PEX-Producing Human Neural Stem Cells Inhibit Tumor Growth in a Mouse Glioma Model (original) (raw)
Cancer Therapy: Preclinical| August 22 2005
1Department of Neurosurgery, Brigham and Women's Hospital and Children's Hospital and
3Department of Neurosurgery, Seoul National University Hospital, Seoul, South Korea;
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1Department of Neurosurgery, Brigham and Women's Hospital and Children's Hospital and
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4Laboratory for Cancer Drug Delivery and Mammalian Cell Technology, Technion-Israel Institute of Technology, Haifa, Israel; and
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1Department of Neurosurgery, Brigham and Women's Hospital and Children's Hospital and
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2Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts;
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2Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts;
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5Division of Neurology, UBC Hospital, University of British Columbia, Vancouver, British Columbia, Canada
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1Department of Neurosurgery, Brigham and Women's Hospital and Children's Hospital and
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1Department of Neurosurgery, Brigham and Women's Hospital and Children's Hospital and
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Requests for reprints: Rona S. Carroll, Department of Neurosurgery, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, MA 02115. Phone: 671-278-0177; Fax: 617-232-9029; E-mail: rcarroll@rics.bwh.harvard.edu.
Received: February 21 2005
Revision Received: April 11 2005
Accepted: April 26 2005
Online ISSN: 1557-3265
Print ISSN: 1078-0432
American Association for Cancer Research
2005
Clin Cancer Res (2005) 11 (16): 5965–5970.
Received:
February 21 2005
Revision Received:
April 11 2005
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Abstract
A unique characteristic of neural stem cells is their capacity to track glioma cells that have migrated away from the main tumor mass into the normal brain parenchyma. PEX, a naturally occurring fragment of human metalloproteinase-2, acts as an inhibitor of glioma and endothelial cell proliferation, migration, and angiogenesis. In the present study, we evaluated the antitumor activity of PEX-producing human neural stem cells against malignant glioma. The HB1.F3 cell line (immortalized human neural stem cell) was transfected by a pTracer vector with PEX. The retention of the antiproliferative activity and migratory ability of PEX-producing HB1.F3 cells (HB1.F3-PEX) was confirmed in vitro. For the in vivo studies, DiI-labeled HB1.F3-PEX cells were stereotactically injected into established glioma tumor in nude mice. Tumor size was subsequently measured by magnetic resonance imaging and at the termination of the studies by histologic analysis including tumor volume, microvessel density, proliferation, and apoptosis rate. Histologic analysis showed that DiI-labeled HB1.F3-PEX cells migrate at the tumor boundary and cause a 90% reduction of tumor volume (P < 0.03). This reduction in tumor volume in animals treated with HB1.F3-PEX was associated with a significant decrease in angiogenesis (44.8%, P < 0.03) and proliferation (23.6%, P < 0.03). These results support the use of neural stem cells as delivery vehicle for targeting therapeutic genes against human glioma.
The treatment of malignant gliomas remains a challenge in the neurosurgical field. Despite some progress in standard treatment, the overall prognosis of this disease remains dismal, with a mean survival period of less than a year. Most treatment failures in malignant gliomas tend to be local, in the region of the original tumor. These tumors rarely form distant metastasis. Therefore, glioma patients with persistent or recurrent disease may be ideal candidates for direct gene transfer intervention. The effectiveness of this delivery system has been a long-standing issue. For a therapeutic delivery system to be effective, it should be able to target the tumor microsatellites that are the ultimate cause of recurrence (1). Recent studies show that neural stem cells administered intracranially exhibit significant migratory behavior and extensive tropism for experimental glioma (2–6). Therefore, neural stem cells have been suggested to be a tumor-targeting delivery system for glioma gene therapy.
PEX is a naturally occurring fragment of human metalloproteinase-2. It acts as an inhibitor of glioma angiogenesis, cell proliferation, and migration (7, 8). We have previously shown that PEX expression correlated with tumor grade and histologic subtype, being highly expressed in glioblastomas (7). Systemic or local administration of PEX results in a 97% to 99% suppression of intracranial tumor growth with no signs of toxicity in a nude mice model (7).
In the present study, we transfected the gene for PEX into human neural stem cells and subsequently injected them into an established glioma in nude mice. The PEX-producing human neural stem cells decreased angiogenesis and proliferation, leading to a significant inhibition of tumor growth. Our findings lend further support to neural stem cell–based gene therapy for malignant gliomas.
