Mesenchymal stem cells display tumor-specific tropism in an RCAS/Ntv-a glioma model

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Mesenchymal Stem Cells Display Tumor-Specific Tropism in an RCAS/Ntv-a Glioma Model 1 Article in Neoplasia (New York, N.Y.) · August 2011 DOI: 10.1593/neo.101680 · Source: PubMed

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Volume 13 Number 8

August 2011

pp. 716–725 716

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Mesenchymal Stem Cells Display Tumor-Specific Tropism in an RCAS/Ntv-a Glioma Model1

Tiffany Doucette*, Ganesh Rao*, Yuhui Yang*, † Joy Gumin*, Naoki Shinojima*, B. Nebiyou Bekele , † ‡ * Wei Qiao , Wei Zhang and Frederick F. Lang *Department of Neurosurgery, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA; † Department of Biostatistics, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA; ‡ Department of Pathology, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA

Abstract Bone marrow–derived mesenchymal stem cells (MSCs) have been shown to localize to gliomas and deliver therapeutic agents. However, the clinical translation of MSCs remains poorly defined because previous studies relied on glioma models with uncertain relevance to human disease, typically xenograft models in immunocompromised mice. To address this shortcoming, we used the RCAS/Ntv-a system, in which endogenous gliomas that recapitulate the tumor and stromal features of human gliomas develop in immunocompetent mice. MSCs were harvested from bone marrow of Ntv-a mice and injected into the carotid artery of Ntv-a mice previously inoculated with RCAS–PDGF-B and RCAS-IGFBP2 to induce malignant gliomas (n = 9). MSCs were labeled with luciferase for in vivo bioluminescence imaging (BLI). After intra-arterial injection, BLI revealed MSCs in the right frontal lobe in seven of nine mice. At necropsy, gliomas were detected within the right frontal lobe in all these mice, correlating with the location of the MSCs. In the two mice without MSCs based on BLI, no tumor was found, indicating that MSC localization was tumor specific. In another cohort of mice (n = 9), MSCs were labeled with SP-DiI, a fluorescent vital dye. After intra-arterial injection, fluorescence microscopy revealed SP-DiI–labeled MSCs throughout tumors 1 to 7 days after injection but not in nontumoral areas of the brain. MSCs injected intravenously did not localize to tumors (n = 12). We conclude that syngeneic MSCs are capable of homing to endogenous gliomas in immunocompetent mice. These findings support the use of MSCs as tumor-specific delivery vehicles for treating gliomas. Neoplasia (2011) 13, 716–725

Introduction Despite multimodal therapy, patients with high-grade gliomas have a poor prognosis, with a median survival of 14 months for grade 4 and 36 months for grade 3 gliomas according to the World Health Organization [1,2]. This poor outcome is due at least in part to an inability to deliver therapeutic agents to the infiltrative tumor cells. Delivery of most conventional therapies is restricted by the blood-brain-tumor barrier, and the efficacy of newer biologic therapies, such as gene and viral therapies, has been limited by the inability to deliver the therapeutic genes to most of the tumor [3]. Consequently, significant efforts have been undertaken to develop new methods for delivering therapeutic agents to infiltrative gliomas [4]. Recent evidence has suggested that stem cells may be effective delivery vehicles for a variety of solid tumors arising throughout the body. Initial studies of brain tumors indicated that neural stem cells

Abbreviations: BLI, bioluminescence imaging; GSC, glioma stem cell; H&E, hematoxylin and eosin; hMSC, human mesenchymal stem cell; IGFBP2, insulin-like growth factor–binding protein 2; MSC, mesenchymal stem cell; RCAS, replicationcompetent, avian leukosis virus, splice acceptor Address all correspondence to: Frederick F. Lang, MD, Department of Neurosurgery, Unit 442, The University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030. E-mail: [email protected] 1 This study was supported by grants from the National Cancer Institute (R01 CA115729 and P50 CA127001) to F.F.L.; the Elias Family Fund for Brain Tumor Research, the Gene Pennebaker Brain Cancer Fund, the Brian McCulloph Research Fund, and the Run for the Roses Foundation to F.F.L.; and the MD Anderson Center for Targeted Therapy, and NINDS (NS070928) to G.R. This research is also supported in part by the National Institutes of Health through MD Anderson’s Cancer Center Support Grant (CA016672). Received 5 December 2010; Revised 22 May 2011; Accepted 25 May 2011 Copyright © 2011 Neoplasia Press, Inc. All rights reserved 1522-8002/11/$25.00 DOI 10.1593/neo.101680

