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

Laboratory investigation

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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.


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.


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.


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:

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.



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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.


  • 1

    Ay TD'Hondt AMPasquet AMelin JAVanoverschelde JL: Heterogeneity of contrast effect during intermittent second harmonic myocardial contrast echocardiography in healthy patients. J Am Soc Echocardiogr 15:144814522002

  • 2

    Chapelon JYRibault MBirer AVernier FSouchon RGelet A: Treatment of localized prostate cancer with transrectal high intensity focused ultrasound. Eur J Ultrasound 9:31381999

  • 3

    Cheng SQZhou XDTang ZYYu YBao SSQian DC: Iodized oil enhances the thermal effect of high-intensity focused ultrasound on ablating experimental liver cancer. J Cancer Res Clin Oncol 123:6396441997

  • 4

    Chomas JEPollard RWisner EFerarra K: Subharmonic phase-inversion for tumor perfusion estimation. IEEE Ultrasonics Symposium 2:171317162001. ( [Accessed November 22 2010]

  • 5

    Clement GT: Perspectives in clinical uses of high-intensity focused ultrasound. Ultrasonics 42:108710932004

  • 6

    Clement GTHynynen K: Correlation of ultrasound phase with physical skull properties. Ultrasound Med Biol 28:6176242002

  • 7

    Cohen ZRZaubermann JHarnof SMardor YNass DZadicario E: Magnetic resonance imaging-guided focused ultrasound for thermal ablation in the brain: a feasibility study in a swine model. Neurosurgery 60:5936002007

  • 8

    Gelet AChapelon JYPoissonnier LBouvier RRouvière OCuriel L: Local recurrence of prostate cancer after external beam radiotherapy: early experience of salvage therapy using high-intensity focused ultrasonography. Urology 63:6256292004

  • 9

    Hanajiri KMaruyama TKaneko YMitsui HWatanabe SSata M: Microbubble-induced increase in ablation of liver tumors by high-intensity focused ultrasound. Hepatol Res 36:3083142006

  • 10

    Hu ZYang XYLiu YSankin GNPua ECMorse MA: Investigation of HIFU-induced anti-tumor immunity in a murine tumor model. J Transl Med 5:342007

  • 11

    Hynynen K: The threshold for thermally significant cavitation in dog's thigh muscle in vivo. Ultrasound Med Biol 17:1571691991

  • 12

    Hynynen KClement GTMcDannold NVykhodtseva NKing RWhite PJ: 500-element ultrasound phased array system for noninvasive focal surgery of the brain: a preliminary rabbit study with ex vivo human skulls. Magn Reson Med 52:1001072004

  • 13

    Hynynen KMcDannold NClement GTJolesz FAZadicario EKilliany R: Pre-clinical testing of a phased array ultrasound system for MRI-guided noninvasive surgery of the brain—a primate study. Eur J Radiol 59:1491562006

  • 14

    Hynynen KMcDannold NVykhodtseva NJolesz FA: Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits. Radiology 220:6406462001

  • 15

    Kaneko YMaruyama TTakegami KWatanabe TMitsui HHanajiri K: Use of a microbubble agent to increase the effects of high intensity focused ultrasound on liver tissue. Eur Radiol 15:141514202005

  • 16

    Kennedy JE: High-intensity focused ultrasound in the treatment of solid tumours. Nat Rev Cancer 5:3213272005

  • 17

    Klotz ARLindvere LStefanovic BHynynen K: Temperature change near microbubbles within a capillary network during focused ultrasound. Phys Med Biol 55:154915612010

  • 18

    McDannold NClement GTBlack PJolesz FHynynen K: Transcranial magnetic resonance imaging–guided focused ultrasound surgery of brain tumors: initial findings in 3 patients. Neurosurgery 66:3233322010

  • 19

    McDannold NVykhodtseva NJolesz FAHynynen K: MRI investigation of the threshold for thermally induced bloodbrain barrier disruption and brain tissue damage in the rabbit brain. Magn Reson Med 51:9139232004

