We examined the merits of combinatorial hMUC1 vaccination and hNIS radioiodine gene therapy and evaluated its tumoricidal effects in an animal tumor model. CMNF (CT26 expressing hMUC1, hNIS, and firefly luciferase) cells were transplanted into 28 mice, and 4 and 11 days after tumor challenge, tumor-bearing mice were immunized i.m. with pcDNA3.1 or pcDNA-hMUC1 vaccine and subsequently administered PBS or 131I i.p. [four groups (7 mice per group): pcDNA3.1 + PBS, phMUC1 + PBS, pcDNA3.1 + 131I, and phMUC1 + 131I groups]. Thirty-two days after tumor challenge, we rechallenged mice in the pcDNA3.1 + 131I and phMUC1 + 131I groups with CMNF cells. Tumor progression and tumor-free mice (%) were monitored by bioluminescence. We investigated hMUC1-associated immune response generated by combination therapy. Marked tumor growth inhibition was observed in the phMUC1 + 131I group by bioluminescence at 32 days after tumor challenge. Mice in phMUC1 + 131I group showed complete hMUC1-expressing tumor suppression after tumor rechallenge, whereas mice in the pcDNA3.1 + 131I group did not. The tumor-free mice (%) were much higher in the phMUC1 + 131I group than in the other three groups. Levels of hMUC1-associated CD8+IFN-γ+ T cells were higher in the phMUC1 + 131I group than in the other three groups. hMUC1-loaded CD11+ cells in the phMUC1 + 131I group were found to be most effective at generating hMUC1-associated CD8+IFN-γ+ T cells. The activities of hMUC1-associated cytotoxic T cells in the phMUC1 + 131I group were higher than in the other three groups. Our data suggest that phMUC1 + 131I combination therapy synergistically generates marked tumoricidal effects against established hMUC1-expressing cancers. [Mol Cancer Ther 2008;7(7):2252–60]

Radiation destroys both cancer cells and other cells within tumor stroma, such as endothelial cells and intratumoral lymphocytes (1). Irradiated cancer cells undergo apoptosis or necrosis and secrete various proteins that stimulate immune cells, such as dendritic cells, macrophages, and T cells. In particular, antigen-presenting cells might recognize proteins generated by irradiating cancer cells and present tumor-specific antigenic peptides to T cells; thus, in turn, effector T cells might identify and kill antigen-expressing cancer cells (24). On the other hand, irradiated cancer or stroma cells show increased expressions of cell surface proteins, such as Fas, MHC class I molecules, and intracellular adhesion molecule-1 (57). These microenvironmental modifications could provide therapeutic advantages: (a) antigen-presenting cells might detect antigenic proteins derived from dying cancer cells and stimulate T cells in a MHC class-restricted manner and (b) activated T cells could migrate to cancer cells or immune-related organs through adhesion molecules in vascular lumen (8). Moreover, when other therapeutic modalities (e.g., immunotherapy and chemotherapy) are combined with radiotherapy, this radiation-induced immune response may be enhanced.

hMUC1 is highly overexpressed in various human cancers (9, 10). Moreover, because high expressions of hMUC1 are related with rapid tumor progression, many researchers have investigated the usefulness of hMUC1 DNA vaccines (1113). However, although the therapeutic effect of hMUC1 immunization against cancer has been well investigated, hMUC1 vaccines alone are limited in terms of their abilities to inhibit cancer progression. Thus, new adjunctive modalities are required to enhance the therapeutic effects induced by hMUC1 immunization.

Sodium/iodide symporter (NIS) is a specialized active iodide transporter, and NIS-expressing cancer cells are known to accumulate several diagnostic or therapeutic radionuclides in vitro and in vivo (14, 15). More specifically, the transfer of the NIS gene and the functional expression of NIS protein enable cancer cells to accumulate therapeutic radionuclides, such as 131I and 188Re, from plasma and thus offer the possibility of radionuclide gene therapy. Some investigators have described the therapeutic effects of NIS-specific radionuclide gene therapy in animal models (1619).

In the previous study, we established the CMNF cell line (a mouse colon cancer cell line; CT26/hMUC1-hNIS-Fluc) expressing the hMUC1, hNIS, and Fluc genes and examined the merits of combinatorial hMUC1 vaccine and radionuclide gene therapy (20). We hypothesized that hMUC1 antigenic peptides released by cancer cells killed by 131I would act as an antigen source and that these antigens would restimulate or maintain immune response against CMNF cells. We designed a combination therapy model that mimics the clinical situation and evaluated the synergistic tumoricidal effects of radiation-induced immune response induced by hNIS radioiodine gene therapy and hMUC1 immunotherapy in tumor-bearing mice.

Animals

Specific pathogen-free 6-week-old female BALB/c mice were obtained from SLC. All animal experiments were done after receiving approval from the Institutional Animal Care and Use Committee of the Clinical Research Institute at Seoul National University Hospital. In addition, the National Research Council guidelines for the care and use of laboratory animals (revised 1996) were observed throughout.

Cell Line

Previously, we established the CMNF cell line, which is a stable clone of CT26 cells (a adenocarcinoma colon cancer cell line) that expresses hMUC1, hNIS, and firefly luciferase genes (CT26/hMUC1-hNIS-Fluc), and examined gene expression in this cell line by fluorescence-activated cell sorting, 125I uptake assays, and in vitro luciferase assays (data not shown; ref. 20).

