Gene Therapy of Intracranial Glioma Using Interleukin 12–Secreting Human Umbilical Cord Blood

Abstract

Clinical trials of gene therapy using a viral delivery system for glioma have been limited. Recently, gene therapy using stem cells as the vehicles for delivery of therapeutic agents has emerged as a new treatment strategy for malignant brain tumors. In this study, we used human umbilical cord blood–derived mesenchymal stem cells (UCB-MSCs) as delivery vehicles with glioma-targeting capabilities, and modified interleukin-12 (IL-12p40N220Q; IL-12M) as a novel therapeutic gene. We also engineered UCB-MSCs to secret IL-12M (UCB-MSC-IL12M) via tetrameric cell-permeable peptide (4HP4)-mediated adenoviral transduction. We confirmed the migratory capacity of UCB-MSC-IL12M toward GL26 mouse glioma cells by an in vitro migration assay and in vivo injection of UCB-MSC-IL12M into the ipsilateral hemisphere of implanted gliomas in C57BL/6 mice. In vivo efficacy experiments showed that intratumoral injection of UCB-MSC-IL12M significantly inhibited tumor growth and prolonged the survival of glioma-bearing mice compared with control mice. Antitumor effects were associated with increased local IL-12M levels, followed by interferon-γ secretion and T-cell infiltration in intracranial gliomas, as well as antiangiogenesis. Interestingly, tumor-free mice after UCB-MSC-IL12M treatment were resistant to ipsilateral and contralateral tumor rechallenge, which was closely associated with tumor-specific long-term T-cell immunity. Thus, our results provide the rationale for designing novel experimental protocols to induce long-term antitumor immunity against intracranial gliomas using UCB-MSCs as an effective delivery vehicle for therapeutic cytokines including IL-12M.

Introduction

G lioblastomas are the most common and aggressive primary brain tumors in humans. Despite technical advances in neurosurgery, radiotherapy, and chemotherapy, the prognosis of most patients with glioblastomas remains very poor (Surawicz et al., 1998; Davis et al., 1999; Legler et al., 1999).

A new approach for targeting tumor cells within the central nervous system (CNS) uses stem cells to efficiently deliver therapeutic genes and their products to brain tumors (Hamada et al., 2005; Lawler et al., 2006; Yip et al., 2006). Neural stem cells (NSCs) possess extensive tropism for experimental gliomas and migrate together with outgrowing tumor microsatellites after intratumoral implantation (Aboody et al., 2000). However, the clinical application of NSCs is limited by ethical concerns associated with their isolation, expansion, and immune response problems. Mesenchymal stem cells (MSCs) have been proposed as an alternative source of NSCs for regeneration and have been shown to also be able to migrate away from the injection site toward tumor beds (Studeny et al., 2002; Phinney and Isakova, 2003; Nakamura et al., 2004). Furthermore, MSCs are easier to obtain and propagate in vivo, and fewer ethical concerns are associated with their use. MSC types differ depending on their source, such as bone marrow (BM), umbilical cord blood (UCB), and so on (Ho et al., 2008). UCB-derived MSCs (UCB-MSCs) are similar to stem cells from BM with respect to cell characteristics and multilineage differentiation potential (Bieback et al., 2004; Gang et al., 2004; Lee et al., 2004). In previous studies, we demonstrated that UCB-MSCs have a strong migratory capacity toward human glioma cells, and that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-secreting UCB-MSCs also have tropism for human gliomas and inhibit glioma growth after intratumoral injection (Kim et al., 2008, 2009). Moreover, UCB-MSCs have clinical advantages because of the immaturity of newborn cells compared with adult cells, large ex vivo expansion capacity, low risk of viral infection, lack of donor attrition, and less pronounced immune response (Kim et al., 2008). Thus, these advantages may result in the replacement of other adult stem cells with UCB-MSCs as the vehicle of choice in gene therapy against glioma.

The brain is traditionally regarded as an immunologically privileged organ site because it possesses a distinct blood–brain barrier and lacks discrete lymphatic structures. Thus, glioma cells derived from brain parenchyma may be more immunogenic than tumors derived from peripheral tissues, indicating that immunogene therapy may be a very effective method for treating malignant glioma (Kanzawa et al., 2003).

