The prognosis of patients with malignant glioma is extremely poor, despite the extensive surgical treatment that they receive and recent improvements in adjuvant radio- and chemotherapy. In the present study, we propose the use of gene-modified mesenchymal stem cells (MSCs) as a new tool for gene therapy of malignant brain neoplasms. Primary MSCs isolated from Fischer 344 rats possessed excellent migratory ability and exerted inhibitory effects on the proliferation of 9L glioma cell in vitro. We also confirmed the migratory capacity of MSCs in vivo and showed that when they were inoculated into the contralateral hemisphere, they migrated towards 9L glioma cells through the corpus callosum. MSCs implanted directly into the tumor localized mainly at the border between the 9L tumor cells and normal brain parenchyma, and also infiltrated into the tumor bed. Intratumoral injection of MSCs caused significant inhibition of 9L tumor growth and increased the survival of 9L glioma-bearing rats. Gene-modification of MSCs by infection with an adenoviral vector encoding human interleukin-2 (IL-2) clearly augmented the antitumor effect and further prolonged the survival of tumor-bearing rats. Thus, gene therapy employing MSCs as a targeting vehicle would be promising as a new therapeutic approach for refractory brain tumor.
The prognosis of patients with malignant gliomas, such as glioblastoma multiforme or anaplastic astrocytoma, is extremely poor, despite their extensive surgical treatment and adjuvant radio- and chemotherapy. This is because of the unique features of gliomas, such as refractoriness to conventional chemo- and radiotherapy and infiltration into the brain.1, 2, 3 Gliomas possess extraordinary migratory ability and readily penetrate adjacent normal tissue. Tumor cells migrating away from the primary tumor site often form tumor microsatellites at distal sites. These outgrowing cells at the borders of the primary tumor frequently remain and present even after extensive surgical resection and are responsible for the eventual recurrence of the tumor after initial treatment.3 Recent developments in molecular biology have made intracranial gene therapy a new option for the treatment of malignant glioma. Although successful treatment of experimental glioma by suicide gene therapy combining herpes simplex virus thymidine kinase with ganciclovir,4, 5 cytosine deaminase with 5-fluorocytosine,6, 7 or cytokine gene therapy has been reported, the results of clinical trials using this approach have been disappointing.8, 9 In these studies, adenoviral vectors were mainly employed as in many current gene therapy strategies because of their high infectivity for nondividing as well as proliferating tumor cells. However, even utilizing adenoviral vectors, therapeutic gene delivery by direct injection into the primary brain tumor or postsurgical tumor cavity has failed to infect outgrowing tumor islands and resulted in insufficient antitumor effects. Furthermore, lack of precise tumor targeting by adenoviral vector infection may cause severe damage to the normal brain parenchyma adjacent to the tumor.
Aboody et al10 demonstrated that neural stem cells (NSCs) administered intracranially possess extensive tropism for experimental glioma and significant migratory behavior. NSCs distribute throughout the primary tumor bed and migrate together with widely outgrowing tumor microsatellites after intratumoral implantation. Moreover, when NSCs are implanted intracranially at sites distant from the tumor, they migrate through the normal parenchyma and localize in the tumor. This behavior of NSCs has been exploited as a tumor-targeting strategy for glioma gene therapy. Strong antitumor effects have been reported following intracranial administration of gene-modified NSCs expressing interleukin (IL) -4,11 IL-12,12 or tumor necrosis factor-related apoptosis-inducing ligand13 in experimental glioma models. Since the preparation of sufficient amounts of autologous NSCs for clinical application is currently technically challenging, fetal brain, adult allogeneic brain, and embryonic stem (ES) cells are being considered as possible substitutes for autologous NSCs.14 However, there are several reasons for hesitating to utilize these sources for clinical application in glioma treatment because of ethical concerns and problems with immune responses directed against them.
Recently, it has been suggested that bone marrow stem cells may represent an alternative source of neural progenitor cells for organ regeneration. Among bone marrow stem cells, much attention has been paid to mesenchymal stem cells (MSCs, also referred to as bone marrow stromal cells) because of their plasticity for differentiation into classical mesenchymal lineages, such as adipocytes, chondrocytes, and osteocytes,15, 16, 17, 18 and also neuronal lineages.19, 20, 21, 22 This neurogenic potential of MSCs was revealed under special culture conditions or following intracranial implantation.23, 24, 25 Furthermore, it has been reported that MSCs can manifest multiple neural phenotypes even without neurogenic stimulation.26 Moreover, in a rat cerebral ischemia model, intracranially implanted MSCs can migrate away from the initial injection site towards an infarction without changing morphologically to a neural phenotype.25 Taken together, these findings imply that NSCs could be replaceable with MSCs as a therapeutic vehicle for gene therapy against glioma. Moreover, MSCs also have the advantage of ease of propagation in vitro and implantation of autologous MSCs into patients with malignant glioma is ethically unproblematic.