Materials and Methods
Cell culture. HB1.F3 is an immortalized human neural stem cell line derived from the human fetal brain (ventricular zone) at 15 weeks of gestation by an amphotropic, replication-incompetent retroviral vector containing v-myc (9–11). HB1.F3 cells express nestin, a cell type–specific marker for neural stem cells (9, 11). They have proven to be a multipotent, migratory, non–tumor-producing line in vivo model (9–14). HB1.F3 cells were maintained as adherent cultures in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mmol/L l-glutamine, and 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL fungizone (Invitrogen, Grand Island, NY). Cells were maintained in T-75 tissue culture flasks in humidified atmosphere containing 5% CO2 at 37°C.
The human glioblastoma cell line U87MG (American Type Culture Collection, Manassas, VA) was cultured in MEMα (Invitrogen) supplemented with 10% FBS, 2 mmol/L l-glutamine, 2 mmol/L nonessential amino acids, 2 mmol/L sodium pyruvate, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL fungizone.
In vitro transfection of neural stem cells. A 594 bp fragment of human PEX was cloned into _Nhe_I and _Eco_RI sites of the pTracer-BsdPEX vector (Invitrogen). HB1.F3 cells were transfected with pTracer-BsdPEX using the SuperFect transfection reagent (Qiagen, Valencia, CA) and selected under 10 μg/mL blasticidin for 4 weeks.
Reverse transcription-PCR. Total cellular RNA from HB1.F3 and HB1.F3-PEX cells was prepared using RNeasy Mini Kit protocol (Qiagen). Single-stranded cDNA was prepared from 1 μg total RNA using oligo-p(dT)15 primer following the First Strand cDNA Synthesis kit for reverse transcription-PCR (AMV) protocol (Roche, Indianapolis, IN). cDNA from the reverse transcription reaction was subject to PCR in the presence of 0.2 μmol/L of each 5′ and 3′ primers, 2.5 units of Taq polymerase, 1.5 mmol/L MgCl2, 0.2 mmol/L deoxynucleotide mix, and 1× PCR buffer. Touch-down PCR for PEX was carried out for 25 cycles, each consisting of denaturation at 94°C for 4 minutes, annealing at 49.2°C in the first cycle and decreased by 0.5°C for each subsequent cycle, and extension at 72°C for 5 minutes. The final extension was continued for 10 minutes. The housekeeping gene β-actin, a positive control, was amplified for 32 cycles (95°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute, with a final extension at 72°C for 10 minutes). The reaction products were analyzed on a 2.5% agarose gel stained with ethidium bromide. The sense and antisense primers, respectively, and the predicted sizes of the reverse transcription-PCR reaction products were as follows: PEX (sense: 5′-TAATACGACTCACTATAGGG-3′, T7 promoter primer in pTracer-CMV/Bsd plasmid, antisense: 5′-GCCTCGTATACCGCATCAAT-3′, 382 bp) and β-actin (sense: 5′-GCCCAGAGCAAGAGAGGCAT-3′, antisense: 5′-GGCCATCTCTTGCTCGAAGT-3′, 513 bp). PEX is a fragment of MMP-2, which is expressed at high levels in HB1.F3 cells; therefore, for PEX amplification, we used a 5′ primer was from the pTracer vector and 3′ primer from PEX.
Assessment of cell viability. The cytotoxic effect of HB1.F3-PEX was analyzed by coculture experiments. U87MG cells (8 × 103 cells/well) were plated in 96-well plates (Corning, Inc., Acton, MA). After 24 hours, variable numbers of HB1.F3 or HB1.F3-PEX cells (0, 2 × 103, 4 × 103, 8 × 103, 16 × 103, and 32 × 103 cells/well) were added to the inside of the inserts (Nalge Nunc International, Rochester, NY) with 0.2 μm pore size. After placing the inserts over the lower chamber, cells were incubated for 96 hours. Quantitation of cell viability was done with colorimetric assays using Cell Counting Kit-8 (Dojindo Molecular Technologies, Gaithersburg, MD). All experiments were conducted in quadruplicate. Viability determination was based on the bioconversion of the tetrazolium compound, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2_H_-tetrazolium (WST-8), into formazan, as determined by absorbance at 450 nm using a multiwell scanning spectrophotometer. Cell viability was expressed as the mean ± SE in percentage of the control viability (=100%).