Interleukin (IL)-12 is a heterodimeric proinflammatory cytokine, secreted primarily from antigen-presenting cells. This cytokine has very strong antitumor and antiangiogenic properties (Trinchieri et al., 2003a). The IL-12 gene, which consists of p40 and p35 subunits, expresses both IL-12p70 heterodimer and free p40 homodimer. IL-12p70 is essential for the induction of T-helper 1 (Th1) and cytotoxic T-cell (CTL) immunity. IL-12p40 acts as a natural antagonist to block the binding of IL-12 to its receptor and thus inhibits IL-12–mediated biological activities. Interestingly, a mutation of the N-glycosylation site in IL-12p40 (IL-12M) reduced secretion of p40 but not p70 (Ha et al., 2002). Therefore, gene therapy using IL-12M was shown to induce and maintain stronger T-cell immune responses against tumors compared with native IL-12 (Jin et al., 2005).

Recently, NSCs and BM-derived MSCs (BM-MSCs) have been used to deliver the IL-12 gene to gliomas in vivo with encouraging results (Ehtesham et al., 2002; Hong et al., 2009). In these studies, glioma-bearing mice treated with IL-12–secreting stem cells had significantly prolonged survival and resulted in antitumor immunity, and transplanted stem cells demonstrated strong tropism for infiltrated glioma. In this study, we first examined whether UCB-MSCs could be used as a novel vehicle for delivering cytokine genes, such as IL-12M, to tumors and evaluated the antitumor activity and long-term protective immunity of IL-12M–expressing UCB-MSCs in intracranial gliomas.

Materials and Methods

Cell cultures and cell lines

Human UCB harvest and expansion of MSCs isolated from UCB was conducted as previously reported (Yang et al., 2004a). Separated MSCs were subcultured at a concentration of 5 × 10⁴ cells/cm² in MEM-α (Invitrogen) and used for experiments during passages 5 to 8. GL26 cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (Invitrogen). All media were supplemented with 2 mmol/l l-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum purchased from Invitrogen. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

Adenoviral vectors and peptide

The recombinant replication-deficient adenoviral vector encoding the gene for EGFP (Ad-EGFP) was constructed and produced using the Ad-Easy vector system, following the manufacturer's instructions (Quantum Biotechnologies). Adenovirus carrying the mouse IL12M gene (Ad-IL12M) was engineered and then transfected as described previously (Jin et al., 2005). Tetrameric cell-permeable peptide (4HP4), which has been reported to enhance rAd delivery into MSCs (Youn et al., 2008; Park et al., 2010), was synthesized by Peptron (www.peptron.com).

Animals and intracranial glioma model

C57Bl/6 mice (6–8 weeks old; Harlan Sprague Dawley) were used in accordance with institutional guidelines under the approved protocols. For the intracranial implantation of syngeneic GL26 tumor cells in the brain of C57BL/6 mice, animals were stereotactically inoculated with 1 × 10⁵ GL26 cells (in 3 μl phosphate-buffered saline [PBS]) into the right frontal lobe (2 mm lateral and 1 mm anterior to bregma, at 2.5 mm depth from the skull base) using a Hamilton syringe (Hamilton Company) and a microinfusion pump (Harvard Apparatus).

In vitro and in vivo migration assay

In vitro and in vivo migration assays were performed as previously reported (Kim et al., 2008). In brief, three different concentrations (1 × 10⁵, 5 × 10⁵, and 1 × 10⁶) of cells were incubated in serum-free medium (SFM) for 48 hr and the resulting conditioned media (CM) were used as chemoattractants. UCB-MSCs or UCB-MSC-IL12M (2 × 10⁴ cells) were suspended in SFM and seeded into the upper well, and 600 μl of CM was placed in the lower well of the Transwell plate. Following incubation for 5 hr at 37°C, cells that had not migrated from the upper side of the filters were scraped off with a cotton swab, and filters were stained with the Three-Step Stain Set (Diff-Quik; Sysmex). The number of cells that migrated to the lower side of the filter was counted under a light microscope with five high-power fields (×100). For the in vivo migration assay, UCB-MSC-IL12M (1 × 10⁵ cells) were implanted into the opposite hemisphere or the tumor mass 2 weeks after tumor cell inoculation. Migration toward the tumor was assessed by direct visualization at 7 days after UCB-MSCs inoculation using fluorescence microscopy.

