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Introduction
Pancreatic cancer is the seventh leading cause of cancer-related death worldwide and the 12th most commonly diagnosed cancer, with more than 510,000 new cases reported globally in 2022 [1]. The 5-year survival rates for patients with pancreatic cancer in the United States and United Kingdom are 13.3% and 7.2%, respectively [2,3]. Symptoms are usually absent in the early stages, and symptoms specific enough to raise suspicion for pancreatic cancer generally do not appear until the disease is advanced [4,5]. Surgical resection is the most effective treatment for pancreatic cancer. However, fewer than 20% of patients are candidates for surgical resection [6], and radiotherapy or chemotherapy alone has limited therapeutic efficacy [7]. Recently, combination regimens such as FOLFIRINOX and albumin-bound paclitaxel/gemcitabine (Gem) have been used in clinical practice [8–10], and immunotherapies based on immune checkpoint inhibitors (ICIs) [11–13] and chimeric antigen receptor T cells have also been investigated [14–16]. Anti-tumor immunotherapy using ICIs has shown greater therapeutic effects than chemotherapy in in vivo studies, but clinical trials have demonstrated low response rates of less than 35%, even when ICIs are combined with other treatments [12,13,17,18]. This response rate appears to be influenced by the tumor microenvironment, which varies across tumors [19–21].
Focused ultrasound (FUS) can induce various bioeffects, and its application has been actively investigated as an alternative treatment modality for diseases such as cancer. The bioeffects induced by FUS include ablative effects, such as thermal ablation (TA) or histotripsy, which involve thermal or non-thermal tissue destruction, and non-ablative effects, such as mechanical stimulation (MS) or hyperthermia, which induce cellular responses through physical stimulation or temperature elevation without tissue ablation [22,23]. More recently, immune modulation has also been recognized as one of the biological effects of FUS [24–26].
In previous work, the authors’ research team showed that repeated MS treatment with FUS enhanced anti-tumor efficacy when combined with anticancer drugs, and that, when total acoustic energy was held constant, sonication with a relatively higher mechanical index was more effective in suppressing tumor growth [27]. However, despite the potential for immune responses, that study did not evaluate the anti-tumor immune responses induced by non-ablative MS treatment. Therefore, the present study was designed to investigate whether repeated MS treatment with FUS alters immune responses in a syngeneic pancreatic cancer model.
Materials and Methods
Compliance with Ethical Standards
All experiments were performed according to procedures approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital (IACUC No. 21-0020).
Study Design
The anti-tumor immune response to FUS treatment was investigated by implanting tumors in one flank and treating the implanted tumor (study 1), whereas the systemic immune effects of FUS treatment were assessed by inoculating tumors in both flanks and treating the tumor on only one side (study 2).
In study 1, animals were randomly divided into five groups: a control group without treatment (control); a group that received a single administration of an anticancer drug without FUS treatment (Gem); a group that received weekly administration of an anticancer drug three times without FUS treatment (Gem×3); a group that received a single TA treatment in combination with anticancer drug during the first week, followed by weekly administration of an anticancer drug alone for the subsequent 2 weeks (TA+Gem); and a group that received weekly combination therapy with MS and anticancer drug for 3 weeks (MS+Gem) (Fig. 1A). A total of 20–23 animals were assigned to each group. Tumor growth was monitored in five animals per group, whereas 15 animals per group were used for weekly collection of tumor, spleen, and blood samples, with three animals sacrificed each week for sample collection. In addition, three animals per group were sacrificed after the first treatment for histological and gene expression analyses. Because the Gem and Gem×3 groups underwent identical conditions in the first week—receiving a single dose of an anticancer drug without FUS treatment—data from the Gem×3 group were not included in the histological or gene expression analyses at week 0. In study 2, the group that received a single administration of an anticancer drug without FUS treatment (Gem) was excluded, and animals were randomly assigned to the remaining four groups (n=20 per group: 5 for tumor growth monitoring and 15 for sample analysis). Data collection and outcome assessment were conducted by investigators blinded to group allocation (Fig. 1B).