In the present study, we evaluated the antitumor effects against rat intracranial 9L glioma of intracranially implanted MSCs engineered to secrete the immunoregulatory cytokine IL-2, and also investigated the intracranial behavior of MSCs, such as their tumor tropism, pattern of distribution, and migratory capacity.
Characterization of primary rat MSCs
We analyzed the surface antigens on rat primary MSCs by flowcytometry. Cultured rat MSCs were CD73 antigen-positive (Figure 1b), which had been reported as one of the typical mesenchymal surface antigens on human MSCs22 Contamination by hematopoietic cells (CD45 or CD11b/c) were not detected in our MSCs culture (Figure 1c, e). We also examined the differentiation of rat MSCs into the classical mesenchymal lineages. Our cultured rat MSCs were capable of differentiating into osteocytes (Figure 1f) and adipocytes (Figure 1h) lineages. The potential for osteogenic and adipogenic differentiation of primary MSCs was not affected by the IL-2 gene-modification with adenoviral vector (Figure 1g, and Figure 1i).
Effect of MSCs on proliferation of 9L cells in vitro
It is not established whether the administration of MSCs into brain tumors in vivo would have any effect on tumor growth, but it is known that MSCs can produce cytokines, such as fibroblast growth factor (FGFs), and other tumor growth factors (TGFs), which can support tumor growth.27 Therefore, we initially evaluated the effect of coculture with MSCs on the growth of 9L glioma cells in vitro. We cocultured Ds-Red2 (humanized Discosoma red fluorescent protein)-labeled 9L cells (9L-DsR) with MSCs or normal rat kidney (NRK) cells and enumerated the total number and the number of DsRed2-positive cells 3 days later by flowcytometry. As shown in Figure 2a, the proliferation of 9L cells was inhibited to a significantly greater extent by cocultivation with MSCs (24.5±1.9% inhibition) than with NRK cells (17.4±1.9% inhibition, P<0.01). To determine whether growth suppression of 9L cells by MSCs was mediated by soluble factors, we utilized a two-chamber culture system. As shown in Figure 2b, significant growth suppression of 9L cells was also effected by MSCs but not NRK cells cultured in a separate chamber (9.8±3.1 and 1.8±1.2%, respectively, P<0.01). These results indicate that MSCs themselves possess a direct antitumor effect against 9L glioma cells in vitro, which is mediated by soluble factors.
Migratory capacity of MSCs in vitro
We evaluated the migratory nature of MSCs towards glioma cells in vitro (Figure 2c). While neither MSCs nor NRK cells migrated spontaneously, both types of cells were stimulated to do so by the addition of 9L cells into the lower chamber. Migratory activity increased in a dose-dependent manner with increasing numbers of 9L cells. Importantly, MSCs were found to possess significantly greater migratory capacity than NRK cells (P<0.01).
Migration and tumor tropism of implanted MSCs
Since the in vitro migratory capacity of MSCs had already been established, we investigated whether implanted MSCs could migrate towards intracranial glioma through normal brain parenchyma in vivo. 9L-DsR glioma cells were inoculated intracranially into the right basal ganglia and subsequently EGFP (enhanced green fluorescent protein)-labeled MSCs (MSCs-EGFP) were injected directly into either the glioma or the contralateral hemisphere 3 days later. Rats were killed 14 days after tumor inoculation, and brain sections were prepared. As shown in Figure 3a and c, enormous glioma masses staining strongly with hematoxylin were observed in all the rats implanted with 9L cells, which occupied the right hemisphere and caused the midline to be shifted towards the left hemisphere. Gene-labeled MSCs were mostly to be found at the border between tumor and normal parenchyma, but had also infiltrated into the tumor bed relatively uniformly after intratumoral injection (Figure 3b). MSCs did not migrate out to distal brain parenchyma or into the contralateral hemisphere. Confocal laser microscopy revealed the accumulation of EGFP-positive MSCs, most of which retained their spindle-like shape, at the edge of the DsRed-positive tumor (Figure 3e). The presence of MSCs coincided with glioma cells, which spread out from the main tumor (Figure 3g). On the other hand, MSCs inoculated into the contralateral hemisphere migrated away from the initial injection site towards the glioma cells along the corpus callosum (Figure 3d). These MSCs were mostly retained at the corpus callosum and the edge of the tumor adjacent to it (Figure 3h). Additionally, infiltration of MSCs into the tumor was observed. Having confirmed the excellent migratory capacity and glioma tropism of these MSCs after intracranial implantation, the next step was to prepare therapeutic gene-modified cells for the treatment of experimental glioma.
