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Introduction
Pancreatic cancer remains one of the deadliest malignancies worldwide. In 2022, an estimated 510,000 individuals were newly diagnosed with pancreatic cancer, and approximately 467,000 deaths from the disease occurred globally; these numbers continue to rise [1]. With a 5-year survival rate of only 13%, the prognosis for pancreatic cancer is poor [2]. Although it accounts for just 2.6% of new cancer cases and ranks 12th by cancer site, pancreatic cancer is the sixth leading cause of cancer-related death worldwide due to its high fatality rate [1]. The high mortality rate is largely attributable to the fact that 80% to 85% of cases are diagnosed at an unresectable stage, by which time the disease is locally advanced or metastatic, making surgery unfeasible [3]. As a result, systemic chemotherapy is now the primary treatment for this condition [4]. However, the dense desmoplastic stroma characteristic of pancreatic cancer hinders drug delivery and reduces the effectiveness of chemotherapy [3]. This challenge underscores the urgent need for new strategies to enhance drug delivery mechanisms in pancreatic cancer treatment.
Therapeutic ultrasound (TUS) has emerged as a promising approach to improve drug delivery [5,6]. TUS can transiently increase cell membrane permeability and enhance drug penetration into cancer cells through mechanical effects such as sonoporation, acoustic radiation force, and shear stress [7]. Additionally, the combination of TUS with microbubbles, commonly used as ultrasound contrast agents, has shown a synergistic effect, further improving drug delivery and therapeutic outcomes [8,9]. TUS can also induce direct mechanical bioeffects, such as acoustic cavitation, which leads to cell membrane disruption, DNA damage, and activation of apoptotic pathways, resulting in cytotoxicity even in the absence of drugs or microbubble assistance [10]. Cell death in cancer can occur via apoptosis, autophagy, or necrosis. Apoptosis is a regulated process of programmed cell death that eliminates damaged or unwanted cells without provoking inflammation, making it a desirable outcome of anticancer treatments. Autophagy enables cells to degrade and recycle their components to survive under stress; while it can promote cancer cell survival during chemotherapy, excessive autophagy may also lead to cell death. Thus, the strategic modulation of autophagy can increase the efficacy of chemotherapy. In contrast, necrosis is an unregulated form of cell death that results from severe cellular injury, leading to cell swelling, membrane rupture, and release of cellular contents that provoke inflammation and damage to surrounding normal tissues [11].
This study aimed to investigate the effects of TUS on pancreatic cancer cells, both as a standalone treatment and in combination with microbubbles and/or the anticancer agent gemcitabine. By examining how different cell death processes—including apoptosis and autophagy—contribute to the cellular response to these treatments, the study sought to elucidate the mechanisms by which TUS and microbubbles enhance drug delivery and therapeutic efficacy. Understanding these mechanisms may provide valuable insights for the development of innovative treatments for pancreatic cancer.
Materials and Methods
Compliance with Ethical Standards
This study used only a commercially available human pancreatic ductal adenocarcinoma cell line (CFPAC-1) and did not involve human participants or live animals. Therefore, institutional review board approval or equivalent ethical oversight was not required.
Cell Preparation
CFPAC-1 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were maintained at 37°C in a humidified incubator with 5% CO2. After culture, 2×106 CFPAC-1 cells in 1 mL of culture medium were transferred to a 2-mL centrifuge tube for treatment.
Treatment Conditions
For TUS treatment, three different modes were employed: continuous wave (CW), 50% duty cycle (DC), and 10% DC. The peak-negative pressure was set at 0.29 MPa for CW, 0.42 MPa for 50% DC, and 0.86 MPa for 10% DC to maintain approximately consistent total acoustic energy delivery across modes. As a result, the peak intensity during each pulse was approximately twice as high in the 50% DC mode compared to the CW mode and approximately 10 times higher in the 10% DC mode compared to the CW mode.
The volumes of microbubbles (SonoVue, Bracco, Milano, Italy) added to the cell suspension were 0, 10, or 50 μL.
Cells were subjected to sonication under the three ultrasound modes. For each mode, treatments were performed with each microbubble volume, both with and without gemcitabine (2 μg). Control groups without ultrasound were included for every combination of microbubble volume and gemcitabine use, resulting in a total of 24 experimental groups.