Animal survival and tumor size evaluation

For survival experiments, intracranial glioma-bearing mice were randomly divided into three groups after tumor implantation and treated with intratumoral injections of saline (PBS), UCB-MSCs infected with Ad-GFP (UCB-MSC-GFP), or AdIL-12M (UCB-MSC-IL12M) in 3 μl of PBS. Tumor size was determined as described previously (Kim et al., 2008). Briefly, brains from mice given therapeutic treatment at a specific time point after tumor inoculation were serially sectioned (20 μm, obtained every 200 μm into the tumor) and then stained with hematoxylin and eosin (H&E). The section with the maximum tumor area was calculated via a computer using the MetaMorph software (Molecular Devices).

Expression analysis of IL-12M and interferon-γ by ELISA and immunohistochemistry

IL-12M protein secreted into brain tissues was quantified by ELISA, as described previously (Kim et al., 2008). For the analysis of IL-12M and interferon (IFN)-γ in tumor-bearing mice in vivo, tumor tissues were harvested and lysed in a RIPA buffer at 3, 6, 12, 18, 24, 30, and 40 days after treatment with UCB-MSC-IL12M. For immunohistochemistry, brain tissues were sectioned (14 μm) and then stained with primary antibody for IL-12 (R&D Systems). The primary antibody was conjugated with the Vectastain Elite ABC Kit (Vector Laboratories). Conjugated slides were developed with 2,4-diaminobutyric acid (Sigma-Aldrich) and counterstained with Harris hematoxylin (Sigma-Aldrich).

Detection of CD4 and CD8 T-lymphocyte infiltration by flow cytometry

For the analysis of T-cell infiltration into intracranial gliomas, mouse brains were harvested from GL26 glioma-bearing mice 1 week after intratumoral inoculations with PBS, UCB-MSC-GFP, or UCB-MSC-IL12M. Tumor-containing hemispheres from each group were dissociated into single cell suspensions by passing them through a Cell Strainer (40 μm; BD Biosciences). Cells were stained with fluorescein isothiocyanate–anti-mouse CD4 and allophycocyanin-anti-mouse CD8 (BD PharMingen). Fluorescence was detected by using a BD FACSCanto II and the percentage of positive cells was determined.

Evaluation of angiogenesis and apoptosis by immunohistochemistry

Slides from each group of mice were incubated at 4°C for 12 hr with polyclonal antibody for von Willebrand factor (vWF). Sections were then washed and incubated with rhodamine-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology).

Apoptotic cells were visualized using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit (Roche) developed using Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories). Briefly, endogenous peroxidase activity was blocked by 3% H₂O₂ for 10 min at a room temperature. After washing with PBS, 50 μl of TUNEL reaction mixture was pipetted onto the sections, which were then incubated in a humidified chamber at 37°C for 1 hr. The reaction was stopped by adding wash buffer. In all sections, nuclei were stained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich) for counterstaining. Vessel density, and apoptotic cells were also measured via a computer using the MetaMorph (Molecular Devices).

Animal rechallenge

For tumor rechallenge experiments, C57BL/6 mice treated with UCB-MSC-IL12M that survived for more than 60 days were rechallenged by ipsilateral or contralateral injection of 1 × 10⁵ GL26 cells. The injection site was proximal to (but not the same as) the initial injection site. Age-matched naïve mice were challenged as controls. After the rechallenge and challenge, animals were followed for survival. Some mice were sacrificed on day 7 after the challenge or rechallenge for H&E staining and FACS analysis. The tumor size and T-cell populations were also measured as described above.

Measurement of tumor-specific T-cell responses

To evaluate tumor-specific immune responses in the cured mice after the GL26 rechallenge, spleens were dissociated into a single cell suspension by passing through a Cell Strainer (40 μm; BD Bioscience) 16 weeks after the rechallenge. Age-matched naïve mice were used as controls (n = 4 in each group). Splenocytes were co-incubated with 150 μg/ml of GL26 tumor lysate prepared by three freeze–thaw cycles on an IFN-γ capture antibody (Ab)-coated (BD PharMingen) 96-well ELISPOT plate (Millipore). After subsequent incubation with biotinylated rat anti-mouse IFN-γ Ab (BD PharMingen), spots were counted using an AID ELISPOT Reader System (Autoimmune Diagnostika).

Statistical analysis

All data are expressed as means ± SEMs. Statistical differences between different test conditions were determined using Student's t test. Probability values less than 0.05 were considered significant. Statistical analysis of survival was performed by a log-rank test.