DNA Vaccine

The human pancreatic mucin1 gene, hMUC1 (accession no. J05582), was cloned into the BamHI site of pcDNA3 vector (Invitrogen). Plasmid DNA was amplified in Escherichia coli DH5α and purified by large-scale plasmid preparation using endotoxin-free Giga Prep columns (Qiagen). DNA was dissolved in endotoxin-free TE buffer for storage purposes.

In vitro Clonogenic Assay

The procedure used has been described previously (21). Briefly, cells were grown in a 75 cm2 flask and incubated for 7 h 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. Cells were then trypsinized, counted, and plated at densities of 250 or 1,000 per well in DMEM in six-well plates. They were then grown for 10 days, fixed with 3:1 methanol/acetic acid, and stained with crystal violet, and macroscopic colony numbers were counted. Survival rates are presented as colony numbers expressed as percentages of colony numbers in plates treated with HBSS only.

Bioluminescence Imaging Acquisition

An IVIS100 imaging system (Xenogen), 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 μL in PBS before use, and mice were injected i.p. with 100 μL of this d-luciferin solution. Mice were placed individually in a specimen chamber containing the CCD camera, and light emitted by luciferase in mice was then measured. Grayscale photographic images and bioluminescent color images were superimposed using LIVINGIMAGE V. 2.12 (Xenogen) and IGOR image analysis software (WaveMetrics). To quantify emitted light, regions of interest were drawn over the tumor region and total photon effluxes over an exposure time of 3 s were determined. Bioluminescent signals were expressed in units of photons per cubic meter per second per steradian.

In vivo Animal Experiments

The four experimental groups are the pcDNA + PBS, phMUC1 + PBS, pcDNA + 131I, and phMUC1 + 131I groups as described previously. The following experimental procedure was done for combined hMUC1 vaccination and hNIS radioiodine gene therapy (Fig. 2).

In 28 mice, 1 × 105 CMNF cells were transplanted s.c. in right thighs. Four and 10 days later, mice were immunized with pcDNA3.1 (50 μg/50 μL) or phMUC1 (50 μg/50 μL) into a quadriceps muscle of the right hind leg and 14 days later were administered PBS or 2 mCi 131I i.p. [four groups (7 mice per group): pcDNA3.1 + PBS, phMUC1 + PBS, pcDNA3.1 + 131I, and phMUC1 + 131I groups]. Mice received a low-iodine diet with T4 supplementation in drinking water for 2 weeks post-tumor challenge to maximize radioiodine uptake in tumors and to reduce iodide uptake by thyroid glands. Tumor sizes were measured using a caliper at 7, 14, 17, 21, 24, 28, and 32 days post-tumor challenge and tumors were weighted in the pcDNA + PBS and phMUC1 + PBS groups at 32 days. For bioluminescence imaging acquisition, mice were repeatedly imaged at 3, 10, 14, 17, 21, 24, 28, and 32 days postchallenge using an optical CCD camera. For scintigraphic imaging acquisition, [99mTc]pertechnetate (0.5 mCi) was injected i.p, and mice were imaged using a γ-ray camera (ON-410) at 14 days postchallenge.

In vivo Tumor Rechallenge Experiment

Mice in the pcDNA + 131I and pMUC1 + 131I groups were rechallenged with 5 × 104 CMNF cells s.c. in right thighs at 32 days postinitial challenge. Mice were repeatedly imaged at 3, 8, 12, and 16 days postrechallenge using an optical CCD camera. Postmortem tumor weights were measured at 16 days postrechallenge.

Intracellular Cytokine Staining and Flow Cytometric Analysis

For the in vivo tumor protection experiments, mouse splenocytes (seven per group) were isolated from treated mice and stimulated in vitro for 72 h using 10 μg/mL hMUC1 peptide (PDTRPAPGSTAPPAHGVTSAPDTRPAPGST) and interleukin-2 (50 units/mL). Stimulated splenocytes were treated with Golgistop (BD PharMingen). Splenocytes were then washed once in fluorescence-activated cell sorting buffer and stained with FITC-conjugated monoclonal rat anti-mouse CD8 (BD PharMingen). Cells were immunostained for cytokines using a Cytofix/Cytoperm kit (BD PharMingen) and with PE-conjugated anti-IFN-γ (BD PharMingen). Flow cytometric analysis was done using a Becton Dickinson FACScan using CellQuest software (Becton Dickinson Immunocytometry Systems).

In vitro Splenocyte Cytotoxicity Assays

For the in vivo tumor protection and long-term tumor growth inhibition experiment, the CytoTox 96 nonradioactive cytotoxicity assay (Promega) was used to measure the cytotoxic activities of splenocytes in treated mice (7 mice per group) according to the manufacturer's protocol with minor modification. Briefly, splenocytes of treated immunocompetent BALB/c mice were incubated in the presence of human interleukin-2 (50 units/mL) and 10 μg/mL hMUC1 peptide (PDTRPAPGSTAPPAHGVTSAPDTRPAPGST). After 3 days, irradiated CT26 and CMNF target cells were plated at 1 × 104 per well on 96-well U-bottomed plates (Costar), and splenocytes (effectors) were added to a final volume of 100 μL in a 1:12 radio. Plates were then incubated for 4 h in a humidified 5% CO2 chamber at 37°C and centrifuged at 500 × g for 5 min. Aliquots (50 μL) were transferred from all wells to fresh 96-well flat-bottomed plates, and an equal volume of reconstituted substrate mix was added to each well. The plates were then incubated in the dark at room temperature for 30 min. Stop solution (50 μL) was then added, and absorbance values were measured at 492 nm. Cell death percentages at each effector-to-target cell ratio were calculated using [A (experimental) - A (effector spontaneous) - A (target spontaneous)] × 100 / [A (target maximum) - A (target spontaneous)].

