Human GBM cells, U87-MG (cat no. HTB-14; GBM of unknown origin), U87-MG-luc2 (cat. no. HTB-14-LUC2; GBM of unknown origin), T98G (cat. no. CRL-1690) and CCF-STTG1 (cat. no. CRL-1718), were purchased from American Type Culture Collection (ATCC). Proneural X01 (21) and Mensenchymal 83 (22) cells were donated by Professor Park Jong Bae's team at the Korea National Cancer Center (23). The passage number at which these cells were supplied was passage 11 and the cells were experimentally used starting from passage 14.

U87-MG, U87-MG-luc2, T98G and CCF-STTG1 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and maintained at 37°C in humidified air with 5% CO₂.

Proneural X01 and were cultured in DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10 ng/ml epidermal growth factor (cat. no. 236-EG; R&D Systems, Inc.), basic fibroblast growth factor (cat. no. 4114-TC; 5 ng/ml for Proneural X01 and 10 ng/ml for Mesenchymal 83; R&D Systems, Inc.). B27 (Invitrogen^™; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.), and maintained at 37°C in humidified air with 5% CO₂.

rNDV-PTEN virus was previously constructed (20). The virus was propagated by infection of vero cells (CCL-81; ATCC) at a multiplicity of infection (MOI) of 0.5 for 2 days prior (20). The virus titer was tested by the 50% tissue culture infective dose (TCID₅₀/ml) method of Spearman and Kärber (24,25).

U87-MG and CCF-STTG1 cells (1×10⁷ cells) seeded in a 175T flask were cultured overnight at 37°C in humidified air with 5% CO₂. The cells were infected with rNDV or rNDV-PTEN viruses at an MOI of 1.0 for 1 h, washed two times with PBS and then incubated for 12–36 h at 37°C in humidified air with 5% CO₂, with DMEM containing 10% FBS and 1% penicillin-streptomycin. The supernatant was removed and cells were collected at 12, 24 and 36 h after virus infection and the cells were subjected to three freezing/thawing cycles at −80°C and 4°C. The cell lysates were used for quantitative (q)PCR and immunoblot analysis.

Female BALB/c nu-/nu- mice (n=40; 5 weeks old), weighing ~18–20 g, were purchased from Orient Bio, Inc. The mice were housed under standard conditions with a 12-h light/dark cycle, a temperature of 22±2°C and a humidity of 55±10%, with food and water provided ad libitum. For anesthesia, each mouse was weighed to calculate the appropriate dose of 2,2,2-tribromoethanol (Avertin^®) via intraperitoneal administration at 250 mg/kg.

U87-MG cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% heat-inactivated FBS (Sigma-Aldrich; Merck KGaA) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.), and maintained at 37°C in humidified air with 5% CO₂. Before injection, U87-MG cells were trypsinized, counted and resuspended in growing media. The cell suspension was kept on ice until the time of injection. Each mouse was injected with 5×10⁴ U87-MG cells in 5 µl (1×10⁴ cells/µl) as follows: A midline incision of ~1.2 cm was made. A small hole was drilled in the skull at the point 0.2 mm back and 2.2 mm to the left of the bregma. Cell suspensions were injected with a Hamilton syringe at a rate of 1 µl/min, and the syringe was left in place for 5 min. The mice were screened using an in vivo imaging system (IVIS) every 7 days. After 40 days, the mice were randomly divided them into three groups (n=4 per group): rNDV (100 µl 10⁷ TCID₅₀/dose, intravenous), rNDV-PTEN (100 µl 10⁷ TCID₅₀/dose, intravenous) and PBS as a negative control.

