genetherapyusingtrail …cancerres.aacrjournals.org/content/canres/68/23/9614.full.pdf · seong muk...

Gene Therapy Using TRAIL-Secreting Human Umbilical Cord Blood–

Derived Mesenchymal Stem Cells against Intracranial Glioma

Seong Muk Kim,1Jung Yeon Lim,

1Sang In Park,

1Chang Hyun Jeong,

1Ji Hyeon Oh,

1

Moonsup Jeong,1,4

Wonil Oh,3Sang-Hoon Park,

5Young-Chul Sung,

5and Sin-Soo Jeun

1,2

1Department of Biomedical Science, College of Medicine, and 2Department of Neurosurgery, Kangnam St. Mary’s Hospital, The CatholicUniversity of Korea; 3Medipost Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul, Korea; 4Research Laboratories of Dong-APharmaceutical Co., Ltd., Yongin, Korea; and 5Division of Molecular and Life Sciences, Pohang University of Science andTechnology, Pohang, Korea

Abstract

Adenovirus-mediated gene therapies against brain tumorshave been limited by the difficulty in tracking glioma cellsinfiltrating the brain parenchyma. Human umbilical cordblood–derived mesenchymal stem cells (UCB-MSC) areparticularly attractive cells for clinical use in cell-basedtherapies. In the present study, we evaluated the tumortargeting properties and antitumor effects of UCB-MSCs asgene delivery vehicles for glioma therapy. We efficientlyengineered UCB-MSCs to deliver a secretable trimeric formof tumor necrosis factor-related apoptosis-inducing ligand(stTRAIL) via adenoviral transduction mediated by cell-permeable peptides. We then confirmed the migratorycapacity of engineered UCB-MSCs toward tumor cells by anin vitro migration assay and by in vivo injection of UCB-MSCsinto the tumor mass or the opposite hemisphere of establishedhuman glioma in nude mice. Moreover, in vitro coculture,experiments on Transwell plates, and in vivo survival experi-ments showed that MSC-based stTRAIL gene delivery has moretherapeutic efficacy compared with direct injection ofadenovirus encoding the stTRAIL gene into a tumor mass.In vivo efficacy experiments showed that intratumoralinjection of engineered UCB-MSCs (MSCs-stTRAIL) signifi-cantly inhibited tumor growth and prolonged the survival ofglioma-bearing mice compared with controls. These resultssuggest that human UCB-MSCs have potential use as effectivedelivery vehicles for therapeutic genes in the treatment ofintracranial glioma. [Cancer Res 2008;68(23):9614–23]

Introduction

Glioblastomas are the most common primary malignant braintumors in humans. The prognosis of patients with malignantgliomas is extremely poor because gliomas are refractory toconventional therapies, such as extensive surgical resection,radiation, and chemotherapy (1). Therapeutic gene delivery withviral vectors by direct injection into the primary brain tumor orpostoperative tumor cavity has failed to reach outgrowing tumorislands because of the migratory abilities of the tumor cells andtheir infiltration into the normal brain parenchyma (2). Neweffective therapeutic tools are needed that specifically target tumorcells, especially those cells that have escaped the main tumor mass.

Recent studies suggest that stem cells can be used as vehiclesfor delivering therapeutic genes to treat brain tumors (3–5). Neuralstem cells exhibit extensive tropism for experimental gliomasand migrate toward outgrowing microsatellites (6–9). However,the clinical application of neural stem cells is limited by ethicalproblems associated with their isolation and immunologicincompatibility in allogenic transplantation. Additionally, a newtherapeutic strategy has been developed that uses mesenchymalstem cells (MSC) for the targeted delivery and local production ofbiological agents in tumors (10, 11). These reports imply that MSCsare particularly attractive for clinical use because they have tumortargeting properties, can be easily isolated and expanded to thenumbers required for use, and can be genetically manipulated withviral vectors.Human umbilical cord blood (UCB) is an alternative source of

adult stem cells. Several studies indicate that UCB-derived MSCs(UCB-MSC) are similar to stem cells from bone marrow withrespect to cell characteristics and multilineage differentiationpotential (12–15). In a previous study, we isolated multipotentUCB-MSCs that have the capacity to differentiate into severalmesodermal tissues (bone, cartilage, tendon, muscle, and adipose),endodermal tissue (hepatocyte), and ectodermal tissue (neurons;refs. 16, 17). Recently, UCB-MSCs were proved to be moreadvantageous in cell procurement, storage, and transplantationthan bone marrow–derived MSCs (18). Moreover, the number anddifferentiation ability of bone marrow–derived MSCs significantlydecrease with age (19). Cells in UCB, or neonatal blood, are lessmature than adult cells and they do not trigger an immenseimmune reaction in unrelated donor transplantation (12, 20). Thesecharacteristics make UCB-MSCs potent candidates for the clinicalapplication of allogenic MSC–based therapies.Tumor necrosis factor-related apoptosis-inducing ligand

(TRAIL)–based cancer therapies, which involve treatment withrecombinant TRAIL (rTRAIL) or an adenovirus bearing the TRAILgene, against glioma have been shown (21, 22); however, clinicaltrials of these therapies are limited. There are potential issues oftoxicity and protein half-life in patients treated with high-doserTRAIL, and in adenoviral gene therapy, limitations arise from therelatively short survival time of the virus caused by an immunereaction (23) and by outgrowing glioma cells infiltrating the brainparenchyma. The artificial TRAIL gene [secretable trimeric TRAIL(stTRAIL)], which encodes a fusion protein composed of threefunctional elements, a secretion signal, a trimerization domain, andan apoptosis-inducing moiety of the TRAIL gene sequence, hasbeen developed (24). Adenoviral vectors delivering the stTRAILgene (Ad-stTRAIL) showed higher tumor suppressor activity thanadenoviral vectors delivering the full-length TRAIL gene in vitroand in vivo (25). To extend the release time of stTRAIL and deliver