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have the capacity to migrate toward and deliver therapeutic agents to human gliomas [5,6]. More recently, mesenchymal stem cells (MSCs), acquired either from bone marrow or from adipose tissue, have been explored as potential delivery vehicles in the treatment of human gliomas [7]. MSCs are defined by their ability to grow as adherent cells in culture, to express an array of positive and negative surface markers, and to differentiate into adipocytes, chondrocytes, and osteocytes under appropriate conditions [8,9]. Compared with all other stem cells [10–12], MSCs have the clinical advantages that they can be easily acquired from patients, are readily expanded ex vivo, can be autotransfused without fear of immune rejection, and are free of ethical concerns [13–15]. We [16] and others [17,18] have demonstrated the feasibility of using human MSCs (hMSCs) as delivery vehicles for glioma therapy. Specifically, hMSCs have been shown to be able to localize to malignant gliomas after systemic delivery and to track infiltrating tumor cells after local delivery [16]. The tropism of hMSCs for glioma seems to be mediated at least in part by specific tumor-derived growth factors, particularly platelet-derived growth factor-BB [16,19]. Importantly, the homing capacity of hMSCs can be translated into therapeutic benefit because hMSCs can be engineered to deliver biologic antiglioma agents to gliomas, including interferon β [16], S-TRAIL [18,20], and oncolytic viruses [21], with demonstrable survival advantages [16,22]. Despite these promising results, the clinical translation of MSCbased therapeutic approaches remains incompletely defined because nearly all published studies analyzing the application of MSCs in gliomas have relied on tumor models that fail to recapitulate the genotypic and/or phenotypic characteristics of human tumor cells and the microenvironment in which they arise. The shortcomings of xenograft models of brain tumors for preclinical testing have been well described [23,24]. To date, most studies analyzing the tropism of MSCs for human gliomas have relied on commercially available human glioma cell lines (e.g., U87, U251) that poorly mimic the invasive growth of gliomas in patients. In addition, because these cell lines are grown as xenografts in nude mice, the interaction of the tumor with the microenvironment or stroma is, by necessity, artificial, raising concerns about the true attraction of exogenously delivered MSCs for human gliomas. Although a few studies have used newer human glioma stem cells (GSCs) [18,25], which are derived directly from patient tumors and grow as highly infiltrative tumors, GSCs must also be grown as xenografts in immunodeficient mice, which obviates the impact of the tumor microenvironment and the immune system on MSC engraftment. Syngeneic rodent models using commercially available murine gliomas implanted in the brain have allowed for some assessment of immune factors influencing MSC migration, but these models poorly recapitulate the genetics of human tumors, and thus their relevance to the human disease is questionable. These concerns call into question the utility of these model systems for evaluating novel therapeutic strategies [26,27]. Lastly, in all previous studies, MSCs have been injected within only a few days of tumor implantation, making it difficult to differentiate the ability of MSCs to localize to areas of injury resulting from the process of cell implantation compared with localization to tumors in their natural setting [28]. Since its initial description by Holland et al. [29], the RCAS/Ntv-a system has been extensively used to model brain tumors (reviewed in Becher and Holland [30]). This system uses a modified avian leukosis virus (the replication-competent\, avian leukosis virus, splice acceptor [RCAS] vector) to deliver genes of interest to brain cells in transgenic mice that express the receptor (TVA) for the virus. TVA expression is

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driven by the Nestin promoter, making glioneuronal precursor cells specifically susceptible to infection and subsequent expression of an inserted gene. This method of somatic cell gene transfer has been used to model a variety of brain tumors [29,31–33]. In particular, somatic transfer of RCAS–PDGF-B and RCAS-IGFBP2 results in murine gliomas with all the histologic features of grade 4 gliomas identical to human gliomas [34]. Activation of the PDGF-B and insulin-like growth factor–binding protein 2 (IGFBP2) signaling pathways is relevant in glioma because up to 30% of human gliomas demonstrate evidence of aberrant PDGF expression [35,36], and most high-grade human gliomas overexpress IGFBP2. Importantly, tumor cells in this model have stem-like features and have been shown to reside within the perivascular niche, indicating that the model faithfully recapitulates the microenvironment and cellular interactions of human gliomas [37,38]. Recent work by our group has shown the benefit of preclinical testing of agents with this model [39]. The RCAS/Ntv-a system is ideal for studying the capacity of MSCs to home to gliomas not only because the gliomas in this model recapitulate the phenotype and genotype of human gliomas but also because the gliomas arise endogenously within the native stroma of the brain in immunocompetent mice and permit natural interactions of MSCs with the surrounding microenvironment [40,41]. Given the advantages of the RCAS/Ntv-a system, we sought to determine the extent to which MSCs isolated from Ntv-a mice are capable of localizing to endogenous high-grade gliomas induced in the brains of Ntv-a mice by somatic transfer of PDGF-B and IGFBP2. Our results demonstrate that MSCs obtained from Ntv-a mice can engraft endogenous brain tumors after systemic delivery, supporting the evolving concept that MSCs have an intrinsic capacity to home to gliomas. Materials and Methods

Harvesting and Characterization of MSCs from Ntv-a Mice MSCs were harvested using the technique described by Peister et al. [42]. Briefly, the cells from the long bones of Ntv-a mice were isolated and centrifuged at 1500 rpm. The pellet was then resuspended in αminimal essential medium (α-MEM) and 20% MSC supplement medium (StemCell Technologies, Vancouver, British Columbia, Canada). Cells were then plated in a T75 flask in α-MEM with 20% MSC supplement medium and incubated at 37°C and 5% CO2. After 24 hours, nonadherent cells were discarded; adherent cells were washed with PBS, and fresh complete isolation medium was added every 3 to 4 days for 4 weeks. Cells were collected after trypsinization and replated in 30 ml of complete isolation medium in 175-cm3 flasks. After 1 to 2 weeks, passage 3 cells were either frozen or further expanded by plating at 50 cells/cm2 and incubating in the complete expansion medium. By passage 4, cells were analyzed using FACS for the mesenchymal markers Sca-1 and CD9, the hematopoietic markers CD45 and CD11b, the lymphocytic marker CD73, the endothelial markers CD31 and CD105, and the GSC marker CD133. Cells were plated in α-MEM with 10% fetal bovine serum (FBS) and supplemented with L-glutamine. Further verification of a mesenchymal lineage was performed by plating the cells in adipogenic, chondrogenic, and osteogenic media.

Differentiation Methods Cells were seeded at 105 cells/μl in a six-well plate. At 100% confluence, cell differentiation was induced with supplemented

Adipogenesis.

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adipogenesis induction medium (Lonza, Walkersville, MD) for 3 days, followed by 3 days of culture in supplemented adipogenesis maintenance medium (Lonza). After four cycles of induction/maintenance, cells were rinsed with PBS, fixed with 10% buffered formalin, and stained with Oil Red O. Cells were seeded at 5 × 104 cells/μl in a six-well plate. After 24 hours, cell differentiation was induced with the osteogenesis induction medium (Lonza). Cells were fed every 3 to 4 days by completely replacing the medium with a fresh osteogenesis induction medium. After 3 to 4 weeks, cells were rinsed in PBS, fixed with 70% ethanol, and stained with Alizarin Red.

Osteogenesis.

