Animal protocols were approved by the INHA Institutional Animal Care and Use Committee (INHA IACUC) at the College of Medicine, Inha University (Incheon, Korea; approval nos. INHA 2211124-848 and INHA 230731-883).

MIA PaCa-2 (cat. no. CRL-1420) and Capan-1 cell lines (cat. no. HTB-79) were obtained from the American Type Culture Collection. MIA PaCa-2 cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Welgene, Inc.), while Capan-1 cell lines were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Gibco; Thermo Fisher Scientific, Inc.). The medium for MIA PaCa-2 was supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin. The medium for Capan-1 was supplemented with 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cell culture reagents, including FBS and penicillin/streptomycin, were purchased from Gibco (Thermo Fisher Scientific, Inc.). The cells were maintained in a CO₂ incubator with 95% air and 5% CO₂ at 37°C.

Aripiprazole and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from MilliporeSigma. For in vitro studies, aripiprazole was dissolved in dimethyl sulfoxide (DMSO) at a 20 mM stock concentration and freshly diluted to working concentrations. For in vivo experiments, the desired doses were prepared by dissolving in a DMSO:Cremophor:DW (1:2:7) mixture. Cisplatin was purchased from Selleck Chemicals.

Cell viability was assessed using the MTT assay. Briefly, MIA PaCa-2 and Capan-1 cells were seeded at densities of 2,500 cells and 5,000 cells per well, respectively, in 96-well plates and treated with aripiprazole and/or cisplatin. After 48 h of incubation, 20 µl of MTT solution (2 mg/ml) was added and incubated for an additional 4 h at 37°C. The plates were read using a microplate reader at a wavelength of 540 nm. The combination index for cisplatin and aripiprazole was calculated using CompuSyn v1.0 (Biosoft), where combination index (CI)<1, CI=1 and CI>1 indicate synergistic, additive and antagonistic effects, respectively (10). Cell proliferation was monitored using the JULI Stage real-time image recording system (NanoEntek).

MIA PaCa-2 and Capan-1 cells (1×10⁶) were treated with cisplatin (1 or 10 mM) and/or aripiprazole (10 mM) for 48 h. The treated cells were seeded in six-well plates at densities of 1×10³ and 3×10³ cells per well with a monolayer and incubated for 14 days. Colonies were washed twice with Dulbecco's phosphate-buffered saline (DPBS), fixed with 4% paraformaldehyde for 15 min at 4°C and stained with 1% crystal violet for 15 min at room temperature.

The TUNEL assay was conducted using the ApopTag peroxidase in situ apoptosis detection kit (MilliporeSigma). MIA PaCa-2 and Capan-1 cells were treated with cisplatin (1 or 10 µM) and aripiprazole (10 µM) for 48 h, fixed and washed three times with phosphate-buffered saline (PBS). After the terminal deoxynucleotidyl transferase enzyme was activated, the reaction was stopped, and the digoxigenin-conjugated antibody was attached overnight. After washing, 10% 3,3′-diaminobenzidine staining was performed to visualize apoptotic cells and stained for 15 min at room temperature.

The cells were fixed in an acetic acid solution (1:2) for 10 min at 4°C and permeabilized with 0.5% Triton X-100 for 10 min and incubated in CAS block solution (Life Technologies) for 1 h at room temperature. Next, the cells were incubated overnight at 4°C with a primary antibody against cleaved caspase-3 (1:50; cat. no. 9661; Cell Signaling Technology). After several washes with PBS, the cells were incubated with fluorescently labeled secondary antibodies (1:60; cat. no. 31460; Invitrogen) for 2 h at room temperature and stained with 4,6-diamidino-2-phenylindole (Antifade mounting medium with DAPI; Vectashield; Vector Laboratories, Inc.) at a 1:100 dilution for 1 h at room temperature. The cells were viewed using a confocal laser scanning microscope (FluoView 1000; Olympus Corporation) at wavelengths of 488 and 568 nm.