Results

Optimal condition of 4HP4 and adenovirus on transduction into UCB-MSCs

To determine the optimal transduction condition of 4HP4 and Ad-IL12M into UCB-MSCs, various concentrations of 4HP4 and multiplicities of infection (MOI) were tested for transduction of Ad-IL12M. The secreted IL-12 in UCB-MSCs was analyzed using ELISA, and adenovirus-induced cytotoxicity was determined by the MTS-based viability assay. Transduction of UCB-MSCs was increased by incorporation of 4HP4 (0.005–0.08 μmol) into the Ad-IL12M infection medium (Fig. 1A). However, cell viability under the same conditions was decreased by incorporation of 4HP4 (0.02–0.08 μmol) at 100 MOI (Fig. 1B). Based on these results, the optimal transduction condition for UCB-MSCs was determined at 0.01 μmol 4HP4 and 100 MOI, which achieved a high level of protein production without affecting cell viability. In addition, to investigate the stability of transduced UCB-MSCs in optimal conditions, the surface phenotype of UCB-MSCs was evaluated in the transduced or nontransduced cells by flow cytometry. Similar to nontransduced UCB-MSCs, IL12M-transduced UCB-MSCs were strongly positive for CD90, CD44, and CD73. However, these cells were negatively stained for HLA-class II (HLA-DR), CD14, CD45, CD51, and hematopoietic marker CD34 (data not shown). These results indicated that the characterization of UCB-MSCs was not affected by IL12M adenoviral transduction.

FIG. 1.

Optimal condition of adenovirus and tetrameric cell-permeable peptide (4HP4) for umbilical cord blood–derived mesenchymal stem cell (UCB-MSC) modification. The effect of 4HP4 on the transduction efficiency of UCB-MSCs infected with Ad-IL12M was evaluated. Cells were transduced at various multiplicities of infection (MOIs) with increasing concentrations of 4HP4. (A) At day 3 after infection, the concentration of secreted IL-12M in culture supernatant was analyzed by ELISA. (B) The viability of transduced cells was also analyzed via MTS assay. Columns and points, mean; bars, SE.

The effect of adenoviral transduction on migration of genetically modified UCB-MSCs in vitro and in vivo

In a previous paper, we showed that UCB-MSCs have migratory properties toward the human glioma cells and intracranial gliomas (Kim et al., 2008, 2009). To test this in genetically modified UCB-MSCs, in vitro migration assays using Transwell plates were conducted. Only a few cells migrated toward SFM, whereas the migration of UCB-MSCs and genetically modified UCB-MSCs were significantly stimulated by three different concentrations of CM from mouse glioma cells. Moreover, the migratory activity of UCB-MSCs occurred in a dose-dependent manner. We confirmed that unmodified UCB-MSCs migrated toward CM from mouse glioma cells in a similar pattern to genetically modified UCB-MSCs (Fig. 2A and B). We also investigated whether genetically modified UCB-MSCs migrated toward intracranial gliomas. UCB-MSC-IL12M inoculated into the ipsilateral hemisphere to the tumor migrated away from the initial injection site toward the tumor mass at 7 days after inoculation. These cells mostly infiltrated into the tumor bed (Fig. 2C, a). Interestingly, these cells were also seen in a satellite tumor at a distance from the main tumor mass (Fig. 2C, b). We also confirmed that the non- or GFP-transduced UCB-MSCs migrated towards intracranial gliomas in a similar pattern to genetically modified UCB-MSCs (data not shown). These results suggest that genetically modified UCB-MSCs have a strong migratory capacity toward gliomas and that the migratory ability of UCB-MSCs is not affected by adenoviral transduction.

FIG. 2.

The effect of adenoviral transduction on migration of UCB-MSC-IL12M in vitro and in vivo. (A) The migration of UCB-MSCs and genetically modified UCB-MSCs in response to three different concentrations of conditioned media (CM) from glioma cell line (GL26) was determined. Representative photomicrographs of stained filters show migrated UCB-MSCs. Magnification, ×100. (B) Cell migration was evaluated after staining by taking photographs and using a light microscope to count cells that had migrated. Serum-free medium (SFM) was used as a negative control (horizontal line). Columns, mean; bars, SE. (C) PKH26-labeled UCB-MSC-IL12M cells (red) were implanted into the tumor mass of intracranial gliomas. At day 7 after implantation, these cells were distributed throughout the tumor mass (a), and also seen in a tumor satellite (b). T, primary tumor; t, tumor satellite; dotted line, tumor edge; Magnification, ×200. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue) for counterstaining. Color images available online at www.liebertonline.com/hum.