Chondrogenesis. Cell pellets were prepared by spinning down 3 × 105 cells in 15-ml polypropylene tubes and growing the cells in the complete chondrogenic medium (Lonza). Cell pellets were fed every 2 to 3 days by completely replacing the medium with a freshly prepared complete chondrogenic medium. After 3 to 4 weeks, pellets were fixed in buffered 10% formalin and embedded in paraffin. Then, 5-μm sections were slide-mounted and stained for glycosaminoglycans with Safranin O. Generation of Gliomas in Ntv-a Mice Vector constructs.

RCAS–PDGF-B was constructed with a hemagglutinin epitope tag as described previously [43]. Details of the creation of RCAS-IGFBP2 are described elsewhere [34]. Briefly, the RCAS-IGFBP2 vector was constructed by subcloning the 1.4-kb complementary DNA fragment of IGFBP2 into a Yap vector, which was then transferred into the RCAS-X vector using NotI and ClaI restriction enzymes. RCAS-GFP was provided by Dr Yi Li (Baylor College of Medicine).

Transfection of DF-1 cells.

DF-1–immortalized chicken fibroblasts were grown in Dulbecco modified Eagle medium with 10% FBS (GIBCO, Carlsbad, CA) in a 5% CO2 humidified incubator at 37°C. To produce live virus, plasmid versions of RCAS vectors were transfected into DF-1 cells using FuGene6 (Roche, Nutley, NJ) and allowed to replicate in culture.

In vivo somatic cell transfer in transgenic mice.

Creation of the transgenic Ntv-a mouse has been previously described [29]; the mice are mixtures of the following strains: C57BL/6, BALB/C, FVB/N, and CD1. To transfer genes through the RCAS vectors, DF-1 producer cells transfected with a particular RCAS vector (105 cells in 12 μl of PBS) were injected into the right frontal lobe of Ntv-a mice from an entry point just anterior to the coronal suture of the skull using a 10-μl gas-tight Hamilton syringe. Equal numbers of DF-1 producer cells for each vector (RCAS–PDGF-B or RCAS-IGFBP2) were coinjected to induce gliomas. Control mice were injected with RCASGFP. To determine whether MSCs were themselves tumorigenic, we coinjected RCAS–PDGF-B with bone marrow–derived MSCs from Ntv-a mice. Escalating concentrations of MSCs (1 × 103, 1 × 104, and 1 × 105) in 1 μl of α-MEM plus 10% FBS were coinjected with RCAS–PDGF-B–infected producer cells (1 × 105 cells in 1 μl of PBS) into cohorts of 30 mice each. A control group of 30 mice was injected with RCAS–PDGF-B alone. We injected mice within 48 to 72 hours after birth because the population of Nestin+ cells producing TVA receptors diminishes progressively with time. To identify the presence or

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absence of tumor, we killed the mice by CO2 inhalation. Their brains were removed, fixed in formalin, embedded in paraffin, stained with hematoxylin and eosin (H&E), and analyzed for tumor formation using light microscopy.

Systemic Delivery of MSCs to Ntv-a Mice Between 6 and 10 weeks after RCAS vector injection, MSCs were delivered into Ntv-a mice systemically either intra-arterially or intravenously. Intra-arterial delivery was performed by injecting MSCs into the right common carotid artery using a technique described previously [16]. Mice were appropriately anesthetized (in accordance with Institutional Animal Care and Use Committee guidelines) with ketamine/ xylazine, and the common carotid artery was visualized microscopically. MSCs were trypsinized and suspended in α-MEM plus 10% FBS. The cells were counted, and 106 MSCs suspended in 100 μl of medium were injected into the carotid artery using a 30-gauge needle attached to a tuberculin syringe. Mice were monitored until awake. For intravenous delivery, MSCs were prepared as above, and 106 cells were injected into the tail vein of mice.

In Vivo Visualization of MSC Engraftment To visualize and track MSCs in live mice, Ntv-a MSCs were transduced with an adenoviral vector containing the complementary DNA of the firefly luciferase gene (Ad-Luc; 1000 virus particles per cell) before injection. Bioluminescence imaging (BLI) was used to detect the MSCs. On the day of imaging, animals were treated with luciferin (150 mg/kg, intraperitoneally) and imaged with the IVIS Imaging System, 200 Series (Xenogen, Alameda, CA). Luciferase in cells converts luciferin to oxyluciferin, generating a light signal that is detected by a CCD camera. Bioluminescence color images were overlaid on grayscale photographic images of the mice to allow for localization of the light source within the animal using the Living Image version 2.11 software overlay (Xenogen) and IGOR image analysis software (version 4.02A; WaveMetrics, Portland, OR). Mice were imaged 1, 4, 7, and 10 days after Ad-Luc MSC injection.

Histologic Confirmation of MSC Migration to Tumor Histologic verification of MSC delivery to tumor-bearing brains was performed by labeling MSCs with a fluorescent dye and analyzing brains after MSC injection with fluorescence microscopy. SP-DiI (Molecular Probes, Eugene, OR), a vital fluorescent dye that incorporates into the cell membrane, was dissolved in dimethylformamide at 21.5 mg/ml. The resulting solution was added to the culture medium at 10 μg/ml. MSCs were incubated with 25 ml of medium with SP-DiI in 175-cm3 flasks for 48 hours. A cohort of Ntv-a mice underwent somatic cell transfer with RCAS–PDGF-B and RCAS-IGFBP2 to generate gliomas and were injected with SP-DiI–labeled MSCs (106 cells) through the carotid artery at least 6 weeks after RCAS vector injection and presumed tumor formation. Animals were killed 1, 4, 7, and 10 days after MSC injection (n = 3 mice per group). Their brains were removed and sectioned in the coronal plane. Half of the brain was fixed, dehydrated, and frozen with cryoembedding medium at −80°C. The other half was fixed in formalin and stained with H&E. The sections stained with H&E were analyzed to determine the presence of tumor. Frozen sections were analyzed by fluorescence microscopy, and five 5-μm sections were obtained every 150 μm. In each of the five sections, the colonies of SP-DiI–labeled MSCs present throughout the tumor mass at 100× magnification were counted. Because it was difficult to distinguish single cells versus groups of cells, high-signal areas were counted

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as one colony. The median colony count for the five sections was calculated for each mouse at days 1, 4, 7, and 10 after intra-arterial injection. To determine whether MSCs were present in other organs, we performed intracarotid injections on mice and killed them at days 1, 4, 7, and 10 after injection (n = 3 mice at each time point). The lungs, livers, and spleens were removed and analyzed by fluorescence microscopy.