Histopathology

Tumor injection sites were removed from mice and preserved in 10% formalin solution (Sigma) until required. All tumor tissues were embedded in paraffin, sectioned at 4 μm, and stained with H&E. Histopathologic reviews of tumor tissues were done independently by two pathologists.

Restimulation of hMUC1-Associated CD8+ T cells by Enriched CD11c+ Cells from Immunized Mice

Ten days after treating tumor-bearing mice (4 mice per group) with 131I, splenocytes were harvested and CD8+ T cells were separated using CD8 (Ly-2) microbeads (Miltenyi Biotec). The enriched CD8+ T cells so obtained were analyzed by fluorescence-activated cell sorting and restimulated in vitro for 72 h with 10 μg/mL hMUC1 peptide (PDTRPAPGSTAPPAHGVTSAPDTRPAPGST) and interleukin-2 (50 units/mL).

Thirteen days after treating tumor-bearing mice (4 mice per group) with 131I, inguinal draining lymphoid cells were harvested and CD11+ cells were enriched using CD11c (N418) Microbeads (Miltenyi Biotec). These enriched cells were then analyzed by fluorescence-activated cell sorting. Restimulated CD8+ T cells [treated with Golgistop for 6 h (BD PharMingen)] were then harvested for coculture with the enriched CD11c+ cells. The enriched CD11+ cells (1 × 105) were then cocultured with restimulated 1 × 106 hMUC1-assoicated CD8+ T cells for 16 h. The restimulated CD8+ T cells were then stained with CD8 and IFN-γ antibody (BD PharMingen) and analyzed by flow cytometry.

Statistical Analysis

All data are expressed as mean ± SD and are representative of at least two separate experiments. Intracellular cytokine staining, flow cytometry analysis, and tumor protection experiment findings were analyzed using the Kruskal-Wallis test and the Wilcoxon rank-sum test with Bonferroni's correction. Kaplan-Meier curves were used to estimate tumor-free mice. P values < 0.02 were considered significant.

In vitro Clonogenic Assay

As shown in Fig. 1, the survival rates of CMNF (hNIS-expressing CT26) cells were markedly reduced to 13.6 ± 1.0% in response to 131I versus CT26 cells (P < 0.001).

Figure 1.

In vitro clonogenic assay after incubation with 131I. CMNF cells were exposed to 37 MBq/10 mL (1 mCi/10 mL) 131I for 7 h at 37°C. Cells were then grown for 10 d and stained with crystal violet, and colony numbers were counted. Survival rates (%) were colony numbers in plates treated with radionuclide expressed as percentages of colony numbers in plates containing buffer only. Columns, mean of triplicate experiments; bars, SD. *, P < 0.001.

Figure 1.

In vitro clonogenic assay after incubation with 131I. CMNF cells were exposed to 37 MBq/10 mL (1 mCi/10 mL) 131I for 7 h at 37°C. Cells were then grown for 10 d and stained with crystal violet, and colony numbers were counted. Survival rates (%) were colony numbers in plates treated with radionuclide expressed as percentages of colony numbers in plates containing buffer only. Columns, mean of triplicate experiments; bars, SD. *, P < 0.001.

Close modal

Therapy of Tumor Xenografts in Animals Treated with hMUC1 Vaccination and/or 131I Therapy

Minor tumor growth inhibition effects were observed in animals vaccinated with hMUC1 (phMUC1 + PBS and phMUC1 + 131I groups) but not in the corresponding pcDNA vaccination groups by scintigraphic imaging (Fig. 2B). A slight tumor growth inhibiting effect was observed in the phMUC1 + PBS group but not in the pcDNA3.1 + PBS at 32 days postchallenge (Fig. 3A-C; P < 0.02, pcDNA + PBS versus phMUC1 + PBS). However, 131I-treated animals (pcDNA + 131I and phMUC1 + 131I) showed rapid tumor growth inhibition (Figs. 2C and 3A-C) by bioluminescence imaging and according to caliper measurements.

Figure 2.

Animal experimental protocol and in vivo monitoring of antitumor effects of combined hMUC1 vaccination and 131I therapy. A, 1 × 105 CMNF cells were transplanted s.c. into the right thighs of immunocompetent BALB/c mice. Tumor-bearing mice were immunized i.m. with each vector (pcDNA3-hMUC1 or pcDNA3.1) at 4 and 10 d post-tumor challenge, and scintigraphic images were acquired at 14 d postchallenge. At 14 d, 131I was injected i.p. Bioluminescent imaging and tumor volume measurements were done at the designated times. Thirty-two days postchallenge, cured mice in the pcDNA + 131I and phMUC1 + 131I groups were rechallenged with 5 × 104 CMNF cells. B, before administering a therapeutic dose of 131I to mice, scintigraphic images were acquired at 14 d postchallenge. T, thyroid; S, stomach; B, urinary bladder; CMNF, CT26/hMUC1-hNIS-Fluc. C, tumor growths were visualized by bioluminescent imaging at the designated times.

Figure 2.