Effects of UCB-MSC-IL12M on tumor growth and survival of glioma-bearing mice

To determine whether the UCB-MSC-IL12M showed antitumor effects on gliomas, tumor sizes in UCB-MSC-IL12M–treated mice were verified by histological staining from glioma-bearing mice (Fig. 3A). The average tumor areas in UCB-MSC-IL12M–treated animals were decreased compared with PBS- or MSC-treated animals. This decrease in tumor size associated with UCB-MSC-IL12M treatment was significant at day 20 (p = 0.042, UCB-MSC-IL12M vs. UCB-MSC-GFP) and at day 40 (p = 0.007, UCB-MSC-IL12M vs. UCB-MSC-GFP) (Fig. 3B). There was no detectable difference in tumor size between animals treated with MSC-GFP and PBS. These results show that UCB-MSC-IL12M reduced the rate of tumor growth in glioma-bearing mice. Next, the survival of UCB-MSC-IL12M–treated mice was significantly prolonged compared with control mice treated with PBS or UCB-MSC-GFP (p < 0.01) (Fig. 3C). It is worth noting that 70% of UCB-MSC-IL12M–treated mice survived more than 90 days after tumor inoculation, whereas in our previous report all TRAIL-secreting UCB-MSC–treated mice died within 30 days (Kim et al., 2008), suggesting that IL12M-mediated immunotherapy of glioma is more potent than TRAIL-mediated direct tumor killing strategy.

FIG. 3.

In vivo effects of UCB-MSCs IL12M on tumor growth and survival of glioma-bearing mice. UCB-MSC-GFP or IL12M (1 × 10⁵ cells) were given intratumorally to glioma-bearing mice at day 14 after GL26 (1 × 10⁵ cells) inoculation. The phosphate-buffered saline (PBS) group was used as a control. (A) Representative photographs of hematoxylin and eosin (H&E) staining from each group show tumor growth. Magnification, ×1. (B) Tumor size was determined by histological analysis at 20 or 40 days after tumor inoculation (n = 3/treatment group). Columns, mean; bars, SE. *p < 0.05; **p < 0.01, Student's t test. (C) Survival curve of intracranial glioma–bearing mice. At day 14 after GL26 inoculation, tumors were injected intratumorally with a single dose (1 × 10⁵ cells) of UCB MSC-EGFP (n = 9) or UCB-MSC-IL12M (n = 9) and PBS (n = 9). Analysis of survival was conducted by a log-rank test based on the Kaplan–Meier method. The results are representative of two independent experiments. Color images available online at www.liebertonline.com/hum

In vivo transgene expression and antitumor immunity of transplanted UCB-MSC-IL12M

To confirm whether transplanted UCB-MSC-IL12M survived in vivo for a sufficient length of time to allow a therapeutic effect to occur, we measured the levels of IL-12M protein and its longevity in intracranial gliomas. ELISA analysis on different days after UCB-MSC-IL12M treatment revealed that the IL-12M protein expressed in tumor tissues peaked on day 3, began to decrease after day 6, and persisted for 2 weeks (Fig. 4A, a). The IL-12M protein was not detected in mouse serum throughout the experimental period (Fig. 4A, b). IFN-γ plays an important role in the activation of T-cells, which are induced by IL-12. IFN-γ in tumor tissues from brain was detected from day 3 and reached maximum levels 6 days after UCB-MSC-IL12M treatment (Fig. 4A, c). Additionally, IL-12M was detected by immunohistochemistry at 7 days after treatment, and transplanted UCB-MSC-IL12M only observed within inoculated tumors and on the border of tumors in the UCB-MSC-IL12M treatment group (Fig. 4B). To determine whether the antitumor effects of UCB-MSC-IL12M treatment on survival and tumor growth were accompanied by T-cell infiltration, brain cells isolated from glioma-bearing mice were analyzed by flow cytometry at 7 days after treatment. The population of CD4 and CD8 T cells, as well as total live cells, in UCB-MSC-IL12M–treated mice was increased compared with controls treated with PBS or UCB-MSC-EGFP (Fig. 4C). There were no statistically significant differences in T-cell populations among animals treated with UCB-MSC-EGFP or PBS (p > 0.05). The mean value for CD4⁺ and CD8⁺ T-cell populations was 15.7 ± 2.45% and 13.8 ± 2.04%, respectively. Taken together, these results suggest that IL12M secreted from UCB-MSC-IL12M could effectively induce infiltration of CD4 and CD8 T cells into tumor sites, which might play an important role in the antitumor immunity of transplanted UCB-MSC-IL12M.

