Epidermal growth factor receptor-transfected bone marrow ... - Nature

Cancer Gene Therapy (2005) 12, 757–768 All rights reserved 0929-1903/05 $30.00

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Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors Hidemitsu Sato,1,a Naruo Kuwashima,1,a Tsukasa Sakaida,1 Manabu Hatano,1 Jill E Dusak,1 Wendy K Fellows-Mayle,1 Glenn D Papworth,2 Simon C Watkins,2 Andrea Gambotto,3 Ian F Pollack,1 and Hideho Okada1,3 1

Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA; 2Department of Cell Biology and Physiology, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA; and 3Department of Surgery and the Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. We have created a novel cellular vehicle for gene therapy of malignant gliomas by transfection of murine bone marrow stroma cells (MSCs) with a cDNA encoding epidermal growth factor receptor (EGFR). These cells (EGFR-MSCs) demonstrate enhanced migratory responses toward glioma-conditioned media in comparison to primary MSCs in vitro. Enhanced migration of EGFR-MSC was at least partially dependent on EGF-EGFR, PI3-, MAP kinase kinase, and MAP kinases, protein kinase C, and actin polymerization. Unlike primary MSCs, EGFR-MSCs were resistant to FasL-mediated cytotoxicity and were capable of stimulating allogeneic mixed lymphocyte reaction, suggesting EGFR-MSCs possess suitable characteristics as vehicles for brain tumor immuno-gene therapy. Following injection at various sites, including the contralateral hemisphere in the brain of syngeneic mice, EGFR-MSCs were able to migrate toward GL261 gliomas or B16 melanoma in vivo. Finally, intratumoral injection with EGFR-MSC adenovirally engineered to secrete interferon-a to intracranial GL261 resulted in significantly prolonged survival in comparison to controls. These data indicate that EGFR-MSCs may serve as attractive vehicles for infiltrating brain malignancies such as malignant gliomas. Cancer Gene Therapy (2005) 12, 757–768. doi:10.1038/sj.cgt.7700827; published online 15 April 2005 Keywords: bone marrow stroma cells; epidermal growth factor receptor; interferon-a; glioma

alignant astrocytomas are highly infiltrative, therefore current treatments that rely on targeting the tumor mass are often ineffective. Recently, neural stem cells (NSCs), the self-renewing precursors of neurons and glia, have attracted particular attention because of their exceptional migratory ability. In rodent brains bearing gliomas, NSCs distribute quickly and extensively throughout the tumor and migrate uniquely in juxtaposition to widely expanding and aggressively advancing tumor cells following implantation of NSCs at sites distant from the tumor.1 Clinical applications of this attractive approach, however, still await further investigations and solutions regarding how to obtain sufficient number of NSCs for individual patients. An alternative source for NSCs may be embryonal stem cells (ES).2 However, this source raises ethical concerns related to the

M

Received October 4, 2004.

Address correspondence and reprint requests to: Dr Hideho Okada, Department of Neurological Surgery, University of Pittsburgh School of Medicine, G12a The Hillman Cancer Center, 5117 Center Avenue, Pittsburgh, PA 15213-1863, USA. E-mail: [email protected] a These authors contributed equally to this work.

use of human fetuses.3 In addition, from the scientific point of view, ES-derived cells, which are allogeneic for the host patients, might cause allogeneic immune responses and may result in their rejection by the host immune system shortly after injection.4 Owing to these difficulties associated with clinical application of NSCs, we have developed an alternative approach to create autologous cellular vehicles that possess migratory capability. Epidermal growth factor receptors (EGFRs) have been shown to regulate migration in a variety of cells through direct and indirect mechanisms. The ligands for EGFR include EGF, heparin-binding EGF (HB-EGF), and transforming growth factor-a (TGF-a).5 In wound-healing models of colonic epithelium, EGF, TGF-a, and HBEGF play critical roles in enhanced migration of epithelial cells and fibroblasts following their binding to EGFR.6,7 In primary culture, corneal epithelial cells that are stimulated by TGF-a or EGF become highly motile, involving EGFR and laminin-5 as key intracellular molecules for mediating the motility.8 In neoplasms, TGF-a stimulation induced sustained MAP-kinase activation in human breast cancer cells, which correlated with enhanced cell motility and in vitro invasion.9 Finally, a

Glioma gene therapy with EGFR-transfected MSCs H Sato et al

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clear correlation between glioma cell motility and tyrosine phosphorylation of EGFR following stimulation with TGF-a was demonstrated.10 Taken together, these findings have led us to hypothesize that ectopic expression of EGFR may provide the target transfected cells with enhanced migratory activity toward the tissues that express TGF-a and EGF at high levels. EGF, TGF-a and their receptor EGFR are thought to be important in the mediation of a variety of biological responses in gliomas via autocrine or paracrine mechanisms.5 Direct consequences of increased expression of a TGF-a/EGF/EGFR autocrine or paracrine loop in glioma cells include the increased motility of glioma cells.10–12 In addition, heparin-binding EGF-like growth factor (HB-EGF) has been found to be overexpressed in most human glioblastoma tissues.13 As HB-EGF is known to promote cell-motility in wound-healing models,7 it is reasonable to hypothesize that gliomaderived HB-EGF also influences EGFR-mediated glioma motility. The ancient Chinese believed bone marrow was the source of brain tissue, as suggested by the maxim ‘‘brain is a sea of marrow’’.14 Marrow stromal cells (MSCs) normally give rise to bone, cartilage, and mesenchymal cells (reviewed in La Russa et al15). Isolation of MSCs that can differentiate to brain cells, such as glial cells16 and neural cells,17 has been demonstrated. Infusion of human MSCs into rat striatum resulted in engrafting, migration, and survival of the cells.18 These findings suggest that bone marrow cells may be a suitable source for the novel cellular vehicles to deliver therapeutic molecules for the treatment of gliomas. In the present study, we tested our hypothesis that EGFR-transfected MSCs would serve as a cellular vehicle that possesses migratory activity towards brain tumors.