Human IL-2 production from genetically modified MSCs
Human IL-2 (hIL-2) was selected as a therapeutic gene because antitumor effects of IL-2 on 9L glioma are well established in the rat model.28, 29 Human IL-2-transduced MSCs (MSCs-IL2) were prepared by infection with modified adenoviral vector as previously described.15 This fiber-mutant vector was employed because rat primary MSCs are relatively resistant to wild-type adenoviral infection due to their low level of expression of the adenoviral receptor, coxsackie-adenovirus receptor (CAR). In agreement with our previous observations, high levels of hIL-2 were detected in the supernatant of MSCs infected with relatively low concentrations of AxCAhIL2-F/RGD (8.6±0.5 and 24.0±1.7 ng/ml/104 cells/72 h at 300 and 1000 particle units/cell, respectively). This high level hIL-2 production was increased further in a dose-dependent manner with increasing adenoviral concentration.
Prolonged survival of glioma-bearing rats implanted with IL-2 gene-transduced MSCs
To determine whether MSCs-IL2 can provide therapeutic benefits in vivo, we inoculated 9L glioma cells admixed with MSCs-IL2 into rat brains (Figure 4a). The results of coinjection of 9L cells with either unmodified MSCs or EGFP gene-modified MSCs (MSCs-EGFP) were also evaluated. The survival of rats coinjected with 9L glioma cells and MSCs-IL2 (26.3±2.2 days, P=0.0003 versus 9L alone, P=0.0008 versus MSCs, P=0.0007 versus MSCs-EGFP) was significantly longer compared with controls injected with 9L alone or either with 9L cells together with unmodified MSCs, or together with MSCs-EGFP (17.1±1.1, 22.0±0.8, 21.3±1.5 days). Both groups of rats injected with 9L cells together with unmodified MSCs (22.0±0.8 days, P=0.0003) or MSCs-EGFP (21.3±1.5 days, P=0.0003) also showed significantly prolonged survival compared to controls, but there was no difference between MSCs and MSCs-EGFP group survival (P=0.5881). IL-2 gene modification of MSCs conferred additional therapeutic benefits on the survival of rats coinjected with MSCs and 9L glioma cells, but gene-modification itself did not affect survival. The therapeutic benefit of MSCs-IL2 was also confirmed in the therapeutic model of glioma-bearing rats. We delivered MSCs-EGFP or MSCs-IL2 into established intracranial 9L glioma in the rat 3 days after tumor inoculation (Figure 4b). Intratumoral inoculation of MSCs-IL2 significantly prolonged survival in 9L glioma-bearing rats (27.7±1.1 days, P=0.0002, versus 9L alone) compared to controls (17.1±1.1 days). The mean survival time of glioma-bearing rats injected with MSCs-EGFP (23.2±0.8 days) was significantly less than that of rats injected with MSCs-IL2 (P=0.0024), but the former survived longer than the nontreated controls (P=0.0006).
Effect of gene-modified MSCs on in vivo tumor growth evaluated by MRI
To assess whether the prolongation of survival observed after injection of MSCs-IL2 or MSCs was associated with inhibition of tumor growth, we monitored tumor volume by magnetic resonance imaging (MRI) every 7 days after intracranial tumor inoculation. The 9L glioma was clearly visible as an enhanced area by T1-weighted imaging in the coronal section of the brain (Figure 5). As shown in Table 1 and Figure 5, progressive growth of the 9L glioma was observed in the brain of untreated rats and reached a lethal volume 14 days after tumor inoculation. In contrast, significantly smaller tumor volumes were present in brains of animals treated with MSCs-IL2 or MSCs (P<0.01 compared to untreated controls 14 days after tumor inoculation). There was no significant difference in tumor volume between groups treated with unmodified MSCs and MSCs-IL2 on day 14. This therapeutic effect of IL-2 gene modification was, however, clearly visible by MRI 21 days after tumor inoculation. At this time, glioma in animals treated with unmodified MSCs had reached a near lethal volume, but treatment with MSCs-IL2 resulted in the tumor remaining small. These observed changes in glioma volume were consistent with the survival duration in the different treatment groups.