For immunoblotting, proteins were extracted using RIPA buffer (Thermo Fisher Scientific, Inc.) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS. Protein concentration was determined using the bicinchoninic acid method. Equal amounts of protein (30 µg) were loaded per lane on a 15% SDS-PAGE gel. Proteins were then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat dry milk in TBS-T (0.1% Tween-20) for 1 h at room temperature. The membranes were incubated overnight at 4°C with the following primary antibodies: Anti-GAPDH (1:3,000; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.); anti-LC3 (1:1,000; cat. no. NB100-2220; Novus Biologicals, LLC); anti-matrix metallopeptidase 9 (MMP9; 1:1,000; cat. no. MA5-15886; Thermo Fisher Scientific, Inc.); anti-proliferating cell nuclear antigen (PCNA; 1:500; cat. no. PC 10; Sigma-Aldrich; Merck KGaA); anti-P-mTOR (Ser2448; 1:1,000; cat. no. 2971S; Cell Signaling Technology, Inc.); anti-mTOR (1:1,000; cat. no. 2972S; Cell Signaling Technology, Inc.); anti-P-Akt (Ser473; 1:1,000; cat. no. 9271S; Cell Signaling Technology, Inc.); anti-Akt (1:1,000; cat. no. 9272S; Cell Signaling Technology, Inc.); anti-cleaved Caspase (Cas)9 (1:1,000; cat. no. 9509S; Cell Signaling Technology, Inc.); anti-cleaved Cas3 (1:1,000; cat. no. 9664S; Cell Signaling Technology, Inc.); anti-cleaved Cas8 (1:1,000; cat. no. 9496S; Cell Signaling Technology, Inc.); anti-B-cell lymphoma 2 (Bcl-2) associated X protein (Bax; 1:1,000; cat. no. 2772S; Cell Signaling Technology, Inc.); anti-p62 (1:1,000; cat. no. 5114S; Cell Signaling Technology, Inc.); anti-Occludin (1:1,000; cat. no. 5506S; Cell Signaling Technology, Inc.); anti-zonula occludens protein 1 (ZO-1; 1:1,000; cat. no. 5406S; Cell Signaling Technology, Inc.); anti-Clauddin-5 (E8F3D; 1:1,000; cat. no. 49564; Cell Signaling Technology, Inc.); and anti-PTEN (1:1,000; cat. no. 9552S; Cell Signaling Technology, Inc.). After washing with TBS-T (0.1% Tween 20), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Anti-Rabbit, cat. no. 7074S; and Anti-Mouse, cat. no. 7076S; 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Proteins were visualized using the Pierce^™ ECL Western Blotting Substrate (cat. no. 32106; Thermo Fisher Scientific, Inc.). Values were normalized to GAPDH as loading controls. Protein levels were semi-quantified using densitometric analysis using Image J software (version 1.49; National Institutes of Health).

Total RNA was isolated using TRIzol^™ Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) based on the acid guanidinium thiocyanate-phenol-chloroform method. Total RNA concentration was determined using a spectrophotometer (Nano Drop^™ 2000/2000c Spectrophotometer; Thermo Fisher Scientific, Inc.). Complementary DNA was prepared from total RNA (1 µg) using the RevertAid First Strand cDNA Synthesis Kit (cat. no. K1622; Thermo Fisher Scientific, Inc.). The thermocycling conditions for cDNA synthesis were as follows: 65°C for 5 min, 55°C for 50 min and 85°C for 5 min. qPCR was then performed using the StepOnePlus^™ Real-Time PCR system (Bio-Rad Laboratories, Inc.) with the SYBR^® Premix Ex Taq^™ kit (cat. no. RR820A; Takara Bio, Inc.). The thermocycling conditions for qPCR were as follows: Initial denaturation, 95°C for 5 sec; followed by 35 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 25 sec and extension at 70°C for 30 sec. The primers used for human PTEN were as follows: Sense, 5′-CAAGATGATGTTTGAAACTAT-3′ and antisense, 5′-CCTTTAGCTGGCAGACCACAA-3′. The primers used for mouse Occludin were as follows: Sense, 5′-ACTGGGTCAGGGAATATCCA-3′ and antisense, 5′-TCAGCAGCAGCCATGTACTC-3′. The primers used for mouse ZO-1 were as follows: Sense, 5′-AGGCTACCTTTGTATTCTC-3′ and antisense, 5′-TAGGGCACAGTATTGTATC-3′. The primers used for mouse Claudin-5 were as follows: Sense, 5′-CTTCCTGGACCACAACATCGTG-3′ and antisense, 5′-CACGTCGGATCATAGAACTCG-3′. The primers for human 18s, used as the internal control, were as follows: Sense, 5′-GTAACCCGTTGAACCCCATT-3′ and antisense, 5′-CCATCCAATCGGTAGTAGCG-3′. The primers for mouse 18s, used as the internal control, were as follows: Sense, 5′-GAGCGACCAAAGGAACCATA-3′ and antisense, 5′-CGCTTCCTTACCTGGTTGAT-3′. Dissociation curves were monitored to assess the aberrant formation of primer-dimers. The fold change in the interest gene expression was calculated using the 2^−ΔΔCq method (26).

