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 2^nd 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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B

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.

(B): Tumor volume also increased with time (C): The density of NSCs decreased inversely with time. Black bars: NSCs implanted after tumor cell inoculation; white bars: inoculation with tumor cells only. (D): Correlation between elapsed time and number of migrating NSCs in the tumor region (Spearman’s rho 0.909; p < 0.001).

חכ 4. Histological analysis

Prior to in vivo histological analysis, when F3.NSCs labeled with 5-bromo-2-deoxyuridine in vitro, BrdU was successfully observed in vitro by DAB stain (Fig.12). To determine if tumor cells release tropic chemotactic signals that cause NSC migration, NSCs were injected in the hemisphere opposite of the tumor site. In control animals, F3 NSCs were implanted in the absence of tumor cell inoculation. F3 NSCs were found in both ipsi- and contra-lateral hemispheres in tumor cell-injected brains but only in the ipsi-lateral hemisphere where F3 NSCs were injected in controls in the absence of tumor cells (Figs. 13, 14).

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Fig.12. In vitro, F3 cells labeled with a 5-bromo-2-deoxyuridine (BrdU).

F3 cells were incubated in feeding medium containing 10 μM BrdU for 3 hrs. (A): F3 cells labeled with a 5-bromo-2-deoxyuridine in vitro (magnification x100), (B): F3 cells labeled with a 5-bromo-2-deoxyuridine in vitro (magnification x200), (C, D): F3 cells labeled with a 5-bromo-2-deoxyuridine in vitro (magnification x400).

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Fig.13. Representative sections of tumor-induced migration of neural stem cells.

(NSCs; F3 cells); Long-range attraction of NSCs labeled with 5-bromo-2-deoxyuridine (BrdU; brown; black arrows) from the left hemisphere (L) across the corpus callosum (cc) in response to a local microinfusion of U373MG cells (0.2 μL/ minute) in the right hemisphere (R) (1 day).

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Fig.14. Representative sections of tumor-induced migration of neural stem cells.

(NSCs; F3 cells); Representative coronal brain sections from different rats with NSCs labeled with BrdU (brown; black arrows) injected in the left hemisphere in the absence of tumor cell inoculation (Scale bar, 2 mm).

בל 5. Proliferation of NSCs in vitro & in vivo

We also investigated the proliferation rate of F3 cells both in vitro and in vivo. In vitro, the doubling time of NSCs was approximately 24 hr, and the proliferative activity of F3, F3.LacZ, and U373MG cells was active for 2 days and decreased thereafter (Fig. 15).

In the case of in vivo, injected F3 cells were continuously found from 50 min to 15 days PI in a same transplantation site (Fig.16). The presence of F3 NSCs was observed in various regions of the whole brain at 15 days after transplantation (Fig. 17). We counted number of BrdU-positive F3 cells in the entire brain, including the cerebellum, in vivo. F3 cells increased in number over time up to 5 days PI and decreased thereafter (Fig. 18). The number of transplanted F3 cells was increased approximately 1.7 fold during the first 24 hrs.

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Fig.15. Quantification of NSC proliferation in vitro.

The graph shows that the proliferative activity of all three cell lines (F3, F3.LacZ and U373MG) increased up to 2 days and decreased thereafter in vitro.

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Fig.16. Engrafted neural stem cells in a transplantation site at time points sequential. (A): Engrafted F3 cells labeled with 5-bromo-2-deoxyuridine in transplantation site at 50 min after injection. (B): Engrafted F3 cells labeled with 5-bromo-2-deoxyuridine in transplantation site at 1 day after injection. (C):

Engrafted F3 cells labeled with 5-bromo-2-deoxyuridine in transplantation site at 5 day after injection. (D): Engrafted F3 cells labeled with

5-bromo-2-deoxyuridine in transplantation site at 15 day after injection.

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Fig.17. Sagittal sections were performed in the entire brain.

