Inhibition of glioma growth by microbubble activation in a subcutaneous model using low duty cycle ultrasound without significant heating

Laboratory investigation

Restricted access

Object

In this study, the authors sought determine whether microbubble (MB) destruction with pulsed low duty cycle ultrasound can be used to reduce brain tumor perfusion and growth through nonthermal microvascular ablation.

Methods

Studies using C57BLJ6/Rag-1 mice inoculated subcutaneously with C6 glioma cells were approved by the institutional animal care and use committee. Microbubbles were injected intravenously, and 1 MHz ultrasound was applied with varying duty cycles to the tumor every 5 seconds for 60 minutes. During treatment, tumor heating was quantified. Following treatment, tumor growth, hemodynamics, necrosis, and apoptosis were measured.

Results

Tumor blood flow was significantly reduced immediately after treatment, with posttreatment flow ranging from 36% (0.00002 duty cycle) to 4% (0.01 duty cycle) of pretreatment flow. Seven days after treatment, tumor necrosis and apoptosis were significantly increased in all treatment groups, while treatment with ultrasound duty cycles of 0.005 and 0.01 inhibited tumor growth by 63% and 75%, respectively, compared with untreated tumors. While a modest duty cycle–dependent increase in intratumor temperature was observed, it is unlikely that thermal tissue ablation occurred.

Conclusions

In a subcutaneous C6 glioma model, MB destruction with low–duty cycle 1-MHz ultrasound can be used to markedly inhibit growth, without substantial tumor tissue heating. These results may have a bearing on the development of transcranial high-intensity focused ultrasound treatments for brain tumors that are not amenable to thermal ablation.

Abbreviations used in this paper: CPS = contrast pulse sequencing; HIFU = high-intensity focused ultrasound; MB = microbubble; TUNEL = terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling.

Article Information

Address correspondence to: Richard J. Price, Ph.D., Department of Biomedical Engineering, University of Virginia Health System, Box 800759, Charlottesville, Virginia 22908. email: rprice@virginia.edu.

Please include this information when citing this paper: published online January 7, 2011; DOI: 10.3171/2010.11.JNS101201.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Schematic illustration of the mouse tumor model with ultrasound exposure.

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    Schematic illustration of the ultrasound pulse sequences. Note that, apart from the number of bursts per pulse and the number of sinusoids per burst, other acoustic variables were kept constant. The asterisks indicate 50 msec between consecutive bursts.

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    Microbubble insonation reduces tumor blood velocity, perfused territory, and blood flow. Bar graphs of perfused area (A), blood velocity (β) (B), and tumor blood flow (C) before and after treatment for duty cycles of 0.00002 (8 animals), 0.0001 (9 animals), 0.005 (8 animals), and 0.01 (8 animals). *Significantly different from pretreatment (p < 0.05).

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    Microbubble insonation increases tumor necrosis and apoptosis. A and B: Photomicrographs obtained in control tumors (A) and in tumors harvested 7 days after treatment with MBs and the 0.005 duty cycle pulsing protocol (B). C: Bar graph showing the percentage of necrotic tumor area for control tumors (6 mice) and tumors treated with ultrasound duty cycles of 0.00002 (5 mice), 0.0001 (5 mice), 0.005 (8 mice) and 0.01 (8 mice). D and E: Photomicrographs from control tumors (D) and tumors harvested 7 days after treatment with an ultrasound duty cycle of 0.005 (E). Arrows denote apoptotic cells (brown). F: Bar graph of apoptotic cells per 50× field of view (F.O.V.) for control tumors (6 mice) and tumors treated with duty cycles of 0.00002 (5 mice), 0.0001 (6 mice), 0.005 (8 mice), and 0.01 (8 mice). *Significantly different from control (p < 0.05). H & E (A and B); TUNEL (D and E). N = necrotic tissue; V = viable tissue.

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    Microbubble insonation inhibits tumor growth. Line graph depicting tumor growth (fold-change over Day 1) as a function of time posttreatment for untreated control tumors (16 mice) and tumors treated with duty cycles of 0.0001 (5 mice), 0.005 (8 mice), and 0.01 (8 mice). *Significantly different from the untreated control group and the 0.0001 duty cycle group at the same time point (p < 0.05). **Significantly different from untreated the control group at the same time point (p < 0.05).

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    A: Tumor temperature increases moderately as duty cycle increases. Line graph depicting changes in temperature increasing above ambient as a function of time during 60-minute treatments for ultrasound duty cycles of 0.0001 (4 mice), 0.005 (7 mice), and 0.01 (4 mice). *Significantly different from all other groups at the same time point (p < 0.05). B and C: Photomicrographs of the skin overlying ultrasound-treated (B) and untreated (C) tumors. The skin appears unchanged histologically, illustrating that no apparent thermal damage occurred in this tissue layer. H & E.

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