depleted for red blood cells and then incubated on plastic dishes in RPMI1640 media containing 15% heat-inactivated (HI)-FBS at 371C with 5% CO2 for 4 hours to deplete adherent mature stromal cells and fibroblasts. The nonadherent cells were then seeded into 10-cm dishes (BD Falcon 3803 Primaria, Franklin Lakes, NJ) in CM containing 15% HI-FBS, 5 105 M 2-mercaptoethanol (2-ME) and 10 ng/ml interleukin-3 at a density of 4 106 cells per dish. After 1 week, the cell phenotype was analyzed and the cells were transfected with pxf-EGFRneo encoding human EGFR and neomycin resistance (NeoR),20 provided by Dr Alan Wells (University of Pittsburgh) using PolyFect Transfection Reagent (Qiagen, Valencia, CA). The stable transfectants were selected with .4 mg/ml G418. At 14 days after addition of G418, completion of selection was confirmed by the absence of live cells in the control nontransfected culture with G418.

Flow-cytometric analyses For phenotypic analysis of MSCs, after incubation with anti-mouse CD16/32 Ab for Fc receptor blocking, the cells were stained with PE- or FITC-conjugated mAbs against mouse cell surface molecules (H-2Kb, I-Ab, CD34, CD44, CD34, CD80, CD86, Fas (CD95), FasL (CD95L), CD135, Sca-1 (all from BD PharMingen, San Diego, CA)) or appropriate isotype controls. Cell surface expression of EGFR was assessed using two-step flow cytometry. First, EGFR-MSCs and control primary MSCs were stained with biotin-conjugated sheep antiEGFR (K20911B Biodesign International, Saco, ME), or appropriate isotype-matched controls. Second, the MSCs were labeled with PE-conjugated streptavidin (Vector Laboratories, Burlingame, CA). Flow-cytometric analysis was performed using a FACscan caliber laser flow cytometry system (Becton Dickinson, San Jose, CA) flow cytometer.

Materials and methods

Cell lines

Migration assays

A mouse glioma cell GL261, a mouse melanoma B16, and TIB81 fibroblasts (American Type Culture Collection, Manassas, VA), all of which are syngeneic to the C57BL/6 background, were maintained in complete medium (CM) (RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 mM L-glutamine (all reagents from Life Technologies, Inc., Grand Island, NY)) in a humidified incubator at 5% CO2 and 371C. Mouse Fas ligand (FasL) transduced L5178Y (L5178Y-FasL) or mock transfected L5178Y effector cells were provided from Dr Hideo Yagita, (Juntendo University, Tokyo, Japan) and maintained in CM.

Cell migration was determined using a 48-well microchemotaxis chamber assay as described previously.21,22 Cell migration was quantified by the number of cells that migrated directionally through a fibronectin-coated 8-mmpore polyvinyl pyrolidone-free polycarbonate filter (Corning Coster, Cambridge, MA) toward the lower wells. Briefly, cells (1 106 cell/ml) were resuspended in serum-free RPMI 1640 containing .1% BSA, and aliquots of 1 105 cells were applied in each of the upper chambers. In some experiments, the cells were also preincubated at room temperature with indicated concentrations of inhibitors (15 minutes) or antibodies (30 minutes). Inhibitors examined in our studies are the following: the tyrosine kinase inhibitor with a selectivity for the EGFR AG1478, the MEK inhibitor PD98059 and the p38 MAP kinase inhibitor SB202190 were purchased from Calbiochem (San Diego, CA); the phosphoinositide 3-kinase (PI3-K) inhibitor Wortmannin, the protein kinase C (PKC) inhibitor Calphostin C and the actin polymerization inhibitor Cytochalasin D were obtained from Sigma

Culture and transfection of MSCs with EGFR Bone marrow stroma-initiating cells were cultured according to the previously described methods19 with slight modifications. Briefly, single-cell suspensions obtained from femoral bones of 8–10-week-old C57BL/6 mice were

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(St Louis, MI). The lower wells contained either base media (RPMI containing .1% BSA), tumor-cell-conditioned media (RPMI containing .1% BSA with which tumor cells had been cultured 48 hours) at serial dilutions with the base media, or recombinant human EGF (rEGF; Invitrogen, Carlsbad, CA) at the indicated concentrations. Chambers were incubated at 371C in a humidified incubator in an atmosphere of 5% CO2 for 4 hours, after which the filters were removed, fixed, and Wright-Giemsa stained, and mounted on glass slides. Nonmigrating cells were removed by wiping with a cotton swab. At least five random fields of vision/well ( objective) were counted for quantification of cell migration. Each group was triplicated, and relative cell numbers in comparison to the average of controls were calculated.

