Human glioma cell lines A172 and LN229 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and authenticated by short tandem repeat profiling within 6 months before use. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific, Grand Island, NY, USA, catalog number 11965092) supplemented with 10% fetal bovine serum (FBS; Gibco, 16140071, Grand Island, NY, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified incubator with 5% CO₂. Cells were routinely tested and confirmed to be mycoplasma-free.

KD01 is a recombinant oncolytic adenovirus type 5 carrying a 27-bp deletion in the conserved E1A region and an engineered E3 region in which ADP is deleted and replaced by a tBID expression cassette. A replication-competent adenovirus with an ADP deletion but without tBID insertion (M20) was used as the control virus. Viral stocks were propagated in HEK293 cells, purified by double cesium chloride gradient ultracentrifugation, and dialyzed against storage buffer (10 mM Tris–HCl, 2 mM MgCl₂, 5% glycerol, pH 8.0). Viral particle (vp) concentrations were determined spectrophotometrically at 260 nm, and infectious titers PFU (Plaque-Forming Units) were determined using plaque assays in HEK293 cells. Viruses were stored at –80 °C until use.

To assess oncolytic activity in vitro, A172 and LN229 cells were seeded into 96-well plates at a density of 5 $\times$ 10³ cells per well and allowed to adhere overnight. Cells were infected with KD01 at the indicated multiplicities of infection (MOIs; 0.1, 1, and 10) in serum-free medium for 2 h, after which the medium was replaced with complete growth medium. Cell viability was measured at 24, 48, and 72 h post-infection using a Cell Counting Kit-8 (CCK-8; Beyotime Biotechnology, Shanghai, China, catalog number C0037), according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA), and cell viability was expressed as a percentage of the mock-treated control.

Mitochondrial membrane potential ($\mathrm{\Delta }$$\psi$m) was assessed using a JC-1 assay kit (Beyotime Biotechnology, Shanghai, China, catalog number C2006). LN229 cells were seeded into 24-well plates, infected with PBS, M20, or KD01 (MOI = 1), and incubated for 24 h. Cells were then incubated with JC-1 working solution at 37 °C for 20 min, washed with JC-1 buffer, and imaged using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Red fluorescence (JC-1 aggregates) and green fluorescence (JC-1 monomers) were recorded. The ratio of red to green fluorescence intensity was quantified using ImageJ (version 1.53t; National Institutes of Health, Bethesda, MD, USA) and used as an index of $\mathrm{\Delta }$$\psi$m.

Total RNA was extracted from LN229 cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), and 1 µg of RNA was reverse-transcribed using a PrimeScript RT kit (Takara Bio Inc., Kusatsu, Shiga, Japan, catalog number RR037A). Quantitative real-time PCR (qRT-PCR) was performed using TB Green Premix (Takara) on a QuantStudio 6 System (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA, USA). Primers for human BID and GAPDH (internal control) were designed using Primer-BLAST. Relative BID mRNA expression was calculated using the 2^{-Δ⁢Δ⁢C⁢t} method. The detailed gene sequences are listed in Table 1.

Protein expression of BID and tBID was examined by western blotting. LN229 cells were infected with KD01 at different MOIs (0, 1, and 10) for 24 h and lysed in RIPA buffer supplemented with protease inhibitors (Beyotime). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Merck Millipore, Burlington, MA, USA). Membranes were blocked with 5% nonfat milk and incubated overnight at 4 °C with primary antibodies against BID (ABclonal Technology, Wuhan, China, catalog number A23234) and $\beta$-actin (ABclonal Technology, Wuhan, China, catalog number AC028), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence (ECL; Millipore, Burlington, MA, USA) and quantified using ImageJ software.

All animal procedures were approved by the Animal Experimentation Ethics Committee of Tongji Medical College (protocol code 4704) and were conducted in accordance with institutional guidelines.