Requests for reprints: Sin-Soo Jeun, Department of Neurosurgery, Kangnam St.Mary’s Hospital, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul137-701, Korea. Phone: 822-590-2568; Fax: 822-3482-1853; E-mail: [email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-08-0451

Cancer Res 2008; 68: (23). December 1, 2008 9614 www.aacrjournals.org

Research Article

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it to infiltrating tumor cells, we evaluated UCB-MSCs secretingstTRAIL as delivery vehicles for brain tumor therapy.In addition, most of the replication-deficient adenoviral vectors

that have been used to transduce MSCs are based on humanadenovirus serotype 5 (Ad5). Cell entry of Ad5-based vectors ismediated through a receptor-mediated biphasic process thatinvolves primary attachment to the cellular coxsackie-adenovirusreceptor (CAR; ref. 26) and internalization via interaction withintegrins present in target cells (27). However, transduction ofMSCs by conventional Ad5 vectors is inefficient even when veryhigh multiplicities of infection (MOI) are used because MSCs donot express CAR (28). Therefore, the development of methods thatachieve comparable adenoviral gene delivery with substantiallylower viral doses is highly desirable to eventually develop therapiesfor the treatment of human glioma. One way to increase cell entryof adenovirus vectors may be through cell-permeable peptides[protein transduction domain (PTD)], small polybasic peptidesderived from the transduction domains of certain proteins thatcross the cell membrane through a receptor-independent mecha-nism (29–31). These cell-permeable peptides have been used tointroduce biologically active cargo molecules, such as DNA,peptides or proteins, and viruses, into cells. Recently, we developeda novel PTD (4HP4), which could significantly enhance adenoviraltransduction into MSCs (32).In the present study, we show that human UCB-MSCs display

tropism for human glioma and that the treatment of stTRAIL-secreting UCB-MSCs has significant antitumor effects comparedwith adenoviral TRAIL gene therapy.

Materials and Methods

Culture of human UCB-MSCs and other cell lines. Human UCB

harvest and expansion of MSCs isolated from UCB were conducted as

previously reported (16). The separated MSCs were subcultured at aconcentration of 5 � 104/cm2 in a-MEM (Invitrogen) and used for

experiments during passages 5 to 8. U-87MG, U-251MG, A172, NIH3T3, and

293 cells were obtained from the American Type Culture Collection.

U-87MG and 293 cells were maintained in Eagle’s MEM (Invitrogen), andU-251MG, A172, and NIH3T3 cells in DMEM (Invitrogen). Normal human

astrocytes were obtained from the Applied Cell Biology Research Institute

and cultured in DMEM. Enhanced green fluorescent protein (EGFP)–expressing U-87MG cells (U87-EGFP) were derived from stable transfection

of U-87MG cells with pEGFP-N1 vector (BD Biosciences) and the stable

transfectants were selected with neomycin analogue G418 (Invitrogen). All

media were supplemented with 2 mmol/L L-glutamine, 100 units/mLpenicillin, 100 Ag/mL streptomycin, and 10% fetal bovine serum (FBS)

purchased from Invitrogen. Cells were incubated at 37jC in a humidified

atmosphere containing 5% CO2.

Western blotting. UCB-MSCs or 293 cells were lysed in a radio-immunoprecipitation assay (RIPA) buffer (Sigma) containing a protease

inhibitor cocktail (Roche). Proteins were separated on a 10% SDS-PAGE and

transferred onto a nitrocellulose transfer membrane (Whatman). After

blocking the membrane with TBS-0.1% Tween 20 containing 5% skim milkfor 1 h at room temperature, the membrane was incubated with rabbit anti-

human CAR antibody (Santa Cruz Biotechnology) overnight at 4jC. Themembrane was incubated for 1 h at room temperature with horseradishperoxidase–conjugated secondary antibody (Santa Cruz Biotechnology) and

the bands were detected using Amersham enhanced chemiluminescence

detection reagents (GE Healthcare Life Sciences).

Adenoviral vectors and transduction conditions. The recombinantreplication-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 stTRAIL gene (Ad-stTRAIL) was engineered as

described previously (25). Ad-c5 was used as a control. Peptide (4HP4) wassynthesized by Peptron.6 To transfect UCB-MSCs or U-87MG cells,

adenoviruses at a specified MOI were pretreated with 4HP4 in serum-free

medium for 30 min at room temperature, and then cells were incubated

with the premixed virus-4HP4 complex for 30 min, washed twice with PBS,and changed to the original growth medium. EGFP expression was analyzed

by flow cytometry using the FACSCalibur system (Becton Dickinson Co.)

or a fluorescence microscope (Axiovert 200, Carl Zeiss).

Animals and brain tumor model.Male athymic nude mice (6–8 wk old;Charles River Laboratories) were used in accordance with institutional

guidelines under the approved protocols. For the intracranial xenografts of

human glioma, animals were anesthetized with ketamine/xylazine i.p. and

stereotactically inoculated with 1 � 105 U-87MG or U87-EGFP cells (in 3 ALPBS) into the right frontal lobe (2 mm lateral and 1 mm anterior to bregma,

at 2.5 mm depth from the skull base) via a Hamilton syringe (Hamilton

Company) using a microinfusion pump (Harvard Apparatus).In vitro migration studies. The migratory ability of UCB-MSCs was

determined using Transwell plates (Corning Costar) that were 6.5 mm in

diameter with 8-Am pore filters. Cells were incubated at three different

concentrations of (1 � 105, 5 � 105, and 1 � 106) in serum-free medium for48 h and the resulting conditioned media were used as chemoattractants.

