OBJECTIVE:Thrombotic occlusions in the setting of stroke, pulmonary embolism and the peripheral vasculature are increasingly being treated with catheter-based aspiration approaches. Unfortunately, aspiration is frequently unsuccessful in extracting more challenging blood clots, which can become corked in the distal tip. In this study, we investigated the feasibility of using histotripsy to mechanically degrade thrombus within the lumen of a hollow cylindrical transducer (HCT), with a view to ultimately using this approach to improve aspiration thrombectomy procedures. METHODS:Retracted porcine blood clots were situated within the lumen of a radially polarized HCT (2.5/3.3 mm inner/outer diameter, 2.5 mm length, PZT). The HCT was stimulated in thickness mode (6.1 MHz) as a function of pulse length (10, 20 and 100 µs), pulse repetition frequency (100, 500 and 1000 Hz) and treatment time (0.1-10.0 seconds). High-frequency ultrasound examination was used to monitor bubble clouds and lesion formation during treatments (2-D) and quantify post-treatment lesion volumes (3-D). RESULTS:Bisected treated clots confirmed the formation of liquified zones. Lesions could form within 0.1 seconds along the central axis of the transducer and then grow in diameter and length with increasing treatment time. The lesion volume was highly dependent on the exposure scheme (n = 5/group), with the largest volume associated with the 10 µs pulse length 1000 Hz pulse repetition frequency scheme. CONCLUSION:Collectively, these results support the feasibility of performing histotripsy on clots situated within an HCT compatible with mounting in the tip of an aspiration format catheter.

Precision oncology for triple-negative breast cancer (TNBC) is urgently needed because not all patients will benefit from PDL1-based immunotherapy. Unfortunately, the expression of PDL1 or abundance of tumor infiltrated lymphocytes (TILs) does not correlate with objective response rates and/or overall survival. We have developed a novel patient-derived xenograft (PDX) model system that offers phenotype-based metrics to predict immunotherapy response prior to receiving treatment. In our PDX model (PDXovo), the patient’s tumour is engrafted onto the chorioallantoic membrane (CAM) of chick embryos. Our PDX model offers significant advantages over mouse PDXs which are expensive, time-consuming, and cannot evaluate immunotherapies. To test the efficacy of this model, we implanted immunocompetent mice with a murine mammary cancer cell line (serving as a surrogate for patients), and implanted tumours into the PDXovo system. We found our model to reliably recapitulate immunotherapy responses observed in mice, with anti-PDL1-treated tumours being significantly smaller in volume as compared to IgG-treated tumours. We also found our model to reliably predict patient response to adoptive cell therapy, wherein TILs were FACS-sorted from murine tumour digests, activated and expanded in vitro, and then engrafted together with cultured murine mammary carcinoma cells. We are now testing this model with patient-derived tumours of a variety of origins, in partnership with the Ontario Institute of Cancer Research. To date we have tested tumour responses to Pembrolizumab in five patient-derived samples from various sites, with an 80% success rate in xenograft formation. We have observed varied responses to immunotherapy, which did not correlate with PDL1 expression on TILs in these samples, highlighting the importance of such a qualitative model for precision oncology. This technology has the potential to make a positive impact in the clinic and improve objective response rates, increase progression-free survival intervals, and increase overall survival. Olivia R. Grafinger, Kabir A. Khan, Esther I. Matus, Sara Mar, Yan Li, David Goertz, Jean Gariépy, Katarzyna J. Jerzak, Robert Kerbel, Hon S. Leong. Ex ovo system for rapid and quantitative modelling of immunotherapy responses in pre-clinical and patient-derived xenografts: PDXovo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1277.

Intravascular aspiration thrombectomy catheters are widely used to treat stroke, pulmonary embolism, and deep venous thrombosis. However, their performance is frequently compromised by clot material becoming lodged within the catheter tip. To address this, we develop a novel ultrasound-enhanced aspiration catheter approach that generates cavitation within the tip to mechanically degrade clots, with a view to facilitate extraction. The design employs hollow cylindrical transducers that produce inwardly propagating cylindrical waves to generate sufficiently high pressures to perform histotripsy. This study investigates the feasibility of self-sensing cavitation detection by analyzing voltage signals across the transducer during treatment. Experiments were conducted for two transmit pulse lengths at varying driving voltages with water or clot in the lumen. Cavitation clouds within the lumen were assessed using 40 MHz ultrasound imaging. Changes in the signal envelope during the pulse body and ringdown phases occurred above the cavitation threshold, the latter being associated with more rapid wave damping in the presence of bubble clouds within the lumen. In the frequency domain, voltage-dependent cavitation signals—subharmonics, ultra-harmonics, and broadband—emerged alongside transmit pulses. This work demonstrates a highly sensitive, sensor-free method for detecting cavitation within the lumen, enabling feedback control to further improve histotripsy-assisted aspiration.

