Glioblastoma is the commonest form of primary brain tumor as well as the most aggressive, hence having the worst prognosis with less than one year of median survival (Vogel et al., 2005). Glioblastoma multiforme remain virtually untreatable and lethal (Imperato et al., 1990; Black et al., 2005; Phillips et al., 2006). Multimodal treatment including radical surgical resection followed by radiation and chemotherapy, has substantially improved the survival rate in patients from glioblastoma, however prognosis of the majority of patients remains poor (Surawicz et al., 1999).
During the past 20 years gene therapy research has advanced greatly, and over 1,340 gene therapy clinical trials have been completed or are ongoing worldwide in 28 countries, and more than 70% of these trials are in cancer gene therapy(Edelstein et al., 2007).
However, clinical trials in cancer gene therapy achieved only a limited success because of the low efficiency of gene transfer by currently available vectors and the inability of these vectors to specifically target cancer cells (Crystal, 1995; Anderson, 1998; Benedetti et al., 2000; Marchisone et al., 2000).
Recently studies have shown that murine and human neural stem cells (NSCs) possess an inherent tumor-tropism that supports their use as a reliable delivery vehicle to target therapeutic gene products to primary and secondary invasive brain tumors (Aboody et al., 2000; Kim et al., 2005; Kim et al., 2006a; Lin et al., 2007; Najbauer J, 2007; Shimato et al., 2007; Aboody et al., 2008; Mercapide et al., 2009). Stem and progenitor cell-mediated gene delivery is emerging as a strategy to improve the efficacy and minimize the toxicity of current gene therapy approaches(Aboody et al., 2008).
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Recently we have generated a stable immortalized cell line of human NSC by introduction of v-myc oncogene, and this human NSC line, HB1.F3 (F3), was successfully utilized in NSC-based gene therapy in animal model of neurological disorders (Kim et al., 2004). There are many studies that have demonstrated the tumor-tropism of NSCs for glioma in rat brain using PET or other image programs. However, little, if any is known about the number of NSCs migrated to glioma sequentially and the rate and pattern of NSCs migration in the brain over time after NSC transplantation. Therefore, in the present study we used unbiased and objective measuring method via the stereological counting system.
The present study as well as previous studies using the same human NSCs, demonstrated that the NSCs serve as powerful and attractive delivery vehicle for tracking glioma. Ten percent of the injected NSCs could migrate into the contralateral tumor site within 1 hour.
The selective and specific nature of NSC’s tumor-tropic property should serve well in suicide/immune therapy for brain tumors. In addition, transplanted NSCs migrated not only to tumor site but also other regions such as hippocampus, auditory cortex and olfactory bulb in other animal models of neurological diseases (Jeong et al., 2003; Lee et al., 2005;
Yasuhara et al., 2006). The neural stem cell-based gene therapy should prove as the powerful therapeutic choice for the patients suffering from neurological diseases.
In the present study, we investigated specific tropism characteristics of human NSCs,
including rate of migration, area of spread, survival pattern, and proliferation in the brain parenchyma in a rat brain tumor model. These results may provide useful information on the nature of tumor-tropism of the NSCs.
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Ⅱ. MATERIALS AND METHODS
1. Cell culture
The human glioblastoma cell line U373MG was obtained from the American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 4 mM L-glutamine, 4.5 g/L 2008) F3 cells were maintained as adherent cultures in DMEM supplemented with 5% FBS, 4 mM L-glutamine, 4.5 g/L glucose, 110 mg/L sodium pyruvate, and 100 units/mL penicillin and 100 μg/mL streptomycin. F3 cells transfected with LacZ gene (HB1.F3.LacZ) were cultured under the same conditions.