Statistical Methods The median colony count of SP-DiI–labeled MSCs detected in the tumor sections at different time points was compared using the KruskalWallis test. P < .05 was considered significant. The Kaplan-Meier method was used to estimate the time to symptomatic tumor development in mice. The log-rank test was used to compare the distributions, and P < .05 was considered significant. Results

Isolation and Characterization of Ntv-a MSCs MSCs were harvested from the long bones of Ntv-a mice. In the initial passages, the cells grew slowly, but by passage 7, the doubling time was 24 to 36 hours and the cells had a spindle shape consistent with the morphology described for human and other murine MSCs (Figure 1A) [44,45]. Successful selection of MSCs was verified by their differentiation into adipocytes or mineralizing osteocytes or chondrocytes when placed in differentiation medium (Figure 1, B-D). FACS was performed on MSCs at passages 4, 6, 10, and 15 (Figure 2). By passage 6, Sca-1 and CD9 were detected on 98.0% and 99.0% of cells, respectively, and this high expression persisted so that by passage 15, Sca-1 and CD9 were detected on more than 99.6% and 99.5% of cells,

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respectively. CD105 was detected on 75.0% of cells at passage 6 but only 10.0% of cells at passage 10. At passage 6, the hematopoietic markers CD45 and CD11b were detected on 2.9% and 0.9% of the cells, respectively, but by passage 10, these markers were not detected on the cells. The endothelial marker CD31 and the GSC marker CD133 were not detected at any passages. Thus, based on their morphology, pattern of surface marker expression, and differentiation capacity, the cells isolated from the bone marrow of the Ntv-a mice were consistent with MSCs.

BLI Evaluation of MSCs after Intra-arterial Delivery Animals were analyzed by BLI 1, 4, 7 and 10 days after MSC injection. BLI signal was detected over the right frontal lobe of seven mice at each time point, whereas no signal was seen in two mice (Figure 3, A and B). In the seven mice in which BLI signal was detected after MSC injection, the median BLI signal intensity at day 1 was 6.3 × 105 photons/sec per squared centimeter (range, 2.9 × 104 to 2.6 × 106 photons/sec per squared centimeter). To verify that the signal arose directly from the brain, brains were removed at day 4 or day 7 after MSC injection and were reimaged. In all seven animals with positive BLI signal after live imaging, the BLI signal was verified as originating from the brain (Figure 3, A and B, right image). The median BLI intensity at day 4 was 5.6 × 105 photons/sec per squared centimeter (range, 2.9 × 105 to 1.5 × 107 photons/sec per squared centimeter) and that at day 7 was 2.5 × 105 photons/sec per squared centimeter (range, 2.5 × 104 to 9.8 × 105 photons/sec per squared centimeter). In the mice imaged at day 10 after injection, we did not observe any BLI signal. Two of the three mice in this group demonstrated tumor formation.

Figure 1. Differentiation of Ntv-a–derived MSCs to mesenchymal tissue subtypes. (A) MSCs in stem cell medium after six passages. (B) Bone formation from MSCs after differentiation to osteoblastic cells after culture in an osteogenic medium. (C) Lipid droplet formation from MSCs after differentiation to adipocytes after culture in an adipogenic medium. (D) Cartilage formation from MSCs after differentiation to chondrocytes after culture in a chondroblastic medium.

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tected) demonstrated tumor formation within the right frontal lobe, whereas in the brains of the two animals in which no signal was detected (i.e., MSCs not present), tumors were not identified (Figure 3, C and D). These results demonstrate that Ntv-a MSCs specifically localized to gliomas generated endogenously in Ntv-a transgenic mice. They also provide evidence that the MSCs that reside within the tumor are viable because generation of a signal requires that the cells express functional luciferase. As a further control to show that Ntv-a MSCs localize specifically to tumors, we injected a cohort of mice (n = 4) with RCAS-GFP and performed intra-arterial injections of Ad-Luc–transfected MSCs 6 weeks later. We did not visualize any BLI signal in the brains of these mice, and histological evaluation showed no evidence of tumor. We also evaluated the ability of intra-arterially injected MSCs to migrate to extracranial organs. A cohort of mice (n = 9) was injected with Ad-Luc–transfected MSCs. At days 1, 4, 7, and 10 after injection, mice (n = 3 at each time point) were analyzed for BLI signal. The mice were also killed, and the internal organs (including lungs, liver, spleen, and abdominal viscera) were again analyzed for BLI signal. At none of these time points did we observe BLI consistent with successful migration of Ntv-a MSCs to these extracranial organs.

Histologic Evaluation of Ntv-a MSCs in Endogenous Gliomas Another cohort of Ntv-a mice (n = 9) underwent somatic cell transfer with RCAS–PDGF-B and RCAS-IGFBP2 and after 6 weeks received intracarotid injection of Ntv-a MSCs stained with SP-DiI. High-grade gliomas developed in all nine of the mice in this cohort. SP-DiI–labeled MSCs were detected throughout the tumors but not in nontumoral areas (Figure 4). At day 1, we identified a median of 7 colonies (range, 3-18 colonies) in the five frozen sections obtained from each of the three mice killed at day 1. The median number of MSC colonies was 9 (range, 3 to 43) at day 4 and also 9 (range, 0-21) at day 7. No colonies were identified in any of the tumor-bearing brains obtained from mice 10 days after injection. When the individual time points were compared, the difference between days 1 and 4 was significant (P = .02), but the difference between days 4 and 7 was not significant (P = .1). The difference among the colony counts at all time points was not significant (P = .07). The extent of tumor engraftment by SP-DiI–labeled MSCs seemed to be associated with BLI signal intensity, with both peaking at around day 4 and disappearing by day 10 (Figure 5). We also determined whether SP-DiI–labeled MSCs migrated to extracranial organs by injecting mice and visualizing the lungs, livers, and spleens harvested from injected mice at days 1, 4, 7, and 10 (n = 3 at each time point). In none of these organs did we observe fluorescence suggesting the presence of SP-DiI–labeled MSCs.