MSCs or MSCs-stTRAIL cells (2 � 104) were suspended in serum-free

medium containing 0.1% bovine serum albumin (Sigma) and seeded into

the upper well; 600 AL of conditioned medium were placed in the lower wellof the Transwell plate. Following incubation for 5 h at 37jC, cells that hadnot 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 had migrated to the lower side

of the filter was counted under a light microscope with five high-power

fields (�400). Experiments were done in triplicate.

In vivo migration studies. Seven days after tumor cell inoculation intothe right frontal lobe, EGFP-expressing MSCs (MSCs-EGFP; 2 � 105) were

implanted into the opposite hemisphere (2 mm lateral and 1 mm anterior

to bregma, at a depth of 2.5 mm from the skull base), or PKH26 (Sigma)–

labeled MSCs-stTRAIL were implanted into the tumor mass. Migrationtoward the tumor was assessed at 4, 7, or 10 d after MSCs inoculation

by direct visualization with a fluorescence microscope or a confocal

microscope (LSM 510 Meta, Carl Zeiss).Assessment of cell viability and terminal deoxyribonucleotidyl

transferase–mediated dUTP nick end labeling staining. UCB-MSCs or

U-87MG cells (5 � 104) were seeded in 24-well plates, and increasing

amounts of Ad-stTRAIL or recombinant human TRAIL (rhTRAIL; R&DSystems) were added to confirm TRAIL tumor-specific cytotoxicity. At 2 or

3 d after treatment, cells were analyzed for viability by the 3-(4,5-dimethyl-

thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H -tetrazolium

(MTS) assay (Promega). For coculture experiments, MSCs-stTRAIL or U87-stTRAIL cells were plated in the Transwell inserts containing 0.4-Am pores

(Corning Costar) with increasing cell concentrations (0.2 � 104, 0.5 � 104,

1 � 104, 2 � 104, and 4 � 104 per well) and then U-87MG cells (2 � 104)

were grown in the lower well of the Transwell plates. For inhibition studies,MSCs-stTRAIL (2 � 104) were seeded in the upper well and U-87MG cells

(2 � 104) were seeded in the lower well of the Transwell plates, and then

neutralizing antihuman TRAIL antibody (R&D Systems) was added to thelower wells as indicated in the figure. After 5 d, the viability of U-87MG cells

in the lower well was analyzed by MTS assay. All experiments were

conducted in triplicate. To detect apoptotic activity, MSCs-stTRAIL and

U-87MG cells (2 � 104 each) were premixed and cultured in four-wellchamber slides (Nalge Nunc International) for 48 h and then stained using

a terminal deoxyribonucleotidyl transferase–mediated dUTP nick end

labeling (TUNEL) assay kit (Roche) developed with Cy3-conjugated

streptavidin (Jackson ImmunoResearch Laboratories).Flow cytometry for TRAIL death receptors. Cells were analyzed for the

surface expression of TRAIL death receptors with phycoerythrin-conjugated

6 http://www.peptron.com

TRAIL-Secreting MSCs in Glioma Therapy

www.aacrjournals.org 9615 Cancer Res 2008; 68: (23). December 1, 2008



antihuman DR4, DR5, DcR1, and DcR2 (R&D Systems). Briefly, cells

(2.5 � 105) were stained with each antibody on ice for 30 min. After washing

with PBS, the expressions of these death receptors were analyzed by flowcytometry using the FACSVantage SE (Becton Dickinson Co.).

ELISA for expressed stTRAIL. stTRAIL protein secreted into the culturesupernatants or brain tissues was analyzed by ELISA, as describedpreviously (25). To investigate the persistence of transgene expression

in vitro , UCB-MSCs or U-87MG cells were seeded at a high density (4 � 104

per well of a 24-well plate) and transduced with Ad-stTRAIL. The virus-

containing medium was removed and additionally incubated in low-serummedium (a-MEM or Eagle’s MEM containing 2% FBS). Culture supernatants

were harvested and changed with fresh medium every 3 d, and secreted

TRAIL was assessed at various time intervals. For the analysis of stTRAIL

expressed in tumor-bearing mice in vivo , tumor tissues were harvested andlysed in RIPA buffer at 1, 4, 7, 10, and 14 d after treatment of MSCs-stTRAIL.

Treatment of mouse experimental glioma. To evaluate the therapeuticeffects of MSCs-stTRAIL in vivo , a single intratumoral (i.t.) injection was

conducted at 7 d after tumor inoculation. Tumor-bearing mice wereinjected with MSCs-EGFP or MSCs-stTRAIL (2 � 105 cells in 5 AL PBS), PBS(5 AL), and Ad-stTRAIL (1 � 1010 viral particles; this is safe in mice and also

a therapeutically effective viral dose) for the survival experiment. To assessinhibition of tumor growth, tumor-bearing mice were injected with

unmodified MSCs or MSCs-stTRAIL (2 � 105 cells in 5 AL of PBS) and

treated with PBS (5 AL).Evaluation of tumor size by histologic analysis. Tumor size was

determined as described previously (33). Briefly, brains with therapeutic

treatment at a specific time point after tumor inoculation were serially

sectioned (20 Am, obtained every 200 Am into the tumor) and then stained

with H&E. The section with the maximum tumor area was identified and

the number of pixels in the delineated area was calculated via a computerusing the NIH Image software.7 The pixel count was then normalized to the

original dimensions (square millimeter) of the scanned sections.

Immunohistochemistry and in vivo apoptosis assay. Mouse brainswere perfused with PBS followed by 4% paraformaldehyde under deep

anesthesia at a specific time point after MSCs-stTRAIL inoculation. The

excised brains were postfixed overnight and then equilibrated in PBS

containing 30% sucrose for 2 d. Fixed brains were embedded, snap frozen inliquid nitrogen, and stored at �70jC until use. Tissues were cryosectioned

(14 Am) and then stained with primary antibodies for anti-TRAIL (R&D

Systems) or antihuman nuclei (Chemicon) using the Vector M.O.M.