2. In vivo protocol of experimental tumor models
Sprague-Dawley (SD) female rats (250g, Samtaco, Osan, Korea) were anesthetized with 10% chloral hydrate (Fluka, Germany) and placed in a stereotaxic apparatus. Three µl of PBS containing 1x106 glioma cells were intracranially transplanted at the rate of 0.2 µl /min
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using a 26 gauge Hamilton micro-syringe. The target was the right caudate-putaman at [antero/posterior (AP) +0.4mm, medial/lateral (ML) -3.1mm, dorsal/ventral (DV) 4 mm].
After tumor cell inoculation, the human neural stem cells transplanted opposite site of hemisphere.
3. In vitro and in vivo cell proliferation assays
In vitro cell proliferation assays were performed with a thiazolyl blue tetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, MO). In brief, F3 cells, F3.LacZ cells, and U373MG cells grown in serum-containing medium for 3 days were isolated, and were plated in 96-well culture plates (1 × 104 cells in a 100-μL volume per well) in DMEM.
After a 2-hour plating period, MTT labeling agent [0.5 mg/mL in 10 μL phosphate-buffered saline (PBS)] was added to each well at 0, 12, 24, and 36 hours and on days 2, 3, 5, and 7 and then incubated for 4 hours. A volume of 100 μL solubilization buffer [10% sodium dodecyl sulfate (Sigma-Aldrich) and 50% N,N-dimethyl formamide (Sigma-Aldrich), pH 7.4] was added to each well and allowed to incubate overnight; the absorbance of each well was then measured at 570–630 nm (Sanfeliu et al., 1999).
In vivo cell proliferation assays were performed with HB1.F3 cell labeled with 10μM BrdU (1 x 105 cells) injected into the left forebrain (~3.1 mm lateral and 0.4 mm anterior to bregma, at a 3-mm depth from the skull surface) of 8-week-old female Sprague-Dawley rats.
The rats (N=20) were received intraperitoneally of 50mg of BrdU/kg of body weight at a concentration of 10mg/ml BrdU (Roche) in sterile 0.9% NaCl solution every 2 days until 24
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hrs before perfusion. After 50 min as well as on days 1, 5, and 15, brains were removed and fixed in 4% paraformaldehyde overnight at 4 °C and soaked in 30% sucrose for an additional 48 hrs. Coronal cryosections (50-μm-thick) of the brain were prepared on a cryostat.
4. In vivo analysis of tumor-tropism of human neural stem cells
U373MG human glioblastoma cells were incubated in 10 μM PKH26 (Sigma-Aldrich) in diluent C solution for 4 min, cells were washed by repeated centrifugation at 400 × g for 10 minutes then harvested for transplantation (Hemmrich et al., 2006). U373MG cells labeled with PKH26 were injected into the striatum of 8-week-old female Sprague-Dawley rats. Rats were anesthetized with 10% chloral hydrate and received 1 × 106 tumor cells in 3 μL Hanks’
balanced salt solution (HBSS; Hyclone, Logan, UT) stereotactically via a 26-gauge Hamilton syringe into the right forebrain (~3.1 mm lateral and 0.4 mm anterior to bregma, at a 4-mm depth from the skull surface). Seven days after tumor cell injection, F3 cells (1 × 106 cells in 3 μL HBSS) were labeled with Hoechst 33258 (10 mg/mL, Molecular Probes, Eugene, OR) for 5 min and injected stereotactically into the opposite hemisphere at the same coordinates.
The migration of F3 cells into the brain parenchyma was observed between 6 hours and 10 days. At each time point (40 and 50 min; 24, 30, 36, and 42 hours; and days 2, 3, 5, 7, and 10; Fig. 1), brains were removed and fixed in 4% paraformaldehyde in 0.1M phosphate buffer overnight at 4 °C and soaked in 30% sucrose for an additional 48 hrs. Coronal
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cryosections (30-μm-thick) were generated. Presence of F3 cells in the transplantation area, the tumor region, as well as the corpus callosum, hippocampus, and auditory cortex was determined but not in the cerebellum. Number of migrated NSCs and the pattern of migration were analyzed with the Computer-Assisted Stereological Toolbox system (CAST-Grid, version 2.1.4; Olympus Denmark, Ballerup, Denmark).