All groups were monitored for 4 weeks after treatment initiation. The monitoring period was limited to remain within the tumor size threshold approved by the IACUC, with a maximum allowable tumor volume of 1 cm³. Based on the results of the authors’ previous study [27], the experimental design for study 1 included five groups. In addition, the TA+Gem group was included to compare its effects with those of the MS+Gem group, based on previous studies suggesting that TA, the conventional form of FUS therapy, has immunomodulatory effects [28]. For anticancer drug treatment, Gem (Gemzar, Eli Lilly Co., Indianapolis, IN, USA), diluted in 0.9% saline, was administered intraperitoneally at a dose of 200 mg/kg.
Animal Model
Murine pancreatic cancer Pan02 cells (Division of Cancer Treatment and Diagnosis, Bethesda, MD, USA) were used to establish a syngeneic model in male C57BL/6 mice (20–25 g; OrientBio, Seoul, Korea). For tumor inoculation, a 0.2-mL cell suspension at a concentration of 1×107 cells/mL was injected subcutaneously into the right flank (study 1) or both flanks (study 2) of each mouse. To control for procedure-related stress, mice in all groups, including the control, Gem, and Gem×3 groups, underwent the same anesthesia and handling procedures on the same schedule as the FUS-treated groups, with sham positioning and preparation matching the FUS sessions. All mice underwent general anesthesia during treatment, induced by intraperitoneal injection of zolazepam hydrochloride (30 mg/kg; Zoletil, Virbac, Seoul, Korea) and xylazine hydrochloride (10 mg/kg; 2% Rompun, Bayer Korea, Seoul, Korea). At the end of the experiment, all animals were euthanized by carbon dioxide (CO₂) inhalation.
FUS Treatment
All FUS treatments were performed using a preclinical FUS system (VIFU 2000, Alpinion Medical Systems, Seoul, Korea). The system consists of three transducer compartments: one for therapeutic treatment, one for image guidance, and one for tumor size monitoring. The therapeutic transducer is composed of a single spherical piezoelectric element with a resonance frequency of 1.1 MHz. A 40-mm-diameter circular opening is located at the center to accommodate the imaging transducer. A linear-array transducer (L8-17, 8-17 MHz) is positioned at the center of the therapeutic transducer to assist with image guidance. In addition, the system includes a phased-array transducer (S12-4, 4–12 MHz) and an imaging module system (E-CUBE9) for tumor volume monitoring. According to the treatment plan programmed in the system, sonication targets were spaced 2 mm apart, and each target was sonicated for 20 seconds. In the TA group, sonication targets were planned to cover approximately 70% of the tumor volume, whereas in the MS group, treatment was applied to the entire tumor volume. In the TA group, coverage exceeding 70% was avoided because of procedure-related mortality caused by excessive systemic hyperthermia. Details of the FUS conditions for TA and MS are listed in Table 1.
Measurement and Analysis
Ultrasound images of tumors were acquired using the VIFU 2000, and tumor volume was calculated by measuring tumor length along three axes on the images. Tumor size in each group (n=5 per group) was reported as mean volume±standard error.
Twenty-four hours after each treatment and during follow-up monitoring, tumors, spleens, and blood samples (n=3 per collection time point per group) were collected. In study 1, additional tumor samples (n=3 per group) were collected at week 0 for histological and gene expression analyses.
H&E, gene, and cytokine analysis
Hematoxylin and eosin (H&E) staining was performed to assess whether histological changes occurred in tumors under each treatment condition. Expression of apoptosis-related genes in tumors was analyzed using real-time polymerase chain reaction. RNA was extracted from bulk tumor tissue; therefore, the measured gene expression profiles reflect contributions from the cellular composition of the tumor microenvironment. γ-H2AX, PLK1, CHEK2, p53, Bax, Bcl-2, caspase-3, and Ki-67 were used as mRNA expression markers. In blood samples collected by cardiac puncture, interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and KC-GRO (IL-8) were measured by enzyme-linked immunosorbent assay. To assess baseline levels for cytokine responses and splenic T-cell analysis, wild-type mice (n=3) were included as an additional control group.