  • 20

    McDannold NJVykhodtseva NIHynynen K: Microbubble contrast agent with focused ultrasound to create brain lesions at low power levels: MR imaging and histologic study in rabbits. Radiology 241:951062006

  • 21

    Meairs SHennerici MGFuture developments in neurovascular ultrasound. Baumgartner RW: Handbook on Neurovascular Ultrasound. Front Neurol Neurosci BaselKarger2006. 21:261268

  • 22

    Miller DLLi PDou CGordon DEdwards CAArmstrong WF: Influence of contrast agent dose and ultrasound exposure on cardiomyocyte injury induced by myocardial contrast echocardiography in rats. Radiology 237:1371432005

  • 23

    Miller DLPislaru SVGreenleaf JE: Sonoporation: mechanical DNA delivery by ultrasonic cavitation. Somat Cell Mol Genet 27:1151342002

  • 24

    Price RJSkyba DMKaul SSkalak TC: Delivery of colloidal particles and red blood cells to tissue through microvessel ruptures created by targeted microbubble destruction with ultrasound. Circulation 98:126412671998

  • 25

    Rabinovici JInbar YRevel AZalel YGomori JMItzchak Y: Clinical improvement and shrinkage of uterine fibroids after thermal ablation by magnetic resonance-guided focused ultrasound surgery. Ultrasound Obstet Gynecol 30:7717772007

  • 26

    Rai RRichardson CFlecknell PRobertson HBurt AManas DM: Study of apoptosis and heat shock protein (HSP) expression in hepatocytes following radiofrequency ablation (RFA). J Surg Res 129:1471512005

  • 27

    Roberts WWHall TLIves KWolf JS JrFowlkes JBCain CA: Pulsed cavitational ultrasound: a noninvasive technology for controlled tissue ablation (histotripsy) in the rabbit kidney. J Urol 175:7347382006

  • 28

    Sadlowski AChomas JPollard RBloch SGriffey SWisner E: Mean flow rate and integrated perfusion estimates obtained with contrast-assisted ultrasound. IEEE Ultrasonics Symposium 2:197719802002. ( [Accessed November 22 2010]

  • 29

    Sokka SDKing RHynynen K: MRI-guided gas bubble enhanced ultrasound heating in in vivo rabbit thigh. Phys Med Biol 48:2232412003

  • 30

    Song JKlibanov ALHossack JAPrice RJ: Acoustic attenuation by contrast agent microbubbles in superficial tissue markedly diminishes petechiae bioeffects in deep tissue. Invest Radiol 43:3223292008

  • 31

    Tachibana KUchida TOgawa KYamashita NTamura K: Induction of cell-membrane porosity by ultrasound. Lancet 353:14091999

  • 32

    Tanter MThomas JLFink M: Focusing and steering through absorbing and aberrating layers: application to ultrasonic propagation through the skull. J Acoust Soc Am 103:240324101998

  • 33

    Tempany CMStewart EAMcDannold NQuade BJJolesz FAHynynen K: MR imaging-guided focused ultrasound surgery of uterine leiomyomas: a feasibility study. Radiology 226:8979052003

  • 34

    ter Haar GR: High intensity focused ultrasound for the treatment of tumors. Echocardiography 18:3173222001

  • 35

    Tran BCSeo JHall TLFowlkes JBCain CA: Microbubble-enhanced cavitation for noninvasive ultrasound surgery. IEEE Trans Ultrason Ferroelectr Freq Control 50:129613042003

  • 36

    Yeh CKKruse DELim MCRedline DEFerrara KW: A new high frequency destruction/reperfusion system. IEEE Ultrasonics Symposium 1:4334362003. ( [Accessed November 22 2010]

  • 37

    Yu TWang GHu KMa PBai JWang Z: A microbubble agent improves the therapeutic efficiency of high intensity focused ultrasound: a rabbit kidney study. Urol Res 32:14192004




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