5. X-gal stain
For immunohistochemical studies, the brains of wild-type rats and experimental group with successive tumor cell-NSC injection were removed and fixed in 4% paraformaldehyde in 0.1M phosphate buffer overnight at 4 °C and soaked in 30% sucrose for an additional 48 hrs. Tissues were embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) and frozen at -20 °C until sectioning. Sagittal cryosections (50 μm-thick) were then prepared on a cryostat. Sections were rinsed three times in PBS for 5 min each, and incubated overnight in X-gal staining solution consisted of PBS containing 2 M MgCl2, 0.2 M potassium ferricyanide, 0.2 M potassium ferrocyanide, and 40 mg/mL X-gal (Amresco, Solon, OH) at 37 °C(Buckner et al., 1999) Sections were counterstained with eosin Y solution, dehydrated in an ethanol series, cleared in xylene, and mounted on slides with Permount (Fisher Scientific, Seoul, Korea). Sections were also stained with Harris hematoxylin solution for 2 min and followed in eosin Y solution for 30 sec, dehydrated in an ethanol series, cleared in xylene, and mounted on slides with Permount solution.
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6. Cell count and unbiased stereology in a tumor region of rats
The rats (N=25) were transplanted with human NSC line expressing LacZ gene (F3.LacZ) contra-lateral side of tumor inoculation after human glioblastoma cell line (U373MG) injection. To perform unbiased stereological estimation, LacZ-positive cells in the tumor region were counted with an optical fractionators (West, 1993). The CAST-grid system was equipped with an Olympus BX51 microscope, a motorized microscope stage run by an IBM-compatible computer, and a microcator (Prior Scientific Instruments, Cambs, UK) connected to the stage to provide distance information for the z-axis(Kim et al., 2006b;
Garcia et al., 2007). The x-and y-frame sliders are used to specify the number of frames per unit in the horizontal and vertical directions. The percentage refers to the area of the counting frame compared to the screen area. If more than on frame is present the percentage refers to the total area of the frames. The tumor region was delineated with a 1.25× objective and a counting grid of 300 × 300 μm. An unbiased counting frame of known area (48.6 × 36.1 μm = 1757 μm2) superimposed on the image was placed randomly over the first counting area and moved systemically over all counting areas until the entire delineated area was sampled. Stereological cell counting method was 3-D image system and cells on green line were counting but cells on red line were non-counting. The areal sampling fraction was 1.95%. The section thickness was 50 μm.
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7. Cell count and unbiased stereology in a entire brain of rats
The entire brain, with the exception of the cerebellum, was assayed with a 1.25×
objective and a counting grid of 1000 × 1000 μm. An unbiased counting frame of known area (60.0 × 44.6 μm = 2673 μm2) superimposed on the image was placed randomly over the first counting area and sampled as described above. The areal sampling fraction was 0.27%.
Actual counting was performed with a 100× oil-immersion objective. Estimation of the total number of BrdU-positive cells was calculated according to the optical fractionator formula (West, 1993). More than 200–300 points over all sections of each specimen were analyzed.
Tumor volume was estimated by Cavalieri’s principle (Larsen et al., 1998). Tumor cells were implanted 3.1 mm lateral and 0.4 mm anterior to bregma, at a 4-mm depth from the skull surface. The entire area of the tumor was assayed. The section thickness was 30 μm.
8. Labeling of F3 Cells with 5-bromo-2-deoxyuridine
For in vitro 5-bromo-2-deoxyuridine (BrdU) labeling experiments, F3 cells were incubated in feeding medium containing 10 μM BrdU (Sigma-Aldrich) for 3 hrs, fixed, permeabilized, and processed for immunostaining with anti-BrdU antibody.