The cells were washed three times with ice-cold DPBS before the lysis. Total cellular proteins were extracted using sterile RIPA lysis buffer (Biosesang; 150 mM sodium chloride, 1% triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5, and 2 mM EDTA), 1X Phosphatase inhibitor (GenDEPOT, LLC; sodium fluoride, sodium orthovanadate, sodium pyrophosphate and sodium glycerophosphate) and 1X Protease inhibitor (GenDEPOT, LLC; PMSF; pepstatin A, leupeptin, benzamidine, bestatin). Equal amounts of proteins (50 µg) were separated by 8% or 12% SDS gel electrophoresis using BCA assay and transferred to PVDF membranes (MilliporeSigma). The membranes were blocked with PBS containing 5% skimmed milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies. After washing, the membranes were incubated with secondary antibodies (1:2,000, Mouse (cat. no. 7076S), Rabbit (cat. no. 7074S); Cell signaling Technology, Inc.) for 1 h at room temperature. Proteins were visualized using Clarity Western ECL Substrate (Amersham Biosciences; Cytiva). Primary antibodies from Cell Signaling Technology, Inc. included cleaved caspase-3 (1:1,000; cat. no. 9661), AKT (1:1,000; cat. no. 9272), phosphorylated (p)-AKT (1:2,000; cat. no. 4060), p-SRC (1:1,000; cat. no. 6943), STAT3 (1:1,000; cat. no. 9139), p-STAT3 (1:1,000; cat. no. 94994) and β-actin (1:10,000; cat. no. 4967). The primary antibody for myeloid cell leukemia-1 (MCL-1, 1:500; cat. no. sc-819) and XIAP (1:500; cat. no. sc-55550) was obtained from Santa Cruz Biotechnology, Inc. The densitometry was quantified using ImageJ software (version 1.54, National Institutes of Health).

The cells were treated with cisplatin (1 or 10 µM) and/or aripiprazole (10 µM) for 48 h at 37°C, followed by incubation with JC-1 solution (Cayman Chemical) for 45 min at 37°C. Following staining with DAPI, the slides were washed twice with DPBS, mounted with Antifade Mounting Medium with DAPI (Vectashield; Vector Laboratories, Inc.) and viewed using confocal laser-scanning microscopy (FluoView 1000; Olympus Corporation).

Cell invasion was analyzed using Transwell permeable support systems (Corning Life Sciences). The inserts were coated with 10% Matrigel for 30 min at 37°C before cell seeding. Cells were seeded at a density of 2×10⁴ cells/well in a serum-free medium. The lower chambers were treated with cisplatin (1 or 10 µM) and/or aripiprazole (10 µM) for 48 h at 37°C. After 48 h, the cells that invaded the PET membrane were stained with 0.5% crystal violet for 15 min at room temperature. The migration assay followed a similar protocol, except Matrigel was not used. The purple regions were quantified using ImageJ software (version 1.54; National Institutes of Health).

The cells were treated with various concentrations of cisplatin and aripiprazole for 48 h. Cells were then trypsinized and resuspended to a concentration of 1×10⁶ cells/ml in culture medium and subjected to three repeated exposures to shear stress through a 30-gauge needle, followed by a constant flow of 100 µl/s. Briefly, 1×10⁶ cells were resuspended in 4 ml PBS and loaded into a 5 ml plastic syringe. A Luer lock fitting was attached to the end of the syringe and connected to a polyether ether ketone tubing (inner diameter, 125 µm) which was connected to a 50 ml centrifuge tube. The syringe plunger was pushed by a syringe pump to flow the cell solution at flow rates corresponding to wall shear stresses of 5, 20, or 60 dyn/cm². The syringe was regularly agitated to maintain cell suspension. After shearing, the cells were centrifuged at 300 × g for 3 min at 4°C and resuspended in DMEM/10% FBS. The cells were then incubated for 11 days at 37°C in 96-well ultra-low cluster round-bottom plates (Costar; Corning, Inc.) in a complement medium supplemented with basic fibroblast growth factor, human epidermal growth factor, N-2 and B-27 at 37°C to form three-dimensional (3D) tumor spheroids. After 11 days, the cells were observed under a phase-contrast microscope and captured images. The size and shape of the 3D tumor spheroids were recorded using an inverted light microscope from day 2 to day 11. Additionally, the cells treated for 48 h were seeded at 8×10⁴ cells per well in collagen-coated 48-well plates, incubated for 1 h at 37°C, washed twice with DPBS, fixed with 4% paraformaldehyde and incubated for 1 h at room temperature with a 1:100 dilution of DAPI.

Relative phosphorylation levels of 43 human protein kinases were determined using a human phospho-kinase array kit (R&D Systems, Inc.). MIA PaCa-2 cells were treated with aripiprazole for 12 h at 37°C and then lysed. After blocking for 1 h at 37°C with Array Buffer 1 (R&D systems, Inc), the membranes were incubated overnight at 4°C with 600 µg of protein lysates, washed and then incubated with a streptavidin-HRP detection antibody for 30 min rocking at room temperature. Membranes were developed using ECL Western blotting detection reagents and protein expression levels were quantified using ImageJ software (version 1.54; National Institutes of Health).