In vitro migration assay. The migratory ability of HB1.F3-PEX on U87MG-derived extracellular matrix was compared with that of HB1.F3 using a modified method previously described by Kaczarek et al. (15). Briefly, U87MG cells (3,000 cells/well) were allowed to grow to confluence on the wells of the Teflon 10-well printed microscope slides (Creative Scientific Methods, Inc., Phoenix, AZ) for 3 days. The cells were lysed from the extracellular matrix using 20 mmol/L of NH4OH and rinsed with PBS. A 1.5 μL cell suspension containing 3,500 cells/μL of HB1.F3 or HB1.F3-PEX was added to each cylinder of the 10-cylinder cell sedimentation manifold (Creative Scientific Methods) over the slide. The cell sedimentation manifold was removed after sedimentation of the cells and the cells were allowed to spread. The area occupied by the attached cells was photographed using an inverted microscope (Nikon Eclipse TE300) connected to a Spot RT Slider digital camera (Diagnostic Instruments, Inc., Houston, TX). The measurements were conducted every 24 hours for 72 hours after the removal of the cell sedimentation manifold in triplicate. The migration was calculated as the increase of the radius beyond the initial radius (the moment when the cell sedimentation manifold was removed) and expressed as the mean ± SE.
In vivo migration assay. To address the relevance of migration in vivo, HB1.F3-PEX cells were inoculated into the hemisphere contralateral to the tumor cell inoculation. Briefly, Swiss nude male mice (n = 4, 6-8 weeks old; Charles River, Wilmington, MA) were anesthetized (100 mg/kg ketamine and 5 mg/kg xylazine) and stereotactically inoculated with 120,000 U87MG cells in 3 μL of PBS via a 30-gauge Hamilton syringe into the left forebrain (2.5 mm lateral and 1 mm anterior to bregma, at a 2.5 mm depth from the skull surface). One week after tumor cell inoculation, HB1.F3-PEX cells (240,000 cells in 3 μL of PBS) were inoculated into the right forebrain (2.5 mm lateral and 1 mm anterior to bregma at a 2.5 mm depth from the skull surface). For this study, the stem cell to tumor cell ratio was 2:1. HB1.F3-PEX was labeled with the lipophilic tracer DiI (D-282; Molecular Probes, Eugene, OR) immediately before injection for 30 minutes according to the protocol of the manufacturer. All animal studies were carried out in the animal facility at Brigham and Women's Hospital in accordance with federal, local, and institutional guidelines.
Inoculation of therapeutic cells into established intracranial gliomas. After anesthesia, animals received stereotactic inoculation of U87MG cells (120,000 in 3 μL of PBS) into the left forebrain at the following coordinates: 2.5 mm lateral and 1 mm anterior to bregma at a 2.5 mm depth from the skull surface previously described. Three days after tumor cell injection, the animals were randomized into three groups and treated with ipsilateral intratumoral inoculations of PBS (8 μL; n = 6), 480,000 HB1.F3 cells (n = 6), or 480,000 HB1.F3-PEX cells (n = 6) in 8 μL of PBS at the established tumor site using the same burr hole and stereotactic coordinates. For this study, stem cell to tumor cell ratio was 4:1. HB1.F3 and HB1.F3-PEX were labeled with the lipophilic tracer DiI (Molecular Probes) immediately before injection.
Magnetic resonance imaging experiments. Seven days after ipsilateral intratumoral inoculation of human neural stem cells, magnetic resonance imaging (MRI) experiments were done on a Bruker 4.7 T system, operating on Paravision (version 3.0.1) software platform (Bruker, Billerica, MA). Mice (n = 18) from all experimental groups were anesthetized with 1% isoflurane in an oxygen/air mixture. Respiratory rate was monitored using a Bruker Physioguard vital sign monitor. The animals were maintained at 37°C inside the magnet using a temperature-controlled water jacket. A T2-weighted image with RARE sequence (TR = 3,000 ms, TE = 50 ms, NEX = 4) was done to acquire 21 coronal slices from the whole brain with a slice thickness of 0.75 mm, a matrix size of 128 × 128 and a field of view of 2.56 × 2.56 cm2. After T2-weighted image was acquired, Gadopentetate dimeglumine (Berlex Laboratories, Wayne, NJ) was administered i.p. (0.8 mL/kg body weight). T1-weighted post-Gd images were obtained 15 minutes after contrast injection using TR = 1,000 ms, TE = 8.8 ms, NEX = 2 and a slice thickness of 0.75 mm, matrix size of 128 × 128, and a field of view of 2.56 × 2.56 cm2. Tumor volumes were estimated using Gd-enhanced T1-weighted spin-echo images, from which three-dimensional renderings of the tumors were generated with in-house three-dimensional software (16, 17).