Tissue sections were incubated in 2 N HCl at 37 °C for 40 min, placed in a borate buffer for 20 min, followed with 0.3% H2O2 in PBS for 30 min, an overnight incubation at 4°C in a 1:200 dilution of mouse monoclonal anti-BrdU (Roche) and 1 hr incubation in HRP-conjugated goat-anti-mouse IgG (Dako, Glostrup, Denmark)(Short et al., 1997). Sections
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were incubated with chromagen 3, 3’-diaminobenzidine for 3 min, counterstained with hematoxylin for 1 min, dehydrated in an ethanol series, cleared in xylene, and mounted on slides with Permount(Kuhn et al., 1996).
9. Hematoxylin and Eosin stain
The sections were dried in room temperature, and sections were rinsed with running tap water for a 5 min. Sections were stained with Harris hematoxylin (sigma, St. Louis, MO) for a 2 min and rinsed with a running tap water. The sections were dipped with 0.5 % HCl in 70 % ethanol and rinsed with running tap water. Thereafter, the sections were dipped with ammonia in distilled water and rinsed with a running tap water, hence, the sections stained with Eosin Y solution (sigma-aldrich) for 30 sec. Finally, the sections were dehydrated with 70 % ethanol, 95 % ethanol, 100 % ethanol, and xylene (DAE JUNG). The sections were covered with cover-glasses using mounting solution (Fisher Scientific)(Titford and Horenstein, 2005) .
10. Beta-galactosidase immunofluorescence
For immunohistochemical studies, the brains of wild-type rats and experimental group with successive tumor cell-NSC injection were removed and fixed in 4% paraformaldehyde in 0.1M phosphate buffer overnight at 4 °C and soaked in 30% sucrose for an additional 48
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hrs. Tissues were embedded in Tissue-Tek OCT compound (Sakura Finetek,Torrance,CA) and frozen at -20 °C until sectioning. Sagittal cryosectons (50 μm-thick) were then prepared on a cryostat. Prior to the stain, sections were dried in 20 °C for 30 min. sections were rinsed three times in PBS for 15 min each, and blocked with 10% horse serum in PBS for 1 hrs.
Sections were incubated overnight at 4 °C in a 1:200 dilution of rabbit-anti-beta-galactosidase (sigma) thereafter sections were rinsed with three times in PBS for 15 min each. Sections were incubated at 20 °C in a 1:200 dilution of rabbit-rhodamine-conjugated 2nd antibody (sigma) for a 2 hrs. Stained cells were viewed with a confocal laser scanning microscope (Olympus, Tokyo, Japan).
11. Statistical Analysis
Statistical analysis was performed in SPSS (version 10.0, SPSS, Chicago, IL).
Spearman’s correlation calculation was used to determine the relationship between the time elapsed and the number of migrated NSCs in the tumor region. To calculate the number of migrated cells, we counted each section stained with X-gal. The numbers of migrated cells into the tumor region were compared with each time point. The significance level for this test was set at 0.01. The comparison of the number of transplanted NSCs in rat brain parenchyma without tumor induction over time was performed using SPSS (version 11.5, SPSS, Chicago,IL) followed by paired t-test. A p value less than 0.05 were considered significant.
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Ⅲ. RESULT
1. Tumor-tropism pattern of NSCs
Tumor-tropism of F3 human NSCs toward U373MG human glioblastoma cells in rat brain was determined at various time points after transplantation of NSCs into brains that were previously implanted with glioblastoma cells. Migration of Hoechst 33258-labeled F3 cells into the tumor region was not observed at 30 min after injection (data not shown). The presence of F3 cells was detected in the tumor region at 40 min post injection (PI), and at 50 min PI, F3 cells were found throughout the tumor region. The accumulation of F3 cells in the tumor region increased up to 2 days PI and then appeared to decrease in number by 10 days PI (Fig. 2A). F3 cells also migrated extensively into the brain parenchyma, tracking to the tumor as well as tumor satellites (Fig. 2A). F3 cells well visualized in high magnification (Fig. 2B), and F3 and U373 cells well co-localized at 30 hours after NSCs injection in a tumor region (Fig.2C). Tumor volume increased significantly at 22 days PI as compared with 8 days after tumor cell inoculation (Fig. 3).