MLR assays Aliquots of 1 104 stimulator cells (EGFR-MSC, primary MSCs from Balb/c or C57BL/6 mice) were g-irradiated (15 Gy) and placed in each well of roundbottom 96-well plates. Red blood cell-depleted splenocytes from Balb/c mice were inoculated in each well at the indicated ratio as responder cells. The cells were cultured with CM containing 10 ng/ml IL-3 at 371 C for 18 hours. Then, [3H]thymidine (5 mCi/ml) was added and the cells were further incubated for 24 hours. The cells were trapped on Packard Unifilter (Perkin-Elmer, Boston MA), dried completely, and the [3H]thymidine incorporation was counted by a scintillation counter.

Cytotoxicity assays Standard 51Cr release assays for evaluation of susceptibility of MSCs to FasL were performed as described before.23,24

Animals and brain tumor models Female 6–8-week-old C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and maintained in microisolator cages within the Central Animal Facility at the University of Pittsburgh. Animals were handled under aseptic conditions as per an Institutional Animal Care and Use Committee-approved protocol and in accordance with recommendations for the proper care and use of laboratory animals. Intracranial tumor inoculation was performed as described previously.25 Briefly, using a 10 ml Hamilton syringe, 1 105 GL261 or 1 104 B16 cells suspended in 2 ml of PBS were stereotactically injected through an entry site at the bregma 2 mm to the right of the sagittal suture and 3 mm below the surface of the skull of anesthetized C57BL/6 mice using a Kopf stereotactic frame (Kopf Instruments, Tujunga, CA).

Migration of MSCs in vivo To evaluate the migration of EGFR-MSC, TIB81 fibroblasts, or primary MSCs in vivo, mice bearing day 14 tumors in the right hemisphere received stereotactic injections with PKH-26 (Sigma, St. Louis, MI)-labeled

cells into the left frontal lobe at the following coordinates: at the bregma 2 mm to the left of the sagittal suture and 3 mm below the surface of the skull. Brain tissues were removed at 4 days after injection and fixed with 4% paraformaldehyde. Sections (1 mm) were imaged for the tumors and PKH-26-labeled nontumor cells using a twophoton microscope comprising a titanium-sapphire ultrafast tunable laser system (Coherent Mira Model 900-F), Olympus Fluoview confocal scanning electronics, an Olympus IX70 inverted system microscope, and custombuilt input-power attenuation and external photomultiplier detection systems. Single-plane image acquisition utilized two-photon excitation at 850 nm with Olympus water-immersion objectives ( 20 UApo .7NA, 60 UplanApo, 1.2NA). Emission filters (Chroma, Brattleboro, VT), comprised an HQ535/50 m filter (green emission), a 565 dclp dichroic mirror and a HQ610/75 m filter (red emission).

Adenoviral vectors and therapeutic experiments The adenoviral vectors Ad-IFN-a and the mock adenoviral vector Ad-c5 were produced and provided by the University of Pittsburgh Cancer Institute’s Vector Core Facility as reported previously.26 Five million EGFRMSCs were infected with Ad-IFN-a (Multiplicity Of Infection: (MOI) ¼ 20) or Ad-c5 (MOI ¼ 20), as reported previously.26 After 1 hour, adenoviral-infected MSCs were used for injection of tumor-bearing animals. Culture supernatants were also collected at 48 hours after transfection for measurement of mouse IFN-a production using a species-specific IFN-a ELISA kit (Research Diagnostics Inc., Flanders, NJ). The level of IFN-a expression from 1 106 transfected EGFR-MSC was determined to be approximately 300 ng per 48 hours, whereas the same number of nontransfected control EGFR-MSC expressed less than .3 ng/48 hours. Syngeneic female C57BL/6 mice bearing day 5 GL261 tumors in the right hemisphere received 1 105 EGFR-MSC transfected with Ad-IFN-a or Ad-c5 at the same injection site used for GL261 tumor inoculation. The animals were monitored daily after treatment for the manifestation of any pathologic signs associated with elevated intracranial pressure, such as hemiparesis, loss of appetite or any grooming habit. Animals with signs described above were killed with CO2 inhalation.

Statistical analysis Survival estimates and median survival times were determined using the method of Kaplan and Meier. Survival data were compared using a log-rank test. Data from migration assays were compared by Student’s t-test for two samples with unequal variances. Statistical significance was determined at the o.05 level.

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Results

referred as EGFR-MSC, and results of some experiments were confirmed using bulk EGFR-MSCs, and indicated in the following texts accordingly.

Generation of EGFR transfected MSCs (EGFR-MSC) and detection of surface EGFR expression Mouse bone marrow derived MSCs were transfected with a retroviral vector pxf-EGFR-neo that encodes cDNAs for human EGFR and neomycin-resistance (NeoR) gene, and stably transfected bulk MSCs were established by G418 selection. Transfection with a backbone- NeoR vector did not result in generation of stable transfectants. Primary MSC cultures actively grew for approximately 2 weeks; however, they gradually stopped proliferating after this point. In contrast, bulk EGFR-MSCs continuously grew after several passages, suggesting that ectopic expression of EGFR might have conferred an additional survival signal in the cells. Flow-cytometric analyses using anti-human EGFR antibody that can also crossreact with mouse EGFR revealed detectable surface expression of EGFR on both bulk EGFR-MSCs and some clones obtained by the limited dilution methods. Figure 1a demonstrates the expression of EGFR on bulk EGFRMSCs and the clone 2G3, which demonstrated the highest expression of EGFR among the clones. Expression of EGFR on primary MSCs, in contrast, was barely detectable on the surface with this method. The cloned 2G3 cells were mainly used in subsequent experiments and