Male BALB/c nude mice (4–6 weeks old; Jicui Biotechnology, Guangdong, China) were anesthetized with isoflurane and positioned in a stereotactic frame. LN229 cells (1 $\times$ 10⁷ cells in 5 µL PBS) were injected into the right striatum (2.0 mm lateral and 1.0 mm anterior to bregma, at a depth of 3.0 mm) at a rate of 0.5 µL/min using a Hamilton syringe. The needle was left in place for 5 min before slow withdrawal to prevent reflux.

Tumor-bearing mice were randomly assigned to four groups (n = 11 per group): PBS control, bevacizumab alone, KD01 alone, and bevacizumab followed by KD01 (Bev$\to$KD01). Bevacizumab (Roche, Basel, Switzerland) was administered intraperitoneally at 10 mg/kg on day 7 after tumor implantation. KD01 was administered via stereotactic intratumoral injection at a dose of 1 $\times$ 10⁹ viral particles (vp) in 5 µL PBS on day 9. Mice in the KD01-alone group received KD01 on day 9 without bevacizumab, those in the bevacizumab-alone group received bevacizumab on day 7 without KD01, and control mice received equal volumes of PBS. Body weight and clinical signs were monitored twice weekly; and survival was recorded from the day of tumor implantation until the humane endpoint was reached.

At the experimental endpoint, mice were humanely euthanized by intraperitoneal (i.p.) injection of an overdose of sodium pentobarbital (200 mg/kg body weight). This method induces rapid, painless anesthesia, followed by the cessation of vital functions, ensuring minimal distress to the animals. Successful euthanasia was confirmed by the absence of corneal reflexes and respiratory and cardiac activity.

Tumor-associated brain edema was assessed by measuring brain water content. On Day 5 after KD01 treatment (day 14 after tumor implantation), three mice from each group were randomly selected and subjected to deep anesthesia followed by decapitation. The brain was rapidly removed, and the tumor-bearing and contralateral hemispheres were dissected and weighed to obtain wet weights. Samples were then dried at 100 °C for 24 h and reweighed to obtain dry weights. Brain water content (%) was calculated as:

Water content = (wet weight – dry weight)/wet weight $\times$ 100%

The difference in water content between the tumor-bearing and contralateral hemispheres ($\mathrm{\Delta }$Water%) was used as an index of tumor-associated edema.

For Ki-67 and CD31 staining, mice were transcardially perfused with PBS, followed by 4% paraformaldehyde. Brains were harvested, fixed overnight, paraffin-embedded, and sectioned at 4 µm. Sections were deparaffinized, rehydrated, subjected to antigen retrieval (citrate buffer, pH 6.0), and blocked with 5% bovine serum albumin. Sections were incubated overnight at 4 °C with primary antibodies against Ki-67 (Abcam, Waltham, MA, USA, catalog number ab15580) or CD31 (Abcam), followed by incubation with HRP-conjugated secondary antibodies. Immunoreactivity was visualized with DAB substrate and counterstained with hematoxylin. The Ki-67 labeling index was calculated as the percentage of Ki-67-positive nuclei counted in $\ge$5 random high-power fields per tumor. CD31 staining was evaluated to assess vascular distribution in the same tissue sections.

To evaluate systemic toxicity, blood samples were collected from the retro-orbital sinus at the experimental endpoint. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and creatinine (CREA) were measured using an automated biochemical analyzer (Cobas 8000 Modular Analyzer; Roche Diagnostics, Switzerland). Platelet counts (PLT) and prothrombin time (PT) were measured using hematology and coagulation analyzers, respectively. Major organs, including the brain, liver, and spleen, were excised and weighed.

Data are presented as mean $\pm$ standard error of the mean (SEM) unless otherwise stated. Comparisons between two groups were performed using unpaired two-tailed Student’s t-tests. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Survival curves were generated using the Kaplan–Meier method and compared using the log-rank (Mantel–Cox) test. Statistical significance was defined as p 0.05. All statistical analyses were performed using GraphPad Prism 10 (version 10.0.3; GraphPad Software, San Diego, CA, USA).

To generate a conditionally replicating oncolytic adenovirus, KD01 was engineered with a small deletion in the conserved region of E1A ($\mathrm{\Delta }$920–926 bp) and with replacement of ADP in the E3 region by a tBID expression cassette (Fig. 1A).