Figure 2. Flow cytometric analysis for Ntv-a–derived MSCs. FACS results for MSCs at passages 6 and 15 for the mesenchymal markers Sca-1 and CD9, the endothelial markers CD105 and CD31, the hematopoietic markers CD45 and CD11b, and the GSC marker CD133.

To determine which of the nine mice harbored tumors, the brains were removed and stained with H&E. Because somatic cell transfer of genes through RCAS vectors does not result in tumors in all mice, we were able to use non–tumor-bearing mice as a control for MSC delivery. All seven animals with positive BLI signal (i.e., MSCs were de-

BLI Evaluation of MSCs after Intravenous Delivery Yang et al. [46] have shown that MSCs localize to U87 xenografts after intravenous delivery. Ntv-a MSCs transduced with Ad-Luc were injected into the tail vein of mice 6 weeks after they were injected with RCAS–PDGF-B and RCAS-IGFBP2. BLI signal was not observed in any of the 12 mice at any of the imaging time points (1, 4, 7, and 10 days after MSC injection). Of the subgroups of animals killed on days 1, 4, 7, and 10 after MSC injection, none demonstrated BLI signal in the brain (Figure 3, E and F). Histologic analysis of the brains (H&E) showed that 10 of the 12 mice demonstrated formation of intracranial tumors (Figure 3G). The visceral organs of three of the mice

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were examined with BLI, and a positive signal was identified in the lungs of each of these mice (data not shown).

MSCs Derived from Ntv-a Mice Do Not Enhance Tumorigenicity To determine whether MSCs derived from Ntv-a mice could enhance tumor formation in the PDGF-B–dependent glioma model, we coinjected RCAS–PDGF-B and MSCs in newborn Ntv-a mice. Escalating doses of MSCs were coinjected with RCAS–PDGF-B. The median survival in the cohort of mice injected with RCAS– PDGF-B alone was 77 days (range, 23-90 days). There was no statistical difference in survival between the cohorts of mice coinjected with

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RCAS–PDGF-B and Ntv-a–derived MSCs compared with the control group injected with RCAS–PDGF-B alone. In the cohort coinjected with 1 × 103 MSCs, the median survival was 90 days (range, 35-90 days; log-rank test, P = .13). In the cohort coinjected with 1 × 104 MSCs, the median survival was 64 days (range, 33-90 days; log-rank test, P = .16). In the cohort injected with 1 × 105 MSCs, the median survival was 67 days (range, 41-90 days; log-rank test, P = .4). Discussion In this report, we exploit the advantages of the RCAS/Ntv-a mouse model system to show for the first time that harvested MSCs are capable of localizing to endogenous high-grade gliomas after intra-arterial

Figure 3. Ad-Luc–transfected MSCs derived from Ntv-a mice track to endogenous brain tumors formed by coexpression of RCAS–PDGF-B and RCAS-IGFBP2 in Ntv-a mice. (A) Whole mouse and brain of mouse at day 1 after intra-arterial MSC injection demonstrating BLI signal over the right frontal region only. (B) Whole mouse and brain of mouse at day 1 after MSC injection. No BLI signal was observed in either the whole mount or the harvested brain. A small, localized area of positive BLI signal was detected over the right ear, a nonspecific finding probably indicating MSCs circulating in the external carotid artery. (C) Coronal section of the brain of the mouse in A, demonstrating tumor in the right frontal lobe of the brain (indicated by arrow). (D) Coronal section (H&E) of the mouse brain in B, without any evidence of tumor formation. (E) Whole mouse at day 1 after intravenous MSC injection, demonstrating absent BLI signal over the right frontal region. (F) Brain of mouse at day 1 after intravenous MSC injection. No BLI signal was observed in the brain. (G) Coronal section (H&E) of the mouse brain in F, demonstrating significant tumor formation (arrow).

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Figure 4. SP-DiI–labeled MSCs distribute throughout endogenously forming gliomas. (A) Whole mount of tumor-bearing mouse brain at day 4 after injection of SP-DiI–labeled MSCs. (B) Photomicrograph (H&E; original magnification, ×200) of glioma induced by RCAS– PDGF-B and RCAS-IGFBP2. (C) Fluorescence photomicrograph (original magnification, ×200) of tumor section, demonstrating SP-DiI– labeled MSCs (red cells). (D) Merged fluorescence photomicrograph (original magnification, ×200) showing distribution of MSCs (red cells) throughout the tumor mass (counterstained blue with DAPI). Scale bars, 100 μm.

delivery. BLI confirmed that the MSCs in the tumors were viable, and histologic studies showed that MSCs display tumor-specific tropism because we did not observe MSCs outside the tumor in the normal brain in this model. The demonstration of the tumor-tropic capacity of MSCs for endogenous gliomas that develop in their native microenvironment provides crucial support for the clinical translation of the strategy of using MSCs to deliver therapeutic agents to human patients with malignant gliomas. Because previous studies have used engrafted tumors, the impact of the stroma on systemically delivered MSCs, particularly from a different species, has been unclear. Our results, however, definitively demonstrate that bone marrow–derived MSCs can be autotransfused into

the donor and that these MSCs localize to tumors that arise endogenously within the natural milieu of the brain; thus, the tropism of MSCs for gliomas is not a result of an artificial interaction between the MSCs, the tumor, and the microenvironment. We used a model of high-grade glioma induced by overexpression of PDGF-B and IGFBP2 to study tumor engraftment by MSCs. Tumors arising from this combination of genes recapitulate the phenotypic features of high-grade glioma, particularly microvascular proliferation. The development of abnormal blood vessels may be critical for MSCs to localize to gliomas, as previous work from our group has suggested that areas of neovascularization in tumors may be necessary for MSCs to gain entry into the tumor after intravascular delivery [22]. The use of

Figure 5. Tumor engraftment by MSCs declines after day 4. (A) Median BLI signal detected in the brains of tumor-bearing Ntv-a mice at days 1, 4, 7, and 10 after intra-arterial Ad-Luc MSC injection. (B) Median SP-DiI–labeled MSC colony count in the brains of tumor-bearing Ntv-a mice at days 1, 4, 7, and 10 after intra-arterial SP-DiI–labeled MSC injection.

