Therapeutic Effects of Microbubbles Added to Combined High-Intensity Focused Ultrasound and Chemotherapy in a Pancreatic Cancer Xenograft Model

Abstract

Objective: To investigate whether high-intensity focused ultrasound (HIFU) combined with microbubbles enhances the therapeutic effects of chemotherapy.

Materials and methods: A pancreatic cancer xenograft model was established using BALB/c nude mice and luciferase-expressing human pancreatic cancer cells. Mice were randomly assigned to five groups according to treatment: control (n = 10), gemcitabine alone (GEM; n = 12), HIFU with microbubbles (HIFU + MB, n = 11), combined HIFU and gemcitabine (HIGEM; n = 12), and HIGEM + MB (n = 13). After three weekly treatments, apoptosis rates were evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay in two mice per group. Tumor volume and bioluminescence were monitored using high-resolution 3D ultrasound imaging and in vivo bioluminescence imaging for eight weeks in the remaining mice.

Results: The HIGEM + MB group showed significantly higher apoptosis rates than the other groups (p < 0.05) and exhibited the slowest tumor growth. From week 5, the tumor-volume-ratio relative to the baseline tumor volume was significantly lower in the HIGEM + MB group than in the control, GEM, and HIFU + MB groups (p < 0.05). Despite visible distinction, the HIGEM and HIGEM + MB groups showed no significant differences.

Conclusion: High-intensity focused ultrasound combined with microbubbles enhances the therapeutic effects of gemcitabine chemotherapy in a pancreatic cancer xenograft model.

Keywords: Animal study; Gemcitabine; High-intensity focused ultrasound; Microbubbles; Pancreatic cancer; Sonoporation.

Fig. 1. Pre-clinical high-intensity focused ultrasound (HIFU) system.

A. HIFU treatment was performed in tank filled with degassed water maintained at temperature of 36.0℃, with tumor-bearing mouse set in animal holder. Microbubbles were injected via tail vein catheter (arrows) before HIFU treatment. B. Target tumor was positioned at center of therapeutic transducer's focal zone according to ultrasound guidance. C. For precise targeting, HIFU system is equipped with three-dimensional target position control (x-, y-, and z-axis). Pulsed HIFU beams are insonated into tumor and cover entire tumor with 2 mm spacing between sonication spots.

Fig. 2. Flow chart of study design.

IVIS = in vivo bioluminescence imaging, Vevo = high-resolution three-dimensional ultrasound for tumor volume measurement

Fig. 3. Tumor volume measurement using high-resolution three-dimensional (3D) ultrasound.

Tumors were scanned using 3D mode under respiratory gating. Tumors in captured 3D images were manually contoured along tumor margin. Then, tumor volume was automatically calculated using parallel segmentation in Vevo2100 software.

Fig. 4. TUNEL assay results and apoptosis rates according to treatment group.

A-E. Apoptotic cells were quantified using TUNEL assay. TUNEL-positive, brown-stained apoptotic cells were visualized under high-power field (× 200 magnification) in each treatment group: control (A), GEM (B), HIFU + MB (C), HIGEM (D), and HIGEM + MB (E). F. Box and whisker plots of tumor apoptosis in each group. There were significant differences in tumor apoptosis between HIGEM + MB group and control, GEM, HIFU + MB, and HIGEM groups (p < 0.05). ^*Significant difference (p < 0.05). Control = no treatment, GEM = gemcitabine treatment alone, HIFU = high-intensity focused ultrasound, HIFU + MB = HIFU with microbubbles treatment, HIGEM = combined HIFU and gemcitabine treatment, HIGEM + MB = combined HIFU and gemcitabine with microbubbles treatment, TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling

Fig. 5. Tumor volume ratio according to treatment group.

Line graphs demonstrating serial change in tumor-volume-ratio (i.e., tumor volume on specific day divided by baseline tumor volume) in each treatment group. Black arrows indicate treatment days. HIGEM + MB group shows slowest growth rate and most delayed growth spurt among study groups. Significant differences were observed starting in 5th week between HIGEM + MB group and control, GEM, and HIFU + MB groups, respectively. ^*Significant difference (p < 0.05). Control = no treatment, GEM = gemcitabine treatment alone, HIFU = high-intensity focused ultrasound, HIFU + MB = HIFU with microbubbles treatment, HIGEM = combined HIFU and gemcitabine treatment, HIGEM + MB = combined HIFU and gemcitabine with microbubbles treatment

Fig. 6. In vivo bioluminescence imaging in treatment groups.

A. Bioluminescence imaging of representative mouse from each treatment group. HIGEM + MB group shows much lower total photonic flux during 8th week. B. Black arrows indicate treatment days. Despite no significant difference between treatment groups, HIGEM + MB group shows lower total photonic flux than other groups. Control = no treatment, GEM = gemcitabine treatment alone, HIFU = high-intensity focused ultrasound, HIFU + MB = HIFU with microbubbles treatment, HIGEM = combined HIFU and gemcitabine treatment, HIGEM + MB = combined HIFU and gemcitabine with microbubbles treatment