Fig. 1.

Structure of KD01 and its oncolytic activity in human glioma cells. (A) Schematic representation of KD01. A 27-bp deletion was introduced into the conserved region of E1A ($\mathrm{\Delta }$920–926 bp), and the ADP in the E3 region was deleted and replaced by a tBID expression cassette. (B,C) Cell viability of human glioma cell lines LN229 (B) and A172 (C) after infection with KD01 at the indicated multiplicities of infection (MOI 0.1, 1, and 10) or mock infection (Control). Cell viability was measured using the CCK-8 assay at 24, 48 and 72 h post-infection and expressed as a percentage of the corresponding control. Data are presented as mean $\pm$ SEM (n = [6]); statistical significance between the indicated groups is shown as **p 0.01 and ***p 0.001. ADP, adenovirus death protein; tBID, truncated BID; MOI, multiplicities of infection; CCK-8, Cell Counting Kit-8.

We first examined the intrinsic susceptibility of human glioma cells to KD01. Infection of LN229 cells with increasing MOIs of KD01 (0.1, 1, and 10) resulted in a clear dose- and time-dependent reduction in cell viability (Fig. 1B). Even at an MOI of 0.1, KD01 significantly reduced cell viability compared with controls at 48 and 72 h. At an MOI of 10, viability decreased to a small fraction of control levels by 72 h (p 0.01). A similar pattern was observed in A172 cells (Fig. 1C), with significant loss of viability at MOIs $\ge$0.1 over time, although the extent of cytotoxicity was slightly less pronounced than in LN229 cells at equivalent doses. Collectively, these results indicate that KD01 exerts potent dose- and time-dependent oncolytic activity against human glioma cell lines in vitro.

To investigate whether KD01 exerts cytotoxicity through mitochondrial apoptosis, we first assessed mitochondrial membrane potential using JC-1 staining. In LN229 cells treated with PBS, strong red JC-1 aggregates and weak green fluorescence were observed, indicating an intact mitochondrial membrane potential (Fig. 2A). Infection with the control virus M20 caused a moderate increase in green fluorescence and a reduction in red aggregates, whereas KD01 infection led to a marked loss of red fluorescence with predominantly green staining, consistent with mitochondrial depolarization. Quantification of the JC-1 aggregate/monomer ratio confirmed that KD01 induced significantly greater disruption of the mitochondrial membrane potential than M20 (Fig. 2B).

Fig. 2.

KD01 induces mitochondrial dysfunction via activation of the BID–tBID pathway in glioma cells. (A) Representative JC-1 staining of LN229 cells treated with PBS, control virus M20 (Del-ADP), or KD01. Red fluorescence indicates JC-1 aggregates in mitochondria with intact membrane potential, and green fluorescence indicates JC-1 monomers in depolarized mitochondria (scale bar, 15 µm). (B) Quantification of mitochondrial membrane potential expressed as the ratio of JC-1 aggregates to monomers (red/green) in LN229 cells after the indicated treatments. (C) Relative BID mRNA expression in LN229 cells infected with KD01 at MOI 0.1 or 1 compared with PBS control, determined by qRT-PCR. (D) Representative Western blot of full-length BID and tBID protein in LN229 cells infected with KD01 at the indicated MOIs; $\beta$-actin served as a loading control. (E) Densitometric analysis of BID and tBID protein levels normalized to $\beta$-actin. Data in (B), (C), and (E) are presented as mean $\pm$ SEM (n = 3 independent experiments). **p 0.01, ***p 0.001.

Given that KD01 encodes tBID, we next examined BID/tBID expression. KD01 infection increased BID transcript levels in a dose-dependent manner (Fig. 2C). At the protein level, full-length BID was progressively reduced, whereas tBID accumulated with increasing MOIs of KD01 (Fig. 2D,E), indicating efficient expression and processing of the tBID cassette. Taken together, these data demonstrate that KD01 potently activates the BID–tBID pathway and induces profound mitochondrial dysfunction in glioma cells, consistent with engagement of the intrinsic apoptotic pathway.