T-cell analysis
Helper T cells (CD4+), cytotoxic T cells (CD8+), and regulatory T cells (Tregs) in splenocytes, as well as CD8+ cells among tumor-infiltrating lymphocytes (TIL), were analyzed in collected spleens (study 1) and tumors (studies 1 and 2) by flow cytometry using the BD FACSymphony A3 (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo software version 10.1 (FlowJo LLC, Ashland, OR, USA). For these analyses, spleens and tumors were harvested and processed into whole single-cell suspensions. Total viable cell numbers were first determined for each sample, and then a fixed number of 1×105 cells was used for staining and acquisition in all experimental groups. Because the primary objective of this study was to compare treatment-induced changes in relative T-cell composition across groups, the immunophenotyping data are presented as frequencies within the lymphocyte or CD45+ compartments rather than as absolute counts per gram of tissue. In addition, tumor samples from all experimental groups were processed for immunofluorescence (IF) staining to analyze CD8+ infiltration. CD8+ cells, secretion of the lytic enzyme granzyme B (GrB), and infiltration of Foxp3+ regulatory T cells were imaged and quantified from the IF-stained sections. Using IF images of tumor samples collected 24 hours after treatment or during follow-up monitoring at week 0, week 2, and week 4, the distribution of CD8+ T cells, GrB secretion, and the proportion of Foxp3+ cells within tumors were determined and quantified. The analyzed samples and assay components for studies 1 and 2 are summarized in Table 2, and additional details for each analysis are provided in the Supplementary Text 1.
Experimental data were analyzed using SPSS version 26 statistical software (IBM Corp., Armonk, NY, USA). All data were analyzed using one-way analysis of variance, and statistical significance was defined as P<0.05.
Results
Anti-tumor Effect of Non-ablative Repeated MS with FUS (Study 1)
Changes in tumor volume
Changes in tumor volume (Fig. 2A) demonstrated a statistically significant suppression of tumor growth in the MS+Gem group compared with the other groups (P<0.001 vs. control, Gem, and TA+Gem, and P=0.003 vs. Gem×3 at week 4).
H&E, gene, and cytokine analysis
At week 0, H&E staining results (Fig. 2B) showed substantial cellular damage in tissues exposed to heat concentration in the TA+Gem group, whereas no apparent tissue damage was observed in the other treatment groups. A significant increase in expression of the DNA damage marker γ-H2AX was observed in the treatment groups compared with the control group (Fig. 2C). In addition, the MS+Gem group exhibited significantly higher p53 expression than the control group (P=0.042), and CHEK2 expression was also significantly higher in the MS+Gem group than in the other groups (P=0.002 vs. control, P=0.032 vs. Gem, and P=0.048 vs. TA+Gem). In contrast, expression of PLK1, a marker associated with tumor growth regulation, was significantly lower only in the MS+Gem group than in the control group (P=0.046). Expression of the tumor growth marker Ki-67 was significantly lower in all treatment groups than in the control group. In addition, expression of the pro-apoptotic genes Bax and caspase-3 in tumor samples was significantly higher in the MS+Gem group (Bax: P=0.001 vs. control, P=0.012 vs. Gem, and P=0.004 vs. TA+Gem; caspase-3: P<0.001 vs. control, P=0.016 vs. Gem, and P=0.054 vs. TA+Gem), whereas expression of the anti-apoptotic gene Bcl-2 was significantly lower in the MS+Gem group (P=0.002 vs. control, P=0.032 vs. Gem, and P=0.001 vs. TA+Gem). Because RNA was quantified from bulk tumor tissue, these changes may reflect shifts in tumor microenvironment cellular composition and therefore cannot be attributed solely to tumor cell-intrinsic regulation.