Immunodetection Kit (Vector Laboratories). The primary antibodies weredetected either Cy3- or fluorescein-conjugated streptavidin (Jackson

ImmunoResearch Laboratories). To detect apoptotic activity, TUNEL

staining was done as described above. In some sections, nuclei were

stained with 4¶,6-diamidino-2-phenylindole (DAPI; Sigma) for counter-staining.

Statistical analysis. All data are expressed as mean F SE. Statistical

differences between different test conditions were determined by Student’st test. P < 0.05 was considered significant. Statistical analysis of survival

was done using log-rank test.

7 http://rsbweb.nih.gov

Figure 1. Effect of 4HP4 on adenoviral transduction into UCB-MSCs. A, CAR expression was determined by Western blot analysis. Cell lysate from the 293 cell lineserved as a positive control. B, the effect of 4HP4 on transduction efficiency of UCB-MSCs infected with Ad-stTRAIL was evaluated. Cells were transduced atvarious MOIs without or with increasing concentrations of 4HP4. At day 3 after infection, the concentration of secreted TRAIL in culture supernatant was analyzed byELISA (a) and the viability of transduced cells was analyzed via MTS assay (b). Columns and points, mean; bars, SE. C, the persistence of transgene expressionin UCB-MSCs or U-87MG cells transduced with Ad-stTRAIL (MOI 20) containing 4HP4 (0.01 Amol/L) was analyzed under conditions of restrained cell division.The concentration of secreted TRAIL was assessed by ELISA at each time point. Columns, mean; bars, SE.

Cancer Research




Results

Effect of PTD on transduction efficiency of UCB-MSCs withadenovirus. Human primary MSCs are relatively resistant to wild-type adenoviral infection because of their low expression level ofthe adenoviral receptor CAR. Thus, we first confirmed whetherhuman UCB-MSCs used in this study expressed CAR protein. Theexpression of CAR from UCB-MSCs was not detected by Westernblot analysis (Fig. 1A). Therefore, we conducted PTD-mediatedadenoviral transduction as a CAR-independent infection method,and the gene delivery efficiency of Ad-stTRAIL into UCB-MSCswas determined (Fig. 1B, a). We found that transduction ofUCB-MSCs could be greatly enhanced by incorporation of 4HP4(0.003–0.03 Amol/L) into the Ad-stTRAIL infection medium.However, along with increased MOI and 4HP4 concentration, celldeath was also augmented, possibly reflecting adenovector cyto-pathogenic effects and/or cell damage due to excessive transgeneexpression (Fig. 1B, b). Taken together, a 4HP4 concentration of0.01 Amol/L and relatively low doses of adenoviruses achievedhigh-level production of proteins without affecting cell viabilityand rendered optimal transduction efficiency of UCB-MSCs.Additionally, a significant dose-dependent increase in the numberof GFP-positive cells was detected, and enhanced transductionefficiency was evident on visual inspection when the Ad-EGFPinfection was carried out in the presence of 0.01 Amol/L 4HP4 (datanot shown). In addition, the persistence of transgene expression inMSCs-stTRAIL or U87-stTRAIL cells (U-87MG cells transduced withAd-stTRAIL under the same conditions as UCB-MSCs; however,the transduction efficiency of U-87MG was not affected byincorporation of 4HP4) was studied under conditions of restrainedcell division. ELISA analysis at different time points aftertransduction revealed that the concentration of secreted TRAILremained fairly constant for 23 days and then began to decline inMSCs-stTRAIL; however, a decrease occurred in U87-stTRAIL cells

after day 8 (Fig. 1C). Based on these results, we used cellsengineered under these transduction conditions (20 MOI of virusesplus 0.01 Amol/L of 4HP4 in infection medium) in all subsequentexperiments.Migratory capacity of UCB-MSCs toward gliomas in vitro

and in vivo. It has been reported that a factor released fromglioma cells may be a potential chemoattractant involved in thetropism of MSCs. To test this in UCB-MSCs, in vitro migrationassays using Transwell plates were conducted. Conditionedmedium from normal human astrocytes was used as a control tobetter mimic the normal brain milieu. Only a few cells migratedtoward serum-free medium and conditioned medium fromfibroblasts or normal human astrocytes, whereas the migrationof MSCs-stTRAIL was significantly (P < 0.001) stimulated byconditioned medium from human glioma cell lines (U-87MG,U-251MG, or A172) compared with conditioned medium fromNIH3T3 cells or astrocytes (Fig. 2A). Moreover, the migratoryactivity of MSCs-stTRAIL was represented in a dose-dependentmanner (Fig. 2B). We also confirmed that unmodified MSCsmigrated to the conditioned medium from human glioma cells in asimilar pattern to MSCs-stTRAIL (data not shown). These resultsindicated that human glioma cells were capable of stimulatingthe migration of MSCs and that the migratory ability of UCB-MSCswas not affected by adenoviral transduction.Next, we investigated whether implanted MSCs could migrate

toward intracranial gliomas in vivo . MSCs-EGFP inoculated intothe contralateral hemisphere to the tumor migrated away from theinitial injection site toward the tumor mass along the corpuscallosum at 7 days after MSCs inoculation (Fig. 3A, a–d), whereasMSCs-EGFP remained within the injection site in the normal brain(Fig. 3A, e–g ). These cells were mostly retained at the corpuscallosum and the border between the tumor and normalparenchyma; they also infiltrated into the tumor bed. We also

Figure 2. Migratory ability of transduced UCB-MSCs in vitro. A, the migratory ability of UCB-MSCs in response to conditioned medium (CM) from different cell lineswas determined using a Transwell plate (8-Am pores). Representative photomicrographs of stained filters show migrated MSCs-stTRAIL. Magnification, �100.B, migration of MSCs-stTRAIL in response to three different concentrations of conditioned medium from three human glioma cell lines (U-87MG, U-251MG, and A172),NIH3T3 cells, or normal human astrocytes was determined. Cell migration was compared and evaluated after staining by taking photographs and counting cells that hadmigrated under a light microscope. Serum-free medium (SFM ) was used as a negative control (horizontal line ). Columns, mean; bars, SE. *, P < 0.001, Student’s t test.