As shown in Fig. 3A, we evaluated the effects of bevacizumab combined with KD01 using a sequential treatment schedule in a nude mouse LN229 intracranial xenograft model. On day 0 (D0), LN229 cells were inoculated into the brain by stereotactic injection; on day 7 (D7), mice received an intraperitoneal injection of bevacizumab or PBS; and on day 9 (D9), KD01 or PBS was administered via stereotactic intratumoral injection. This design generated four treatment groups: PBS, KD01, bevacizumab, and KD01 + bevacizumab, and animals were monitored until death.

Fig. 3.

Bevacizumab priming enhances antitumor efficacy of KD01 in an orthotopic LN229 glioma model. (A) Schematic of the treatment schedule. LN229 cells were implanted intracranially on day 0 (D0). Mice received intraperitoneal bevacizumab or PBS on day 7 (D7) and stereotactic intratumoral injection of KD01 or PBS on day 9 (D9), generating four groups: PBS, KD01, bevacizumab, and KD01 + bevacizumab. (B) Body weight changes in each group at the indicated time points. Data are shown as mean $\pm$ SEM (n = [5]); the combination group maintained significantly higher body weight than the KD01 group at D14, D21, and D28. (C) Kaplan–Meier survival curves for tumor-bearing mice in the four treatment groups. Sequential bevacizumab + KD01 treatment markedly prolonged survival compared with PBS, KD01, or bevacizumab alone (log-rank test). (D) Representative Ki-67 immunohistochemistry of intracranial tumors from each group (scale bar, 100 µm). Strong, diffuse nuclear Ki-67 staining is seen in the PBS group, reduced staining in the KD01 and bevacizumab groups, and the weakest staining in the KD01 + bevacizumab group. (E) Tumor-associated brain edema assessed as the difference in water content between tumor-bearing and contralateral hemispheres ($\mathrm{\Delta }$Water%). $\mathrm{\Delta }$Water% was highest in the PBS group, slightly reduced in the KD01 and bevacizumab groups, and significantly lowest in the KD01 + bevacizumab group. (F) CD31 immunohistochemical staining of tumor vasculature in each treatment group (scale bar, 100 µm). (G) Statistical analysis of CD31-positive area percentage (n = 5). Data are presented as mean $\pm$ SEM (n = [3]); ICCI, Intracranial Injection; IP, Intraperitoneal Injection; IT, Intratumoral Injection. *p 0.05, **p 0.01, ***p 0.001.

Body weight changes were compared among groups as an indirect measure of systemic toxicity and overall condition. Throughout the observation period, mice in the PBS and KD01 groups showed a gradual decline in body weight, whereas body weight in the bevacizumab group remained largely stable. Notably, in the combination group, body weight did not decrease and was consistently higher than that in the PBS and KD01 groups from D10 onward (Fig. 3B). On D14, D21, and D28, body weight in the KD01 + bevacizumab group was significantly higher than that in the KD01 monotherapy group (p 0.05), indicating that, at the doses used, the combination regimen did not increase systemic toxicity and was associated with better clinical status than KD01 alone.

Kaplan–Meier survival analysis further confirmed that KD01 combined with bevacizumab significantly prolonged survival in tumor-bearing mice (Fig. 3C). Animals in the PBS group developed neurological deterioration shortly after implantation and died sequentially. Bevacizumab or KD01 monotherapy delayed death to some extent, but survival curves declined rapidly. In contrast, the survival curve of the KD01 + bevacizumab group shifted markedly to the right, with most mice surviving longer than those in the three control groups. Log-rank testing revealed statistically significant differences between the combination group and the KD01, bevacizumab, and PBS groups, indicating that sequential bevacizumab priming markedly enhanced the in vivo antitumor efficacy of KD01.