Animal experimental protocol and in vivo monitoring of antitumor effects of combined hMUC1 vaccination and 131I therapy. A, 1 × 105 CMNF cells were transplanted s.c. into the right thighs of immunocompetent BALB/c mice. Tumor-bearing mice were immunized i.m. with each vector (pcDNA3-hMUC1 or pcDNA3.1) at 4 and 10 d post-tumor challenge, and scintigraphic images were acquired at 14 d postchallenge. At 14 d, 131I was injected i.p. Bioluminescent imaging and tumor volume measurements were done at the designated times. Thirty-two days postchallenge, cured mice in the pcDNA + 131I and phMUC1 + 131I groups were rechallenged with 5 × 104 CMNF cells. B, before administering a therapeutic dose of 131I to mice, scintigraphic images were acquired at 14 d postchallenge. T, thyroid; S, stomach; B, urinary bladder; CMNF, CT26/hMUC1-hNIS-Fluc. C, tumor growths were visualized by bioluminescent imaging at the designated times.

Close modal
Figure 3.

Quantification or measurement of tumor growth. A, to quantify light intensities, regions of interest were drawn over tumor regions and total photon effluxes were measured. n = 7 mice per group. *, P < 0.02 (pcDNA + PBS versus phMUC1 + PBS); **, P < 0.01 (pcDNA + 131I versus phMUC1 + 131I). B, tumor volumes were measured using a caliper. Tumor volumes were defined as length (mm) × width (mm). *, P < 0.02 (pcDNA + 131I versus phMUC1 + 131I); **, P < 0.02 (phMUC1 + 131I versus phMUC1 + PBS); ***, P < 0.02 (pcDNA + PBS versus phMUC1 + PBS). C, tumors were extracted from animals in the pcDNA + PBS and phMUC1 + PBS groups at 32 d postchallenge and weighted. *, P < 0.02, at 16 d post-tumor rechallenge of cured mice in the pcDNA + 131I and phMUC1 + 131I groups, tumor masses were extracted and weighted; *, P < 0.001. D, numbers of tumor-free mice before and after rechallenge are expressed as percentages. Experiments were done in triplicate. Columns, mean (n = 7 mice per group); bars, SD.

Figure 3.

Quantification or measurement of tumor growth. A, to quantify light intensities, regions of interest were drawn over tumor regions and total photon effluxes were measured. n = 7 mice per group. *, P < 0.02 (pcDNA + PBS versus phMUC1 + PBS); **, P < 0.01 (pcDNA + 131I versus phMUC1 + 131I). B, tumor volumes were measured using a caliper. Tumor volumes were defined as length (mm) × width (mm). *, P < 0.02 (pcDNA + 131I versus phMUC1 + 131I); **, P < 0.02 (phMUC1 + 131I versus phMUC1 + PBS); ***, P < 0.02 (pcDNA + PBS versus phMUC1 + PBS). C, tumors were extracted from animals in the pcDNA + PBS and phMUC1 + PBS groups at 32 d postchallenge and weighted. *, P < 0.02, at 16 d post-tumor rechallenge of cured mice in the pcDNA + 131I and phMUC1 + 131I groups, tumor masses were extracted and weighted; *, P < 0.001. D, numbers of tumor-free mice before and after rechallenge are expressed as percentages. Experiments were done in triplicate. Columns, mean (n = 7 mice per group); bars, SD.

Close modal

H&E analysis showed different degrees of tumor necrosis (%) in the pcDNA + 131I and phMUC1 + 131I groups (70% and 95%, respectively; Supplementary Fig. S1A)5

5

Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

at 32 days postchallenge. Bioluminescence imaging (Fig. 2C; 6 tumor-bearing mice in pcDNA + 131I group, 1 tumor-bearing mouse in phMUC1 + 131I group, and 7 mice per group) and total photon efflux findings were significantly different in the pcDNA + 131I and phMUC1 + 131I groups at this time (Fig. 3A; P < 0.01, pcDNA + 131I versus phMUC1 + 131I group). Seven 131I-treated mice were tumor free (one in the pcDNA + 131I group and six in the phMUC1 + 131I group), but no mouse was tumor free in the non-131I-treated groups (Fig. 3D).

Tumor Rechallenge Experiment

Aggressive tumor growth was observed in the pcDNA + 131I group but not in the phMUC1 + 131I group from 4 to 16 days after tumor rechallenge by bioluminescent imaging (Figs. 2C and 3A; pcDNA + 131I versus phMUC1 + 131I, P < 0.01). A significant difference in tumor weight was observed between pcDNA + 131I and phMUC1 + 131I groups (Fig. 3C; P < 0.001) at 16 days post-tumor rechallenge. We also found a difference between these two groups in terms of degrees of necrosis (pcDNA + 131I and phMUC1 + 131I, 60% and 95% to 100%, respectively; Supplementary Fig. S1B).5

hMUC1-Associated CD8+IFN-γ+ T cells and CTL Activity

The number of T cells expressing CD8+IFN-γ+ in the phMUC1 + 131I group was markedly higher than in the other three groups (pcDNA + PBS, phMUC1 + PBS, pcDNA + 131I, and phMUC1 + 131I groups, 53.5 ± 1.2, 91.7 ± 1.7, 676.5 ± 3.6, and 1278.7 ± 3.4, respectively, *, P < 0.02 and **, P < 0.01; Fig. 4A and B). As shown in Fig. 5A, the killing activities of CTL cells against hMUC1-expressing cancer was greatest in phMUC1 + 131I group among four therapy groups (pcDNA + PBS, phMUC1 + PBS, pcDNA + 131I, and phMUC1 + 131I groups, 16 ± 2%, 20 ± 1%, 30 ± 2%, and 60 ± 2%, respectively, *, P < 0.02 and **, P < 0.01).