In sagittal brain sections, F3 cells were found migrating far from the injection site, but not to the cerebellum, at 1 day PI (Fig. 4E). F3 cells (blue) were observed in the area of the tumor (Fig.5a), as well as in the corpus callosum, hippocampus (Figs. 5B, C) but not found in a cerebellum (Figs.5D), at 1 day PI. Ten days PI, F3 cells were still observed in the hippocampus and auditory cortex (Fig. 6).
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Fig.1. Scheme of the experimental protocol. 1 × 106 U373MG cells labeled with PKH26 were injected into the right striatum of rats. Seven days later, when the tumors were well established,1 × 106 HB1.F3 neural stem cells (NSCs) labeled with Hoechst 33258 were
injected into the opposite striatum, and the rats were killed at various time points after HB1.F3 (F3) cell implantation.
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Fig.2. Images of NSCs in the tumor region.
(A):F3 NSCs (blue) in the tumor mass (red) at each time point (40 and 50 minutes; days 2, 3, 5, and 10) after NSC injection. (Bar indicates 100 μm). (B): F3 NSCs (blue) (Bar indicates 50 μm). (C): Merged F3 NSCs (blue) and U373 cells (red) at 30 hours after injected NSCs (bar indicates 100 μm).
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Fig.3. Tumor cells inoculation
(A): Tumor size at 8 days after tumor cell inoculation (hematoxylin and eosin staining, (B): Tumor size at 22 days after tumor cell inoculation (hematoxylin and eosin staining).
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Fig.4. Sagittal section of the transplanted site with F3 cells.
U373MG cells (red) were injected into the right hemisphere and F3 cells (blue; white arrows) were injected into the left hemisphere 7 days later. Sagittal sections are shown 24 hours after F3 cell injection (hematoxylin and eosin staining). Sagittal section of the F3 transplantation site at 24 hours (magnification, ×200; bar indicates 100 μm); (A): The anterior end of the F3 injection site. (B): F3 injection site. (C, D): The posterior end of the HB1.F3 injection site. (E): Cerebellum.
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Fig.5. Sagittal section within the tumor area (hematoxylin and eosin staining).
U373MG cells are labeled with PKH26 (red) and F3 cells are labeled with Hoechst 33258 (blue); (A): merged pictures of j and k. (B): corpus callosum. (C): hippocampus.
(D): cerebellum. (E): U373 cell (red) in a tumor region. (F): F3 cell (blue) in a tumor region (magnification, ×200; bar indicates 100 μm).
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Fig.6. Coronal section through at 10 days after F3 cell [neural stem cell (NSC)]
injection. (A): Hematoxylin and eosin staining. NSCs are visible in the hippocampus and auditory cortex in the hemisphere of the F3 injection. (B): F3 cells are labeled with Hoechst 33258 (blue) a: Hippocampus on the F3 injection side. b: Hippocampus on the tumor side.
c: Auditory cortex on the F3 injection side. d: Auditory cortex on the tumor side. (Scale bars, 100 μm).
טי 2. Migration rate of NSCs
To investigate the pattern of migration and the number of NSCs that migrated into the tumor mass, F3 cells encoded with LacZ gene were utilized. F3.LacZ cells migrated along the corpus callosum to the contralateral hemisphere 1 day PI and F3 LacZ cells found in corpus callosum as well as tumor region (Figs.7, 8A). The distance between the injection site and the tumor site was approximately 7 mm (Fig. 8B). Hoechst 33258-labeled NSCs were first detected in the tumor site 40 min PI (Fig.2A) but not found at 10 min and 30 min PI.
The migration rate of the NSCs was calculated to be approximately 175μm/min (Fig.8B).