Male BALB/c nude mice (five-weeks-old; 18-20 g) were purchased from Orient Bio, Inc. All animal experiments were conducted in accordance with the guidelines of the INHA IACUC (approval no. INHA 2211124-848). The mice were acclimated for one week and then injected with 3×10⁶ MIA PaCa-2 cells into the flank. When the tumor size reached 50-100 mm³, the mice were randomly assigned to four groups (control, cisplatin, aripiprazole, cisplatin and aripiprazole; n=9). The treatment group received cisplatin [2 mg/kg, intraperitoneally (i.p.)] once a week and/or aripiprazole [10 mg/kg, orally (p.o.)] three times a week. Tumor size was calculated using Vernier calipers with the formula: 0.5x length × (width)². After 28 days, the tumors were carefully dissected out and fixed in 10% paraformaldehyde at 4°C overnight, embedded in paraffin and sectioned at 3 µm. All animals had ad libitum access to food and water and were maintained in a stable environment at 25±1°C, 60±5% humidity and a 12-h light/dark cycle. The health of the mice was monitored by observing the temperature, humidity, noise and lighting conditions in the animal room by monitoring the weight and general condition of mice to assess the health and behavioral status of the mice. At the end of study, the mice were then anesthetized using Ketamin:Rompun (9:1) mixture and sacrificed by collecting blood sample from the heart. The mice were confirmed dead by no spontaneous breathing for 2-3 min and no blink reflex. The blood, lung, liver, pancreas, spleen and tumors of mice were collected for sample preparation. During the experiment, all mice were anesthetized and sacrificed according to the experimental plan and all measures were taken to decrease the pain of the experimental animals. If the mice showed persistent pain behavior, severe dehydration, inability to eat, extreme fatigue or even severe infection during the research process, they were sacrificed; however, no animals in the present study reached these humane endpoints.

Male C57BL/6 mice aged 5 weeks (18-20 g) were purchased from Orient Bio Animal, Inc. All animal experiments were conducted in accordance with the guidelines of the INHA IACUC (approval no. INHA 230731-883). The mice (n=7) were injected with 1×10⁴ KPC cells into the pancreas. After two weeks, the treatment groups received cisplatin (2 mg/kg, i.p.) once a week and/or aripiprazole (10 mg/kg, p.o.) three times a week. After 25 days, the tumors were carefully dissected to avoid contamination from surrounding tissues. At the end of study, the mice were then anesthetized using Ketamin:Rompun (9:1) mixture and sacrificed by collecting blood sample from the heart. The mice were confirmed dead by no spontaneous breathing for 2-3 min and no blink reflex. The blood, lung, liver, pancreas, spleen and tumors of mice were collected for sample preparation. During the experiment, all mice were anesthetized and sacrificed according to the experimental plan and all measures were taken to decrease the pain of the experimental animals. If the mice showed persistent pain behavior, severe dehydration, inability to eat, extreme fatigue or even severe infection during the research process, they were sacrificed; however, no animals in the present study reached these humane endpoints.

Tumor samples were fixed in 10% buffered formaldehyde at 4°C overnight, embedded in paraffin, and sectioned, Immunostaining was performed on 3-µm sections of tumor samples after deparaffinization. Antigen retrieval was performed by incubation of 0.2 mg/ml Proteinase K (Thermo Fisher Scientific, Inc.) in PBS for 15 min at room temperature. After gently washing twice with PBS, tissue sections were permeabilized with 0.5% Triton X-100 for 10 min and endogenous peroxidase was blocked with 0.3% H₂O₂ in distilled water for 15 min at room temperature, followed by incubation in CAS block solution (Life Technologies) for 1 h at room temperature. The tissue sections were incubated with primary antibodies (1:50 dilution) following antigen retrieval at 4°C overnight. Subsequently, sections were incubated with a biotinylated secondary antibody (1:60 dilution; cat. no. 31460) for 1 h at room temperature. Primary antibodies from Cell Signaling Technology, Inc. included cleaved caspase-3 (1:50; cat. no. 9661), BCL-2 (1:50; cat. no. 15071), p-STAT3 (1:50; cat. no. 94994). Primary antibody from Abcam included Ki-67 (1:50; cat. no. ab16667). Immunoreactive proteins were detected by incubating sections with an avidin-biotin peroxidase complex solution ABC kit (Vector Laboratories, Inc.). After washing with PBS, proteins were visualized by incubating sections with DAB for 15 min at room temperature, followed by counterstaining with hematoxylin solution for 40 sec at room temperature. At least three random fields of each section were examined at ×400 magnification and analyzed using a computer image analysis system (Olympus Corporation).