Tumor cell proliferation was next assessed using Ki-67 immunohistochemistry. In the PBS group, strong brown nuclear Ki-67 staining was diffusely distributed throughout the tumor, resulting in the highest Ki-67 labeling index. The proportion of Ki-67–positive cells was clearly reduced in both the KD01 and bevacizumab groups, although numerous positive cells remained. In the KD01 + bevacizumab group, Ki-67–positive cells were markedly decreased, with only scattered weakly positive nuclei and much lower overall staining intensity. Semi-quantitative analysis showed that the Ki-67 labeling index was lowest in the combination group and was significantly lower than that in the PBS and monotherapy groups (Fig. 3D).

To evaluate the effect of combination therapy on tumor-associated brain edema, we measured the difference in water content between the tumor-bearing and contralateral hemispheres ($\mathrm{\Delta }$Water%). Quantitative analysis revealed that $\mathrm{\Delta }$Water% was highest in the PBS group, slightly reduced in the KD01 and bevacizumab groups, and significantly lower in the KD01 + bevacizumab group compared with all three control groups (p 0.05, Fig. 3E). These findings indicate that bevacizumab priming not only enhances the survival benefit of KD01 but also cooperatively alleviates tumor-associated brain edema, likely contributing to the improved overall condition of treated animals.

Tumor vascular density was evaluated using CD31 (an endothelial cell marker) staining (Fig. 3F,G). The PBS group exhibited dense, disorganized CD31-positive vessels. KD01 monotherapy mildly reduced vascular density, whereas bevacizumab alone induced a more pronounced decrease. Notably, the KD01 + bevacizumab combination group displayed the sparsest CD31 staining, with only scattered vascular structures, consistent with synergistic vascular remodeling by bevacizumab-mediated normalization and KD01-induced oncolytic disruption.

To further evaluate systemic safety of sequential KD01 and bevacizumab treatment, serum biochemical and coagulation parameters were measured at the study endpoint, and major organ weights were compared among groups. Serum AST, ALP, and CREA levels remained within physiological ranges in all groups, with no statistically significant differences between the KD01, bevacizumab, or KD01 + bevacizumab groups and the PBS control group (Fig. 4A–C). ALT levels were slightly higher in the KD01 and bevacizumab monotherapy groups than in the PBS group; however, the increase was modest, and ALT levels in the KD01 + bevacizumab group were comparable to PBS (Fig. 4D). These findings suggest that either virus or anti-VEGF monotherapy may cause mild, tolerable fluctuations in liver function that are not exacerbated by the combination regimen.

Fig. 4.

Systemic safety of sequential KD01 and bevacizumab treatment in tumor-bearing mice. (A‒D) Serum biochemical parameters at the end of the study: AST (A), ALP (B), CREA (C), and ALT (D) in mice treated with PBS, KD01, Bev, or KD01 + Bev. AST, ALP, and CREA remained within the physiological range with no significant intergroup differences; ALT showed a mild increase in the KD01 and Bev monotherapy groups but returned to control levels in the combination group. (E,F) PLT (E), and PT (F) among the four groups, showing no significant effects of KD01, Bev, or their combination on coagulation function or platelet production. (G) Relative weights of major organs (brain, spleen, and liver) at sacrifice. No significant differences were observed among groups, and no obvious organ atrophy or enlargement was detected. Data are presented as mean $\pm$ SEM (n = [3]). Bev, bevacizumab; AST, aspartate aminotransferase; ALP, alkaline phosphatase; CREA, creatinine; ALT, alanine aminotransferase; PLT, Platelet counts; PT, prothrombin time. *p 0.05; ns, not significant.

PLT and PT values were also similar across all four groups (Fig. 4E,F), indicating that neither KD01 nor bevacizumab, alone or in combination, significantly affected coagulation function or platelet production. Likewise, relative weights of major organs, including the brain, spleen, and liver, did not differ significantly among the PBS, KD01, bevacizumab, and KD01 + bevacizumab groups (Fig. 4G), and no obvious organ atrophy or enlargement was observed.

Taken together, these results indicate that sequential bevacizumab priming combined with KD01 oncolytic therapy did not induce hepatic or renal injury, coagulation abnormalities, or major organ toxicity at the doses and schedules used in this study, supporting a favorable systemic safety profile for this combination strategy.