Figure 4.

Levels of hMUC1-associated CD8+IFN-γ+ T-cell immune response in immunocompetent mice treated with phMUC1 + 131I. A, representative flow cytometry data. B,columns, numbers of IFN-γ secreting hMUC1-associated CD8+ T cells per 1 × 105 cells. Splenocytes were stained with PE-labeled IFN-γ and FITC-labeled CD8 antibody and analyzed by flow cytometry. *, P < 0.02; **, P < 0.01. Experiments were done in triplicate. Columns, mean (n = 8 mice per group); bars, SD.

Figure 4.

Levels of hMUC1-associated CD8+IFN-γ+ T-cell immune response in immunocompetent mice treated with phMUC1 + 131I. A, representative flow cytometry data. B,columns, numbers of IFN-γ secreting hMUC1-associated CD8+ T cells per 1 × 105 cells. Splenocytes were stained with PE-labeled IFN-γ and FITC-labeled CD8 antibody and analyzed by flow cytometry. *, P < 0.02; **, P < 0.01. Experiments were done in triplicate. Columns, mean (n = 8 mice per group); bars, SD.

Close modal
Figure 5.

Cytotoxic effects of T cells against hMUC1-expressing cancer cells generated by combination therapy (phMUC1 + 131I). A, splenocytes of treated mice were prepared and restimulated with interleukin-2 and hMUC1 peptides for 3 d. Irradiated CMNF cells (target cells) were then incubated with splenocytes (effectors) at an effector-to-target ratio of 12:1 for 4 h in 96-well plates. *, P < 0.02; **, P < 0.01. B, to investigate long-term cytotoxic T-cell activity, splenocytes of mice were prepared at 16 d post-tumor rechallenge and restimulated with interleukin-2 and hMUC1 peptides for 3 d. Irradiated CMNF cells (target cells) were incubated with splenocytes (effectors) at an effector-to-target ratio of 12:1 for 4 h in 96-well plates. Supernatants were collected and amounts of LDH were determined using LDH assays. *, P < 0.001. Experiments were done in triplicate. Columns, mean (n = 8 mice per group); bars, SD.

Figure 5.

Cytotoxic effects of T cells against hMUC1-expressing cancer cells generated by combination therapy (phMUC1 + 131I). A, splenocytes of treated mice were prepared and restimulated with interleukin-2 and hMUC1 peptides for 3 d. Irradiated CMNF cells (target cells) were then incubated with splenocytes (effectors) at an effector-to-target ratio of 12:1 for 4 h in 96-well plates. *, P < 0.02; **, P < 0.01. B, to investigate long-term cytotoxic T-cell activity, splenocytes of mice were prepared at 16 d post-tumor rechallenge and restimulated with interleukin-2 and hMUC1 peptides for 3 d. Irradiated CMNF cells (target cells) were incubated with splenocytes (effectors) at an effector-to-target ratio of 12:1 for 4 h in 96-well plates. Supernatants were collected and amounts of LDH were determined using LDH assays. *, P < 0.001. Experiments were done in triplicate. Columns, mean (n = 8 mice per group); bars, SD.

Close modal

Numbers of CD8+IFN-γ+-Expressing T Cells Generated by hMUC1-Loaded CD11c+ Cells

As shown in Fig. 6, enriched CD11c+ cells from the phMUC1 + 131I group most effectively generated CD8+IFN-γ-expressing T cells among four therapy groups (pcDNA + PBS, phMUC1 + PBS, pcDNA + 131I, and phMUC1 + 131I groups, 7 ± 3, 31 ± 9, 86 ± 19, and 139 ± 15, respectively, *, P < 0.02 and **, P < 0.01).

Figure 6.

Levels of hMUC1-associated CD8+IFN-γ+ T cells stimulated by hMUC1 antigen-loaded dendritic cells in the draining lymph nodes of mice treated with combination therapy. A, representative flow cytometry data. B,columns, numbers of IFN-γ-secreting hMUC1-associated CD8+ T cells per 5 × 104 cells. Restimulated cells were stained with PE-labeled IFN-γ and FITC-labeled CD8 antibody and analyzed by flow cytometry. *, P < 0.02; **, P < 0.01. Experiments were done in triplicate. Columns, mean; bars, SD.

Figure 6.

Levels of hMUC1-associated CD8+IFN-γ+ T cells stimulated by hMUC1 antigen-loaded dendritic cells in the draining lymph nodes of mice treated with combination therapy. A, representative flow cytometry data. B,columns, numbers of IFN-γ-secreting hMUC1-associated CD8+ T cells per 5 × 104 cells. Restimulated cells were stained with PE-labeled IFN-γ and FITC-labeled CD8 antibody and analyzed by flow cytometry. *, P < 0.02; **, P < 0.01. Experiments were done in triplicate. Columns, mean; bars, SD.

Close modal

Long-term CTL Activity

The killing activity of CTLS against hMUC1-expressing cancer in the phMUC1 + 131I group was 12 times that in the pcDNA + 131I group (Fig. 5B; pcDNA + 131I and phMUC1 + 131I, 8 ± 2% and 96 ± 5%, respectively, P < 0.001).