Determination of intracranial distribution and in vivo tumor size. Two weeks after therapeutic intratumoral, ipsilateral inoculation of human neural stem cell, animals were perfused with 4% paraformaldehyde under deep anesthesia. These animals were the same animals previously used in the MRI experiments. The brains were removed, placed in sucrose gradient solution, embedded in optimum cutting temperature compound (Tissue-Tek, Miles, Elkhart, IN), and stored at −80°C. Brains were sectioned coronally using a cryostat into 10-μm-thick slices that were mounted on slides and then stained with H&E or 4′,6-diamidino-2-phenylindole (DAPI) as per standard protocol. Using fluorescence microscopy, intracranial distribution of DiI-labeled human neural stem cells was assessed. Tumor volumes were estimated using the formula for ellipsoid and expressed as a mean ± SE as previously described (16).
Immunohistochemistry. Immunohistochemistry was carried out using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Brain sections were fixed in cold acetone. Primary antibodies included anti-CD31 (1:100; BD Biosciences PharMingen, San Jose, CA) for blood vessel density, anti-Ki67 nuclear antigen (1:100; DAKO, Carpinteria, CA) for proliferating cells, and anti–cleaved caspase-3 (1:100; Cell Signaling Technology, Beverly, MA) for the detection of apoptosis. Sections were counterstained with hematoxylin, and negative control slides were obtained by omitting the primary antibody. Microvessel count was assessed according to the method described by Leon et al. (18) and Weidner et al. (19). The proliferation and apoptotic indices were defined as the percentage of positively stained cells of 100 nuclei from five randomly chosen high-power fields.
Statistics. All of the values were calculated as mean ± SE or were expressed as percentage of control ± SE. Significant differences between assessment of cell viability, tumor volume, proliferation, apoptosis index, and microvessel density were determined using the Mann-Whitney U test. Values of P < 0.05 were considered significant.
Results
Effect of HB1.F3-PEX on viability of U87MG cells in vitro. HB1.F3-PEX cells were obtained by transfection of the pTracer-BsdPEX vector with SuperFect followed by blasticidin selection. Expression of the PEX transcript was confirmed by reverse transcription-PCR. Reverse transcription-PCR showed that the PEX transcript was present only in HB1.F3-PEX cells, but not in HB1.F3 cells (Fig. 1A) To confirm the biological activity of the PEX released from the transfected HB1.F3 cells, cell viability studies were done (Fig. 1B). U87MG cells cultured in the presence of HB1.F3-PEX cells, but not HB1.F3 cells, showed significant growth inhibition from the ratio of HB1.F3-PEX cells to tumor cells of 1:2. These results confirmed the ability of HB1.F3-PEX cells to produce and secrete biologically relevant quantities of the PEX protein.
Fig. 1.
The analysis of expression and bioactivity of PEX from HB1.F3-PEX. A, HB1.F3 cells were transfected by SuperFect pTracer-BsdPEX. Reverse transcription-PCR products from HB1.F3 and HB1.F3-PEX cells were analyzed on a 2.5% agarose gel stained with ethidium bromide. Actin controls confirmed equal protein loading. B, U87MG cells (8 × 103 cells/well) were plated in 96-well cell plates. After 24 hours, HB1.F3 or HB1.F3-PEX cells (0, 2 × 103, 4 × 103, 8 × 103, 16 × 103, and 32 × 103 cells/well) were added to the inside of the inserts with 0.2 μm pore size. After 96 hours of incubation, quantitation of cell viability was done with colorimetric assays using Cell Counting Kit-8. Columns, mean cell viability as a percentage of the control viability; bars, SE (*P < 0.05, Mann-Whitney U test).
Fig. 1.
The analysis of expression and bioactivity of PEX from HB1.F3-PEX. A, HB1.F3 cells were transfected by SuperFect pTracer-BsdPEX. Reverse transcription-PCR products from HB1.F3 and HB1.F3-PEX cells were analyzed on a 2.5% agarose gel stained with ethidium bromide. Actin controls confirmed equal protein loading. B, U87MG cells (8 × 103 cells/well) were plated in 96-well cell plates. After 24 hours, HB1.F3 or HB1.F3-PEX cells (0, 2 × 103, 4 × 103, 8 × 103, 16 × 103, and 32 × 103 cells/well) were added to the inside of the inserts with 0.2 μm pore size. After 96 hours of incubation, quantitation of cell viability was done with colorimetric assays using Cell Counting Kit-8. Columns, mean cell viability as a percentage of the control viability; bars, SE (*P < 0.05, Mann-Whitney U test).
Migratory capacity of HB1.F3-PEX in vitro and in vivo. The effects of PEX transfection on the migration of HB1.F3 were assessed in vitro and in vivo. The migration pattern of the HB1.F3-PEX cells on the U87MG-produced extracellular matrix was indistinguishable from the migration pattern of HB1.F3 cells. The incremental expansion of the advancing rim of migrating neural stem cells followed a linear relationship over time regardless of PEX transfection (Fig. 2).
