Insonation of targeted microbubbles produces regions of reduced blood flow within tumor vasculature.
OBJECTIVE
In ultrasound molecular imaging, a sequence of high-pressure ultrasound pulses is frequently applied to destroy bound targeted microbubbles, to quantify accumulated microbubbles or to prepare for successive microbubble injections; however, the potential for biological effects from such a strategy has not been fully investigated. Here, we investigate the effect of high-pressure insonation of bound microbubbles and the potential for thrombogenic effects.


METHODS
A total of 114 mice carrying either Met-1 or neu deletion mutant (NDL) tumors was insonified (Siemens Sequoia system, 15L8 transducer, 5-MHz color-Doppler pulses, 4 or 2 MPa peak-negative pressure, 8.1-millisecond pulse repetition period, 6-cycle pulse length, and 900-millisecond insonation). Microbubbles conjugated with cyclic-arginine-glycine-aspartic acid (cRGD) or cyclic-aspartic-acid-glycine-tyrosine (3-NO)-glycine-hydroxyproline-asparagine (LXY-3) peptides or control (no peptide) microbubbles were injected, and contrast pulse sequencing was used to visualize the flowing and bound microbubbles. An anti-CD41 antibody was injected in a subset of animals to block potential thrombogenic effects.


RESULTS
After the accumulation of targeted microbubbles and high-pressure (4 MPa) insonation, reduced blood flow, as demonstrated by a reduction in echoes from flowing microbubbles, was observed in 20 Met-1 mice (71%) and 4 NDL mice (40%). The area of low image intensity increased from 22 ± 13% to 63 ± 17% of the observed plane in the Met-1 model (P < 0.01) and from 16 ± 3% to 45 ± 24% in the NDL model (P < 0.05). Repeated microbubble destruction at 4 MPa increased the area of low image intensity to 76.7 ± 13.4% (P < 0.05). The fragmentation of bound microbubbles with a lower peak-negative pressure (2 MPa) reduced the occurrence of the blood flow alteration to 28% (5/18 Met-1 tumor mice). The persistence of the observed blood flow change was approximately 30 minutes after the microbubble destruction event. Dilated vessels and enhanced extravasation of 150 kDa fluorescein-isothiocyanate (FITC)-dextran were observed by histology and confocal microscopy. Preinjection of an anti-CD41 antibody blocked the reduction of tumor blood flow, where a reduction in blood flow was observed in only 1 of 26 animals.


CONCLUSIONS
High-pressure fragmentation of microbubbles bound to tumor endothelial receptors reduced blood flow within 2 syngeneic mouse tumor models for ∼30 minutes. Platelet activation, likely resulting from the injury of small numbers of endothelial cells, was the apparent mechanism for the flow reduction.
Department of Biomedical Engineering, University of California, Davis 95616, USA.
Xiaowen
X
Hu
Azadeh
A
Kheirolomoom
Lisa M
LM
Mahakian
Julie R
JR
Beegle
Dustin E
DE
Kruse
Kit S
KS
Lam
Katherine W
KW
Ferrara
D000818:Animals; D001943:Breast Neoplasms / Q000098:blood supply* / Q000000981:diagnostic imaging / Q000473:pathology; D003287:Contrast Media; D004195:Disease Models, Animal; D005260:Female; D051379:Mice; D045423:Microbubbles; D057054:Molecular Imaging; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging*; D010974:Platelet Aggregation; D014463:Ultrasonography / Q000379:methods*
D003287:Contrast Media