Transplanted HB1.F3 cells labeled with 5-bromo-2-deoxyuridine (black arrows) were dispersed in a whole brain parenchyma after 15 days injection Scale bar, 100 μm.

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Fig.18. Quantification of NSC proliferation in vivo.

(A): The histogram shows that the proliferative activity of the F3 cells increased by 5 days, thereafter NSCs decreased until 15 days after NSC injection in vivo. (B): Numerical value of statistics (n=5 per condition, mean±SD.).*p <0.05; **p<0.01 verse other groups by a paired t-test.

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Ⅳ. DISCUSSION

Because of their high rate of cell proliferation and diffuse infiltrating properties into surrounding brain parenchyma, glioblastomas are known to be fatal. Indeed, radical surgical resection is practically impossible because of the disseminated infiltration and growth beyond the tumor boundaries, visible even on modern neuro-radiological imaging(Giese and Westphal et al., 1996). Therefore, selective targeting to treat the infiltrating tumor cells may be the goal for a new therapeutic approach. NSCs distribute throughout the primary tumor bed and migrate together with widely outgrowing tumor microsatellites after intratumoral implantation. Moreover, when NSC is implanted intracranially at sites distant from the tumor, they migrate through the normal parenchyma and localize in the tumor sites, known as the ‘chasing down’ phenomenon(Aboody et al., 2006).

Previous studies have demonstrated that a significant reduction in tumor size by CD-encoded F3 NSCs with 5-FC pro-drug application in medulloblastoma model (Marchisone et al., 2000; Kim et al., 2006a), and F3 NSCs transfected with PEX, fragment of human metalloproteinase-2, and an inhibitor of tumor proliferation, have reduced glioblastoma tumor size (Anderson et al., 1998; Kim et al., 2005). NSCs encoded with Herpes simplex virus thymidine kinase (HSV-tk) gene also resulted in decreases in tumor volume (Costantini et al., 2000; Pulkkanen and Yla-Herttuala et al., 2005), indicating that NSCs can be used as highly effective therapeutic tools for brain tumors (Forsyth and Cairncross et al., 1995; Flax

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et al., 1998; Noble et al., 2000; Bourbeau et al., 2004; Glass et al., 2005; Hadaczek et al., 2005).

Previously we have demonstrated the potential of human NSCs as an effective delivery system to target and disseminate therapeutic agents to glioma and we used gene-modified human NSCs as a new tool for gene therapy of glioblastoma. There are many studies which reported 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 migrating to glioma at various time points. In the present study, therefore we used unbiased and objective measuring method via the stereological counting system. These results may provide useful information on the nature of tumor-tropism of the NSCs.

In the present study, we injected human NSCs into the rat’s opposite hemisphere to the tumor site and observed the migration of NSCs to the tumor area. To identify the migration of NSCs from the transplantation site to the tumor region, we observed extensive migration of BrdU-labeled NSCs along the corpus callosum and in the tumor mass. The corpus callosum appears to be the main pathway for NSC migration into the tumor mass, consistent with a previous study (Shah et al., 2005). We calculated the migration rate for NSCs as approximately 175 μm/min. NSCs were clearly tumor-tropic, but smaller number of NSCs migrated to areas other than the tumor site such as hippocampus and auditory cortex.

Migration of NSCs and tumor regression in response to gene targeting has been studied by real-time imaging with dual luciferase/substrate imaging (Shah et al., 2005). However, little is known about the number of NSCs that migrate to the tumor site over time, the rate of

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migration from the injection site to the tumor region, where NSCs migrate within the brain parenchyma, and how long NSCs remain in these regions. In the present study, the number of NSCs observed in the tumor region 15 days PI was approximately 10% greater than the number of injected NSCs. The number of NSCs in the tumor region increased slowly up to 5 days after injection and increased dramatically thereafter up to 15 days. Tumor volume was gradually increased by 15 days after inoculation. The density of LacZ-positive human NSCs in the tumor region increased during the first 24 hrs after injection and decreased thereafter up to 15 days after injection, indicating that the tumor growth rate as faster than the NSC migration rate in vivo.