The TUS transducer used in this study was a custom-built device with a frequency of 715 kHz, a diameter of 10 cm, and a radius of curvature of 8 cm. The -3 dB focal size was 9.8 mm in the axial direction and 1.8 mm in the lateral direction. For all experiments, 2-mL centrifuge tubes with an inner diameter of approximately 8.5 mm were used. The duration of TUS exposure was 20 seconds for each treatment. After sonication, cells were transferred to 6-well plates and cultured for 24 hours (Fig. 1).
Cell Viability
Cell viability after each treatment condition was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (CGD1, Cell Growth Determination Kit-MTT-based, Sigma-Aldrich, St. Louis, MO, USA). This colorimetric method measures cell viability based on mitochondrial activity. Cells were seeded in a 96-well plate and treated according to the specific experimental conditions for the designated period. After treatment, MTT reagent (0.5 mg/mL) was added to each well and incubated at 37°C for 2 to 4 hours, allowing viable cells to reduce MTT to insoluble formazan. The formazan crystals were then dissolved in dimethyl sulfoxide, and absorbance was measured at 570 nm using a microplate reader. Absorbance values were normalized to those of the control group to determine relative cell viability. A decrease in MTT reduction capacity indicated increased cell death, allowing comparative analysis of cytotoxic effects across different treatment conditions. Five independent replicates were performed for each condition to improve reliability and statistical significance.
Assessment of Apoptosis
Apoptosis in treated cells was evaluated using an annexin V/propidium iodide staining kit (BML-AK120, ApoStrand ELISA Apoptosis Detection Kit, Enzo Life Sciences, Farmingdale, NY, USA) in combination with flow cytometry fluorescence-activated cell sorting analysis. Cells were harvested by trypsinization and washed twice with cold phosphate-buffered saline (PBS). The cell pellet was resuspended in 1× binding buffer at a concentration of 1×106 cells/mL.
To stain the cells, 5 μL of annexin V-FITC and 5 μL of propidium iodide solution were added to 100 μL of cell suspension and incubated at room temperature in the dark for 15 minutes. After incubation, 400 μL of 1× binding buffer was added to each sample, and apoptotic cell populations were immediately analyzed using a FACS system (SA3800, Sony, Tokyo, Japan). The percentage of apoptotic cells was quantified to assess the extent of apoptosis under different experimental conditions. Three independent replicates were performed for each condition.
Induction of Autophagy
Autophagy activation in treated cells was assessed using the Sony SA3800 FACS system and an autophagy detection kit (ENZ-51031, CYTO-ID autophagy detection kit, Enzo Life Sciences). Cells were harvested and washed with PBS. After detachment by trypsinization and centrifugation, the cell pellet was resuspended in 1× staining buffer. An autophagy probe was then added, followed by incubation at 37°C for 30 minutes in the dark. After staining, the cells were washed and resuspended in 1× staining buffer. FACS analysis was performed using the Sony SA3800 system with a 488-nm laser to detect fluorescence in the FL1 channel (FITC detector). Fluorescence intensity was compared across samples to quantify autophagy activation. Changes in mean fluorescence intensity between the control and experimental groups were used to evaluate autophagy induction, providing reliable quantification of autophagy levels under different treatment conditions. Three independent replicates were performed for each condition.
Statistical Analysis
Three-way analysis of variance (ANOVA) and the post hoc Tukey honestly significant difference (HSD) test were used to assess cell viability, apoptosis, and autophagy. The independent variables were gemcitabine presence (0 μg or 2 μg), microbubble volume (0 μL, 10 μL, or 50 μL), and ultrasound parameters (control, 0.29 MPa CW, 0.42 MPa at 50% DC, or 0.86 MPa at 10% DC). All statistical analyses were conducted using the open-source programming language Python version 3.12.7 within the Jupyter Notebook environment, ensuring robust and reproducible computational processing of the data.
Results
Cell Viability
Three-way ANOVA was performed to evaluate the effects of gemcitabine use, microbubble volume, and ultrasound parameters on cell viability. The ANOVA results (Table 2) indicated that all main effects—gemcitabine treatment, microbubble volume, and ultrasound parameters—significantly affected cell viability (P<0.001). The significant main effect of gemcitabine demonstrated that gemcitabine effectively reduced pancreatic cancer cell viability. The significant main effect of microbubble volume suggested that the addition of microbubbles enhanced the reduction in cell viability. Furthermore, increasing the microbubble volume from 0 μL to 50 μL resulted in progressively lower cell viability. The significant main effect of the ultrasound parameters demonstrated that ultrasound intensity and DC played important roles in affecting cell viability. Specifically, higher ultrasound intensities and lower DCs (e.g., 0.86 MPa/10% DC) were associated with greater reductions in cell viability. In addition, all interaction terms, including two-way and three-way interactions, were statistically significant (P<0.001), indicating that the effect of each independent variable on cell viability depended on the levels of the other variables.