a

Bulk EGFR-MSC

Both EGFR-transfected and primary MSCs express markers distinct from hematopoietic stem cells EGFR-MSC and control primary MSCs were analyzed for their phenotype with flow cytometry. Since transfection of primary MSCs with pxf-neo control vectors did not result in generation of G418-resistant, stably transfected cell lines, we compared EGFR-transfected and primary MSCs. As shown in Figure 1b and Table 1, Sca-1, CD34 and CD135 (Flk2/Flt3), all of which are known as markers for hematopoietic stem cells,27 were negative on both EGFR-transfected and primary MSCs. With regard to the markers that are related to recognition by the immune system, mouse major histocompatibility complex (MHC) class I (H-2Kb) and class II (I-Ab) were below undetectable level (Table 1). Primary MSCs but not EGFR-MSC expressed CD80, and both expressed CD86. Interestingly, while both cell types were negative for FasL (CD95L), only primary MSCs, but not EGFR-MSC, expressed Fas (CD95) (Fig 1), suggesting that primary MSCs might be susceptible for apoptotic signals from FasL that is expressed by brain tumor cells and

Clone 2G3

120

Primary MSC

120

120

Counts

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b

101

102 FL2-H

103

104

100

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EGFR-MSC

102 FL2-H

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0 100

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103 102 FL2-H

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Primary MSC

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Line Color Marker Isotype control Sca1 CD135 (Flk2/Flt3)

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CD86 (B7.2)

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Line Color Marker Isotype control CD80 (B7.1) CD95L (Fas L) CD95 (Fas)

0

0 10

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1

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10

4

10

0

10

1

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Figure 1 Surface phenotypic characterization of EGFR-transfected vs primary MSCs. (a) Cell surface expression of EGFR was assessed using two-step flow cytometry. Bulk EGFR-MSCs (left), one of the clones (2G3) that expressed high levels of EGFR (middle) and primary MSCs (right), were stained with biotin-conjugated sheep anti-EGFR (green lines), or appropriate isotype-matched controls (black lines). The cells were then labeled with PE-conjugated streptavidin. (b) EGFR-MSCs (2G3) and primary MSCs were compared for other hematopoietic markers with fluorochrome-linked antibodies to CD80, CD86, CD95, CD95L, CD135, and Sca-1. Negative controls for antibody type were performed on MSCs using appropriate isotype controls. Similar results were obtained in three independent experiments.

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761

+ + + +

EGFR-MSC pMSC

200 150 100 50 0 B1 6

+ +

250

25 %

Primary MSC

b 120 Relative Migration (%)

astrocytes.28 Importantly, Fas expression was also undetectable on bulk EGFR-MSCs, excluding the possibility of the undetectable Fas expression on the 2G3 clone being a unique event associated with the cloning (data now shown). CD44, which has been known to mediate cell migration in various cell types including gliomas,29,30 expressed on both primary and EGFRtransfected MSCs (Table 1). These data indicate that EGFR-MSC were a homogenous cell population devoid of hematopoietic cells.

C

on

tro

EGFR-MSC

300 Relative Migration (%)

H-2Kb/I-Ab CD34 CD44 CD80 (B7.1) CD86 (B7.2) CD135 (Flk2/Flt3) CD95 (Fas) CD95 L (Fas L) Sca-1

a

lM ed 25 ia % G L2 50 61 % G L2 10 61 0% G L2 61

Table 1 Surface phenotypic characterization

100 80 60 40 20 GL261

100

50

10

5

1

0

EGF (ng/ml)

c

120 Relative Migration (%)

In order to test our hypothesis that EGFR transfection enhances migration of MSCs toward tumor cells, we employed in vitro two-chamber migration assays. First, we tested migratory responses of primary MSCs and EGFR-MSC toward GL261- or B16-conditioned media (Fig 2a). The numbers of EGFR-MSC detected on the bottom side of the chamber increased proportionally to the concentration of GL261-conditioned media up to 50%, and then plateaued. GL261-conditioned media also enhanced the migration of primary MSCs, but the numbers were lower than those observed with EGFRMSC in all concentration ranges (Po.01). B16 melanoma-conditioned media also enhanced the migration of EGFR-MSC when it is tested as 25%. However, at higher concentrations of B16-conditioned media, there was no significant difference in the migration rate between the primary and EGFR-MSC (data not shown). Subsequently, EGFR-MSC demonstrated a characteristic bellshaped chemomigratory curve toward rEGF (1–100 ng/ ml) (Fig 2b). Optimal migration of the cells was observed at a concentration of 10 ng/ml rEGF. The maximum migratory response with rEGF, though, was still lower than that observed with GL261-conditioned media, suggesting the presence of other chemoattractive ligands in GL261-conditioned media. As reverse transcriptasepolymerase chain reaction (RT-PCR) revealed that both GL261 glioma and B16 melanoma cells expressed TGF-a and EGF (data not shown), both of which are major ligands for EGFR, the direct role of EGFR in the migratory response toward GL261-conditioned media was examined by addition of a EGFR inhibitor AG1478