Weekly cytokine analysis of blood samples (Fig. 2D) showed significant changes in circulating cytokines in the MS+Gem group, suggesting systemic immune modulation. Among the cytokines analyzed, IL-6, a pro-inflammatory cytokine commonly associated with immune activation and inflammation, showed a marked increase (P<0.001 vs. control, Gem, Gem×3, and TA+Gem at week 0), highlighting a strong inflammatory response. The chemokine KC-GRO, which is involved in neutrophil recruitment and plays a key role in inflammation, was also significantly higher than in the other groups. Similarly, TNF-α, a central cytokine in systemic inflammation and immune regulation, was significantly elevated compared with the other groups. An increase in IL-10, an anti-inflammatory cytokine involved in suppressing excessive immune responses and maintaining immune homeostasis, was also observed. IL-10 levels were significantly higher in the TA+Gem group after the first treatment than in the other experimental groups; however, a gradual decrease in IL-10 levels was observed as treatment progressed.
T-cell analysis
Analysis of immune cells in splenocytes showed that, compared with the other groups, the MS+Gem group exhibited increased proportions of CD4+ and CD8+ T cells and a trend toward decreased Foxp3+ T cells (Fig. 3A). IF staining of tumor samples collected at week 2 showed a higher proportion of CD8+ cells and GrB in the MS+Gem group than in the control group, whereas the proportion of Foxp3+ cells was significantly lower (Fig. 3B). The distribution of CD8+ T cells and GrB, along with the proportion of Foxp3+ cells, was quantified from IF images of tumor samples (Fig. 3D). At week 0, both the TA+Gem and MS+Gem groups exhibited similar effects, including increased CD8+ cells (P<0.001) and decreased Foxp3+ cells compared with the control group (P<0.001), whereas GrB did not differ significantly among treatment groups relative to the control group. Differences between the TA+Gem and MS+Gem groups became apparent by week 2. The TA+Gem group exhibited a gradual decrease in CD8+ and GrB expression and an increase in Foxp3+ as the tumor grew, showing a temporal pattern similar to that of the control group. In the Gem group, the proportions of CD8+ cells and GrB increased during treatment (week 0 and week 2), whereas a marked reduction was observed at week 4, suggesting an inability to sustain the treatment effect.
In contrast, the MS+Gem group showed sustained proportions of CD8+ cells and GrB at week 4, similar to those observed at week 2, compared with the other treatment groups (CD8+: P<0.001 vs. Gem×3 and P=0.005 vs. TA+Gem; GrB: P<0.001 vs. Gem×3 and vs. TA+Gem) and the control group (P<0.001). Foxp3+ cells increased slightly relative to the first treatment, but their proportion remained comparatively low and was significantly lower than in the other groups (P<0.001 vs. control, P=0.034 vs. Gem×3, and P<0.001 vs. TA+Gem at week 4). The potent anti-tumor effect of MS+Gem associated with intratumoral infiltration of cytotoxic T cells was further analyzed by flow cytometry (Fig. 3C). The MS+Gem group exhibited a significantly higher proportion of CD8+ cells within TIL than the other groups (P=0.004 vs. control, P=0.002 vs. Gem×3, and P=0.003 vs. TA+Gem at week 2), and this elevated level was maintained even after treatment completion (P=0.039 vs. control at week 4).
Abscopal Anti-tumor Effect of FUS Treatment (Study 2)
Changes in tumor volume and immune cell infiltration in the distant tumor that did not receive direct treatment are presented in Fig. 4. The MS+Gem group showed significant suppression of tumor growth in both the distant tumor and the treated tumor (Fig. 4A). Fig. 4B shows IF images of the distant tumor collected 24 hours after the last treatment (week 2). At week 0, immediately after the first treatment, there were no significant differences in CD8+ or Foxp3+ levels between the treatment groups and the control group (Fig. 4D). However, at week 2, high expression of CD8+ cells and GrB was observed in the MS+Gem group (CD8+: P=0.005 vs. control, P=0.014 vs. Gem×3, and P=0.041 vs. TA+Gem; GrB: P<0.001 vs. control, P=0.043 vs. Gem×3, and P=0.046 vs. TA+Gem), whereas Foxp3+ expression was lower in the MS+Gem group than in the control group (P<0.001) and the other treatment groups (P=0.016 vs. Gem×3 and P=0.001 vs. TA+Gem). Furthermore, results at week 4 showed that, in the Gem and TA+Gem groups, CD8+ and GrB expression decreased to levels similar to those of the control group as the tumors enlarged. In contrast, the MS+Gem group exhibited higher expression of CD8+ cells and GrB than the other treatment groups (CD8+: P<0.001 vs. Gem×3 and P=0.01 vs. TA+Gem; GrB: P<0.001 vs. Gem×3 and vs. TA+Gem), whereas Foxp3+ expression was markedly lower than in the other groups (P<0.001 vs. control, P=0.027 vs. Gem×3, and P=0.033 vs. TA+Gem).