Fig. 2.
The migratory ability of HB1.F3-PEX on U87MG-derived extracellular matrix. A 10-cylinder cell sedimentation manifold was placed over the slide on U87MG-derived extracellular matrix. A 1.5 μL cell suspension (3,500 cells/μL) of HB1.F3 or HB1.F3-PEX was added to each cylinder of the cell sedimentation manifold. Migration value was quantified as the increase in the radius of the circle occupied by the cell population beyond that measured on time 0 and every 24 hours for 72 hours after the removal of the cell sedimentation manifold.
Fig. 2.
The migratory ability of HB1.F3-PEX on U87MG-derived extracellular matrix. A 10-cylinder cell sedimentation manifold was placed over the slide on U87MG-derived extracellular matrix. A 1.5 μL cell suspension (3,500 cells/μL) of HB1.F3 or HB1.F3-PEX was added to each cylinder of the cell sedimentation manifold. Migration value was quantified as the increase in the radius of the circle occupied by the cell population beyond that measured on time 0 and every 24 hours for 72 hours after the removal of the cell sedimentation manifold.
In vivo studies showed that HB1.F3-PEX cells could migrate toward intracranial glioma through normal brain parenchyma in a similar pattern of untransfected cells. U87MG glioma cells were inoculated into the left forebrain, and 1 week later DiI-labeled HB1.F3-PEX cells were inoculated into the contralateral hemisphere. HB1.F3-PEX cells migrated away from the initial injection site and targeted the tumor (Fig. 3). DiI-labeled HB1.F3-PEX cells, inoculated intracranially, were found mostly at the border between tumor and normal parenchyma. These findings confirmed that the potential for migration of HB1.F3-PEX was not affected by the PEX transfection.
Fig. 3.
Intracranial distribution of DiI-labeled HB1.F3-PEX. HB1.F3-PEX were labeled with the lipophilic tracer DiI immediately before injection into contralateral (A-C) or ipsilateral hemisphere (D-F). Brains were sectioned coronally into 10-μm-thick slices and then stained with DAPI. Using fluorescence microscopy, intracranial distribution of DiI-labeled HB1.F3-PEX was assessed; arrow, tumor margin; arrowhead, corpus callosum (original magnification, ×100).
Fig. 3.
Intracranial distribution of DiI-labeled HB1.F3-PEX. HB1.F3-PEX were labeled with the lipophilic tracer DiI immediately before injection into contralateral (A-C) or ipsilateral hemisphere (D-F). Brains were sectioned coronally into 10-μm-thick slices and then stained with DAPI. Using fluorescence microscopy, intracranial distribution of DiI-labeled HB1.F3-PEX was assessed; arrow, tumor margin; arrowhead, corpus callosum (original magnification, ×100).
Effect of HB1.F3-PEX on in vivo tumor growth. To assess the therapeutic efficacy of HB1.F3-PEX in glioma animal models, we monitored tumor volume by MRI 7 days after intracranial human neural stem cell inoculation. T2-weighted images showed heterogeneous high signal intensity areas in the left hemisphere, indicating the presence of human glioblastoma xenografts. The U87MG glioma was clearly visible as an enhanced area by T1-weighted imaging in the coronal section of the brain of control animals (9.7 ± 2.20 mm3 in PBS-treated and 8.7 ± 2.07 mm3 in HB1.F3-treated respectively; P > 0.1, PBS versus HB1.F3). In contrast, significantly smaller tumor volumes were measured in the brains of animals treated with HB1.F3-PEX (Fig. 4; 1.00 ± 1.53 mm3; P < 0.03, PBS versus HB1.F3-PEX; P < 0.03, HB1.F3 versus HB1.F3-PEX).
Fig. 4.
Tumor volumes as determined by MRI at 7 days after therapeutic intratumoral inoculation of human neural stem cells and histologic analysis at 14 days after inoculation of human neural stem cells (n = 18). A, representative images from MRI (Gd-T1–weighted images). B, representative photographs from histology (H&E staining, original magnification, ×1). C, tumor volumes were estimated from MRI; columns, mean; bars, SE (P < 0.03, PBS versus HB1.F3-PEX; P < 0.03, HB1.F3 versus HB1.F3-PEX, Mann-Whitney U test). D, tumor volumes were estimated from histologic analysis; columns, mean; bars, SE (P < 0.03, PBS versus HB1.F3-PEX; P < 0.03, HB1.F3 versus HB1.F3-PEX, Mann-Whitney U test).
Fig. 4.