To address existing challenges with intravascular mechanical thrombectomy devices, a novel ultrasound-enhanced aspiration approach is being developed to mechanically degrade clots using cavitation. This method employs standing waves within a mm-scale hollow cylindrical transducer to generate high pressures sufficient to perform histotripsy on clots situated within the transducer lumen and generate substantial lesions. The objective of this study is to assess the feasibility of self-sensing cavitation detection by analyzing voltage signals across the transducer during treatment pulses. External ultrasound imaging of the transducer lumen validated cavitation detection. Impedance was also altered by the presence of clot material within the lumen. Experiments varying the driving voltage in water-filled lumens demonstrated changes in the relative amplitudes of the envelopes of the pulse body and ringdown portions of the voltage signals above the cavitation threshold, as well as changes in the spectral domain. In particular both broadband and ultraharmonic signals showed an increase in amplitude above the cavitation threshold. Similar temporal and spectral voltage signal changes in the presence of cavitation were also observed when treating clots within the lumen. This work demonstrates a highly sensitive method for detecting cavitation within the lumen, enabling monitoring with readily acquired signals without additional sensors in the catheter configuration. These findings hold significant potential for improving the efficacy of ultrasound-enhanced aspiration thrombectomy procedures.

Design strategies that lead to a more focused in vivo delivery of functionalized nanoparticles (NPs) and their cargo can potentially maximize their therapeutic efficiency while reducing systemic effects, broadening their clinical applications. Here, we report the development of a noncovalent labeling approach where immunoglobulin G (IgG)-decorated NPs can be directed to a cancer cell using a simple, linear bispecific protein adaptor, termed MFE23-ZZ. MFE23-ZZ was created by fusing a single-chain fragment variable domain, termed MFE23, recognizing carcinoembryonic antigen (CEA) expressed on tumor cells, to a small protein ZZ module, which binds to the Fc fragment of IgG. As a proof of concept, monoclonal antibodies (mAbs) were generated against a NP coat protein, namely, gas vesicle protein A (GvpA) of Halobacterium salinarum gas vesicles (GVs). The surface of each GV was therapeutically derivatized with the photoreactive agent chlorin e6 (Ce6GVs) and anti-GvpA mAbs were subsequently bound to GvpA on the surface of each Ce6GV. The bispecific ligand MFE23-ZZ was then bound to mAb-decorated Ce6GVs via their Fc domain, resulting in a noncovalent tripartite complex, namely, MFE23.ZZ-2B10-Ce6GV. This complex enhanced the intracellular uptake of Ce6GVs into human CEA-expressing murine MC38 colon carcinoma cells (MC38.CEA) relative to the CEA-negative parental cell line MC38 in vitro, making them more sensitive to light-induced cell killing. These results suggest that the surface of NP can be rapidly and noncovalently functionalized to target tumor-associated antigen-expressing tumor cells using simple bispecific linkers and any IgG-labeled cargo. This noncovalent approach is readily applicable to other types of functionalized NPs.

Rationale:The acoustic stimulation of microbubbles within microvessels can elicit a spectrum of therapeutically relevant bioeffects from permeabilization to perfusion shutdown.These bioeffects ultimately arise from complex interactions between microbubbles and microvascular walls, though such interactions are poorly understood particularly at high pressure, due to a paucity of direct in vivo observations.The continued development of focused ultrasound methods hinges in large part on establishing links between microbubble-microvessel interactions, cavitation signals, and bioeffects.Methods: Here, a system was developed to enable simultaneous high-speed intravital imaging and cavitation monitoring of microbubbles in vivo in a chorioallantoic membrane model.Exposures were conducted using the clinical agent Definity TM under conditions previously associated with microvascular damage (1 MHz, 0.5-3.5 MPa, 5 ms pulse length).Results: Ultrasound-activated microbubbles could be observed and were found to induce localized wall deformations that were more pronounced in smaller microvessels and increased with pressure.A central finding was that microbubbles could extravasate from microvessels (from 34% of vessels at 1 MPa to 79% at 3 MPa) during insonation (94% within 0.5 ms) and that this occurred more frequently and in progressively larger microvessels (up to 180 µm) as pressure was increased.Following microbubble extravasation, transient or sustained red blood cell leakage ensued at the extravasation site in 96% of cases for pressures ≥1 MPa. Conclusions:The results here represent the first high-speed in vivo investigation of high-pressure focused ultrasound-induced microbubble-microvessel interactions.This data provides direct evidence that the process of activated microbubble extravasation can occur in vivo and that it is linked to producing microvessel wall perforations of sufficient size to permit red blood cell leakage.The association of red blood cell leakage with microbubble extravasation provides mechanistic insight into the process of microvessel rupture, which has been widely observed in histology.