Implantation of MSCs-IL2 induces lymphocytes infiltration into glioma
We evaluated whether implantation of MSCs-IL2 into 9L gliomas could elicit immunological reaction in vivo. MSCs-IL2 were intratumorally injected into established 9L gliomas 3 days after tumor inoculation and the histology of the glioma was evaluated 10 days after tumor inoculation. In the histological analysis of the hematoxylin and eosin (H–E) staining of the MSC-treated 9L glioma, a massive mononuclear cell infiltration was observed in the 9L glioma treated with the IL-2 gene-modified MSCs (Figure 6c, d). In contrast, inflammatory cell infiltration was minimum in the glioma transplanted with unmodified MSCs (Figure 6a, b). Few CD4 and CD8 cells infiltrated in the specimens of tumor implanted with the unmodified MSCs (Figure 6e, g). In clear contrast, obvious infiltration of both CD4- and CD8-positive lymphocytes was detected in the tumor inoculated with the IL-2 gene-modified MSCs (Figure 6f, h).
In the present study, we documented prolongation of survival and antitumor effects mediated by intracranial administration of IL-2 gene-modified MSCs in the rat 9L glioma model, and in addition have demonstrated migratory ability of MSCs towards glioma cells in vitro and in vivo.
In our in vitro study, the migration of MSCs was clearly stimulated using separately cultured 9L cells in Transwells in a dose-dependent manner varying with 9L cell density. This result indicates that soluble factors released from 9L glioma cells could mediate the activation of MSC migration. Cytokines such as vascular endothelial cell growth factors, TGFs, FGFs, platelet-derived growth factors, monocyte chemoattractant protein-1, and IL-8 released from the neoplasm or inflammatory tissue are all possible candidates.27, 30, 31, 32, 33, 34 It is known that these factors released from cancer cells promote the migration of endothelial cell and stromal cell progenitors from the bone marrow towards the cancer bed35, 36 or tissue surrounding the tumor, enhancing the formation of tumor-stroma.37 Similar mechanisms would be anticipated for tumor-stromal formation in glioma, and for the migration of implanted MSCs. MSCs injected intratumorally were largely distributed at the border zone between tumor and normal parenchyma. They developed a capsule-like structure and also infiltrated into the tumor bed relatively uniformly, as shown in Figure 3. Moreover, we found an association of MSCs with outgrowing glioma cells, known as the ‘chasing down’ phenomenon, which is reported in the case of intratumoral injection of NSCs into gliomas. These intracranial distribution patterns of MSCs were quite similar to those of NSCs reported by Aboody et al.10 In agreement with previous observations in the rat cerebral infarction model,25 after inoculation into the contralateral hemisphere, MSCs migrated through the corpus callosum and targeted 9L gliomas. Thus, MSCs possess great migratory ability and glioma tropism; clearly, these attributes would be useful for in vivo targeting of gliomas.