0.5

0

EGFR-MSC demonstrated enhanced migratory responses toward tumor-conditioned media, which was partially dependent on EGF and EGFR

P=0.038

100 80 60 40 20 0 No Inhibitor

AG

Figure 2 In vitro migratory responses of EGFR-MSC toward tumorconditioned media, and the role of EGF pathways. EGFR-MSC were analyzed for their migratory responses to GL261 glioma or B16 melanoma-conditioned media, or recombinant human EGF using two-chamber migration assays. Dose responses for tumor-conditioned media were analyzed using tumor-conditioned media diluted in the same media that was used for culturing tumor at indicated ratios (a). The migratory ability of EGFR-MSC to recombinant human EGFR was assessed, and compared with that toward 100% GL261conditioned media (b). The role of EGFR in the migration of EGFRMSC toward GL261-conditioned media was examined by preincubation of EGFR-MSC with, and addition of a specific inhibitor against EGFR, AG1478 (2 mM) in the upper chamber (c). The graph indicates relative numbers of the cells detected in the bottom chambers in comparison to: control media (a), GL261-conditioned media (b) and GL261-conditioned media without AG1478 (c). The graphs represent the results of one out of three similar experiments with comparable results.

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Relative Migration (%)

100 80 60 40 P=0.028 20 0 0

b

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100

100 80 60 40 P=0.006 20

0

c

120 100

0.05 0.1 SB202190 (µM)

0.5

80 60 40 20

D si n

tin

ra ha

ph

C

yt

oc

al C

W

or

os

tm

bi

an

C

in

to r

0

o

As we propose to apply this cellular vehicle system for delivery of immunostimulatory cytokines, we further characterized EGFR-MSC for immunological aspects. It has been reported that human primary bone marrowderived MSCs suppress allogeneic mixed lymphocyte reaction (MLR), thereby placing MSCs as suitable cells for allogeneic cellular transplantation.33 We have compared murine primary and EGFR-MSC in this light. As demonstrated in Figure 4a, in accordance with the observations in human MSCs,33 primary MSCs completely suppressed proliferation of allogeneic, Balb/c micederived responder cells. In contrast, EGFR-MSC stimulated proliferation of the allogeneic responder cells in a dose-dependent manner. Bulk EGFR-MSCs also stimu-

5 10 50 PD98059 (µM)

0

N

EGFR-MSCs are able to stimulate allogeneic T cells and resistant to FasL-mediated cytotoxic effects

1

120

In

The binding of EGF or TGF-a to the extracellular domain of EGFR activates its cytoplasmic tyrosine kinase, which undergoes autophosphorylation and recruits downstream effectors including phosphoinositide 3kinase (PI3-K), mitogen-activated protein kinases (MAPK) including extracellular signal-regulated protein kinase (ERK) 1 and 2, and PKC signaling pathways, among others.31,32 All of these pathways have been shown to be involved in the regulation of EGFR-mediated cell proliferation, protease secretion, and cell migration.31 To identify the signal transduction pathways involved in the tumor-conditioned media-stimulated migratory response in the EGFR-BMSC, we added a series of specific signaling inhibitors in the assays of GL261-conditioned media-induced migration of EGFR-MSC (Fig 3). A MAP kinase kinase (MEK) inhibitor PD98059 and a p38 MAP kinase inhibitor SB202190 in the migration assays inhibited the migration of EGFR-MSC in a dosedependent manner (Fig 3a and b). PI3-K, PKC, and actin polymerization pathways also appear to be responsible, as treatment of the EGFR-MSC with pharmacological doses of a PI3 K inhibitor, Wortmannin (50 nM), a PKC inhibitor, Calphostin C (500 mM), or an actin polymerization inhibitor, Cytochalasin D (100 ng/ml) significantly inhibited their migration in response to GL261-conditioned media (Fig 3c).

120


Migration of EGFR-MSC is dependent on PI3-K, MAPK, protein kinase C (PKC), and actin polymerization

a

hi

(Fig 2c). There was a significant reduction of the migratory response with 2 mM AG1478. However, abrogation of the migration was still only partial, suggesting that other molecules that had been secondarily induced by EGFR transfection might have played some roles for enhanced migratory responses of the EGFR-MSC. In addition, EGFR-MSC demonstrated enhanced migratory responses when they were tested with U-87 human glioma cell-conditioned media (data not shown), suggesting the presence of cross-reacting chemoattractive substances between species. Bulk EGFR-MSCs also demonstrated similar results in three independent experiments (data not shown).


762

Figure 3 MAPK pathways, PKC, and actin polymerization mediate migratory responses of EGFR-MSC toward GL261-conditioned media. Aliquots of 1 105 EGFR-MSC were applied in each of the upper chambers in the presence of indicated concentrations of inhibitors. The bottom chambers contained GL261-conditioned media. After incubation for 4 hours, the cells that had migrated to the bottom chambers were counted. Roles of MEK and MAPK signaling pathways were examined by addition of an MEK inhibitor (PD98059, Calbiochem, La Jolla, CA) and a p38 MAP kinase inhibitor (SB202190 Calbiochem, La Jolla, CA) in the migration assays (a and b, respectively). Roles of PI3K, PKC and actin polymerization were assessed by addition of a PI3K inhibitor, Wortmannin (50 nM; P ¼ .001 against control no inhibitor), a PKC inhibitor, Calphostin C (500 mM; P ¼ .007 against control no inhibitor) or an actin polymerization inhibitor, Cytochalasin D (100 ng/ml; P ¼ .003 against control no inhibitor) (c).


a

EGFR-MSC

EGFR-MSC +CM

pMSC

pMSC syngeneic

3000 2500

c.p.m.