FIG. 4.

In vivo transgene expression and antitumor immunity of transplanted UCB-MSC-IL12M in gliomas. (A) At day 14 after GL26 (1 × 10⁵ cells) inoculation, UCB-MSC-GFP or IL12M (1 × 10⁵ cells) were treated intratumorally. (a) For the quantification of secreted IL-12M and longevity of IL-12M expression in tumor tissues, brain tissues were homogenized at 3, 6, 12, 18, 24, 30, and 40 days (n = 3/each group) after treatment and then assessed by ELISA. (b) Serum IL-12M was also measured at the same time. (c) For the longevity of IFN-γ induction by IL-12M in tumor tissues, brain tissues were also homogenized at the same time (n = 3/each group) and then assessed by ELISA. Control, UCB-MSC-EGFP–treated brain tissues; Columns, mean; bars, SE. (B) Brain tissues were removed from C57BL/6 mice at 7 days after transplantation of UCB-MSC-GFP or UCB-MSC-IL12M. The PBS group was used as a control. Cryosections were stained with antibody for IL-12. Magnification, ×100. (C) For the detection of infiltrated T cells in tumor tissues, brain tissues were dissociated into a single cell suspension at 7 days (n = 3 per group) after treatment. The populations of CD4 and CD8 T cells in brain tissues were analyzed by flow cytometry. Representative plots are shown with respective percentages of CD4 or CD8 T cells within total live cells. Columns, mean; bars, SE. *p < 0.05; **p < 0.01, Student's t test. The Results are representative of three independent experiments.

Antiangiogenic activity and apoptosis induction of transplanted UCB-MSC-IL12M in intracranial glioma

It has been reported that IL-12 inhibits growth in experimental tumors in vivo and exerts anti-angiogenic effects thought to be mediated by IFN-γ, which induces apoptosis, and later by hypoxia-induced apoptosis (Sgadari et al., 1996; Gee et al., 1999). To determine whether the antiangiogenic effects were involved in the antitumor activity of transplanted UCB-MSC-IL12M, tissue sections from glioma-bearing mice were analyzed by immunocytochemistry at 7 days after treatment. Vessels in brain tumors were identified based on the endothelial cell staining for vWF. The tumor vessels in UCB-MSC-IL12M–treated mice were significantly decreased compared with PBS- or MSC-treated mice (Fig. 5A). TUNEL staining also demonstrated a significant increase in the number of apoptotic cells in the group treated with UCB-MSC-IL12 compared with controls treated with PBS or UCB-MSC-GFP (Fig. 5B). These results demonstrate that the antitumor activity of transplanted UCB-MSC-IL12M is mediated by antiangiogenesis and apoptosis.

FIG. 5.

Antiangiogenic and apoptotic effects of UCB-MSCs IL12M in gliomas. Brain tissues were removed from glioma-bearing mice at 7 days after transplantation of UCB-MSC-GFP or UCB-MSC-IL12M (2 × 10⁵ cells). The PBS group was used as a control. (A) For the detection of tumor vessels in brain tissues, cryosections from each group were stained with antibody for von Willebrand factor (vWF). Nuclei were stained with DAPI (blue) for counterstaining. Magnification, ×100. The vWF staining intensity (vessel density) was also quantified by computerized image analysis. Columns, mean; bars, SE. (B) Apoptotic cells were also detected by TUNEL staining in cryosections and quantified by the above method. TUNEL-positive nuclei (red) were stained and counterstaining was conducted with DAPI (blue). Magnification, ×200. T, tumor mass; N, normal tissue. Columns, mean; bars, SE. *p < 0.05; **p < 0.01, Student's t test. The results are representative of three independent experiments.