confirmed that MSCs-stTRAIL inoculated into the contralateralhemisphere migrated toward the tumor mass (data not shown).More importantly, PKH26-labeled MSCs-stTRAIL injected directlyinto the tumor bed distributed extensively throughout the tumormass at 4 or 10 days after MSCs inoculation (Fig. 3B). Although theMSCs-stTRAIL remained within the injection site, they largelymigrated at the border of the tumor where it interfaced withnormal tissue (Fig. 3B, b and c). Interestingly, these cells were alsoseen in tumor satellites along the corpus callosum at a distancefrom the main tumor mass (Fig. 3B, d–g ). We showed that themigration of MSCs-stTRAIL occurs within 4 days, with appreciablelevels of TRAIL secretion being present after i.t. administration (seeFig. 6A), and the migrating human MSCs remain at the border ofthe tumor where it interfaced with normal tissue at day 10. We alsoconfirmed that the mock-transduced or nontransduced cellsmigrated toward intracranial gliomas in a similar pattern totransduced MSCs (data not shown). Thus, this result showed that

UCB-MSCs have an excellent migratory capacity and gliomatropism in vivo .TRAIL induces apoptosis in U-87MG cells but not in UCB-

MSCs. To quantitatively assess the cytotoxic effect of TRAIL onU-87MG cells and UCB-MSCs, we cultured these cells in mediacontaining various concentrations of rhTRAIL and infected bothcell types with an increasing MOI of Ad-stTRAIL. There was asignificant dose-dependent decrease in the viability of U-87MGcells that were supplemented with rhTRAIL or infected withAd-stTRAIL (P < 0.01). In contrast, UCB-MSCs did not show anydecrease in viability with exposure to rhTRAIL protein or TRAILgene transfer, even at high concentrations or MOIs (Fig. 4A). Toinvestigate the cause of resistance to TRAIL in UCB-MSCs, the cellsurface expression of TRAIL receptors possessing a death domain(DR4 and DR5) or not (DcR1 and DcR2) was evaluated by flowcytometry (Fig. 4B). U-87MG cells, sensitive to TRAIL, expressedhigh levels of DR5, the major receptor involved in TRAIL-induced

Figure 3. Migration of UCB-MSCs toward gliomas in vivo . MSCs-EGFP or PKH26-labeled MSCs-stTRAIL cells were implanted into the opposite hemisphere tothe U-87MG tumor (A ) or i.t. into the U87-EGFP tumor mass (B ). A, MSCs-EGFP (green ) cells were implanted into the opposite hemisphere to the tumor inglioma-bearing mice (a–d ) or normal mice as a control (e–g ). At day 7 after implantation, MSCs-EGFP were seen through the corpus callosum (b, box 1 from a)and in the tumor mass (c, box 2 from a ). d, H&E staining shows the interface of the tumor and normal brain in a contiguous section of c . MSCs-EGFP were only seen atthe injection site (f, box 1 from e ) and were not found within the corpus callosum (g, box 2 from e) or the opposite hemisphere in normal mice. T, tumor mass;dotted line, tumor edge; arrowheads, MSCs-EGFP cells located in the tumor mass; insets, higher magnification (�400) of the area indicated by arrow. Magnification,�200. B, PKH26-labeled MSCs-stTRAIL cells (red) were implanted into the tumor mass (green ) in an established glioma (a , H&E staining). At day 4 after implantation,PKH26-labeled MSCs-stTRAIL cells can be seen extensively interspersed among the green tumor cells (b, box 1 from a ) or distributed throughout the tumor mass(c, box 2 from a ), and also seen in a tumor satellite (f, box 4 from a ) through the corpus callosum (d and e, box 3 from a). A higher magnification (�400) ofboxed area in d shows the migrating MSCs (e ). The cells stained with human nuclei antibody (hNA ; green ) of boxed area in f revealed the injected human MSCs(g , �800). At day 10 after implantation, MSCs-stTRAIL were mostly to be found at the border between tumor and normal parenchyma (h and i). H&E staining shows theinterface of the tumor and normal brain (h , �100). T, primary tumor; t, tumor satellite; arrow, injection site of PKH26-labeled MSCs-stTRAIL. Magnification, �200.Nuclei were stained with DAPI (blue ) for counterstaining.

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apoptosis. DR5 was also detected in UCB-MSCs but at lower levelscompared with the expression in U-87MG cells. UCB-MSCsexpressed significant levels of DcR1 decoy receptor that have highaffinity to TRAIL, suggesting that the overexpression of DcR1

blocks DR5 functions in TRAIL-induced apoptosis by competingwith DR5 for TRAIL. We then cultured premixed MSCs-stTRAILand U-87MG cells to determine whether TRAIL secretion fromtransduced MSCs could induce apoptosis in U-87MG cells. This