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a c.c
Tumor
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Fig.7. Migration of NSCs into the tumor region.
(A):F3 cells (LacZ-labeled) migrate to the tumor region along the corpus callosum.
Rats were killed at 1 day after F3.LacZ cell (1 × 106 cells) injection, and the corpus callosum was stained with X-gal. (B): Migrating F3.LacZ cells in the tumor region.
(Scale bar, 100 μm; C.C indicates a corpus callosum).
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בכ Fig.8. Migration rate of NSCs.
(A): F3.LacZ cells (5 × 106 cells, white arrows) were injected on the contra-lateral side and animals were stained at 1 day later; beta-galactosidase staining of the corpus callosum was performed as described in the Methods. (Bar indicates 100 μm). (B): Atlas of rat brain with tumor taken from (Paxinos G, 1998). Hoechst 33258 labeled with F3 cells migrated in the tumor region from 10 min to 40 min PI. The calculated migration rate of F3 cells is shown.
גכ 3. NSC migration studied by stereology
We designed experiment of the stereological analysis (Fig. 9). To evaluate the migration patterns of the NSCs, we counted the numbers of F3.LacZ cells in the tumor region 50 min and on 1, 3, 5, 10, and 15 days PI (Fig.10). F3.LacZ cells were injected at a depth 1.0 mm above that of the tumor cells because NSCs migrate readily into the tumor site along the corpus callosum. Most of rats died 3 -4 weeks after tumor cell inoculation; therefore we observed migration patterns of F3.LacZ cells up to 15 days after tumor cell inoculation.
Approximately 10% of injected F3.LacZ cells migrated into the tumor region by 50 min after injection (Fig.11).
The number of F3.LacZ cells in the tumor region increased slowly up to 5 days PI and increased dramatically between 5 to 15 days PI. Changes in tumor volume showed a similar pattern (Fig. 11). Average tumor volume was 7.0 ± 1.5 mm3 (n = 3) at 50 min PI, 7.7 ± 0.8 mm3 (n = 3) on day 1, 17.0 ± 1.5 mm3 (n = 3) on day 3, 26 ± 4 mm3 (n = 5) on day 5, 66 ± 16 mm3 (n = 5) on day 10, and 230 + 56 mm3 (n = 2) on day 15 PI. The density of LacZ-positive cells in the tumor region increased up to 1 day PI and decreased thereafter up to 15 days PI (Fig. 11), indicating that the tumor growth rate was greater than the NSC migration rate in vivo. Average tumor volume was 7.7 + 0.8 mm3 8 days PI of U373MG cells but was 230 + 56 mm3 22 days PI (Fig. 3). Thus, the tumor volume increased approximately 30 fold over 14 days. By 15 days after F3.lacZ cell injection, F3.lacZ cells were found in the tumor
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region as well as along the corpus callosum and in the hippocampus and auditory cortex.
Thus, NSCs survived until 15 days after transplantation and migrated to various brain areas as well as to the tumor mass.
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Fig.9. Scheme of the stereological experiments. 1×106 U373MG cells were transplanted into the right striatum of rats. Seven days later, when the tumors were well established, 1 × 106 F3 cells transfected with LacZ gene (HB1.F3.LacZ) were injected into the opposite striatum and the rats were killed at various time points after HB1.F3.LacZ cell implantation.
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Fig.10. NSCs migration studied by stereology.
(A): Hematoxylin and eosin staining of a coronal section containing the tumor (magnification: x12.5): (B) F3.LacZ cells were counted only the cells marked with red triangles in the tumor region with the use of a computer-assisted stereological toolbox system as described in the Methods (magnification: x1000, Scale bar, 100 μm).
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Fig.11. The number of NSCs in the tumor region increased with time.
(A): The numbers of F3.LacZ cells were increased in the tumor region with time.
(A): The numbers of F3.LacZ cells were increased in the tumor region with time.