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.

Sononeoperfusion effect by ultrasound and microbubble promotes nitric oxide release to alleviate hypoxia in a mouse MC38 tumor model.

Tumor hypoperfusion not only impedes therapeutic drug delivery and accumulation, but also leads to a hypoxic and acidic tumor microenvironment, resulting in tumor proliferation, invasion, and therapeutic resistance. Sononeoperfusion effect refers to tumor perfusion enhancement using ultrasound and microbubbles. This study aimed to further investigate hypoxia alleviation by sononeoperfusion effect and explore the characteristics and mechanism of sononeoperfusion effect. To stimulate the sononeoperfusion effect, mice bearing MC38 colon cancers were included in this study and diagnostic ultrasound for therapy was set at a mechanical index (MI) of 0.1, 0.3, and 0.5, frequency of 3 MHz, pulse length of 5 cycles, and pulse repetition frequency of 2000 Hz. The results demonstrated that a single ultrasound and microbubble (USMB) treatment resulted in tumor perfusion enhancement at MI = 0.3, and nitric oxide (NO) concentration increased at MI = 0.3/0.5 (P < 0.05). However, there were no significant difference in the hypoxia-inducible factor-1α (HIF-1α) or D-lactate (D-LA) (P > 0.05) levels. Multiple sononeoperfusion effects were observed at MI = 0.3/0.5 (P < 0.05). For each treatment, USMB slightly but steadily improved the tumor tissue oxygen partial pressure (pO2) during and post treatment. It alleviated tumor hypoxia by decreasing HIF-1α, D-LA level and the hypoxic immunofluorescence intensity at MI = 0.3/0.5 (P < 0.05). The sononeoperfusion effect was not stimulated after eNOS inhibition. In conclusion, USMB with appropriate MI could lead to a sononeoperfusion effect via NO release, resulting in hypoxia amelioration. The tumors were not resistant to multiple sononeoperfusion effects. Repeated sononeoperfusion is a promising approach for relieving tumor hypoxia and resistance to therapy.