Induction of tumor-specific long-term memory by UCB-MSC-IL12M

One of the beneficial characteristics of T-cell–mediated immunity is the development of an effective memory response (Sprent and Surh, 2002; Sallusto et al., 2004). To determine whether a tumor-specific memory response was established in animals that survived after UCB-MSC-IL12M treatment of intracranial glioma, the surviving mice were rechallenged with GL26 glioma cells in the ipsilateral or contralateral side of brain. All of the rechallenged mice survived beyond 90 days after rechallenge, whereas age-matched naïve mice died of brain tumors within 35 days of challenge (Fig. 6A). The tumor growth of challenged or rechallenged mice was assessed by measuring tumor size at 1 week. No tumor cells were observed in the injection sites of tumor cells in rechallenged mice (Fig. 6B). These results indicate that IL12M-treated tumor-free mice were resistant to ipsilateral or contralateral tumor rechallenge, presumably due to the establishment of tumor-specific immunological memory. To examine whether complete protection from tumor rechallenge was associated with intratumoral T-cell infiltration and effector function, brain cells isolated from glioma-bearing mice were analyzed by flow cytometry at 1 week after tumor inoculation. The population of CD4 and CD8 T cells in brains of rechallenged mice were significantly increased compared with those of control mice challenged with GL26 for the first time (Fig. 6C). The mean value for CD4 and CD8 T-cell populations of rechallenged mice was 23.7 ± 3.3% and 8.8 ± 0.7%, respectively. When the tumor-specific T-cell response was investigated by ELISPOT assay using splenocytes, about 18-fold higher GL26-specific IFN-γ–secreting T cells were detected in the cured mice compared to age-matched naïve mice, indicating that the cured mice transplanted with UCB-MSC-IL12M before 6 months had well-established tumor-specific T-cell memory (Fig. 6D). Thus, these results suggest that UCB-MSC-IL12M treatment of intracranial glioma induces tumor-specific memory T cells and has a long-term antitumor effect. This also implies that this therapeutic strategy could elicit protection for glioma patients from cancer relapse or metastasis, as well as eliminate the primary tumor.

FIG. 6.

Induction of tumor-specific long-term immunity by UCB-MSC-IL12M in gliomas. Naïve or cured mice (long-term survivors) were inoculated with GL26 (1 × 10⁵ cells) in the ipsilateral or contralateral hemisphere at day 50 after treatment. (A) Survival curve of challenged (n = 7) or rechallenged mice (n = 7). Analysis of survival was conducted by a log-rank test based on the Kaplan–Meier method. (B) Mice were sacrificed at 7 days after tumor inoculation, and the brains were harvested for H&E staining. Representative photographs of H&E staining from each group show tumor growth. Magnification, ×1. (C) Brain tissues were dissociated into a single cell suspension (n = 3/each group) at 7 days after tumor inoculation. Cell population of CD4 and CD8 T cells in brain tissues were analyzed by flow cytometry. (D) To investigate tumor-specific immune response in rechallenged mice, spleens were dissociated into a single cell suspension (n = 4/each group) at 16 weeks after tumor inoculation. Splenocytes were stimulated by GL26 tumor lysate and the number of GL26-specific IFN-γ producing cells were determined by ELISPOT assay. Age-matched naïve mice were used as controls. Columns, mean; bars, SE. *, p < 0.05; **, p < 0.01, Student's t test. The results are representative of two independent experiments. Color images available online at www.liebertonline.com/hum

Discussion

In this study, we optimized transduction condition of UCB-MSCs, which are known to lack the Coxsackie B-Ad receptor (CAR) (Conget and Minguell, 2000), using recombinant adenovirus and 4HP4. In addition, recombinant adenovirus expressing the novel therapeutic gene, IL12M, which induces more potent Th1 and antitumor immunity than wild-type IL12 (Ha et al., 2002), was used to produce UCB-MSC-IL12M for glioma therapy. Taken together, we have prepared UCB-MSCs that optimally secrete a potent anticancer cytokine without any adverse effects on viability and migration capacity, resulting in prolonged survival of glioma-bearing mice by inducing tumor-specific T-cell memory.

We used xenogenic UCB-MSCs for evaluating migratory ability and therapeutic efficacy in a mouse glioma model. Recently, many studies have examined the therapeutic effects of MSCs and genetically modified MSCs in xenotransplantation without immune suppressor (Li et al., 2002; Djouad et al., 2003; Nomura et al., 2005). In Fig. 4C, the population of CD4 and CD8 T cells, as well as total live cells, was slightly increased in UCB-MSC-GFP–treated mice. However, there were no statistically significant differences in T-cell populations among animals treated with UCB-MSC-EGFP or PBS (p > 0.05). Similarly, there were no detectable differences in tumor growth and survival between animals treated with MSC-GFP and PBS (Fig. 3). Therefore, UCB-MSCs, which have no immune rejection response, can be used as a vehicle in stem cell–based therapy.