Figure 4. Tumor-specific cytotoxicity of TRAIL and therapeutic efficacy of MSC-based gene delivery in vitro. A, U-87MG cells and UCB-MSCs were infected withdifferent doses of Ad-stTRAIL or treated with varying concentrations of rhTRAIL. Two days after rhTRAIL treatment or 3 d after Ad-stTRAIL infection, cell viability wasanalyzed using an MTS assay. *, P < 0.01, Student’s t test. B, the cell surface expression of TRAIL death receptors in U-87MG cells and UCB-MSCs wasanalyzed by flow cytometry (green lines ). Mouse isotype IgG1 antibody served as a control (black lines ). Results are representative of three independent determinationsfor each receptor and cell line. Percent of positive cells was determined. C, U-87MG cells premixed with MSCs-stTRAIL were cultured for 48 h and then analyzed usingthe TUNEL assay. A coculture of MSCs-c5 and U-87MG cells was used as a negative control. TUNEL-positive nuclei (red) were stained and counterstaining wasconducted with DAPI (blue ; a ). Magnification, �200. Secreted TRAIL–mediated apoptosis was confirmed by blocking with neutralizing antihuman TRAIL antibody incoculture of MSCs-stTRAIL and U-87MG cells using Transwell plates containing semiporous membranes (0.4-Am pores). After 5 d, the viability of U-87MG cellsin the lower well was assayed by MTS (b). Control, 2 � 104 cells of MSCs-c5 in the upper wells. Columns, mean; bars, SE. D, effect of MSCs-stTRAIL compared withU87-stTRAIL on survival of U-87MG cells was determined using Transwell plates (0.4-Am pores). After 5 d, the viability of U-87MG cells in the lower well was assayedby MTS (a) and the concentration of secreted TRAIL was determined using an ELISA assay (b ). Control, 4 � 104 cells of MSCs-c5 or U87-c5 in the upper wells.Columns and points, mean; bars, SE. *, P < 0.05; **, P < 0.001, Student’s t test.





resulted in significant apoptosis in U-87MG cells premixed withMSCs-stTRAIL; however, U-87MG cells premixed with Ad-c5-infected MSCs (MSCs-c5) did not undergo apoptosis (Fig. 4C, a).In addition, neutralization of secreted TRAIL from transducedMSCs fully abolished apoptosis induction of U-87MG cells incoculture system using Transwell plates, suggesting that the killingis mediated by secreted TRAIL (Fig. 4C, b). These findingsconfirmed that MSCs-stTRAIL secreted biologically relevantquantities of TRAIL protein, which affected only transformed cellswithout damaging normal cells.

Therapeutic potential of TRAIL-secreting UCB-MSCs asdelivery vehicles in vitro. To determine the therapeutic benefitof MSCs-stTRAIL compared with U87-stTRAIL cells, which couldmimic the effects of injecting the adenovirus encoding stTRAIL

Figure 5. Effects of MSCs-stTRAIL on tumor growth and survival ofglioma-bearing mice in vivo. A, MSCs-stTRAIL or MSCs (2 � 105 cells) and PBSwere given i.t. to glioma-bearing mice at day 7 after U-87MG (105 cells)inoculation. a, tumor size was determined by histologic analysis at 14 or 21 dafter tumor inoculation (n = 5 per treatment group). Columns, mean; bars, SE.*, P < 0.01; **, P < 0.05, Student’s t test. b, representative photographs of H&Estaining from each group. Magnification, �1. B, survival curve of intracranialglioma-bearing mice. At day 7 after U-87MG (105 cells) inoculation, tumors wereinjected with a single dose (2 � 105 cells) of MSCs-stTRAIL (n = 10) orMSCs-EGFP (n = 10) and inoculated with Ad-stTRAIL (1 � 1010 viral particles;n = 10). The PBS-treated group was used as a control (n = 10). Analysis ofsurvival was conducted by a log-rank test based on the Kaplan-Meier method.Representative result of three independent experiments.

Figure 6. TRAIL expression and apoptosis in MSCs-stTRAIL–treated gliomasin vivo . At day 7 after U-87MG (105 cells) inoculation, MSCs-stTRAIL orMSCs-EGFP (2 � 105 cells) were treated i.t. A, for the quantification of stTRAILlevels and longevity of stTRAIL expression in tumor tissues, brain tissueswere homogenized at 1, 4, 7, 10, and 14 d (n = 3 per group) after treatmentand then assessed by ELISA. Control, MSCs-EGFP–treated brain tissues.Columns, mean; bars, SE. B, at day 7 after MSCs-stTRAIL inoculation, treatedbrains were stained for TRAIL. a, H&E staining shows the interface of the tumorand normal brain (�100). b, sections from MSCs-stTRAIL–treated brainsshowed positive staining for TRAIL (red ) in the tumor mass and at the tumorborder line, indicating the presence of MSCs-stTRAIL (�400). c to f, a highermagnification image of the TRAIL-stained cells (arrows ) illustrated in b showsthat the TRAIL-positive cell nuclei were stained with human nuclei antibody(hNA, green ; �800). Nuclei were stained with DAPI (blue ) for counterstaining.Arrowheads, human nuclei antibody–stained cell nuclei. C, MSCs-stTRAIL–treated brains stained with TUNEL (red) show the specific staining of apoptosisin the tumor and the lack of staining in adjacent normal tissue (a); however, asection from MSCs-EGFP–treated brains shows negligible staining (b ; �100).c, apoptotic cells were detected in the main tumor mass (�200); inset, ahigh-power view (�800) of TUNEL-stained nuclei from the area indicated witharrow. d, additionally, apoptotic cells were seen in close proximity to invadingtumor islands (�200). Nuclei were stained with DAPI (blue ) for counterstaining.T, tumor mass; dotted line, tumor edge; arrowheads, tumor island.