Coinjection of 9L gliomas either with unmodified MSCs or those transduced with a nontherapeutic gene (EGFP) prolonged the survival of tumor-bearing rats. This antitumor effect of MSCs was also confirmed in a therapeutic model treated where MSCs were injected 3 days after tumor inoculation. Taken together, these results on the suppression of 9L cell growth by cocultivation with MSCs in vitro and delayed tumor growth after MSCs injection seen by MRI monitoring in vivo imply that the prolonged survival in glioma-bearing rats treated in this way might depend on a direct antitumor effect of the MSCs themselves. Although the mode of action of the antitumor effect exhibited by MSCs is largely unknown, we demonstrated that coculture of MSCs in isolated chambers inhibited the growth of 9L cells. This result is consistent with reports by Hombauer38 and Maestroni,39 who demonstrated growth suppression of lung cancer cells by soluble factors released from MSCs. MSCs produce several neurotrophic factors including nerve growth factor (NGF),23 which can induce the differentiation and the growth-inhibition of C6 glioma cells in vitro.40 In our 9L glioma model, NGF produced by MSCs could contribute to the induction of differentiation and growth suppression as a mechanism of the antitumor effect exerted by MSCs. In the cerebral infarction model, it has been reported that implanted MSCs mediate neural protection through the inhibition of neuronal apoptosis and this protective effect is thought to be due to neurotrophic factors, such as NGF, released from MSCs.23 The protective effect of MSCs for normal brain parenchyma may also contribute to prolonged survival of 9L-bearing rats treated by MSCs implantation. We also found that MSCs secreted large amounts of angiogenic factors, such as angiopoietin-1 (Ang1) (data not shown). Recently, it has been reported that Ang1 can inhibit tumor-vascular leakage and also tumor growth in vivo.41 Ang1 released from MSCs could represent another component of the antitumor effect of MSCs. Ang1 may also protect brain parenchyma from lethal cerebral edema through suppression of vascular leakage. As shown in Figure 3, MSCs mainly localized to the edge of the tumor and formed capsule-like structures. This unique intracranial distribution of MSCs may act as a barrier preventing spread of glioma cells into normal parenchyma. Therefore, MSCs implantation for the treatment of gliomas might be beneficial both because of an antitumor effect as well as a protective effect for normal brain.
Since MSCs possess high affinity for glioma and exert organ-protective effects, utilizing MSCs as a vehicle for gene therapy in the treatment of brain cancer represents a rational clinical approach. However, the efficiency of exogenous gene transfer is relatively low using physiochemical methods,42 and even employing viral vectors, such as retroviral43, 44 or adenoviral vectors,15, 42 the method is not efficient. Refractoriness to viral vector infection may be mainly due to a deficit of native viral receptors.15 We previously found that resistance against adenoviral infection could be bypassed by using a genetically modified adenovirus with fiber mutated by inserting the integrin-binding motif RGD into the HI-loop of the fiber-knob protein.15, 45, 46 Indeed, as shown in the present study, MSCs infected with a low concentration of AxCAhIL2-F/RGD produced large amounts of the recombinant protein and revealed strong antitumor effects in glioma-bearing rats.
Although prolonged survival of the glioma-bearing rat by transplantation with IL-2 gene modified MSCs was demonstrated, we did not achieve complete tumor regression by this therapeutic approach. One of the possible explanations for this unsatisfactory result is early elimination of engineered MSCs. It is generally believed that gene expression accomplished by adenoviral transduction is diminished within a few weeks by immune-mediated elimination of virus-infected cells. However, we found that a significant amount of EGFP-expressing MSCs persisted in the rat brain until at least 24 days after tumor inoculation (data not shown) and also confirmed a long-lasting 9L glioma growth-suppressive effect after a single intratumoral administration of engineered MSCs. These findings indicate that the majority of intracranially implanted gene-modified MSCs retain and express the transgene for longer periods without rejection. This might be because of the immunologically privileged nature of the brain.47 However, the immunologically privileged nature of the brain may also negatively impact the therapeutic effect of intracranially administered MSCs-IL2. Iwadate et al29 reported a progressive growth of IL-2 gene-transduced 9L glioma after intracranial implantation. They concluded that subsequent subcutaneous tumor vaccination was required for effective brain tumor regression.29 Likewise, our intracranial administration of IL-2 gene-modified MSCs was insufficient for the induction of specific and curative anti-9L glioma immunity. Intensification of the specific antitumor immunity, by combining a cancer vaccine that produces cytokines such as GM-CSF,48 might be advantageous to improve antitumor effects of our approach. On the other hand, a further control of the local lesion by MSCs with other therapeutic genes that possess direct antitumor effects, such as interferons (IFN), or tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), might be promising. Recently, a similar approach was reported by Studeny et al49 utilizing MSCs modified with adenoviral vector encoding β-IFN in a nonbrain tumor setting. Therefore, adenoviral gene-modified MSCs could be beneficial as therapeutic tools for the treatment of brain tumors.
In conclusion, MSCs show tropism and high migratory capacity towards 9L glioma in tumor-bearing rats; therefore, gene therapy employing MSCs as a targeting vehicle would be promising as a new therapeutic approach for refractory brain tumor.