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PDGF-B to induce tumors is also of interest because we have recently shown that PDGF-BB may be a critical mediator of MSC tropism to gliomas [20,21]. Because the RCAS/Ntv-a glioma model is dependent on PDGF-B to initiate tumor formation, it is possible that recruitment of MSCs to the tumor was facilitated by PDGF-B expression in the tumor. Further studies are needed to decipher the specific mechanism underlying the tropism of MSCs for gliomas. Nevertheless, the potential for PDGF-B to mediate the tropism of MSCs for gliomas is relevant to the human disease because approximately a third of gliomas are defined by the PDGF pathway activation [35]. Thus, the model described herein to study the migration of MSCs to brain tumors is relevant to a significant subset of human glioma patients. Another strength of our study is that the time between RCAS vector injection and the introduction of MSCs was at least 6 weeks. Because these tumors were formed well in advance of MSC injection, it is unlikely that the MSC migration observed in this study is a consequence of the known ability of MSCs to track to areas of injury [47]. Because all other studies to date have relied on intracranial injection of tumor cell lines, it has always been uncertain whether the injury induced by the injection was at least partly responsible for the localization of MSCs to glioma. Because of the long latency in this study between injection of the RCAS vector and injection of the MSCs, this is the first study to demonstrate MSC migration to gliomas that is independent of tissue injury in the brain. Thus, our study shows that the tropism of MSCs for gliomas is a direct response to the tumor and not to brain injury. Successful harvesting of functional MSCs from Ntv-a mice is also significant because these mice are a mixture of different genetic backgrounds and supports the translational application of this therapeutic strategy to humans. In an extensive report describing the isolation of MSCs from five independent strains of mice, Peister et al. [42] noted that each strain demonstrated variable expansion in different types of culture media. Ntv-a mice are derived from four strains: C57BL/6, BALB/C, FVB/N, and CD1. We determined that the optimal growth medium was α-MEM, which resulted in robust cell growth. Although previous studies have shown that hMSCs migrate to xenograft tumors in mice, we are the first, to our knowledge, to show that it is possible to obtain MSCs from a mouse, expand them in culture, and deliver these MSCs into the same mouse bearing an endogenously formed intracranial glioma. This strategy exactly mimics the approach that will be used in humans and, therefore, is clinically important. The intra-arterial route of delivery was required for a successful systemic delivery of MSCs to brain tumors in our model. We observed BLI signal in the region of the tumor (i.e., the right frontal lobe) from Ad-Luc MSCs in 100% of mice harboring tumors. As we have demonstrated previously, BLI can be used to track murine MSCs in brain tumors in vivo [16]. We were able to demonstrate BLI signal in tumorbearing mice through day 7 after injection but not at day 10. We suspect that the cells lose their viability over time, and this observation is supported by our work showing only rare visualization of MSCs beyond 14 days after injection of human MSCs into nude mice bearing U87 xenografts [unpublished data]. Because the decline in the MSC activity over time has been observed in both the immunocompetent RCAS/Ntv-a and immunodeficient systems, the loss of viability appears to be independent of the immune status of the host. This may be an advantage to using MSCs as delivery vehicles because they may deliver a therapeutic agent and then gradually disappear. Although a positive BLI signal verified delivery of MSCs to the region of the tumor (i.e., right frontal area), we relied on SP-DiI–labeled MSCs to confirm engraftment of MSCs throughout the tumor mass.

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Mice injected with SP-DiI–labeled MSCs after RCAS vector injection had labeled MSCs distributed throughout the tumor, but these did not appear in non–tumor-bearing areas. The number of MSCs engrafted into the tumor declined after day 4, consistent with our observation from the mice injected with Ad-Luc MSCs that these cells have decreased viability over time. This result confirms the tumor-specific tropism of MSCs and their ability to insinuate themselves throughout the tumor mass. In contrast, intravenous delivery of MSCs did not result in successful engraftment of Ad-Luc MSCs. We did detect Ad-Luc MSCs in the thoracic cavity of the animals, and we interpret this result to mean that MSCs were trapped in the lung parenchyma. It is possible that if we had imaged at later time points, MSCs would have been able to engraft the tumor after passing into the arterial circulation. We suspect, however, that this would be a much smaller population of cells compared with intraarterial delivery and that their viability would be questionable. Other investigators have had success with intravenous delivery of MSCs. For example, Yang et al. [46] recently demonstrated that hMSCs migrate to brainstem glioma xenografts in nude mice after intravenous injection. However, in other studies using human neural stem cells to track to intracranial xenografts in nude mice, migration to tumors was observed after intravenous delivery but at a much lower efficiency compared with direct intratumoral injection [10]. Although our group has had success with intra-arterial delivery of hMSCs, we have been unable to demonstrate effective delivery of hMSCs through intravenous injection to intracranial xenografts in nude mice [16]. The ability of MSCs to be engineered to deliver therapeutic agents has been well described. MSCs have been engineered to secrete a variety of therapeutic proteins, including interferon β, S-TRAIL, and others [16,22,46,48–50]. These studies have demonstrated that delivery of these agents through MSCs confers a survival benefit compared with control animals. Virtually all of these studies have relied on xenograft models in immunocompromised mice, however. Limitations to using gene-based therapies through MSC delivery remain, however, particularly with respect to the successful expression of a potentially therapeutic gene from a short-lived MSC. Although our study was not designed to evaluate a therapeutic benefit from MSC-delivered therapy, the results suggest that such a strategy would be viable in endogenous tumors in an immunocompetent host. Studies evaluating the therapeutic benefit of MSCs in the RCAS/Ntv-a glioma model are currently underway. Conclusions In this study, MSCs from Ntv-a mice were successfully derived from a heterogeneous population of bone marrow cells. These cells were carried from harvest, and characterization through injection and engraftment of tumors generated endogenously in an immunocompetent host. These results lend credence to the concept of using MSCs as delivery vehicles for glial neoplasms in humans. Acknowledgments The authors thank Karen Muller and David M. Wildrick PhD, for editing the article. References [1] Cairncross G, Berkey B, Shaw E, Jenkins R, Scheithauer B, Brachman D, Buckner J, Fink K, Souhami L, Laperierre N, et al. (2006). Phase III trial of chemotherapy plus radiotherapy compared with radiotherapy alone for pure and mixed anaplastic oligodendroglioma: Intergroup Radiation Therapy Oncology Group Trial 9402. J Clin Oncol 24, 2707–2714.