The post hoc Tukey HSD test (Table 3) was applied to identify specific differences in microbubble volume and ultrasound conditions among the treatment groups. Gemcitabine was not assessed in this manner because only two concentrations (0 μg and 2 μg) were used. Significant differences (P<0.01) were observed between the 0 μL and 10 μL conditions, as well as between the 0 μL and 50 μL conditions, with higher microbubble volumes resulting in greater reductions in cell viability. However, no significant difference was found between the 10 μL and 50 μL conditions. All ultrasound conditions (0.29 MPa/CW, 0.42 MPa/50% DC, and 0.86 MPa/10% DC) significantly reduced cell viability compared with the control (no ultrasound), with higher intensities and lower DCs being significantly more effective (P<0.001) except for the comparison between 0.29 MPa/CW and 0.42 MPa/50% DC.
Assessment of Apoptosis
Apoptosis was evaluated using flow cytometry with annexin V/propidium iodide staining under the same treatment conditions. The percentages of apoptotic cells are summarized in Table 1 and Fig. 3.
Three-way ANOVA was performed to analyze the effects on apoptosis. The ANOVA results (Table 4) showed that all main effects—gemcitabine, microbubble volume, and ultrasound parameters—significantly influenced the apoptosis rate (P<0.001). The significant main effect of gemcitabine confirmed that gemcitabine effectively induced apoptosis in pancreatic cancer cells. Compared with untreated cells, those treated with 2 μg of gemcitabine exhibited higher percentages of apoptosis. The significant main effect of microbubble volume indicated that microbubbles enhanced apoptosis, with higher microbubble volumes resulting in increased apoptosis rates. The significant main effect of ultrasound parameters demonstrated that ultrasound exposure induced apoptosis, with higher ultrasound intensities and lower DCs associated with higher apoptosis rates. All two-way and three-way interaction terms were also significant (P<0.001), indicating that the effect of each independent variable on apoptosis depended on the levels of the other variables.
The post hoc Tukey HSD test (Table 5) was used to identify specific differences in microbubble volume and ultrasound conditions among the treatment groups. Significant differences (P<0.01) were observed between the 0 μL and 10 μL conditions and between the 0 μL and 50 μL conditions, indicating a dose-dependent increase in the degree of apoptosis induced by microbubbles. However, no significant difference was found between the 10 μL and 50 μL conditions. Compared with the control (no ultrasound), all ultrasound conditions (0.29 MPa/CW, 0.42 MPa/50% DC, and 0.86 MPa/10% DC) significantly increased apoptosis (P<0.01), and lower DCs induced significantly more apoptosis (P<0.001), except for the comparison between 0.29 MPa/CW and 0.42 MPa/50% DC.
Induction of Autophagy
Autophagy levels were measured using FACS with an autophagy kit. The percentages of cells undergoing autophagy are presented in Table 1 and Fig. 4.
Three-way ANOVA was conducted to analyze the effects on autophagy. The ANOVA results (Table 6) showed that all main effects—gemcitabine, microbubble volume, and ultrasound parameters—significantly influenced autophagy levels (P<0.001). The significant main effect of gemcitabine indicated that gemcitabine affected autophagy in pancreatic cancer cells, with treatment using 2 μg of gemcitabine generally resulting in lower percentages of autophagy compared to no gemcitabine treatment. The significant main effect of microbubble volume suggested that microbubbles also influenced autophagy induction; however, the effect varied, with some conditions increasing and others decreasing autophagy levels. Similarly, the effect of ultrasound on autophagy was inconsistent. Significant two-way and three-way interactions were observed (P<0.001), indicating that the effect of each independent variable on autophagy depended on the levels of the other interacting variables.