Brain tissues from the orthotopic GBM model were fixed with 4% (w/v) paraformaldehyde at room temperature for 24 h. The fixed tissues were then embedded in paraffin and sectioned into 5 µm-thick slices. The sections were deparaffinized using xylene, followed by rehydration through a graded series of alcohols (100, 80 and 70%), and finally rinsed in PBS. Next, hematoxylin and eosin staining was performed by incubating the sections in hematoxylin for 5 min at room temperature, followed by eosin for 2 min at room temperature. For immunohistochemistry staining, tumor tissue sections were fixed with 10% neutral buffered formalin at room temperature for 24 h. After fixation, the sections were embedded in paraffin using standard procedures. Tumor sections of a 5-µm thickness were cut and mounted on slides. For antigen retrieval, sections were treated with sodium citrate buffer (pH 6.0; cat. no. C999; MilliporeSigma) and heated in a microwave for 3 min at 95°C. After retrieval, the sections were rehydrated through a descending alcohol series (100, 80 and 70% ethanol) and washed in PBS. The sections were blocked with 1% bovine serum albumin (cat. no. 4378; MilliporeSigma) in PBS for 1 h at room temperature, and then stained with the following primary antibodies: Anti-MMP9 (1:100; cat. no. MA5-15886; Thermo Fisher Scientific, Inc.), anti-PTEN (1:200; cat. no. 9559S; Cell Signaling Technology, Inc.) anti-NDV hemagglutinin-neuraminidase protein (1:200; HN; cat. no. sc-53562; Santa Cruz Biotechnology, Inc.) and anti-Ki-67 (1:100; cat. no. MA5-14520; Thermo Fisher Scientific, Inc.) overnight at 4°C. HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (cat. nos. AP160P and 12–348; MilliporeSigma) were then applied for 60 min at room temperature. Color was developed for 30 sec by incubation with DAB. Sections were counterstained with hematoxylin at room temperature for 3 min and observed under a light microscope (Motic Instruments) at ×100 magnification.

CCF-STTG1, U87-MG, T98G, Mesenchymal 83 and Proneural X01 cells were seeded at 1×10⁴ cells/well in 96-well plates (cat. no. 34096; SPL Life Sciences). On the next day, the cells were treated with rNDV-PTEN or rNDV (0.3, 1 or 3 MOI) for 24 h. Cell proliferation was measured using a CCK-8 kit (cat. no. CK04-1000; Dojindo Laboratories, Inc.) according to the manufacturer's instructions. Briefly, cells were washed with PBS and suspended in growth medium including CCK-8 reagent added at 1/100 the media volume. Cells were then incubated at 37°C for 1 h in the dark. Cell proliferation was measured at a wavelength of 450 nm.

A TUNEL assay was used to detect DNA fragmentation, such as apoptosis. U87-MG cells were seeded at 1×10⁵ cells/well in a 6-well plate (cat. no. 30006; SPL Life Sciences). Cells were treated with rNDV-PTEN or rNDV (1 MOI) for 24 h. After 24 h of incubation at 37°C with 5% CO₂, the cells were washed twice with PBS, detached from the plate using trypsin and collected in a 15 ml tube. These cells were fixed in 100% ethanol overnight at 4°C. A TUNEL assay was performed according to the manufacturer's instructions (TUNEL Assay Kit-FITC; cat. no. ab66108; Abcam). Following fixation, the cells were permeabilized with 0.1% Triton X-100 in PBS for 2 min on ice. The cells were then incubated with FITC-labeled dUTP in the presence of terminal deoxynucleotidyl transferase at 37°C for 1 h. Stained cells were analyzed using flow cytometry and fluorescence for FITC using a NovoCyte Quanteon flow cytometer (Agilent Technologies, Inc.) and fluorescence microscope (Zeiss Axio Imager M1; Zeiss GmbH) as per the manufacturer's instructions (Agilent Technologies, Inc.). Data acquisition was performed using a flow cytometer (FACS; NovoCyte Quanteon flow cytometer; Agilent Technologies, Inc.), measuring PE-A fluorescence intensity, and ~1,000 cells per sample were analyzed to determine the extent of apoptosis. Flow cytometry data were analyzed using NovoExpress software (version 1.6.2; http://www.agilent.com/ko-kr/product/research-flow-cytometry/flow-cytometry-software/novocyte-novoexpress-software-1320805). After completing the FACS experiment, 100 µl of the stained cells were transferred onto a cover slide. The cells were then assessed using fluorescence microscopy (ZEISS LSM 980; Zeiss GmbH) to evaluate and visualize the expression and localization of the TUNEL (FITC).