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[2] Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, et al. (2005). Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352, 987–996. [3] Lang FF, Bruner JM, Fuller GN, Aldape K, Prados MD, Chang S, Berger MS, McDermott MW, Kunwar SM, Junck LR, et al. (2003). Phase I trial of adenovirusmediated p53 gene therapy for recurrent glioma: biological and clinical results. J Clin Oncol 21, 2508–2518. [4] Ahmed AU, Alexiades NG, and Lesniak MS (2010). The use of neural stem cells in cancer gene therapy: predicting the path to the clinic. Curr Opin Mol Ther 12, 546–552. [5] Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, and Andreeff M (2002). Bone marrow–derived mesenchymal stem cells as vehicles for interferon-β delivery into tumors. Cancer Res 62, 3603–3608. [6] Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira-Hansen M, Bekele BN, Champlin RE, and Andreeff M (2004). Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst 96, 1593–1603. [7] Kosztowski T, Zaidi HA, and Quinones-Hinojosa A (2009). Applications of neural and mesenchymal stem cells in the treatment of gliomas. Expert Rev Anticancer Ther 9, 597–612. [8] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, and Marshak DR (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147. [9] Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, and Horwitz E (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8, 315–317. [10] Aboody KS, Brown A, Rainov NG, Bower KA, Liu S, Yang W, Small JE, Herrlinger U, Ourednik V, Black PM, et al. (2000). Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci USA 97, 12846–12851. [11] Ehtesham M, Kabos P, Gutierrez MA, Chung NH, Griffith TS, Black KL, and Yu JS (2002). Induction of glioblastoma apoptosis using neural stem cell– mediated delivery of tumor necrosis factor–related apoptosis-inducing ligand. Cancer Res 62, 7170–7174. [12] Ehtesham M, Kabos P, Kabosova A, Neuman T, Black KL, and Yu JS (2002). The use of interleukin 12–secreting neural stem cells for the treatment of intracranial glioma. Cancer Res 62, 5657–5663. [13] Bianco P, Riminucci M, Gronthos S, and Robey PG (2001). Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19, 180–192. [14] Caplan AI and Bruder SP (2001). Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends Mol Med 7, 259–264. [15] Tocci A and Forte L (2003). Mesenchymal stem cell: use and perspectives. Hematol J 4, 92–96. [16] Nakamizo A, Marini F, Amano T, Khan A, Studeny M, Gumin J, Chen J, Hentschel S, Vecil G, Dembinski J, et al. (2005). Human bone marrow–derived mesenchymal stem cells in the treatment of gliomas. Cancer Res 65, 3307–3318. [17] Nakamura K, Ito Y, Kawano Y, Kurozumi K, Kobune M, Tsuda H, Bizen A, Honmou O, Niitsu Y, and Hamada H (2004). Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther 11, 1155–1164. [18] Sasportas LS, Kasmieh R, Wakimoto H, Hingtgen S, van de Water JA, Mohapatra G, Figueiredo JL, Martuza RL, Weissleder R, and Shah K (2009). Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer therapy. Proc Natl Acad Sci USA 106, 4822–4827. [19] Cassiede P, Dennis JE, Ma F, and Caplan AI (1996). Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-beta 1 or PDGF-BB as assayed in vivo and in vitro. J Bone Miner Res 11, 1264–1273. [20] Cheng P, Gao ZQ, Liu YH, and Xue YX (2009). Platelet-derived growth factor BB promotes the migration of bone marrow–derived mesenchymal stem cells towards C6 glioma and up-regulates the expression of intracellular adhesion molecule-1. Neurosci Lett 451, 52–56. [21] Hata N, Shinojima N, Gumin J, Yong R, Marini F, Andreeff M, and Lang FF (2010). Platelet-derived growth factor BB mediates the tropism of human mesenchymal stem cells for malignant gliomas. Neurosurgery 66, 144–156, discussion 156–157. [22] Yong RL, Shinojima N, Fueyo J, Gumin J, Vecil GG, Marini FC, Bogler O, Andreeff M, and Lang FF (2009). Human bone marrow–derived mesenchymal stem cells for intravascular delivery of oncolytic adenovirus Delta24-RGD to human gliomas. Cancer Res 69, 8932–8940.