Materials and methods
The 9L rat glioma cell line (syngeneic with the Fisher 344 rat) and NRK cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Inc., St Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen-Life Technologies Inc., Grand Island, NY, USA), 2 mM L-glutamine, 50 μg of streptomycin, and 50 U/ml penicillin. For biolabeling of 9L cells, the pDsR2-N1 plasmid encoding humanized Discosoma red fluorescent protein (DsRed2) under the control of the CMV promoter was purchased from BD Biosciences Clontech (Palo Alto, CA, USA). Cells were transfected with pDsR2-N1 at 50–60% confluence with NeuroPORTER (Gene Therapy Systems Inc., San Diego, CA, USA) using a DNA complex prepared at the ratio of 1 μg DNA: 2.5 μl NeuroPORTER reagent. DsRed2-positive cells were isolated by sorting with a FACScalibur (Becton Dickinson Co., Franklin Lakes, NJ, USA) 24 h after transfection and further purified by repeated sorting 72 h after transfection. Isolated DsRed2-positive 9L cells were selected in DMEM supplemented with 10% FBS and 1 mg/ml G418 (Invitrogen-Life Technologies) for 14 days to establish stable lines (9L-DsR).
MSCs were prepared from bone marrow as described previously.15 Briefly, Fischer 344 rats (9 weeks old, male) were killed by cervical dislocation. Femurs and tibias were dissected free of soft tissue, and the epiphyses removed with a rongeur. Femoral and tibial midshaft bone marrow tissue was flushed out into normal medium (DMEM supplemented with 10% FBS, 100 U/ml of penicillin, 100 μg/ml of streptomycin, 0.25 μg/ml amphotericin-B, and 2 mM L-glutamine). A single cell suspension was obtained by sequentially drawing the marrow into syringes through needles of decreasing size (gauge 18, 20, 22, respectively). Primary cultured MSCs were seeded in normal medium at a density of 5 × 107 cells/10-cm dish. In order to remove nonadherent cells, the medium was replaced with fresh normal medium 4 days after initial culture. MSCs were maintained at 37°C in 5% CO2 by exchanging the spent medium with fresh medium at 4-day intervals.
In vitro differentiation of MSCs
Evaluation of the in vitro differentiation ability of rat primary MSCs or gene-modified MSCs into classical mesenchymal lineages was carried out as previously described15, 22 Briefly, MSCs were treated with either osteogenic differentiation medium supplemented with 80 μg/ml vitamin C phosphate (WAKO Pure Chemical Industries, Osaka, Japan), 10 mM sodium β-glycerophosphate (Calbiochem, San Diego, CA, USA), and 0.1 μM dexamethasone (Sigma-Aldrich Inc.), or with adipogenic differentiation medium supplemented with 0.5 μM hydrocortisone, 500 μM isobutylmethylxanthine, and 60 μM indomethacin. The differentiation medium was changed every 3 days until day 21. The cells were stained with 5% silver nitrate (Sigma-Aldrich Inc.) to detect mineral deposition (von Kossa staining) for 15 min after fixation with 10% formalin for 10 min in order to confirm osteogenic differentiation. The lipid droplet formation in cell culture was also stained with a freshly prepared oil red O solution (mixture of three parts of an oil red O stock solution in 0.5% in isopropanol with two parts of distilled water) after fixation with 10% formalin for 15 min for the detection of adipogenic differentiation.
Phenotypical analysis of primary MSCs was performed by Flowcytometry using FACScalibur (Becton Dickinson Co.). Briefly, cells were washed twice with PBS containing 0.1% bovine serum albumin (BSA). Cells were labeled with anti-rat CD73 (SH3), CD45, or CD11b/c (Pharmingen, San Diego, CA, USA) monoclonal antibody, and then labeled with goat anti-mouse IgG antibody conjugated with fluorescent isothiocyanate (Immunotech, Marseilles, France) as a secondary antibody. As an isotype-matched control, mouse IgG1 (Immunotech)- or mouse IgG2a-labeled cells were analyzed.