A Transwell assay was used to assess cell migration. U87-MG cells were seeded at 1×10⁵ cells/well, with uninfected cells (CON) or rNDV (1 MOI) or rNDV-PTEN (1 MOI), into 6-well tissue culture plates for 24 h, followed by transfer of 5×10⁵/ml cells in the upper Transwell chamber (24-well plate; Corning, Inc.) and cultured with FBS-free medium at 37°C, with 5% CO₂. Complete growth medium with 10% FBS (Merck KGaA) was added to the lower chamber and incubated for another 24 h at 37°C, with 5% CO₂. Cells on the upper side (non-migrating cells) were then removed and migrated cells on the lower face were washed with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and stained with DAPI at room temperature for 10 min. The cells were counted in 5 random high-power fields (×200 magnification) under a microscope (ZEISS LSM 980; Zeiss GmbH) and averaged.

Mice were anesthetized with 2.5% isoflurane for induction and maintained with 1.5% isoflurane until the completion of IVIS imaging. Luciferase imaging was performed using an in vivo optical imaging system (IVIS Lumina XR; PerkinElmer, Inc.) 15 min after intraperitoneal injection of 100 µl Luciferin (30 mg/ml). The images were captured and then the signal was displayed as Radiant Efficiency (Photons/sec/cm²/steradium (sr) or µW/cm²). Images of the region-of interest were analyzed using the Living imaging 4.4 software (PerkinElmer, Inc.).

Mice were transferred to the MRI unit using individual portable cages within 30 min of anesthesia with 2.5% isoflurane. Using a 32-channel phased array sensitivity encoding head coil, MRI imaging was performed on the anesthetized mice with a 7.0 Tesla Philips MR scanner (Ingenia; Philips Healthcare). Each mouse was scanned in the upright position with a coil over its head. Mice were mainlined under anesthesia with 1% isoflurane. The following parameters were used for acquisition of the multi-shot echo-planar imaging fast spin echo, with image reconstruction using image-space sampling functions with b-values of 0 and 1,000 s/mm², and 3 orthogonal directions of diffusion gradients: Echo time, 45 msec; repetition time, 5,000 msec; slice thickness, 8 mm; interslice gap, 1 mm; number of averaging, =2; bandwidth, 936 Hz/pixel; echo train length, 35; field of view, 25.0×25.0 cm; and matrix size, 256×256 pixels.

Statistical analysis was performed using Prism 8 software (Dotmatics). Data are presented as mean ± standard deviation. Differences between two groups were evaluated using unpaired t-tests. For multiple comparisons, one-way analysis of variance was performed followed by Tukey's multiple comparison test. P<0.05 were considered to indicate a statistically significant difference. Data are representative of at least three independent experiments.

A significant decrease in PTEN expression was demonstrated in the U87-MG GBM cell line in comparison with normal brain cells (astrocytes: CCF-STTG1). PTEN expression in other GBM cell lines (T98G, Proneural X01 and Mesenchymal 83 cells) did not demonstrate a significant decrease compared with astrocytes (Fig. 1A and B). Therefore, U87-MG cells were chosen as the primary focus for assessing the anticancer effects of PTEN restoration.