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[23] de Vries NA, Beijnen JH, and van Tellingen O (2009). High-grade glioma mouse models and their applicability for preclinical testing. Cancer Treat Rev 35, 714–723. [24] Hu X and Holland EC (2005). Applications of mouse glioma models in preclinical trials. Mutat Res 576, 54–65. [25] Wakimoto H, Kesari S, Farrell CJ, Curry WT Jr., Zaupa C, Aghi M, Kuroda T, Stemmer-Rachamimov A, Shah K, Liu TC, et al. (2009). Human glioblastoma– derived cancer stem cells: establishment of invasive glioma models and treatment with oncolytic herpes simplex virus vectors. Cancer Res 69, 3472–3481. [26] Garber K (2006). Realistic rodents? Debate grows over new mouse models of cancer. J Natl Cancer Inst 98, 1176–1178. [27] Garber K (2009). From human to mouse and back: “tumorgraft” models surge in popularity. J Natl Cancer Inst 101, 6–8. [28] Burns TC, Verfaillie CM, and Low WC (2009). Stem cells for ischemic brain injury: a critical review. J Comp Neurol 515, 125–144. [29] Holland EC, Hively WP, DePinho RA, and Varmus HE (1998). A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice. Genes Dev 12, 3675–3685. [30] Becher OJ and Holland EC (2006). Genetically engineered models have advantages over xenografts for preclinical studies. Cancer Res 66, 3355–3358, discussion 3358–3359. [31] Holland EC, Li Y, Celestino J, Dai CK, Schaefer L, Sawaya RA, and Fuller GN (2000). Astrocytes give rise to oligodendrogliomas and astrocytomas after gene transfer of polyoma virus middle T antigen in vivo. Am J Pathol 157, 1031–1037. [32] Rao G, Pedone CA, Coffin CM, Holland EC, and Fults DW (2003). c-Myc enhances sonic hedgehog–induced medulloblastoma formation from nestinexpressing neural progenitors in mice. Neoplasia 5, 198–204. [33] Rao G, Pedone CA, Valle LD, Reiss K, Holland EC, and Fults DW (2004). Sonic hedgehog and insulin-like growth factor signaling synergize to induce medulloblastoma formation from nestin-expressing neural progenitors in mice. Oncogene 23, 6156–6162. [34] Dunlap SM, Celestino J, Wang H, Jiang R, Holland EC, Fuller GN, and Zhang W (2007). Insulin-like growth factor binding protein 2 promotes glioma development and progression. Proc Natl Acad Sci USA 104, 11736–11741. [35] Brennan C, Momota H, Hambardzumyan D, Ozawa T, Tandon A, Pedraza A, and Holland E (2009). Glioblastoma subclasses can be defined by activity among signal transduction pathways and associated genomic alterations. PLoS One 4, e7752. [36] Phillips HS, Kharbanda S, Chen R, Forrest WF, Soriano RH, Wu TD, Misra A, Nigro JM, Colman H, Soroceanu L, et al. (2006). Molecular subclasses of highgrade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell 9, 157–173. [37] Bleau AM, Hambardzumyan D, Ozawa T, Fomchenko EI, Huse JT, Brennan CW, and Holland EC (2009). PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem–like cells. Cell Stem Cell 4, 226–235. [38] Charles N, Ozawa T, Squatrito M, Bleau AM, Brennan CW, Hambardzumyan D, and Holland EC (2010). Perivascular nitric oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells. Cell Stem Cell 6, 141–152. [39] Kong LY, Wu AS, Doucette T, Wei J, Priebe W, Fuller GN, Qiao W, Sawaya R, Rao G, and Heimberger AB (2011). Intratumoral mediated immunosuppression is prognostic in genetically engineered murine models of glioma and correlates to immunotherapeutic responses. Clin Cancer Res 16, 5722–5733. [40] Binning MJ, Niazi T, Pedone CA, Lal B, Eberhart CG, Kim KJ, Laterra J, and Fults DW (2008). Hepatocyte growth factor and sonic Hedgehog expression in cerebellar neural progenitor cells costimulate medulloblastoma initiation and growth. Cancer Res 68, 7838–7845. [41] Hu X, Pandolfi PP, Li Y, Koutcher JA, Rosenblum M, and Holland EC (2005). mTOR promotes survival and astrocytic characteristics induced by Pten/AKT signaling in glioblastoma. Neoplasia 7, 356–368. [42] Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, and Prockop DJ (2004). Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103, 1662–1668. [43] Dai C, Celestino JC, Okada Y, Louis DN, Fuller GN, and Holland EC (2001). PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo. Genes Dev 15, 1913–1925.

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[44] Pittenger MF and Marshak DR (2001). Mesenchymal stem cells of human adult bone marrow. In Stem Cell Biology. DR Marshak, RL Gardner, and D Gottlieb (Eds). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp. 349–373. [45] Azizi SA, Stokes D, Augelli BJ, DiGirolamo C, and Prockop DJ (1998). Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—similarities to astrocyte grafts. Proc Natl Acad Sci USA 95, 3908–3913. [46] Yang B, Wu X, Mao Y, Bao W, Gao L, Zhou P, Xie R, Zhou L, and Zhu J (2009). Dual-targeted antitumor effects against brainstem glioma by intravenous delivery of tumor necrosis factor–related, apoptosis-inducing, ligand-engineered human mesenchymal stem cells. Neurosurgery 65, 610–624, discussion 624.

Doucette et al.

725

[47] Karp JM and Leng Teo GS (2009). Mesenchymal stem cell homing: the devil is in the details. Cell Stem Cell 4, 206–216. [48] Hong X, Miller C, Savant-Bhonsale S, and Kalkanis SN (2009). Antitumor treatment using interleukin-12–secreting marrow stromal cells in an invasive glioma model. Neurosurgery 64, 1139–1146, discussion 1146–1147. [49] Menon LG, Kelly K, Yang HW, Kim SK, Black PM, and Carroll RS (2009). Human bone marrow–derived mesenchymal stromal cells expressing S-TRAIL as a cellular delivery vehicle for human glioma therapy. Stem Cells 27, 2320–2330. [50] Sonabend AM, Ulasov IV, Tyler MA, Rivera AA, Mathis JM, and Lesniak MS (2008). Mesenchymal stem cells effectively deliver an oncolytic adenovirus to intracranial glioma. Stem Cells 26, 831–841.

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