IL-12 is a potent cytokine that acts against tumors; however, its administration has been associated with severe toxicity in mice and humans (Leonard et al., 1997). It has been reported that systemic expression of IL-12 causes acidosis, hepatitis, renal failure, and death in clinical trial (Cohen, 1995). Furthermore, local delivery of the recombinant IL-12 protein does not yield systemic toxicity and appears to be effective at inhibiting tumors (Tare et al., 1995). Here, IL-12 was not observed in serum after administration of UCB-MSC-IL12M, although it was detected in local brain tumors. This implies that intratumoral administration of UCB-MSC-IL12M leads to high local IL-12M concentrations without systemic toxicity.

Recently, stem cells were used as a form of immunotherapy to elicit an immune response against tumor cells by locally secreting IL-12 (Yang et al., 2004b). One study reported that treatment of mice bearing intracranial gliomas with NSCs secreting IL-12 experienced increased survival. Over 30% of the mice treated with IL-12-secreting NSCs had a 60-day survival rate, whereas untreated mice did not survive, even for 30 days (Ehtesham et al., 2002). Under similar conditions, over 70% of UCB-MSC-IL12M–treated mice experienced prolonged survival of 100 days compared with controls treated with PBS or UCB-MSC-GFP. Although survival analysis conditions were not exactly the same, UCB-MSC-IL12M treatment may provide better therapeutic and prolonged efficacy than NSC-IL12 treatment.

Several reports have described that infiltration of natural killer (NK) and T cells, which are known to play a crucial role in killing tumor cells, into gliomas is enhanced by IL-12 therapy (Colombo and Trinchieri, 2002; Trinchieri et al., 2003b). Consistent with previous reports, CD4 and CD8 T-cell populations in tumor sites were significantly increased by UCB-MSC-IL12M therapy. However, the number of NK cells, which are key effector cell types in IL-12–mediated anticancer therapy (Klein and Mantovani, 1993; Del Vecchio et al., 2007), was not altered at 1 week after UCB-MSC-IL12M therapy in our experiment (data not shown). This discrepancy might be caused by differences in assay time point and high background level of NK cells induced by the tumor challenge itself. Although the CNS is classically regarded as an immunologically privileged site, several studies showed that the tumor or CNS-related antigens could be recognized and presented to immune cells and activated lymphocytes migrated into the CNS through cervical lymph nodes and the blood–brain barrier, implying that brain tumors are possibly subjected to cytokine-mediated immunotherapy (Weller et al., 1996; Matyszak et al., 1998). In the present study, we directly elucidated that GL26 tumor-specific T-cell responses were highly induced in cured mice even 16 weeks after rechallenge, demonstrating that UCB-MSC-IL12M treatment in intracranial glioma could elicit strong T-cell memory response against tumors. In conclusion, we demonstrate that UCB-MSCs can be used as a new delivery vehicle for therapeutic cytokine, and UCB-MSC-IL12M exhibits long-term antitumor activity by inducing tumor-specific T-cell memory in an intracranial glioma model.

Footnotes

Acknowledgments

This study was supported by grants from the National R&D Program for Cancer Control (0820040) and the Korea Healthcare technology R&D Project (A092258), Ministry of Health, Welfare & Family Affairs, Republic of Korea, and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0021527).

Author Disclosure Statement

No competing financial interests exist.

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Gene Therapy of Intracranial Glioma Using Interleukin 12–Secreting Human Umbilical Cord Blood–Derived Mesenchymal Stem Cells

Abstract

Introduction

Materials and Methods

Cell cultures and cell lines

Adenoviral vectors and peptide

Animals and intracranial glioma model

In vitro and in vivo migration assay

Animal survival and tumor size evaluation

Expression analysis of IL-12M and interferon-γ by ELISA and immunohistochemistry

Detection of CD4 and CD8 T-lymphocyte infiltration by flow cytometry

Evaluation of angiogenesis and apoptosis by immunohistochemistry

Animal rechallenge

Measurement of tumor-specific T-cell responses

Statistical analysis

Results

Optimal condition of 4HP4 and adenovirus on transduction into UCB-MSCs

The effect of adenoviral transduction on migration of genetically modified UCB-MSCs in vitro and in vivo

Effects of UCB-MSC-IL12M on tumor growth and survival of glioma-bearing mice

In vivo transgene expression and antitumor immunity of transplanted UCB-MSC-IL12M

Antiangiogenic activity and apoptosis induction of transplanted UCB-MSC-IL12M in intracranial glioma

Induction of tumor-specific long-term memory by UCB-MSC-IL12M

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References