Although we did not measure the number of proliferated NSCs which were migrated into the tumor region, we supposed that most of NSCs in the tumor are the migrated NSCs from injection site rather than proliferated NSCs. Because of the total number of NSCs in the tumor at 15 days after injection was not much more than the number of total injected NSCs. It means that NSCs were not proliferating continuously after injection of NSCs.

NSCs and neural progenitor cells have the biologic potential to differentiate into CNS cell types, including neurons, astrocytes, and oligodendrocytes. Multipotent NSCs are found in the developing and adult mammalian CNS, including that of humans (Culver et al., 1992;

Hurelbrink et al., 2002; Hemmrich et al., 2006). The human NSC line F3 carries the normal human karyotype (46XX) and has the ability to self-renew, differentiate into cells of neuronal or glial lineage, and integrate into the damaged CNS upon transplantation in animal models of various neuronal diseases (e.g., Parkinson’s disease, Huntington disease,

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stroke, and brain tumors). This cell line was generated from the human fetal telencephalon with the use of a retroviral vector encoding v-myc gene (Kim et al., 2004; Lee et al., 2007;

Kim et al., 2008). A number of gene therapy trials have been performed in an attempt to improve survival rates for patients with glioblastoma with only limited gains. Nonetheless, gene therapy will continue to be used in combination with classical treatment strategies.

Gene therapy can modify the genetic make-up of target cells, which is not possible with other therapeutic modalities. Gene therapy that involves suicide genes such as cytosine deaminase (CD) or thymidine kinase (TK) is an attractive approach for the treatment of

cancer. The CD gene locally converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which interferes with DNA synthesis and results in the death of dividing cells (Culver et al., 1992; Barba et al., 1993; Barresi et al., 2003). Similarly, the TK gene converts the non-toxic drug ganciclovir into a phosphorylated metabolite that acts as a potent killer of dividing cancer cells(Moolten et al., 1986). To deliver these therapeutic genes to tumors, viral and non-viral delivery vectors are necessary. An optimal gene delivery system would comprise a vector capable of encoding large DNA inserts and efficiently transducing the target tissue, resulting in the selective targeting of the expression of the therapeutic gene for a sufficient duration. In addition, the vector should be easily manufactured, able to be delivered systemically, and be non-cytotoxic, preferably allowing for repeated administration.

NSCs have recently been recognized for their ability to migrate throughout the adult CNS and become normal constituents of the host cytoarchitecture (Snyder et al., 1995;

Hurelbrink et al., 2002). Since the reports of the ability of NSCs to migrate into glial tumor

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masses (Snyder et al., 1995; Ourednik et al., 1999; Aboody et al., 2000), several studies have investigated the mechanism involved. There are many molecules and factors associated with the tropism of NSCs for tumor cells. Tumor cells secrete various chemoattractant molecules, such as vascular endothelial growth factor (VEGF), scatter factor/hepatocyte growth factor (SF/HGF), fibroblast growth factor 2 (FGF-2), stromal derived factor-1 (SDF-1), and chemokine receptor 4 (CXCR4) (Machein and Plate et al., 2000; Heese et al., 2005; Schmidt et al., 2005; Ghosh and Maity et al., 2006; Ratajczak et al., 2006; Menon et al., 2007; Bao et al., 2009).

It is well known that certain angiogenic factors, such as VEGF, SF/HGF, and placenta growth factor (PLGF), are associated with the attraction for NSCs (Schmidt et al., 2005). In addition, NSCs express CXCR4, and the inhibition of CXCR4 dramatically decreases the

It is well known that certain angiogenic factors, such as VEGF, SF/HGF, and placenta growth factor (PLGF), are associated with the attraction for NSCs (Schmidt et al., 2005). In addition, NSCs express CXCR4, and the inhibition of CXCR4 dramatically decreases the