The post hoc Tukey HSD test (Table 7) was applied to compare the microbubble volume and ultrasound conditions. Significant differences (P<0.01) were observed between the 0 μL and 10 μL conditions, as well as between the 0 μL and 50 μL conditions, with higher microbubble volumes associated with decreased autophagy. However, no significant difference was found between the 10 μL and 50 μL conditions. Across all ultrasound conditions (including the control), no significant pairwise differences were observed.
Discussion
This study investigated the combined effects of TUS, microbubbles, and gemcitabine on pancreatic cancer cells, focusing on cell viability, apoptosis, and autophagy. The results demonstrated that, compared with the control (0 μg), treatment with 2 μg of gemcitabine significantly reduced cell viability, increased apoptosis, and decreased autophagy. The addition of microbubbles further enhanced these effects, with a significant benefit observed at 10 μL, but no additional effect at 50 μL. Ultrasound exposure exhibited a dose-dependent relationship, with higher intensities and lower DCs (e.g., 0.86 MPa at 10% DC) leading to greater reductions in cell viability and increased apoptosis, while effects on autophagy were variable. The significant two-way and three-way interactions among gemcitabine treatment, microbubble volume, and ultrasound parameters highlight a synergistic interplay, offering a promising avenue for addressing the notorious therapeutic resistance of pancreatic cancer.
The observed synergy likely arises from the mechanical interplay between TUS and microbubbles, which enhances the cytotoxic potential of gemcitabine. In this in vitro study, the absence of the desmoplastic stroma—characteristic of pancreatic tumors—may amplify the mechanical effects of TUS and microbubbles, as cells are directly exposed to ultrasound-induced cavitation and microstreaming. As a result, the reductions in cell viability and increases in apoptosis observed here may be primarily attributable to these mechanical effects. Thus, in this simplified model, the therapeutic effects may stem more from mechanical damage than from enhanced drug delivery. Nevertheless, in vivo studies suggest that TUS and microbubbles can improve drug delivery by overcoming stromal barriers, which were absent in the present model.
The concentration of gemcitabine used in this study was chosen to induce a moderate level of cytotoxicity, allowing the effects of additional treatments such as ultrasound and microbubbles to be clearly observed without being masked by excessive drug-induced cell death. Preliminary dose-finding experiments revealed that 1 μg exhibited minimal cytotoxic effects, while 3 μg resulted in more than 50% cell death, potentially saturating the system and obscuring the therapeutic benefits of the adjunctive treatments. Therefore, an intermediate concentration of 2 μg was selected to establish optimal conditions for evaluating the synergistic effects of combination therapy.
In addition to the microbubble volumes added to the cell suspension (0, 10, or 50 μL), a higher volume of 100 μL was initially tested but excluded because it led to predominant necrotic cell death across all treatment conditions, obscuring the specific effects of ultrasound and gemcitabine. The selected volumes (10 and 50 μL) still induced significant mechanical effects through TUS and microbubble interactions, which likely contributed substantially to the observed therapeutic outcomes.
Although the present results generally demonstrated a dose-dependent relationship in the in vitro setting, higher treatment parameters may not necessarily translate into better clinical outcomes. Safety considerations are paramount, as excessive ultrasound intensity or microbubble dosage can increase the risk of microvascular damage and inflammation, potentially leading to collateral tissue injury [12,13]. The absence of a significant difference between 10 μL and 50 μL microbubbles suggests a therapeutic plateau, indicating that lower doses may minimize the risk of microbubble- or ultrasound-related tissue damage. Optimizing ultrasound parameters, such as balancing intensity and DC, could further reduce collateral effects and help ensure that synergistic effects enhance efficacy without compromising safety. For example, using lower DCs with higher intensities (e.g., 0.86 MPa at 10% DC) may maximize cell death without causing thermal injury, which is a critical balance for in vivo applications. These insights could inform preclinical dosing studies to establish a safe therapeutic window. In a clinical case study using combined ultrasound, microbubbles, and gemcitabine, the authors reported an increased number of treatment cycles and reduced tumor size in some patients with pancreatic cancer, without major adverse effects [14]. Similarly, a recent proof-of-concept study showed that combining TUS with chemotherapy was safe and effective, resulting in significant tumor reduction and prolonged survival in patients with unresectable pancreatic cancer. Notably, that study found that the intermediate-intensity group exhibited better outcomes than both the low- and high-intensity groups, highlighting the importance of parameter optimization for clinical translation [15].
