Inducing antivascular effects in tumors with ultrasound stimulated micron-sized bubbles

It has been demonstrated that relatively low intensity ultrasound in combination with microbubbles is capable of inducing a vascular shutdown in preclinical tumor models, and that this can lead to growth inhibition. In previous reports we have shown that this effect can be coupled with anticancer agents to achieve potent antitumor effects, by a means that is not linked to increases in drug uptake. Work to date has been conducted with commercial agents such as Definity or experimental formulations with bubble size distributions designed for resonance at the exposure frequencies employed (0.5-3 MHz). Here we investigate the potential of an experimental agent comprised of micron to submicron sized bubbles to induce antivascular effects in tumors. Experiments were conducted with KHT sarcoma tumors initiated intramuscularly in the hind legs of C3H mice and grown to approximately 200 mm3 in size. Control and treated groups were evaluated (n=5-6/group), with ultrasound exposures conducted at 1 MHz with a peak negative pressure of 1.6 MPa. This experimental, lipid-encapsulated, perfluorobutane microbubble agent had a volume peak at approximately 1 micron. The agent was injected into mice with injection gas volume fractions matched to Definity doses, and monitored with a Philips L12-5 probe in contrast mode. Tumor tissue was harvested after a pre-sacrifice perfusion stain injection (DiOC7). Agent characterization experiments included frequency dependant attenuation measurements (1-40 MHz) and cavitation detection and analysis. The agent frequency response was such that insonations occurred well below the resonant frequency and exposures were at a level that produced wideband acoustic emissions associated with inertial cavitation. Rapid perfusion reductions were observed in the tumors after treatments as evidenced by perfusion staining and contrast imaging. The particular characteristics of micron-sized formulations may offer advantages in the context of clinical implementations compared to commercial bubbles with larger size distributions.