Cancer vaccinations using cancer DNA, tumor-derived cell, or dendritic cell vaccines have been applied to preclinical/clinical disease models (2228). Of these, cancer DNA vaccines provide several advantages compared with the others: (a) DNA vaccines have the same effects as live attenuated vaccines in terms of their ability to induce CD8+ T cell response but reduce safety concerns associated with live vaccines (29, 30). (b) DNA vaccines can be manipulated in a relatively cost-effective manner and are easily stored (31). Several researchers have reported that DNA vaccines can induce protection from tumors in immunocompetent mice and increase mouse survival (23, 24, 3133). However, many researchers have not obtained satisfactory results in terms of long-term tumor protection and inhibition from recurrence in various cancer models due to the low immunogenicities of DNA vaccines or to the disruptions of regulator T cells or cytokine balances in the host (34, 35). Thus, we considered that cancer DNA vaccine immunotherapy may be augmented by other therapies.

Radionuclide therapy based on β-ray irradiation is a conventional cancer treatment. Current research is focusing on the use of radionuclide gene therapy to concentrate therapeutic radionuclides (131I, 188Re) in specific cancer cells (14, 21, 36). β-rays emitted from 131I travel 0.2 to 2.4 mm in tissue (37), which results in the death of cells near hNIS-expressing cancer cells by the crossfire effect.

Cancer cells escape the attack of cancer-specific immune cells generated by cancer DNA vaccine because of no expression of tumor antigen or MHC class molecules and hindrance of regulatory T cells, etc. Therefore, cancer researchers have investigated new combination therapy to overcome weak therapeutics effects of cancer DNA vaccine. Recently, it was reported that radiation therapy plus cancer DNA vaccine is a promising strategy. Local radiation is the standard treatment for care of various type of cancer and has ability to directly not only kill cancer cells but also induce radiation-induced immune response. Particularly, gene modification of cancer cell by radiation could provide cancer DNA vaccine the opportunity to overcome weak antitumor immunity. For example, Chakraborty et al. examined the phenotype marker and biological effects of irradiated cancer cell on killing activity of CD8+ T cells (7). Irradiated cancer cell highly expresses Fas receptor and intracellular adhesion molecule-1 in a dose-dependent manner. Also, radiation provides increased the killing activity of tumor-associated CTL against cancer cell. Other group reported on similar finding with human cancer cell line (12 colon, 7 lungs, and 4 prostate). It was found that when those human cancer cell lines were irradiated with 10 Gy radiation, they observed the change of the surface gene expression (Fas, intracellular adhesion molecule-1, MUC1, CEA, and MHC class I) in human cancer (38).

Based on the recent fascinated reports, we have considered that hMUC1 vaccination plus hNIS radioiodine gene therapy may induce synergistic therapeutic effects similar with external local radiation in combination with cancer vaccine in established tumor model. Particularly, it was expected that hMUC1 vaccination plus hNIS radioiodine gene therapy could effectively inhibit tumor recurrence following complete tumor inhibition.

In this study, the therapeutic effects of a combined hMUC1 cancer DNA vaccine and hNIS radioiodine gene therapy were evaluated. Minor tumor growth inhibition was observed in the hMUC1 + PBS group, and slightly greater tumor growth inhibition was observed in the pcDNA3.1 + 131I group by bioluminescence imaging and according to caliper measurements. The weak therapeutic effect of DNA vaccines has been substantially reviewed (31), and the inadequacy of radionuclide NIS gene single therapy has been well reported (1719), which suggests that monotherapies based on these modalities are probably insufficient to induce complete remission.

Theoretically, combinatorial cancer vaccine and radiotherapy may have a synergistic antitumor effect. Radiation would induce cell death through apoptosis or necrosis and these destroyed cells and their byproducts provide antigens that stimulate immune-related cells. Moreover, the radiation-induced tumor microenvironment could give various immune-related cells the opportunity to home in on affected tissues (8, 39). In the present study, marked tumor growth inhibition was observed in the phMUC1 + 131I group but not in the monotherapy groups (Figs. 2C and 3A). Moreover, at 36 days postchallenge, 6 of 7 mice were tumor free in the phMUC1 + 131I group.

This combination therapy generated the highest number of hMUC1-assoicated CD8+IFN-γ+ T cells (Fig. 4) and most enhanced the killing activity of cytotoxic T cells among four therapy groups (Fig. 5A). Moreover, this combination therapy was found to generate more hMUC1 antigen-loaded CD11c+ cells than the other treatments (Fig. 6). Based on these findings, we suggest that the synergistic therapeutic effects of phMUC1 + 131I therapy proceeds via the following mechanisms; there may have been many more hMUC1-loaded dendritic cells and hMUC1-associated effectors in the hMUC1 vaccination group than in the mock (pcDNA3.1) vaccination group. hMUC1-loaded dendritic cells could effectively recognize and cross-present hMUC1 antigenic peptides derived from apoptotic or necrotic cells induced by 131I to effectors in the phMUC1 + 131I group.

In our tumor rechallenge experiment, mice completely rejected tumor recurrence in combination therapy group (phMUC1 + 131I) but not in monotherapy group (pcDNA + 131I; Figs. 2C and 3A). hMUC1-associated CTL activity was also greater in the phMUC1 + 131I group than in the pcDNA + 131I group (Fig. 5B).

We already described the mechanism of the effects of therapy in tumor rechallenge study. Please refer to following description below.