2000 1500 1000 500 0 10000 20000 40000 Number of Stimulators

b

mFasL/L5178 vs EGFR-MSC

Percent Specific Lysis

40

mock/L5178Y vs EGFR-MSC mFasL/L5178 vs primary MSC

30

mock/L5178Y vs primary MSC 20

As demonstrated in Figure 1, primary MSCs, but not EGFR-MSC, expressed a remarkable level of Fas (CD95). As Fas and its physiological ligand, FasL system, have been implicated in induction of apoptotic death of Fas-expressing cells34 and both neurons and astrocytoma cells are known to express FasL,28 we examined whether Fas-positive primary MSCs would be susceptible to the cytotoxic effects of FasL, and whether EGFR-MSC would acquire resistance against FasL-mediated cytotoxicity. Mouse T-cell lymphoma L5178Y transfected with mFasL were used as cytotoxic effector cells in 51Cr release assays in which they were incubated with MSCs as targets. As demonstrated in Figure 4b, primary MSCs but not EGFR-MSC were killed by FasL þ effectors at indicated E/T ratios. Bulk EGFR-MSCs were not susceptible to the cytotoxic effect of FasL either (data not shown). Control mock-transfected L5178Y cells elicited no cytotoxicity in either cells, supporting a FasL-specific effect in these assays. These data demonstrate that lysis depended upon high levels of Fas expression by primary MSCs, thus suggesting the possibility that EGFR-MSC may possess a survival advantage over primary MSCs in environments in which significant FasL is expressed, namely the central nervous system (CNS) and CNS tumors.

EGFR-MSC demonstrated a migratory capacity toward intracranial tumors

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25 12.5 6.2 E/T Ratio

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Figure 4 EGFR-MSCs, but not primary MSCs, were able to stimulate proliferation of alloreactive T-cells and were resistant to the cytotoxic effect of FasL. Aliquots of 1 105/well responder splenocytes (SPCs) derived from Balb/c mice were stimulated with irradiated primary allogeneic (C57BL/6 mice-derived) MSCs (crosses), syngeneic (Balb/c mice derived) MSCs (closed diamond), allogeneic EGFR-MSC (closed rectangles) or allogeneic EGFR-MSC in the presence of allogeneic primary MSC-conditioned media (open rectangles) as stimulator cells to determine responsiveness to allogeneic stimulator cells (a). Susceptibility of primary and EGFRMSC against the cytotoxic effect of FasL was examined by a 51Crrelease assay using 51Cr-labeled primary (K) or EGFR-MSC (m) as targets and mouse FasL-transfected L5178 cells as effector cells. Specificity of response was also assessed using mock-transfected L5178 cells as control effector cells and primary (J) or EGFR-MSC (n) as targets (b). E/T ratio stands for effector/target ratio.

lated the allogeneic responders (data not shown). Interestingly, addition of primary MSC-conditioned media suppressed the EGFR-MSC-induced allogeneic response, suggesting that primary MSCs secrete soluble substances that can suppress allogeneic immune responses. Although the underlying mechanisms for this reversion of immunosuppressive effect by EGFR-gene insertion remain to be clarified, the results support that EGFR-MSC may serve as more suitable cellular vehicle system than primary MSCs for delivery of immunostimulatory cytokines or chemokines to the brain tumor site.

To examine whether EGFR-MSCs have the superior migratory capacity over primary MSCs toward the tumors in the brain, PKH-labeled EGFR-MSC were injected into the left basal ganglia of the animals bearing day 14 GL261 glioma on the right basal ganglia. Animals were killed 4 days following the EGFR-MSC injection, and brain sections were prepared for analyses by the twophoton microscope system. As demonstrated in Figure 5, numerous PKH-labeled cells were detected in the tumor located in the contralateral hemisphere (Fig 5c). In contrast, primary MSCs, which demonstrated comparatively weak migratory responses toward GL261- and B16conditioned media in vitro, also showed little migration toward a CNS tumor site (Fig 5d). In addition, fibroblasts grafted as controls never showed this dispersion or tropism, consistent with previous reports35 (data not shown). It is noteworthy that PKH-labeled cells were also observed migrating across the corpus callosum (Fig 5b), as well as a small number of PKH-labeled cells in the original injection site (Fig 5a). Very few EGFR-MSCs were found in normal brain tissue beyond the injection site, except when tracking toward the main tumor mass or near infiltrating tumor cells, suggesting that, whereas EGFR-MSC migrated toward the tumor, the normal adult brain parenchyma presented a less permissive environment for migration. When we used B16 melanoma inoculated in the right hemisphere, PKH-labeled EGFRMSCs were also detected in the tumor and corpus callosum on day 4 following the injection into the contralateral, left basal ganglia (data not shown).