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gene to the tumor mass directly, U-87MG cells were coculturedwith MSCs-stTRAIL or U87-stTRAIL. To determine whether thisgrowth inhibition was specifically due to the release of solubleTRAIL, we used Transwell plates containing semiporous mem-branes that separated the cells. Whereas treatment with MSCs-stTRAIL resulted in a dose-dependent inhibition of U-87MG cellgrowth, treatment with U87-stTRAIL was limited in its effects ontumor cell death (Fig. 4D, a). To verify that this effect was due tothe release of TRAIL, the concentration of TRAIL in the mediumwas determined (Fig. 4D, b). A dose-dependent increase in theconcentration of TRAIL directly correlated with tumor cell deathin the MSCs-stTRAIL treatment group; however, an increase inTRAIL concentration was not observed in the U87-stTRAILtreatment group and there was no correlation with increasing cellnumbers. This was because transduced U-87MG cells were killedby the TRAIL secreted from them, which correlates with the resultsshown in Fig. 1C ; thus, they could not continuously secret thetherapeutic gene.Effects of MSCs-stTRAIL on tumor growth and survival of

glioma-bearing mice in vivo. To assess whether inoculationof MSCs-stTRAIL showed antitumor effects in vivo , maximaltumor surface areas were determined by histologic analysis fromglioma-bearing mice. The average maximal tumor areas in MSCs-stTRAIL–treated animals were decreased compared with PBS- orMSCs-treated animals. This decrease in tumor size associated withMSCs-stTRAIL treatment was significant at day 14 (P = 0.007,MSCs-stTRAIL versus MSCs; P = 0.0016, MSCs-stTRAIL versus PBS)and at day 21 (P = 0.022, MSCs-stTRAIL versus MSCs; P = 0.039,MSCs-stTRAIL versus PBS; Fig. 5A). There was no detectabledifference in tumor size between animals treated with MSCs andPBS. These results show that stTRAIL-secreting MSCs, when i.t.implanted in established glioma, reduce the rate of tumor growth.Next, the survival of MSCs-stTRAIL–treated mice was signifi-

cantly prolonged compared with controls treated with PBS orMSCs-EGFP (Fig. 5B). There were no detectable differences insurvival among animals treated with MSCs-EGFP, unmodifiedMSCs (data not shown), or PBS. Statistical analysis revealedthat the effects of MSCs-stTRAIL and Ad-stTRAIL were bothsignificantly different from that of MSCs-EGFP (P = 0.0001,MSCs-stTRAIL versus MSCs-EGFP; P = 0.001, Ad-stTRAIL versusMSCs-EGFP). However, MSCs-stTRAIL–treated mice showedprolonged survival compared with Ad-stTRAIL–treated mice(P = 0.0241), which was evaluated for the comparison betweenMSC-based therapy and viral gene therapy at a single i.t. injection.These data indicated that the treatment of stTRAIL-secreting UCB-MSCs has a strong antitumor effect compared with adenoviralTRAIL gene therapy.In vivo expression of TRAIL and induction of apoptosis in

established intracranial brain tumors by inoculated MSCs-stTRAIL. We quantitatively measured the levels of stTRAIL proteinand its longevity in vivo to determine whether the antitumoractivity of MSCs-stTRAIL correlated with the levels of stTRAILexpressed in tumor tissues. ELISA analysis on different days aftertreatment revealed that the TRAIL protein expressed in tumortissues was detectable on day 1, peaked on day 4, began to decreaseafter day 7, and persisted for 2 weeks (Fig. 6A). In the control group(MSCs-EGFP), TRAIL protein was not detected throughout theexperimental period. Additionally, immunohistochemical analysisof tissues prepared from mice at day 7 showed strong staining forTRAIL within inoculated tumors and on the border of tumors,indicating the presence of TRAIL-secreting human UCB-MSCs

(Fig. 6B). Furthermore, to evaluate the apoptosis-inducing abilityof TRAIL secreted from UCB-MSCs in vivo , TUNEL staining wasdone (Fig. 6C). MSCs-stTRAIL–treated tumors were almostcompletely apoptotic, and apoptosis was confined to the tumormass and not normal brain parenchyma, indicating that MSCs-stTRAIL inoculated i.t. was inducing tumor cell death. Apoptoticactivity detected in MSCs-EGFP– or PBS-treated tumors wasnegligible. Importantly, apoptotic cells were detected in the maintumor mass and were seen in proximity to invading tumor islands,indicating that MSCs-stTRAIL migrated through the outgrowingtumor from the primary tumor site into adjacent normal tissue.

Discussion

In this study, we efficiently engineered human MSCs derivedfrom UCB to secret stTRAIL via adenoviral transduction mediatedby cell-permeable peptides and provided evidence that these cellscan migrate toward human gliomas. Additionally, considerablereduction in tumor growth and prolongation of survival occur inglioma-bearing mice treated with TRAIL-secreting UCB-MSCs.We used a xenogenic model of human glioblastoma grown in

nude mice for evaluating the migratory ability and the therapeuticefficacy of human MSCs. Recently, it has been reported that thehuman xenograft glioblastoma models in immunodeficient miceexhibit histopathologic features compatible with tumor invasioninto the normal/nonneoplastic brain parenchyma, although thetumor borders in the murine models are not as diffuse as those ofthe spontaneous glioblastoma in human (34). Therefore, murinemodels of glioblastoma seem to recapitulate several of the humanglioblastoma histopathologic features, and considering theirreproducibility and availability, they constitute a valuable in vivosystem for preclinical studies.The migration of UCB-MSCs in vitro was stimulated by

conditioned medium from cultured glioma cells in a dose-dependent manner. This indicates that soluble factors releasedfrom glioma cells could induce the migration of MSCs. It is knownthat these factors released from cancer cells promote therecruitment of endothelial cells and endogenous stromal cellsfrom the bone marrow toward the tumor mass, and such responsesclosely resemble tissue remodeling after injury or inflammation(35, 36). Similar mechanisms would be mediated in the migrationof UCB-MSCs in glioma; however, these tumor-specific migratoryproperties require further elucidation in relation to their potentialuse in therapeutic applications. In our in vivo experiments, wefound that when injected contralaterally or i.t. to the tumor mass,UCB-MSCs show a tracking ability through outgrowing gliomacells. Although this is consistent with reports describing the tumor-specific migratory abilities of MSCs derived from bone marrow(11), UCB-MSCs have many advantages, such as the immaturity ofnewborn cells compared with adult cells, large ex vivo expansioncapacity, low risk of viral infection, lack of donor attrition, and lesspronounced immune response. Thus, it is important that thestrategies using the tracking ability of UCB-MSCs to achievewidespread distribution of therapeutic agents throughout out-growing gliomas could improve brain tumor therapy.In the present study, we engineered UCB-MSCs expressing