The present study constructed rNDV-PTEN (20) to induce PTEN mRNA and protein expression in the cytoplasm of U87-MG and CCF-STTG1 cells. In normal cells, such as astrocytes, the Type I interferon (IFN) pathway, particularly IFN-α, is active and effectively inhibits viral replication by inducing an antiviral state, preventing the proliferation of NDV. By contrast, cancer cells like those in GBM often have a compromised IFN-α signaling pathway due to the deletion of the cyclin-dependent kinase inhibitor 2A and Type I IFN gene cluster (15). This impairment allows NDV to replicate efficiently within cancer cells, leading to selective oncolysis. Therefore, IFN-α serves a critical role in maintaining antiviral defenses in normal cells, whereas its dysfunction in cancer cells enables the oncolytic activity of the virus (27). The results of the present study demonstrate that in CCF-STTG1 cells, the virus did not exhibit oncolytic activity, resulting in no significant change in cell proliferation compared with GBM cell lines. However, in GBM cell lines, there was a greater reduction in cell proliferation with increased virus exposure time or when treated with rNDV-PTEN, which contains the inserted PTEN gene. Statistical analysis revealed a significant reduction in cell proliferation in GBM cell lines compared with that in untreated cells (Fig. 1C). The reason for using the PTEN gene is that 40% of patients with GBM have PTEN gene mutations, as well as the U87-MG cell line we used. These mutations lead to the dysfunction of the PTEN protein, which is associated with a worse prognosis in GBM (28–30). Therefore, the present study aimed to enhance the therapeutic effect on GBM by delivering and expressing the PTEN gene through rNDV-PTEN. Treatment with rNDV-PTEN significantly suppressed the proliferation of U87-MG cells in comparison with its effects in the CCF-STTG1 and other GBM cell lines (Fig. 1C). Furthermore, the present study assessed the NDV-HN protein in astrocyte cells (CCF-STTG1) and GBM cell lines in samples that were either untreated (0 h, no treatment) or treated (36 h, rNDV-PTEN virus). The results revealed that in astrocytes (CCF-STTG1), the expression of the NDV-HN protein was significantly lower compared with that in the other GBM cell lines (Fig. S1). Taken together, U87-MG cells were selected for further assessment of the anticancer effects of rNDV-PTEN.

A Transwell assay to evaluate U87-MG cell migration revealed that most of the rNDV-PTEN-treated cells did not migrate to the lower chamber containing the complete medium. Further analysis, including DAPI staining of migrated cells, followed by microscopic counting, demonstrated significantly decreased migration (Fig. 1D). To assess the mechanism through which PTEN restoration induces apoptosis in U87-MG cells, a TUNEL assay was performed (Fig. 1E). TUNEL-positive cells were observed in both rNDV and rNDV-PTEN, with rNDV-PTEN treatment associated with ~2.5× more positive cells compared with rNDV. The TUNEL assay (FITC) was performed using FACS and cell staining was evaluated with a fluorescence microscope on a cover slide (Fig. S2). These findings indicate that rNDV-PTEN treatment inhibited the migration of U87-MG cells and induced DNA fragmentation, leading to apoptosis.

The deactivation (dephosphorylation) of AKT/mTOR and apoptosis-associated signaling pathways in U87-MG cells were also analyzed using immunoblot analysis. Specifically, these cells were infected with rNDV or rNDV-PTEN at a MOI of 1 and collected for analysis at 12, 24 and 36 h post-infection (hpi). The results revealed that in comparison with the PTEN bands in rNDV-infected cells, those in rNDV-PTEN-infected cells gradually increased between 12 and 36 hpi and peaked at 36 hpi, indicating active virus replication, with a significant increase in PTEN expression over time (Fig. 2A). Additionally, the levels of PCNA were assessed, a well-conserved protein in eukaryotes and a proliferation marker expressed in cells undergoing division (31). The level of MMP9 was also evaluated, which serves an essential role in local proteolysis of the extracellular matrix and in leukocyte migration (32,33). The results demonstrated decreased PCNA and MMP9 expression levels in rNDV-PTEN-infected U87-MG cells compared with those in uninfected cells (Fig. 2A). Additionally, the activation of AKT/mTOR, as the endpoint of the PI3K pathway, contributes to the malignant transformation of cells in several cancers (34). In the present study, AKT and mTOR phosphorylation in cells infected with rNDV or rNDV-PTEN were assessed. The results revealed that rNDV-PTEN treatment decreased the levels of phosphorylated AKT and mTOR in a dose-dependent manner (Fig. 2B). Furthermore, an increase in the levels of Bax and cleaved caspases 3, 8 and 9 were demonstrated, along with a decrease in the level of Bcl-2 in rNDV-PTEN-infected cells, compared with that in uninfected cells, indicating that this treatment induced apoptosis in GBM cells (Fig. 2C).