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Figure 5 EGFR-MSCs migrate toward contralateral GL261 glioma through corpus callosum. C57BL/6 mice bearing day 14 GL261 glioma in the right hemisphere received stereotactic injections with PKH-26-labeled EGFR-MSC (a–c) or PKH-26-labeled primary MSC (d), or PBS (e) into the left frontal lobe. Brain tissues were removed at 4 days after injection and fixed with 4% paraformaldehyde. Sections (1 mm) were imaged for PKH26-labeled cells using a two-photon microscope system. EGFR-MSCs were detected at the injection site in the left hemisphere (a), corpus callosum (b), and within the contralateral tumor (c). White arrows in (a) and (b) indicate clusters of PKH-26-labeled EGFR-MSC. GL261 glioma tissues from control animals that had received PKH-26-labeled primary MSC (d) or PBS (e) did not contain any detectable PKH-26-positive cells. White arrows in (c–e) indicate the margin of the tumor tissue within the brain parenchyma.

In order to examine whether systemic i.v. administration of EGFR-MSC could result in distribution in intracranial tumors, animals with established GL261 tumors received 1 106 PKH-26-labeled EGFR-MSC via tail vein. At 4 days after i.v. injection, two-photon microscopic analyses revealed the presence of PKH-26labeled cells in the lung and spleen, but not in normalappearing brain tissue, or in the tumors in the brain (data not shown).

Treatment of established GL261 glioma with IFN-a-transfected EGFR-MSC prolonged the survival of animals Finally, the efficacy of EGFR-MSC as vehicles for carrying a therapeutic gene was tested in GL261 gliomabearing animals. On day 5 after inoculation of 1 105 GL261 cells in the right hemisphere, the animals received an intratumoral injection of 1 105 EGFR-MSC that had been transfected ex vivo with adenoviral vector (Ad) encoding interferon (IFN)-a cDNA. IFN-a was chosen as

Cancer Gene Therapy

a therapeutic agent because we have been shown IFN-a gene transfer to be effective in controlling preclinical brain tumors36 and inducing effective antiglioma immune responses (manuscripts submitted). These mIFN-a transfected EGFR-MSC expressed approximately 300 ng/106 cells/48 hours IFN-a based on a specific ELISA, while EGFR-MSC transfected with a backbone-control Ad-c5 did not express detectable amount of IFN-a. Control animals received either phosphate-buffered saline (PBS) only, or EGFR-MSC transfected with Ad-c5. Figure 6 demonstrates survival of the glioma-bearing animals treated with these EGFR-MSC. None of 15 animals that had received PBS only survived longer than 36 days (median survival 32 days). Though injection of mocktransfected MSCs prolonged the survival significantly in comparison to PBS controls (P ¼ .0076), none of these animals survived longer than 57 days (n ¼ 15, median survival 34 days). In striking contrast, the animals that had received mIFN-a-transfected EGFR-MSC demonstrated a prolonged survival in comparison to both PBS controls and mock-transfected EGFR-MSC controls


100 PBS

Percent Survival

EBM-Ψ5 EBM-IFNα 50

0 0

25

50 75 Days After i.c. Injection

100

Figure 6 Intratumoral injection with IFN-a-transfected EGFR-MSC conferred long-term survival in mice bearing established GL261 tumors. Mice bearing day 5 GL261 tumors (1 105) in the right hemisphere were injected intratumorally with 1 105 EGFR-MSC that had been transfected ex vivo with Ad-IFN-a (n ¼ 16) or backbone vector Ad-c5 (n ¼ 15). The other group of animals received injection of PBS (n ¼ 15). The data shown are cumulative survival curves pooling results in two independent experiments. J, PBS; m, EGFRMSC-c5; K, EGFR-MSC-IFN-a.

(Po.0001 and P ¼ .0079, respectively) with the median survival of 53 days. Furthermore, four of 16 animals in this group survived longer than 80 days. With regard to the therapeutic potential of IFN-a transfected primary MSC, injections of the same number (1 105/animal) of these cells did not significantly prolong the survival of GL261 glioma-bearing animals (data not shown).

EGFR-MSC did not possess tumorigenicity Although overexpression of EGFR is related to the tumorigenicity in some types of cancers,37 injections of 1 106 EGFR-MSC or bulk EGFR-MSCs in the brain or 1 107 in the subcutaneous space of each of total 10 C57BL/6 background athymic mice failed to form tumors during 100 days of our careful observation following the tumor injection, supporting the lack of tumorigenicity of the cells (data not shown).

Discussion

In the present study, we demonstrated that mouse bone marrow-derived MSCs are amenable to genetic engineering with retroviruses encoding EGFR. These EGFRMSCs, in comparison to primary MSCs, demonstrated higher migration responses toward tumor-conditioned media, as well as tumors inoculated in the brain. Enhanced migration of EGFR-MSC was at least partially dependent on PI3-K, MEK and MAP kinases, PKC, and actin polymerization. Unlike primary MSCs, EGFRMSCs did not suppress allogeneic immune response, and are resistant to the cytotoxic effects by FasL. Finally,