Flow-cytometric analysis was performed to investigate whether induction of CD8+ T-cell responses within the distant tumor was associated with the tumor growth-inhibitory effect observed in a manner similar to that in the treated tumor (Fig. 4C). In the MS+Gem group, the proportion of CD8+ cells was relatively higher than that in the control group as treatment was repeated (P=0.007, P=0.004, and P=0.002 at weeks 0, 1, and 2, respectively). In addition, even at week 4, an increased proportion of CD8+ cells was observed in tumor tissue compared with the control group.
Discussion
This study demonstrated that combination therapy with non-ablative MS by FUS (MS+Gem) produced favorable therapeutic outcomes in a syngeneic pancreatic cancer model. Specifically, tumors in the MS+Gem group showed increased expression of tumor-suppressive and apoptosis-related transcripts. In addition, the MS+Gem group showed changes in circulating cytokines, suggesting systemic immune modulation. This treatment also promoted intratumoral infiltration of cytotoxic T cells and was associated with immune changes in the contralateral untreated tumor. One limitation of the present study is that an MS-only arm was not included. However, the Gem-only group showed minimal immune activation in the present assays, whereas MS+Gem consistently increased CD8+ infiltration and reduced Foxp3+ in both treated and contralateral tumors. Therefore, the immune modulation observed in this study is likely driven, at least in part, by the MS component.
Genes related to the γ-H2AX/ATM/CHEK2/p53 pathway were examined to evaluate treatment-associated changes in DNA damage responses and apoptotic signaling. The MS+Gem group showed increased p53 expression and higher Bax and caspase-3 levels than the other groups, suggesting enhanced pro-apoptotic signaling at the bulk tumor tissue level.
Cytokine analysis of mouse plasma revealed significant increases in IL-6, KC-GRO, and TNF-α in the MS+Gem group. These cytokine changes were interpreted as evidence of systemic immune modulation rather than a direct measure of adaptive immunity. In contrast, the TA+Gem group showed increased IL-10, an anti-inflammatory cytokine, which may reflect a distinct inflammatory environment after thermal treatment. The spleen plays an important role in both the circulatory and immune systems. Measurements of CD4+, CD8+, and Treg populations in splenocytes demonstrated treatment-associated immune changes after MS+Gem treatment. These findings support systemic immune modulation rather than a purely local effect.
Analysis of IF staining and flow cytometry in tumors showed that the MS+Gem group had a significant increase in the frequency of CD8+ cells and a decrease in Foxp3+ cells after the initial treatment. These patterns persisted during follow-up monitoring compared with the other groups. These findings suggest that sustained enrichment of cytotoxic T cells within the tumor microenvironment is associated with improved tumor suppression. In contrast, after single TA+Gem treatment, CD8+ cells and GrB gradually decreased after treatment at week 0, and the three-cycle Gem treatment showed a similar pattern, suggesting that immune activation was less sustained in these groups. An important limitation of this study is the absence of absolute immune cell counts per spleen or per gram of tumor tissue. Although total viable cell yields were measured during processing to standardize input for flow cytometry, these values were not archived for final reporting. Consequently, the flow-cytometric data should be interpreted as reflecting compositional shifts in immune subsets rather than changes in total immune cell burden. Future studies should prospectively record absolute yields or incorporate counting beads to provide a more comprehensive quantification of treatment-induced immune modulation.