Tumor volumes as determined by MRI at 7 days after therapeutic intratumoral inoculation of human neural stem cells and histologic analysis at 14 days after inoculation of human neural stem cells (n = 18). A, representative images from MRI (Gd-T1–weighted images). B, representative photographs from histology (H&E staining, original magnification, ×1). C, tumor volumes were estimated from MRI; columns, mean; bars, SE (P < 0.03, PBS versus HB1.F3-PEX; P < 0.03, HB1.F3 versus HB1.F3-PEX, Mann-Whitney U test). D, tumor volumes were estimated from histologic analysis; columns, mean; bars, SE (P < 0.03, PBS versus HB1.F3-PEX; P < 0.03, HB1.F3 versus HB1.F3-PEX, Mann-Whitney U test).
Tumor volumes were determined from glioma-bearing brains harvested from mice 14 days after neural stem cell inoculation by histologic analysis. The average tumor volume in controls, PBS-treated, and HB1.F3-treated animals were 31.6 ± 20.89 and 21.7 ± 11.75 mm3, respectively, compared with only 3.29 ± 3.44 mm3 in HB1.F3-PEX–treated brains. This decrease in tumor size associated with HB1.F3-PEX treatment was highly significant as has also been shown by a MRI study (P < 0.03, PBS versus HB1.F3-PEX; P < 0.03, HB1.F3 versus HB1.F3-PEX; Fig. 4).
We next sought to determine biological action of PEX on glioma. Histologic analysis revealed a significant decrease in tumor vascularization by 44.8% and a significant decrease in the proliferative index by 23.6% in those treated with HB1.F3-PEX compared with the respective control groups (P < 0.03, PBS versus HB1.F3-PEX; P < 0.03, HB1.F3 versus HB1.F3-PEX; Fig. 5). In contrast, the apoptotic indices revealed no significant differences among groups.
Fig. 5.
Treatment effects assessed by immunohistochemistry (n = 18). Primary antibodies included anti-CD31 for blood vessels, anti-Ki67 nuclear antigen for proliferating cells, and anti–cleaved caspase-3 for the detection of apoptosis. Sections were counterstained with H&E (original magnification, ×200). Columns, mean microvessel count, proliferation index, and apoptotic index as percentages of the control; bars, SE (microvessel count and proliferation index, P < 0.03, PBS versus HB1.F3-PEX; P < 0.03, HB1.F3 versus HB1.F3-PEX, Mann-Whitney U test).
Fig. 5.
Treatment effects assessed by immunohistochemistry (n = 18). Primary antibodies included anti-CD31 for blood vessels, anti-Ki67 nuclear antigen for proliferating cells, and anti–cleaved caspase-3 for the detection of apoptosis. Sections were counterstained with H&E (original magnification, ×200). Columns, mean microvessel count, proliferation index, and apoptotic index as percentages of the control; bars, SE (microvessel count and proliferation index, P < 0.03, PBS versus HB1.F3-PEX; P < 0.03, HB1.F3 versus HB1.F3-PEX, Mann-Whitney U test).
Discussion
The discovery of multipotential, self-renewing neural stem cells offers new approaches to brain tumor researchers. In neuro-oncology, the biology of neural stem cells has been pursued in two ways: (a) as cancer stem cells to understand the origin and maintenance of brain tumors (20–24) and (b) as a potential cell-based vehicle for gene therapy (2–6, 25–29).
Regarding the first issue, some groups have described the existence of a cancer stem cell population in human brain tumors using separation techniques with selective neural stem cell markers (20–22, 24). The identification and characterization of brain tumor stem cells provides a valuable tool to understand their biology more clearly and to develop therapies targeted to the brain tumor stem cells. On the other hand, several studies have focused on tumor-tropic capacities of neural stem cells. Neural stem cell lines have been shown to be effective for delivering transgenes to brain tumors based on their unique migratory properties within the central nervous system (2–5, 28, 29). Migratory genetically modified neural stem cells have been reported to promote tumor regression and prolong survival of glioma-bearing animals (3–6, 27, 29). Genes encoded for neural stem cell–based therapy included immune enhancing genes, such as interleukin-4 (29), interleukin-12 (4, 25); apoptosis promoting gene, such as tumor necrosis factor-related apoptosis-inducing ligand (2, 3); prodrug converting enzymes, such as cytosine deaminase (5, 27) and herpes simplex virus thymidine kinase (30); and herpes simplex viral therapy (6). Moreover, it was found that the unmodified stem cells also had a potential for tumor outgrowth inhibition, indicating that neural stem cells themselves may produce some factors with an antitumor activity (31).