All experiments were repeated three times. The data were analyzed using one-way ANOVA with Tukey's post hoc tests for multiple comparisons. Results were presented as mean ± standard deviation (SD). All analyses were performed with GraphPad Prism (version 8.00, GraphPad Software, Inc.; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

To identify the synergistic effects of cisplatin and aripiprazole, PDAC cells (MIA PaCa-2 and Capan-1) were treated with both drugs for 48 h. The co-treatment exhibited significant synergistic effects, with CI<1 for the combination of 10 µM cisplatin and 10 µM aripiprazole in MIA PaCa-2 cells (CI=0.884) and 1 µM cisplatin and 10 µM aripiprazole in Capan-1 cells (CI=0.867; Fig. 1A). The cell proliferation curves showed similar trends (Fig. 1B). Additionally, the clonogenic survival of PDAC cells treated with this combination was inhibited in a dose-dependent manner compared with single-agent treatments (Fig. 1C). These findings indicated that aripiprazole enhanced the anticancer efficacy of cisplatin and synergistically inhibits the proliferation of PDAC cells.

Since the co-treatment significantly reduced cell proliferation, it was investigated whether it could induce apoptosis in PDAC cells. TUNEL staining revealed an increase in the percentage of TUNEL-positive cells following treatment (Fig. 2A and B). Furthermore, the combination treatment significantly increased the expression levels of cleaved caspase-3 (Fig. 2C and D). These apoptotic effects were confirmed by the increase in cleaved caspase-3 compared with single-agent treatments, as demonstrated by western blotting (Fig. 2E). Taken together, these results indicated that the combination of cisplatin and aripiprazole synergistically induced apoptosis in pancreatic cancer cells.

Given that the combined treatment of cisplatin and aripiprazole markedly increased apoptotic cell death, the present study next investigated whether it affects the mitochondrial membrane potential (MMP), which is associated with apoptosis. MMP was examined using JC-1 staining. The control cells exhibited strong red fluorescence in the cytoplasm, whereas the combined treatment showed a clear decrease in red fluorescence and an increase in green fluorescence, indicating changes in MMP associated with apoptosis (Fig. 3A and B). Additionally, the combination treatment decreased the expression of XIAP and MCL-1, prominent anti-apoptotic proteins (Fig. 3C). The results suggested that the synergistic effects of aripiprazole and cisplatin are mediated by mitochondria-mediated apoptosis in PDAC cells.

Cancer cell migration and invasion are critical steps in metastasis. To investigate the potential inhibitory effects of co-treatment with aripiprazole and cisplatin on these processes, migration and invasion assays were conducted using MIA PaCa-2 and Capan-1 cell lines. Transwell assays demonstrated that the combination of aripiprazole and cisplatin synergistically inhibited both cell migration and invasion compared with the effects of either agent alone (Fig. 4A and B). Additionally, cell spheroid growth and attachment to collagen-coated plates were significantly diminished by the combined treatment, in contrast to the effects of single-agent treatments (Fig. 4C and D).

Among the 43 kinases analyzed, aripiprazole inhibited STAT3 expression by 40% (Fig. 5A). To validate these findings, cells were treated with aripiprazole (10 µM) for 12 h, followed by western blot analysis to examine the expression of p-STAT3, p-AKT and p-SRC. As shown in Fig. 5B, aripiprazole decreased p-STAT3 expression, and the combination of aripiprazole and cisplatin almost completely abolished its expression in MIA PaCa-2 cells. These findings suggested that the combination treatment could induce pancreatic cancer cell death by inhibiting STAT3 signaling.

Tumor growth was delayed in mice treated with either aripiprazole or cisplatin alone compared with the control group. Notably, the combination treatment significantly reduced tumor volume by 78% relative to the control (Fig. 6A). These results were corroborated using an orthotopic pancreatic cancer model (Fig. 6B). All treatments were well-tolerated, with no significant differences between groups. Tumor formation incidence was >95%. Treatment with aripiprazole or cisplatin inhibited primary pancreatic tumor growth by 33 and 23%, respectively, compared with the control. The combination treatment markedly enhanced the anti-tumor effect, with a 73% growth inhibition observed compared with control. Furthermore, the combined treatment reduced the expression of Ki-67 and Bcl-2, markers of cell proliferation and apoptosis, respectively and increased the expression of cleaved caspase-3 and the number of TUNEL-positive cells. The combination treatment also significantly reduced p-STAT3 expression (Fig. 6C).