Intravital imaging and cavitation monitoring of antivascular ultrasound in tumor microvasculature

Abstract

Rationale: Focused ultrasound-stimulated microbubbles have been shown to be capable of inducing blood flow shutdown and necrosis in a range of tissue types in an approach termed antivascular ultrasound or nonthermal ablation. In oncology, this approach has demonstrated tumor growth inhibition, and profound synergistic antitumor effects when combined with traditional platforms of chemo-, radiation-and immune-therapies. However, the exposure schemes employed have been broad and underlying mechanisms remain unclear with fundamental questions about exposures, vessel types and sizes involved, and the nature of bubble behaviors and their acoustic emissions resulting in vascular damage -impeding the establishment of standard protocols. Methods: Here, ultrasound transmitters and receivers are integrated into a murine dorsal window chamber tumor model for intravital microscopy studies capable of real-time visual and acoustic monitoring during antivascular ultrasound. Vessel type (normal and tumor-affected), caliber, and viability are assessed under higher pressure conditions (1, 2, and 3 MPa), and cavitation signatures are linked to the biological effects. Results: Vascular events occurred preferentially in tumor-affected vessels with greater incidence in smaller vessels and with more severity as a function of increasing pressure. Vascular blood flow shutdown was found to be due to a combination of focal disruption events and network-related flow changes. Acoustic emissions displayed elevated broadband noise and distinct sub-and ultra-harmonics and their associated third-order peaks with increasing pressure. Conclusions: The observed vascular events taken collectively with identified cavitation signatures provide an improved mechanistic understanding of antivascular ultrasound at the microscale, with implications for establishing a specific treatment protocol and control platform.

Synchronous Intravital Imaging and Cavitation Monitoring of Antivascular Focused Ultrasound in Tumor Microvasculature Using Monodisperse Low Boiling Point Nanodroplets

“…In the literature, it has been proposed that with endothelial cell targeted MBs and shorter pulses at high pressures antivascular ultrasound damages the endothelium to induce platelet aggregation and thrombosis. 25,138 However, clots were rarely observed with MBs with long pulses at higher pressures (6.8% at 3 MPa) 85 and even more rarely with NDs (0.2% at 3 MPa) in the current study, suggesting that they may be occurring incidentally rather than being causal for flow shutdown. Another mechanism proposed in the context of combined antivascular ultrasound and radiotherapy is the upregulation of ASMase and ceramide due to endothelial perturbation, resulting in endothelial cell death.…”

“…Another mechanism proposed in the context of combined antivascular ultrasound and radiotherapy is the upregulation of ASMase and ceramide due to endothelial perturbation, resulting in endothelial cell death. 42,139 The time scale of shutdown observed in the present model with MBs 85 and NDs is far shorter (seconds) than that of apoptosis induction, and it is therefore not implicated as being a cause of shutdown here. However, it should be noted that the radiosensitization study was conducted at lower pressures and with higher MB doses; different mechanisms for shutdown may underly the varying exposure conditions.…”

“…However, it has been hypothesized that flow cessation in vessels without focal disruption is due to a cascade of discrete events within the microvascular network. 85 Here focal disruption/leakage events overlapped with shutdown in 31% of microvessels with NDs at 3 MPa (compared to 38% with MBs, Table S3). It is notable that vessels exhibiting blood flow shutdown could remain shutdown (termed "sustained shutdown") or recover flow when reassessed at 60 min after ultrasound.…”

“…This suggests the possibility of using broadband noise as well as the relative sub-and ultraharmonics and their complex-order counterparts (1/3, 2/3 sub-and 4/3, 5/3 ultraharmonics) as indicators of biological effects. Inertial cavitation has previously been linked to vascular damage in large mm-sized vessels 14 as well as in microvessels 33,85,145 and has recently been associated with nonthermal (ischemic) necrosis regions caused by vascular shutdown in brain tissue 24 and in tumors. 18,36 The complex order harmonics have been reported in the context of focused ultrasound in subcutaneous mouse tumors, 36 in the chorioallantoic membrane of a duck embryo, 145 and in a dorsal window chamber tumor model, 85 all under therapeutically relevant (>2 MPa high pressure, 5 ms long pulse, ∼1 MHz low frequency) schemes with MBs.…”

“…Vessel type, caliber, and viability are assessed under therapeutic antivascular ultrasound conditions (1−3 MPa, 5 ms pulse length), and cavitation signatures are analyzed and linked to the biological effects. Results are discussed relative to those obtained with Definity MBs in the same context, 85 systematically comparing MBs and NDs under controlled dose and exposure conditions. We demonstrate that NDs can induce the same or more vascular shutdown relative to MBs with no activity below the ND vaporization threshold and fewer other microvascular events, demonstrating the greater sensitivity and specificity achievable with NDs.…”