TRAIL, which selectively induce apoptosis in a wide variety oftransformed cells without damaging normal cells and tissues (37),to investigate the therapeutic potential of MSCs deliveringproapoptotic proteins. UCB-MSCs could be used as therapeuticvehicles to deliver TRAIL because they are resistant to





TRAIL-mediated apoptosis, which would extend the release time ofour stTRAIL gene and could lead to delivery of the therapeutic geneto outgrowing tumor cells because of the cell tropism for gliomas.The coculture experiments in vitro showed that treatment ofMSCs-stTRAIL is more effective than U87-stTRAIL treatment,which mimics adenoviral gene therapy that is injected directly intothe tumor mass. Furthermore, the survival experiment in vivorevealed that direct injection of Ad-stTRAIL did not result inenhanced survival compared with treatment of MSCs-stTRAIL ata single dose of i.t. administration. These results indicate thatMSC-based TRAIL gene therapy is more advantageous than viralgene therapy, which involves direct injection of an adenovirus-encoding TRAIL gene into tumors. However, further work needsto be done in relation to the safety and brain toxicity of solubleTRAIL at high local concentrations before this MSCs-stTRAILbecomes a candidate for a human clinical glioma therapy trial.In agreement with a study showing that the efficiency of

exogenous gene transfer into MSCs using adenoviral vectors isrelatively low (28), we confirmed that UCB-MSCs lack CARexpression and show poor transduction of exogenous genes. Inflow cytometry analysis, when UCB-MSCs were infected with Ad-EGFP, about 20% to 30% of cells were transduced, even at a highMOI (300–500). At higher MOIs (z1,000), f80% of cells becametransduced, suggesting that adenovirus entry occurs via low-affinity interactions with nonspecific attachment molecules;however, we found that cell viability was severely hampered byviral cytopathogenic effects (data not shown). To circumvent thisproblem, several groups have reported that fiber-modifiedadenoviral vectors can result in expanded tropism compared withwild-type adenoviral vectors (38, 39). In the present study, weengineered TRAIL-secreting UCB-MSCs by PTD-mediated adeno-viral transduction using a novel PTD (4HP4), which improvesadenoviral gene expression at reduced titers of the virus in variouscells in vitro and the efficacy of therapeutic gene transductionin vivo (32). Our data show that UCB-MSCs can be efficientlytransduced by adding 4HP4 to the infection medium (f90%represented in flow cytometry analysis) and migration capacitywas not affected by PTD-mediated adenoviral transduction.Therefore, addition of 4HP4 during adenoviral infection of MSCscan enhance the transduction efficiency, facilitating the use ofadenoviral vectors in MSC-mediated gene therapies.Recently, it has been reported that MSCs could support tumor

growth (40) and promote cancer metastasis (41). Although thesefinding suggest that MSCs could favor tumor growth underphysiologic conditions, the antitumor effects of TRAIL secretedfrom engineered MSCs can lead to reversal of tumor growth inour therapy. Moreover, the supportive effect of MSCs alone onefficacy experiments in the present study was not observed, whichimplies that the proportion of inoculated UCB-MSCs, which

reached only about 5% in established tumor masses after a singleinjection, was insufficient to support tumor growth.Although prolonged survival of glioma-bearing mice by treat-

ment with MSCs-stTRAIL was shown, we did not achieve completetumor regression by single i.t. administration. We found that theexpression from MSCs-stTRAIL inoculated into glioma-bearingmice persisted for 14 days but began to decline after 7 days. This isone of the possible explanations for incomplete tumor regressionin relation to the elimination of engineered UCB-MSCs and theprogressive growth of glioma cells. These results suggest thatfurther investigations on the repeated administration of MSCs-stTRAIL with controlled doses and appropriate time intervals andthe expression level of stTRAIL in tumor tissues will be needed forthe clinical application. Systemic administration has the advantagethat repeated injections are clinically feasible. Although systemicadministration by tail vein injection has been reported for mouseneural stem cells (42), we found that the human MSCs were filteredby the lung and few cells were detected in the intracranial tumorof nude mice (data not shown), which may have been due tospecies incompatibilities (11). In addition, a wide surgical resectionof tissue in malignant gliomas is highly limited because this mightresult in an unwanted loss of brain function. Therefore, a certainamount of tumor mass might remain in the primary site aftersurgical resection. Thus, it will be useful to evaluate thecombination of surgery, which is accompanied by other therapiessuch as chemotherapy and radiation therapy, and repeated MSCs-stTRAIL therapy in human patients.In conclusion, we have shown for the first time the migratory

capacity of UCB-MSCs toward gliomas and show an efficientadenoviral transduction system for MSCs. The engineered UCB-MSCs secreting stTRAIL are effective in regressing tumor growth inthe intracranial xenograft mouse model. Thus, the use of UCB-MSCs as delivery vehicles of therapeutic genes will be of greatinterest for the clinical application of stem cell–based cancertherapy.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Received 2/5/2008; revised 9/2/2008; accepted 9/4/2008.Grant support: National R&D Program for Cancer Control (0820040) and Korea

Health 21 R&D Project grant 0405-DB01-0104-0006, Ministry of Health and Welfare,Republic of Korea, and the Korea Research Foundation Grant funded by the KoreanGovernment (MOEHRD, Basic Research Promotion Fund, KRF-2007-313-E00419;S.S. Jeun).

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

We thank Dong-A Pharmaceutical Co., Ltd. (Yongin, Korea) for providing us withAd-stTRAIL.

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2008;68:9614-9623. Cancer Res Seong Muk Kim, Jung Yeon Lim, Sang In Park, et al. Glioma

Derived Mesenchymal Stem Cells against Intracranial−BloodGene Therapy Using TRAIL-Secreting Human Umbilical Cord

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