EGFR-MSC ex vivo transfected with IFN-a demonstrated a remarkable therapeutic effect following intratumoral injections to the GL261 tumor-bearing mice. The mechanisms by which EGFR-gene transfection conferred enhanced migratory responses in MSCs have been partially elucidated. In vitro migration assays using rEGF and an EGFR inhibitor AG1478 demonstrated a direct effect of the EGF and EGFR pathway in the migratory responses. Although we employed human EGFR for genetic manipulation of MSC, CNS cells that were transfected with human EGFR have demonstrated chemotactic migration in response to endogenous EGF in the rodent brain.38 Hence, we believe human EGFRinserted MSCs were responding to factors derived from mouse glioma cells. However, inhibition of migration by an EGFR inhibitor was only partial, suggesting other mechanisms, which were secondarily activated by EGFRgene insertion, play significant roles in the enhanced motility. Studies with hematopoietic stem cells revealed their strong migratory response to stromal derived factor1 (SDF-1)39 and secondary lymphoid chemokine (SLC).40 Although EGFR-MSCs expressed messages for CXC chemokine receptor 4 and CC chemokine receptor 7, which are the receptors for SDF-1 and SLC, respectively, we were unable to demonstrate specific migration of EGFR-MSC to SDF-1 or SLC (data not shown). A recent study has indicated that overexpression of EGFR led to sustained cell motility to TGF-a.41 Although our in vitro migration assays were performed with a relatively short period of incubation (4 hours), if EGFR-MSCs possess more sustained motility compared to the primary MSC, this may have contributed to more efficient in vivo migration of EGFR-MSC than primary MSCs in tumorbearing brains. The resistance of EGFR-MSC against FasL-mediated apoptosis was associated with downregulated expression of Fas in comparison to the primary MSCs. EGFR overexpression suppressed Fas-induced apoptosis in human breast cancer cells,42 and EGF-EGFR exerts its antiapoptotic action partially through the tyrosine kinase activity of EGFR in mouse hepatocytes.43 However, further analyses are warranted to address how the expression of Fas on the cell surface was reduced in EGFR-MSC. In vivo assessment of PKH-labeled MSCs demonstrated that only EGFR-MSCs, but not primary MSCs, were able to migrate toward distant intracranial tumors. Based on our in vitro data, it is thought that this superior migratory response of EGFR-MSC over primary MSCs in vivo resulted from several biological events that were induced by EGFR gene insertion: (1) direct enhancement of migration by interaction of EGFR and its ligands as demonstrated in vitro; (2) resistance to FasL-induced apoptosis, because FasL is known to be expressed in both normal and neoplastic CNS tissues,44 and (3) EGFR may have provided survival signals and other signals to the MSCs, thereby inducing secondary effects that can contribute to enhanced migration, particularly in vivo. With regard to the last point, EGFR is known to activate the serine/threonine kinase Akt through various

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766

mechanisms including activation of PI3-K.45 Akt provides a powerful survival signal in many systems,46 and a recent study has demonstrated that Akt-engineered MSCs are more resistant to apoptosis and can enhance cardiac repair after transplantation into the ischemic rat heart.47 Indeed, both bulk and cloned EGFR-MSCs were maintained as a cell line over 30 passages, whereas primary MSCs died after three to four passages within a span of a month after their harvesting from bone marrow. Although the introduction of growth-stimulating receptor into a stem cell raises concern for potential tumorigenicity, our results demonstrated that EGFRMSC were not tumorigenic. This fits with other observations in the literature that disruption of a single tumorsuppressor gene or activation of one proto-oncogene does not appear to be sufficient for induction of tumors.48 With regard to the role of EGFR transfection in stem cells, a recent study demonstrated that NSCs with a combined loss of both p16 (INK4a) and p19(ARF), but not of p53, p16(INK4a), or p19(ARF), undergo dedifferentiation in response to EGFR transfection.49 Therefore, insertion of the EGFR gene is thought to potentially cause transformation of MSCs that already harbor other critical genetic lesions. In view of this concern, for projected human trials, concurrent transfection with a suicide gene, such as the Herpes Simplex-thymidine kinase (HSVTK),50 and on/off regulatable systems51 for EGFR expression may be considered for warranting an additional level of safety. In addition, it is likely that EGFR-MSC will have their greatest applicability as a vehicle for delivering other therapeutically relevant genes into the tumor site. Our studies showed that EGFR-MSC engineered ex vivo to secrete IFN-a demonstrated therapeutic efficacy following direct intratumoral injections in GL261-bearing animals. It was also noteworthy that even MSCs transfected with a backbone Ad-c5 demonstrated a statistically significant prolongation of survival in comparison to the control animals that had received PBS. Human and mouse bone MSCs inhibit growth of MCF-7 breast cancer cells52 and B16 melanoma cells,53 respectively. Although stroma cells are also known to promote tumor growth in certain conditions,54 our results may support that bone-marrow-derived, genetically engineered MSCs suppress tumor growth in vivo. Furthermore, longterm survival of the animals that received IFN-a transfected EGFR-MSC supports the efficacy and feasibility of this approach for future clinical translation. In addition to their cytotoxic effects on selected tumor cells,55 type I IFNs are known to have broader effects on the immune system by influencing the function and phenotype of dendritic cells (DCs) and T cells.56–58 The fact that EGFR-MSC did not suppress allogeneic MLR responses may be a favorable characteristic for their application in immunotherapy approaches. In conclusion, EGFR gene transfection conferred enhanced migratory ability to intracranial tumors and other favorable biological properties on MSCs as vehicles for immuno-gene therapy approaches for refractory brain tumors.

Cancer Gene Therapy

Acknowledgments

This work was supported by P01 NS40923 (IFP, SCW, HO) and a grant from the Copeland Fund of the Pittsburgh Foundation (HO). We thank Dr Manabu Hatano for helpful discussions in the review of this manuscript.

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