For the treatment of gliomas, we combined the extensive tumor tracking capability of neural stem cells with the antiangiogenic and antiproliferative potency of PEX. Our cellular vehicle system has two very unique characteristics. First, the neural stem cells used in our study are of human origin, as opposed to other reported groups with murine–origin neural stem cells. In the clinical perspective, human-origin neural stem cells could be used more safely with less immune rejection than murine-origin neural stem cells, avoiding the potential dangers of xenograft. Second, the neural stem cells we used as a therapeutic vehicle were engineered to have the ability to significantly reduce glioma angiogenesis and migration. This is the first experimental study in which neural stem cells were used to deliver an antiangiogenic gene into an established human xenograft. This resulted in the potent reduction of angiogenesis and proliferation, leading to a highly significant inhibition of tumor growth compared with neural stem cell only or PBS-inoculated controls. In this paradigm, the neural stem cells have the unique ability to engulf the tumor and concentrate in the tumor boundary where angiogenesis is most active. Treatment effects of HB1.F3-PEX in our animal model were confirmed by magnetic resonance imaging. This noninvasive technique can monitor dynamic changes of tumor growth during treatment and provide an independent measurement of tumor volume (17). It has a wide range of applications for the study of novel therapeutics for brain tumors.
We showed a 90% reduction of tumor growth in a xenograft nude mice model by inoculation of HB1.F3-PEX cells, suggesting stable expression of PEX in vivo. However, we did not achieve complete tumor regression by this therapeutic approach. One possible explanation of this is that, in our study, no increase in apoptosis was observed. Our previous study (7) also showed that to achieve the proapoptotic activity of PEX, much higher concentrations were needed than those required to inhibit glioma growth or migration (25 versus 1 versus 10 μg/mL). In our study, HB1.F3-PEX cells may not be able to produce sufficient amount of PEX protein to induce apoptosis of glioma cells. However, high concentration of PEX gene could induce apoptosis of neural stem cells themselves and inhibit their migratory ability. For PEX-producing neural stem cells to be an effective therapeutic delivery vehicle, the amount of PEX produced must be carefully balanced. Although therapeutic effect was assessed 2 weeks after inoculation of human neural stem cell, we did not observe any signs of local or systemic toxicity in HB1.F3- and HB1.F3-PEX–treated groups.
The HB1.F3-PEX could migrate to tumor site and inhibit angiogenesis without inducing apoptosis or inhibiting cell motility. One way to increase therapeutic efficiency of HB1.F3-PEX would be using a construct that has an inducible promoter, such as the tetracycline silencer system (32). This could allow a higher level of PEX protein expression after neural stem cells migrate within and beyond the tumor mass.
In conclusion, human-origin neural stem cells engineered to produce PEX can migrate toward glioma and have strong antitumor effect. Our finding strengthens the potential strategies aimed at malignant gliomas using human neural stem cells as a targeting vehicle for therapeutic gene transfer. Neural stem cells might be used with standard therapies in parallel to reduce the incidence of recurrence and improve the survival of patients. After tumor removal surgery, PEX-producing human neural stem cells could be grafted into tumor resection cavity to target residual invading tumor cells and to inhibit tumor angiogenesis and proliferation.
Grant support: NIH, Postdoctoral Fellowship Program of Korea Science and Engineering Foundation (S.K. Kim), and Whitaker Foundation grant RG-01-0251 (Y. Sun).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
References
1
Dunn IF, Black PM. The neurosurgeon as local oncologist: cellular and molecular neurosurgery in malignant glioma therapy.
Neurosurgery
2003
;
52
:
1411
–24.
2
Shah K, Bureau E, Kim DE, et al. Glioma therapy and real-time imaging of neural precursor cell migration and tumor regression.
Ann Neurol
2005
;
57
:
34
–41.
3
Ehtesham M, Kabos P, Gutierrez MA, et al. Induction of glioblastoma apoptosis using neural stem cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand.
Cancer Res
2002
;
62
:
7170
–4.
4
Ehtesham M, Kabos P, Kabosova A, Neuman T, Black KL, Yu JS. The use of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma.
Cancer Res
2002
;
62
:
5657
–63.
5
Aboody KS, Brown A, Rainov NG, et al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas.
Proc Natl Acad Sci U S A
2000
;
97
:
12846
–51.
6
Herrlinger U, Woiciechowski C, Sena-Esteves M, et al. Neural precursor cells for delivery of replication-conditional HSV-1 vectors to intracerebral gliomas.
Mol Ther
2000
;
1
:
347
–57.
7
Bello L, Lucini V, Carrabba G, et al. Simultaneous inhibition of glioma angiogenesis, cell proliferation, and invasion by a naturally occurring fragment of human metalloproteinase-2.
Cancer Res
2001
;
61
:
8730
–6.