Adenoviral vectors and ex vivo gene transduction
The adenoviral vector with modified fiber (AxCAhIL2-F/RGD) encoding human IL-2 was described previously.45 Adenoviral vectors (AxCAEGFP-F/RGD) carrying a humanized variant of Aequoria victoria green fluorescent protein (enhanced GFP: EGFP) together with RGD-mutated fiber under the control of a CA promoter (CMV-IE enhancer, with the chicken β-actin promoter) were constructed as described previously.45, 46 The EGFP gene fragment was isolated from the pEGFP vector (BD Biosciences Clontech, Palo Alto, CA, USA) and inserted into the pCAcc vector50 (pCAEGFP). An expression cassette containing the EGFP gene was isolated by restriction enzyme digestion with ClaI and inserted into the ClaI site of cosmid vector pL. The cosmid vector pLEGFP thus generated, together with ClaI and EcoT22I-digested DNA-TPC from Ad5dlx-F/RGD, was cotransfected into human embryonic kidney 293 cells. Plaques arising from the transfected 293 cells were isolated and evaluated by restriction enzyme digestion of the viral genome. The adenoviral EGFP expression vector with RGD-fiber, AxCAEGFP-F/RGD, obtained from isolated plaques was expanded in 293 cells. All adenoviral vectors were propagated in 293 cells and purified by a cesium chloride ultracentrifugation method. After purification, virus was dialyzed against phosphate-buffered saline (PBS) with 10% glycerol and stored at −80°C. The viral titer was determined, in terms of particle units (pu), via spectrophotometric measurement at A260 nm.45
Ex vivo adenoviral gene transduction of primary MSCs was described previously.15 Briefly, 5 × 105 MSCs were plated in 10 cm dishes 1 day before adenoviral infection. Cells were infected by incubation with 5 ml of stocked viral solution containing either 1000 pu/cells of AxCAEGFP-F/wt or AxCAhIL2-F/RGD at 37°C in 5% CO2 for 1 h. After infection, the cells were washed twice with PBS (pH 7.4), and then supplemented with 10 ml of normal medium.
Cell proliferation assay
9L-DsR cells (5 × 104/well) were cultured either alone or mixed with MSCs (5 × 103/well) or NRK cells (5 × 103/well) in six-well plates for 72 h. Cells were then trypsinized and counted. The numbers of 9L-DsR cells were determined using a flowcytometer (FACScalibur). To assess the effect of soluble factor released from MSCs on the proliferation of 9L cells, MSCs or NRK cells were seeded onto Transwell inserts (pore size 0.4 μm, Costar, Cambridge, MA, USA) at a density of 1 × 105 cells/Transwell in DMEM containing 10% FBS. 9L cells were plated in the well at a density of 5 × 103 cells/well in DMEM containing 10% FBS. Cocultures were incubated for 72 h and cells were counted directly to determine proliferation in the coculture system. All data are expressed as the percentage inhibition calculated as follows: % growth inhibition=[1−(the number of 9L-DsR cells cocultured with MSCs or NRK cells/the number of 9L-DsR cells cultured alone)] × 100.
In vitro cell migration assay
The cell migration assay was performed using double-chamber culture dishes, Transwell (Costar). Cells were metabolically labeled with 125I-deoxyuridine (125I-IUDR, Amersham Biosciences Corp., Piscataway, NJ, USA). Briefly, 1 × 105 cells/ml were cultured for 24 h in the medium containing 0.1 μCi/ml 125I-IUDR. Then cells were washed three times with DMEM and resuspended in the same medium. 125I-IUDR-labeled cells (5 × 104 cells) were placed in the upper chamber with 8-μm pores, and 9L cells were placed in the lower chamber. The Transwell was placed at 37°C, 5% CO2 for 24 h, then cells in the lower chamber were lysed with 1 N NaOH. Radioactivity in the cell lysate was assessed with a gamma counter. Results of cell migration assays are expressed as a percentage (count in the lower chamber as % of total cell count).
Intracranial distribution of implanted MSCs
To evaluate intracranial distribution of MSCs, 4 × 104 9L-DsR cells were inoculated and subsequently 4 × 105 MSCs-EGFP were implanted intratumorally or into the contralateral hemisphere 3 days after tumor inoculation. At 14 days after tumor inoculation, the rat brains were perfused with PBS followed by 4% paraformaldehyde under deep anesthesia. The excised brains were postfixed with 4% paraformaldehyde overnight and then equilibrated in PBS containing 30% sucrose for 48 h. Fixed brains were embedded in OTC compound (Miles Inc., Elkhart, IN, USA), snap frozen in liquid nitrogen, and stored at −70°C. Tissue was cryosectioned at 20-μm thickness and stained with hematoxylin and eosin (H–E), or immunohistochemically with anti-GFP monoclonal antibody (BD Sciences Clontech). The section stained with the first antibody was visualized by using the Vectastain kit from Vector Laboratories (Burlingame, CA, USA). Imaging was performed with a Zeiss-Pascal microscope (Carl Zeiss, Inc., Thornwood, NY, USA).
