scope of the radioiodine/immunotherapy concept.
We demonstrate for the first time that limited tumor growth inhibition achieved using a hMUC1 DNA vaccine can be augmented by radioiodine gene therapy based on sodium iodide symporter over an extended period (52 days) in a preclinical model. We hope that this combination therapy will find a place as a treatment for preventing the occurrence or recurrence of cancer.