8
Brooks PC, Silletti S, von Schalscha TL, Friedlander M, Cheresh DA. Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity.
Cell
1998
;
92
:
391
–400.
9
Cho T, Bae JH, Choi HB, et al. Human neural stem cells: electrophysiological properties of voltage-gated ion channels.
Neuroreport
2002
;
13
:
1447
–52.
10
Ourednik V, Ourednik J, Flax JD, et al. Segregation of human neural stem cells in the developing primate forebrain.
Science
2001
;
293
:
1820
–4.
11
Flax JD, Aurora S, Yang C, et al. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes.
Nat Biotechnol
1998
;
16
:
1033
–9.
12
Chu K, Kim M, Chae SH, et al. Distribution and in situ proliferation patterns of intravenously injected immortalized human neural stem-like cells in rats with focal cerebral ischemia.
Neurosci Res
2004
;
50
:
459
–65.
13
Chu K, Kim M, Jeong SW, Kim SU, Yoon BW. Human neural stem cells can migrate, differentiate, and integrate after intravenous transplantation in adult rats with transient forebrain ischemia.
Neurosci Lett
2003
;
343
:
129
–33.
14
Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage.
Stroke
2003
;
34
:
2258
–63.
15
Kaczarek E, Zapf S, Bouterfa H, Tonn JC, Westphal M, Giese A. Dissecting glioma invasion: interrelation of adhesion, migration and intercellular contacts determine the invasive phenotype.
Int J Dev Neurosci
1999
;
17
:
625
–41.
16
Schmidt KF, Ziu M, Schmidt NO, et al. Volume reconstruction techniques improve the correlation between histological and in vivo tumor volume measurements in mouse models of human gliomas.
J Neurooncol
2004
;
68
:
207
–15.
17
Sun Y, Schmidt NO, Schmidt K, et al. Perfusion MRI of U87 brain tumors in a mouse model.
Magn Reson Med
2004
;
51
:
893
–9.
18
Leon SP, Folkerth RD, Black PM. Microvessel density is a prognostic indicator for patients with astroglial brain tumors.
Cancer
1996
;
77
:
362
–72.
19
Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma.
N Engl J Med
1991
;
324
:
1
–8.
20
Galli R, Binda E, Orfanelli U, et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.
Cancer Res
2004
;
64
:
7011
–21.
21
Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells.
Nature
2004
;
432
:
396
–401.
22
Yuan X, Curtin J, Xiong Y, et al. Isolation of cancer stem cells from adult glioblastoma multiforme.
Oncogene
2004
;
23
:
9392
–400.
23
Recht L, Jang T, Savarese T, Litofsky NS. Neural stem cells and neuro-oncology: quo vadis?
J Cell Biochem
2003
;
88
:
11
–9.
24
Singh SK, Clarke ID, Terasaki M, et al. Identification of a cancer stem cell in human brain tumors.
Cancer Res
2003
;
63
:
5821
–8.
25
Yang SY, Liu H, Zhang JN. Gene therapy of rat malignant gliomas using neural stem cells expressing IL-12.
DNA Cell Biol
2004
;
23
:
381
–9.
26
Yip S, Aboody KS, Burns M, et al. Neural stem cell biology may be well suited for improving brain tumor therapies.
Cancer J
2003
;
9
:
189
–204.
27
Barresi V, Belluardo N, Sipione S, Mudo G, Cattaneo E, Condorelli DF. Transplantation of prodrug-converting neural progenitor cells for brain tumor therapy.
Cancer Gene Ther
2003
;
10
:
396
–402.
28
Brown AB, Yang W, Schmidt NO, et al. Intravascular delivery of neural stem cell lines to target intracranial and extracranial tumors of neural and non-neural origin.
Hum Gene Ther
2003
;
14
:
1777
–85.
29
Benedetti S, Pirola B, Pollo B, et al. Gene therapy of experimental brain tumors using neural progenitor cells.
Nat Med
2000
;
6
:
447
–50.
30
Uhl M, Weiler M, Wick W, Jacobs AH, Weller M, Herrlinger U. Migratory neural stem cells for improved thymidine kinase-based gene therapy of malignant gliomas.
Biochem Biophys Res Commun
2005
;
328
:
125
–9.
31
Staflin K, Honeth G, Kalliomaki S, Kjellman C, Edvardsen K, Lindvall M. Neural progenitor cell lines inhibit rat tumor growth in vivo.
Cancer Res
2004
;
64
:
5347
–54.
32
Freundlieb S, Schirra-Muller C, Bujard H. A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells.
J Gene Med
1999
;
1
:
4
–12.
American Association for Cancer Research
2005