Measurement of human IL-2 produced by engineered MSCs
For measurement of human interleukin-2 (IL-2) production from MSCs transduced with the human IL-2 gene, MSCs were plated in 24-well plates in triplicate at a density of 104 cells/well 12 h prior to adenoviral infection. The cells were then infected with AxCAhIL2-F/RGD and incubated for 72 h. Human IL-2 concentrations in culture supernatants were measured by ELISA (IL-2 immunoassay kit; R&D Systems, Minneapolis, MN, USA).
Treatment of rat experimental glioma
Male Fisher 344 rats (7–8 weeks old, 200–240 g) were purchased from Japan SLC Inc. (Hamamatsu, Japan). Animals were anesthetized and placed in a stereotaxic apparatus (Narishige Scientific Instrument Laboratory, Tokyo, Japan). A burr hole was made at an appropriate location (1 mm posterior to bregma and 3 mm right to midline). A 26-gauge needle was inserted 4 mm ventral from the dura, where 9L tumor cells in 5 μl of PBS were inoculated using a 10-μl microsyringe (Hamilton Company, Reno, NV, USA). In coinjection experiments, 4 × 104 9L cells were mixed with 4 × 105 MSC or IL-2-transduced (infected with AxCAhIL2-F/RGD at 1000 pu/cells) MSCs (MSCs-IL2) in 5 μl of PBS and then the cell suspension was intracranially injected as described above. In experiments for intratumoral injection of MSCs, 4 × 104 9L glioma cells were implanted. Subsequently, 4 × 105 MSCs or MSCs-IL2 in 5 μl PBS were intratumorally implanted 3 days after tumor inoculation.
Evaluation of tumor volume by magnetic resonance image
Magnetic resonance imaging was performed in all animals every 7 days to estimate intracerebral tumor volumes. The animals were anesthetized by intraperitoneal injection of ketamine (2.7∼3 mg/100 g) and xylazine (0.36∼0.4 mg/100 g). They were then injected with 0.2 ml of Gd-DTPA (0.8∼1.0 ml/kg, Magnevist, Schering Japan, Tokyo, Japan), and coronal T1-weighted spin-echo images (TR 500 ms, TE 10 ms, field of view 50 × 50 mm, slice thickness 1.5 mm, gapless) were obtained with a 7 T, 18 cm-bore, superconducting magnet interfaced to a UNITYINOVA console (Oxford Instruments KK, Tokyo, Japan). Tumor volumes (mm3) were calculated as the sum of the Gd-DTPA-enhanced portion of each MR-imaged area (mm2) times imaged thickness. The estimated tumor volumes on MRI have a linear correlation with actual tumor weights obtained immediately after the imaging study.51
CD4- or CD8-positive cell infiltration in glioma implanted with MSCs-IL2
For the detection of CD4- or CD8-positive cell infiltration into gliomas after MSCs-IL2 treatment, 4 × 104 of 9L-DsR cells were implanted, then subsequently 4 × 105 of MSCs-EGFP or MSCs-IL2 were injected intratumorally 3 days after tumor inoculation. Rats were killed and excised brains were embedded in paraffin 7 days after tumor inoculation. Brain specimens with a 6-μm-thickness were immunohistochemically stained with anti-rat CD4 (clone W3/25, Serotec Inc., Oxford, UK) or anti-rat CD8 (clone OX-8, Serotec Inc.) monoclonal antibody and then visualized by the Vectastain ABC kit (Vector Laboratories).
Statistical analysis for the cell proliferation assays and the migration assay was performed by Student's t-test. Scheffe's test for the tumor volume assessment on day 7 and 14, and Student's t-test for that on day 21 were performed. A P-value less than 0.05 considered as significant in Student's t-test and Scheffe's test. Statistical analysis of survival was performed by the Log-rank test.
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We thank H Isogai at the Institute for Animal Experimentation of Sapporo Medical University for help in animal experiments. This work was supported in part by a grant to HH from the Ministry of Education and Science.
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Nakamura, K., Ito, Y., Kawano, Y. et al. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther 11, 1155–1164 (2004). https://doi.org/10.1038/sj.gt.3302276
- mesenchymal stem cell
- adenoviral vector
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