This combination therapy generated the highest number of hMUC1-assoicated CD8+IFN-γ+ T cells (Fig. 4) and most enhanced the killing activity of cytotoxic T cells among four therapy groups (Fig. 5A). Moreover, this combination therapy was found to generate more hMUC1 antigen-loaded CD11c+ cells than the other treatments (Fig. 6). Based on these findings, we suggest that the synergistic therapeutic effects of phMUC1 + 131I therapy proceeds via the following mechanisms; there may have been many more hMUC1-loaded dendritic cells and hMUC1-associated effectors in the hMUC1 vaccination group than in the mock (pcDNA3.1) vaccination group. hMUC1-loaded dendritic cells could effectively recognize and cross-present hMUC1 antigenic peptides derived from apoptotic or necrotic cells induced by 131I to effectors in the phMUC1 + 131I group. Importantly, these results suggest that combination therapies have the potential to prevent cancer recurrence after complete remission.

Our findings indicate that hNIS radioiodine gene therapy could be improved by several modifications. Using targeted gene therapy under the control of a tissue-specific promoter, the hNIS gene could be expressed in target tumor cells only, which would maximize therapeutic effect in cancerous tissues and minimizing damage to normal tissues (16, 18, 19). Moreover, more powerful therapeutic radionuclides like Re-188 could be used, and cotransfection with the thyroid peroxidase gene offers another means of enhancing radionuclide retention in cancer cells (14, 40, 41).

Further study is required before the efficacy of combined hMUC1 vaccination and 131I therapy can be examined in a patients. First, we should determine the proper dose (minimum dose) of 131I to maximize the immune response and cytotoxic activity against cancer cells and to minimize the damage of normal organs. Second, other immune modulators should be examined to improve on the weak immune response shown to hMUC1. For example, it has been reported that when adjuvants, such as CpG motif and a cytokine (granulocyte macrophage colony-stimulating factor, Flt3 ligand, and macrophage inflammatory protein-1α), were added to combination therapy to attract or cause the proliferation of antigen-presenting cells, antigen-specific immune responses against cancer were enhanced (31).

In summary, the present study shows that combined hMUC1 vaccination and hNIS radioiodine gene therapy can synergistically enhance hMUC1-associated immune response against hMUC1-expressing cancer cells and effectively inhibit tumor growth in an immunocompetent mouse model. In addition, this combination therapy seems to prevent tumor recurrence after complete remission. Further more experiments should be followed to be applied in caner patients.

No potential conflicts of interest were disclosed.

Grant support: Cancer Research Center, Korean Science & Engineering Foundation through the Tumor Immunity Medical Research Center at Seoul National University College of Medicine; BK21 Project for Medicine, Dentistry, and Pharmacy 2007 (Y.H. Jeon and Y. Choi).

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

Note: Y.H. Jeon and Y. Choi contributed equally to this study.