Study 2 showed that MS+Gem treatment was associated with systemic immune effects accompanied by abscopal tumor control in untreated distant tumors. Repeated MS+Gem treatments resulted in increased CD8+ abundance and sustained low Foxp3+ levels in distant tumors. These findings suggest that local MS treatment can be associated with systemic anti-tumor immune effects. Based on prior studies, one possible explanation is that MS increases tumor antigen availability and promotes antigen presentation, leading to activation and trafficking of cytotoxic T cells.
Recent preclinical studies support the concept that ultrasound-based local tumor interventions can elicit immune-mediated control of distant untreated lesions in bilateral tumor models. FUS ablation using boiling histotripsy showed abscopal tumor control and demonstrated antigen-dependent dendritic cell activation, linking local mechanical destruction to systemic anti-tumor immunity [29]. In addition, cavitation bubble-assisted high-intensity FUS increased infiltration of CD8+ T cells and CD11c+ dendritic cells in both treated and contralateral untreated tumors, and CD8+ T-cell depletion abrogated both local and distant tumor control, supporting a CD8-dependent mechanism for the abscopal response [30]. These reports support the concept that mechanical or cavitation-associated ultrasound treatment can induce systemic anti-tumor immunity, which is consistent with the findings of study 2, showing sustained CD8+ enrichment and Foxp3+ suppression in contralateral tumors after repeated MS+Gem treatment.
Notably, the higher expression and prolonged presence of cytotoxic T cells after repeated MS+Gem treatments suggest a potential link to the development of memory-associated T-cell responses. Conversely, in the TA+Gem group, the low level of CD8+ cell infiltration accompanied by increased Tregs and IL-10 suggests that thermal treatment may predominantly trigger innate immune responses related to thermal tissue damage and inflammation in surrounding tissues. Further investigation is needed to verify this hypothesis and to explore the relationship between the therapeutic effects of MS+Gem treatment and memory T cells.
Taken together, these findings suggest that repeated non-ablative MS treatment combined with gemcitabine induces an abscopal therapeutic effect by inhibiting pancreatic tumor growth and promoting infiltration of cytotoxic T cells into both locally treated and untreated tumors through systemic immune modulation.
This study involved a relatively short monitoring period under IACUC constraints, which may limit conclusions regarding the long-term immune efficacy of MS+Gem treatment. Although the study suggests that MS+Gem therapy can induce a systemic immune response, the precise mechanisms underlying this response, particularly the contributions of memory T cells and specific cytokines, remain unclear. In addition, Ki-67 was assessed only at the bulk tumor level, and future studies should evaluate Ki-67 in TIL subsets by flow cytometry to better define treatment-associated T-cell proliferation. Another important limitation involves the tumor model used. Human pancreatic ductal adenocarcinoma is characterized by a dense desmoplastic microenvironment that acts as a physical barrier to drug delivery and immune cell infiltration. However, the subcutaneous syngeneic Pan02 model used in this study does not fully recapitulate this desmoplastic stroma. Consequently, the drug penetration and immune cell infiltration observed here may be more favorable than those seen in the clinical setting. Therefore, further research using clinically relevant models is needed to assess translational relevance.
In the clinical treatment of pancreatic cancer, maintaining an induced immune response may be as important as inducing a strong anti-tumor immune response to prevent recurrence. Given the potential clinical relevance of this concept, it is meaningful that the present study demonstrates induction of anti-tumor immune responses and sustained effects after repeated non-ablative MS treatment through experimental procedures and analyses. In addition, the MS treatment method used in this study has been validated for safety in a proof-of-concept clinical study [31]. Therefore, repeated non-ablative MS treatment by FUS may be rapidly translatable to clinical application and could influence pancreatic cancer treatment. Further studies are needed to explore the potential of MS-based combination therapy for sustaining and improving pancreatic cancer treatment.
Combination therapy using MS stimulated both local and systemic anti-tumor immune responses while also suppressing tumor growth. Because this approach is feasible for repeated treatment, it holds promise for enhancing therapeutic efficacy and may have clinical relevance.
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