Additionally, the significant three-way interactions revealed by ANOVA provide a statistical foundation for optimizing this therapy. In this study, combinations such as 2 μg of gemcitabine, 10 μL of microbubbles, and 0.86 MPa at 10% DC yielded robust reductions in cell viability, increases in apoptosis, and decreased autophagy, suggesting a potential optimal parameter set for efficacy. This data-driven approach could ultimately enable standardized treatment protocols; however, to improve generalizability, these findings should be extended to additional cell lines [16]. More importantly, further in vivo studies are needed to strengthen translational value and refine these combinations for clinical application.
Autophagy plays a dual role in carcinogenesis. It can suppress early tumorigenesis by reducing reactive oxygen species, DNA damage, and inflammation, while also promoting advanced tumor progression by supporting cancer cell survival, migration, invasion, and cancer stem cell pluripotency under hypoxic and nutrient-deprived conditions [17,18]. The autophagic response observed in this study highlights the complex, context-dependent role of autophagy in cancer therapy. The suppressive effect of gemcitabine on autophagy likely redirected cellular fate toward apoptosis, preventing cancer cells from utilizing autophagy as a survival mechanism under chemotherapeutic stress [11]. This mechanism potentially explains the crosstalk between autophagy and apoptosis in therapeutic contexts. However, ultrasound-mediated autophagy exhibits context-dependent effects, ranging from cytoprotective responses that diminish treatment efficacy to enhanced drug sensitivity via endoplasmic reticulum stress pathways [19,20]. As the present study did not demonstrate a clear dose-dependent relationship between microbubble volume or TUS intensity and autophagy, it appears that, under certain conditions, ultrasound or microbubble exposure may induce autophagy as a protective response to mechanical stress, potentially opposing the intended degree of cell death. Notably, the combination of gemcitabine with 10 μL of microbubbles, together with increasing ultrasound intensity, progressively reduced autophagy, suggesting a potential optimal point at which mechanical disruption does not provoke survival pathways. This dual nature underscores the need for precise calibration of treatment parameters to favor apoptosis over autophagy-mediated resistance. Future investigations could explore the use of autophagy inhibitors, such as chloroquine analogs [21], to increase therapeutic efficacy, although the incorporation of these inhibitors warrants careful evaluation of tumor-specific autophagic dynamics.
The in vitro nature of this study limits its ability to reflect in vivo complexity, including stromal-immune interactions and vascular dynamics. The use of a single-cell line (CFPAC-1) restricts generalizability across the heterogeneity of pancreatic cancer, and short-term assessments may not capture long-term outcomes such as resistance or recurrence. Future research should incorporate multiple cell lines, patient-derived models, and extended timelines to address these gaps and ensure robust translational potential.
The clinical potential of this combination therapy is compelling, especially given the poor prognosis and resistance of pancreatic cancer to conventional treatments. Early clinical evidence supports this approach; notably, a 2016 phase I trial demonstrated that gemcitabine treatment combined with ultrasound and microbubbles was safe and nearly doubled the median survival, from 8.9 to 17.6 months, in patients with inoperable pancreatic cancer [22].
While the present in vitro findings demonstrate clear mechanistic effects, translating these results into clinical applications presents substantial challenges due to the limitations of conventional cell culture models, which do not replicate the complex tumor microenvironment of pancreatic cancer. The dense desmoplastic stroma characteristic of pancreatic ductal adenocarcinoma—a feature absent in vitro—serves as a major barrier to effective drug delivery. TUS combined with microbubbles may help overcome this barrier by mechanically disrupting the stromal structure and enhancing vascular permeability, potentially allowing deeper penetration of gemcitabine into tumor regions [23,24]. Furthermore, the hypovascular and hypoxic nature of pancreatic tumors impairs both microbubble distribution and drug accessibility—factors not reflected in standard cell culture systems. These challenges highlight the gap between in vitro efficacy and in vivo feasibility and underscore the need for more advanced preclinical models such as patient-derived xenografts, which better mimic the stromal barriers and microenvironmental complexity of pancreatic cancer.
In summary, this study demonstrated that TUS, microbubbles, and gemcitabine synergistically reduce pancreatic cancer cell viability by promoting apoptosis and, under optimized conditions, suppressing autophagy. These findings, supported by prior clinical evidence, provide a strong rationale for advancing this multifaceted strategy through translational research. Continued efforts to address mechanistic, clinical, and safety challenges are essential for developing innovative therapies for this devastating disease.
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