1
Watters D. Molecular mechanisms of ionizing radiation-induced apoptosis.
Immunol Cell Biol
1999
;
77
:
263
–71.
2
Chakravarty PK, Alfieri A, Thomas EK, et al. Flt3-ligand administration after radiation therapy prolongs survival in a murine model of metastatic lung cancer.
Cancer Res
1999
;
59
:
6028
–32.
3
Gulley JL, Arlen PM, Bastian A, et al. Combining a recombinant cancer vaccine with standard definitive radiotherapy in patients with localized prostate cancer.
Clin Cancer Res
2005
;
11
:
3353
–62.
4
Nikitina EY, Gabrilovich DI. Combination of γ-irradiation and dendritic cell administration induces a potent antitumor response in tumor-bearing mice: approach to treatment of advanced stage cancer.
Int J Cancer
2001
;
94
:
825
–33.
5
Klein B, Loven D, Lurie H, et al. The effect of irradiation on expression of HLA class I antigens in human brain tumors in culture.
J Neurosurg
1994
;
80
:
1074
–7.
6
Sheard MA, Vojtesek B, Janakova L, Kovarik J, Zaloudik J. Up-regulation of Fas (CD95) in human p53wild-type cancer cells treated with ionizing radiation.
Int J Cancer
1997
;
73
:
757
–62.
7
Chakraborty M, Abrams SI, Camphausen K, et al. Irradiation of tumor cells up-regulates Fas and enhances CTL lytic activity and CTL adoptive immunotherapy.
J Immunol
2003
;
170
:
6338
–47.
8
Demaria S, Bhardwaj N, McBride WH, Formenti SC. Combining radiotherapy and immunotherapy: a revived partnership.
Int J Radiat Oncol Biol Phys
2005
;
63
:
655
–66.
9
Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, et al. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin.
J Biol Chem
1990
;
265
:
15286
–93.
10
Gendler S, Taylor-Papadimitriou J, Duhig T, Rothbard J, Burchell J. A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats.
J Biol Chem
1988
;
263
:
12820
–3.
11
Mushenkova N, Moiseeva E, Chaadaeva A, Den Otter W, Svirshchevskaya E. Antitumor effect of double immunization of mice with mucin 1 and its coding DNA.
Anticancer Res
2005
;
25
:
3893
–8.
12
Snyder LA, Goletz TJ, Gunn GR, et al. A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice.
Vaccine
2006
;
24
:
3340
–52.
13
Kontani K, Taguchi O, Ozaki Y, et al. Novel vaccination protocol consisting of injecting MUC1 DNA and nonprimed dendritic cells at the same region greatly enhanced MUC1-specific antitumor immunity in a murine model.
Cancer Gene Ther
2002
;
9
:
330
–7.
14
Chung JK. Sodium iodide symporter: its role in nuclear medicine.
J Nucleic Med
2002
;
43
:
1188
–200.
15
De La Vieja A, Dohan O, Levy O, Carrasco N. Molecular analysis of the sodium/iodide symporter: impact on thyroid and extrathyroid pathophysiology.
Physiol Rev
2000
;
80
:
1083
–105.
16
Spitzweg C, O'Connor MK, Bergert ER, Tindall DJ, Young CY, Morris JC. Treatment of prostate cancer by radioiodine therapy after tissue-specific expression of the sodium iodide symporter.
Cancer Res
2000
;
60
:
6526
–30.
17
Spitzweg C, Dietz AB, O'Connor MK, et al. In vivo sodium iodide symporter gene therapy of prostate cancer.
Gene Ther
2001
;
8
:
1524
–31.
18
Chen L, Altmann A, Mier W, et al. Radioiodine therapy of hepatoma using targeted transfer of the human sodium/iodide symporter gene.
J Nucleic Med
2006
;
47
:
854
–62.
19
Scholz IV, Cengic N, Baker CH, et al. Radioiodine therapy of colon cancer following tissue-specific sodium iodide symporter gene transfer.
Gene Ther
2005
;
12
:
272
–80.
20
Jeon YH, Choi Y, Kim HJ, et al. Human sodium iodide symporter gene adjunctive radiotherapy to enhance the preventive effect of hMUC1 DNA vaccine.
Int J Cancer
2007
;
121
:
1593
–9.
21
Mandell RB, Mandell LZ, Link CJ, Jr. Radioisotope concentrator gene therapy using the sodium/iodide symporter gene.
Cancer Res
1999
;
59
:
661
–8.
22
Tobery TW, Siliciano RF. Targeting of HIV-1 antigens for rapid intracellular degradation enhances cytotoxic T lymphocyte (CTL) recognition and the induction of de novo CTL responses in vivo after immunization.
J Exp Med
1997
;
185
:
909
–20.
23
Boyle JS, Brady JL, Lew AM. Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction.
Nature
1998
;
392
:
408
–11.
24
King CA, Spellerberg MB, Zhu D, et al. DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma.
Nat Med
1998
;
4
:
1281
–6.
25
Livingston PO, Kaelin K, Pinsky CM, Oettgen HF, Old LJ. The serologic response of patients with stage II melanoma to allogeneic melanoma cell vaccines.
Cancer
1985
;
56
:
2194
–200.
26
Berd D, Maguire HC, Jr., McCue P, Mastrangelo MJ. Treatment of metastatic melanoma with an autologous tumor-cell vaccine: clinical and immunologic results in 64 patients.
J Clin Oncol
1990
;
8
:
1858
–67.
27
Mayordomo JI, Zorina T, Storkus WJ, et al. Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.
Nat Med
1995
;
1
:
1297
–302.
28
Paglia P, Chiodoni C, Rodolfo M, Colombo MP. Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vivo.
J Exp Med
1996
;
183
:
317
–22.
29
Ulmer JB, Fu TM, Deck RR, et al. Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA.
J Virol
1998
;
72
:
5648
–53.
30
Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein.
Science
1993
;
259
:
1745
–9.
31
Gurunathan S, Klinman DM, Seder RA. DNA vaccines: immunology, application, and optimization.
Annu Rev Immunol
2000
;
18
:
927
–74.
32
Kim TW, Hung CF, Ling M, et al. Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins.
J Clin Invest
2003
;
112
:
109
–17.
33
Kim TW, Hung CF, Juang J, He L, Hardwick JM, Wu TC. Enhancement of suicidal DNA vaccine potency by delaying suicidal DNA-induced cell death.
Gene Ther
2004
;
11
:
336
–42.
34
Rosenberg SA, Yang JC, Sherry RM, et al. Inability to immunize patients with metastatic melanoma using plasmid DNA encoding the gp100 melanoma-melanocyte antigen.
Hum Gene Ther
2003
;
14
:
709
–14.
35
Wang R, Doolan DL, Le TP, et al. Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine.
Science
1998
;
282
:
476
–80.
36
Spitzweg C, Harrington KJ, Pinke LA, Vile RG, Morris JC. Clinical review 132: the sodium iodide symporter and its potential role in cancer therapy.
J Clin Endocrinol Metab
2001
;
86
:
3327
–35.
37
Vallabhajosula S. Radiopharmaceuticals in oncology. In: Khalkhali IMJ, Goldsmith SJ, editors. Nuclear oncology: diagnosis and therapy. Philadelphia (PA): Lippincott, Williams & Wilkins; 2001. p. 31–62.
38
Garnett CT, Palena C, Chakraborty M, Tsang KY, Schlom J, Hodge JW. Sublethal irradiation of human tumor cells modulates phenotype resulting in enhanced killing by cytotoxic T lymphocytes.
Cancer Res
2004
;
64
:
7985
–94.
39
Larsson M, Fonteneau JF, Bhardwaj N. Dendritic cells resurrect antigens from dead cells.
Trends Immunol
2001
;
22
:
141
–8.
40
Kang JH, Chung JK, Lee YJ, et al. Establishment of a human hepatocellular carcinoma cell line highly expressing sodium iodide symporter for radionuclide gene therapy.
J Nucleic Med
2004
;
45
:
1571
–6.
41
Lee YJ, Chung JK, Shin JH, et al. In vitro and in vivo properties of a human anaplastic thyroid carcinoma cell line transfected with the sodium iodide symporter gene.
Thyroid
2004
;
14
:
889
–95.

Supplementary data