Augmentation of tumour perfusion by ultrasound and microbubbles: A preclinical study

Background: Hypoperfusion and the resulting hypoxia in solid tumours are critical causes of treatment resistance. Ultrasound-stimulated microbubbles (USMB) enhance tumour perfusion in a mechanism named the "sononeoperfusion" effect, which may relieve tumour hypoperfusion and hypoxia. The aim of this study was to determine the optimal mechanical index (MI) and therapeutic ultrasound exposure time for the sononeoperfusion effect and preliminarily explore the mechanism of sononeoperfusion and its effect on tumours.

Methods: A total of 155 mice bearing MC38 tumours were included in this study. A modified diagnostic ultrasound and microbubbles (Zhifuxian) was used for USMB treatment. Tumour perfusion was evaluated by contrast-enhanced ultrasound (CEUS) and Hoechst 33342. The therapeutic pulse was operated with MIs of 0.1 to 0.5. The ultrasound exposure time was set from 150 s to 600 s. Endothelial nitric oxide synthase (eNOS) inhibition and NO, ATP, and phospho-eNOS (p-eNOS) detection were performed to explore the mechanisms of sononeoperfusion. Hypoxia-inducible factor-1α (HIF-1α) and tumour oxygen partial pressure (pO₂) represent hypoxic tumour conditions.

Results: Tumour perfusion was increased after USMB treatment at MIs of 0.1-0.4 and ultrasound exposure times of 150 s to 600 s, with optimal augmentation achieved at an MI of 0.3 and ultrasound exposure time of 450 s. The mean fluorescence intensity of Hoechst 33342 after USMB treatment was stronger than that of the control group. Biochemical assays showed a significant increase in ATP, p-eNOS and NO after USMB treatment. PO₂ in tumour tissue increased significantly after USMB treatment and was maintained for more than 20 min.

Conclusions: The best sononeoperfusion effect was obtained with an MI of 0.3 and an ultrasound exposure time of 450 s. The effect is most likely related to NO and ATP increases. The sononeoperfusion effect might be a novel way to ameliorate tumour hypoperfusion and hypoxia.