To determine whether PTEN restoration significantly suppresses U87-MG cell proliferation in an in vivo preclinical mouse model and in vitro, U87-MG-Luc2 cells were orthotopically xenografted into BALB/c nude mice (Fig. 3A). A total of 40 days after tumor injection, virotherapy was initiated using intravenous injections of rNDV-PTEN, rNDV or PBS (CON). The body weight (Fig. 3B) and survival rates (Fig. 3C) of the mice were also monitored during the treatment period. To measure the in vivo efficacy of PTEN restoration for tumor growth suppression in GBM mouse models, the MRI findings and IVIS assessments of luciferase activity in tumor scans were compared at 1–8 weeks after tumor injection.

The results indicated weight loss was associated with tumor progression rather than survival rate in the GBM orthotopic mouse model (Fig. 3B). The treatment group that received virotherapy experienced significantly less weight loss compared with the control group mice (PBS). Furthermore, whilst all control mice died by day 60 post-tumor establishment, the rNDV and rNDV-PTEN treated mice had survival rates of 50 and 70%, respectively (Fig. 3C). MRI and IVIS assessments also demonstrated markedly lower tumor growth in rNDV-PTEN-treated mice than in rNDV- and PBS-treated mice (Fig. 3D and E). Moreover, the level of NDV-HN in the brain tumor tissues of rNDV- and rNDV-PTEN-treated mice was significantly greater than that in the brain tissue of PBS-treated mice (Fig. 4A). This was further supported by the higher infection rate of rNDV-PTEN observed through HN staining in Fig. 4A. The mRNA and protein expression levels of tight junction proteins such as ZO-1, claudin-5 and occludin were also assessed; however, the expression levels did not significantly change in the brain tissue of mock mice models or tumor tissues in rNDV-PTEN, rNDV and PBS-treated mice (Fig. S3). This indicates that NDV crosses the BBB to reach the tumor tissue without disrupting tight junctions.

Immunohistochemical analysis was used to assess whether PTEN expression was upregulated in GBM tissues. The brains of rNDV-PTEN-treated mice exhibited significantly higher PTEN expression levels than those of PBS-treated mice (Fig. 4A). This finding indicates that the treatment with rNDV and rNDV-PTEN successfully led to the presence and activity of the NDV virus within the brain tumor tissues. The increased levels of NDV-HN in rNDV- and rNDV-PTEN-treated mice compared with PBS-treated mice is shown in Fig. 4A. These increases were statistically significant, indicating a meaningful difference in NDV-HN and PTEN levels between the treated and control groups, suggesting effective viral targeting and replication in the tumor environment. This indicates that the virus actively engaged with the tumor cells, potentially leading to oncolytic effects, which were absent in the PBS-treated group. The elevated presence of the viral protein in the treated mice demonstrates the potential of rNDV and rNDV-PTEN as therapeutic agents capable of reaching and affecting tumor sites within the brain. Immunoblotting performed to assess PTEN expression in cancerous and normal tissues in the brain and other tissues revealed that NDV injected into the tail vein did not affect PTEN expression in other organs compared with that in PBS-treated mice, but significantly upregulated PTEN expression in brain cancer cells (Fig. 4B and C).

Staining of brain sections with antibodies against Ki-67 and MMP9 significantly markedly lower cancer cell proliferation and migration indices (brown) in rNDV-PTEN-treated mice than in rNDV- and PBS-treated mice (Fig. 5A). Furthermore, analysis of PCNA and MMP9 protein expression levels demonstrated similar results (Fig. 5B). To elucidate the mechanism by which rNDV-PTEN treatment induces apoptosis, the regulation of the AKT/mTOR signaling pathway in GBM mouse models was evaluated. The phosphorylation levels of AKT and mTOR were significantly reduced in rNDV-PTEN-treated mice compared with those in PBS-treated or rNDV-treated mice (Fig. 5C). Changes in apoptotic protein markers were also assessed and significantly increased levels of cleaved caspases 3, 8 and 9 and Bax, and decreased levels of Bcl-2 were demonstrated in rNDV-PTEN-treated mice compared with those in PBS-treated mice. This observation indicates the activation of apoptosis in the rNDV-PTEN-treated group (Fig. 5D). Taken together, the results suggest that PTEN restoration induces apoptosis during GBM cell proliferation and migration by disrupting the AKT/mTOR signaling pathway.