Thrombotic and chronic occlusions of large blood vessels are a major cause of mortality and morbidity, and so there is a need for improved treatments in many clinical circumstances. Endovascular ultrasound approaches have been shown to hold considerable potential to treat large vessel thrombotic occlusions. Here, we report the development of a novel forward-looking therapeutic ultrasound catheter approach. The design concept centers on the use of a radially polarized hollow cylindrical transducer situated at the distal tip. This approach enables a compact configuration where the central lumen can accommodate a guidewire during navigation as well as provide a route to release cavitation seeds and therapeutic agents adjacent to the site of occlusion. PZT-5H transducers with outer/inner diameters of 1.35/0.73 mm were evaluated using simulations and experiments for their capacity to project forward-looking ultrasound. A length of 2.5 mm operating in the 3rd harmonic of the length mode resonance (1.85 MHz) was selected. Catheters (1.55 mm outer diameter) were designed and fabricated with a liner, outer jacket, and braiding, where the transducers were incorporated with air-backing and a front-face matching layer. Forward-looking pressures of 2.5 MPa (peak negative) at 0.5 mm were achieved. Proof of principle vessel phantom experiments were performed demonstrating the ability to eject microbubbles in proximity to an occlusion and stimulate inertial cavitation. This approach holds potential for treating thrombotic and chronic total occlusions.

Lipid coated nanobubbles (NBs) have attracted a great level of interest as ultrasound (US) contrast agents due to their ability to extravagate through leaky tumor vasculature. Their linear resonance frequency is in the range of ∼50 M Hz–200 MHz, leading to confusion over their observed strong contrast in diagnostic US frequencies. By solving the Marmottant model, the dynamics of uncoated and lipid coated NBs and microbubbles (MBs) are studied over the frequency and pressure ranges (6–12 MHz, 0.1–1.2 MPa) generally used in diagnostic US. A novel bifurcation analysis in tandem with the analysis of the frequency component of the scattered pressure are conducted. Results show that despite the increased linear resonance frequency and viscous damping due to the lipid shell, buckling and rupture of the shell enhances the generation of the 2nd and 3rd harmonic resonances at pressures as low as 0.2 MPa, not observed with uncoated NBs. The generation of the harmonic resonances are concomitant with an abrupt increase in the 2nd and 3rd harmonic frequency component of the scattered pressure with their pressure threshold (PT) increasing with decreasing NBs size. For the same gas volume, and above the PT, the maximum non-destructive 2nd and 3rd harmonic powers of NBs can become higher than the 2–4 μm MBs. Similar to the lower subharmonic pressure threshold of MBs, the dynamic variation of the NBs effective surface tension due to buckling and rupture may be the potential reason behind the observed harmonic echogenicity.

Use of nanodroplets as alternative to bubbles is limited to high pressure applications due to their high vaporization threshold (>1 MPa). For low pressure applications (e.g <800 kPa) and stable bubble activity, size isolated micron or sub-micron bubbles may be used to tackle the pre-focal bubble activity and attenuation. Numerical simulations of the Marmottant model were ran for bubble sizes of 0.45, 1, 2, and 4 μm in response to 1 MHz ultrasound with pressures between 10 and 700 kPa. All agents were volume matched to 20 μl/kg of Definity considering inter-bubble interactions. The pressure-dependent attenuation and the total acoustic power (TAP) were calculated for each population. Finite element simulations (FEMS) were run by taking account the pressure dependent attenuation and sound speed. Using size isolated bubbles an experimental passive cavitation case study was performed for the same exposure conditions and sizes. Numerical results show that TAP and attenuation of the bubbles are size dependent. Bigger bubbles have stronger responses at lower pressures. However, smaller agents exhibit a size-dependent pressure threshold behavior (PT) above which their attenuation and TAP grow stronger than their bigger counterparts in qualitative agreement with experiments. FEMS show that the PT of oscillations may be used to reduce pre-focal attenuation for ultrasound propagation with minimal loss.

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.

Focused ultrasound-stimulated microbubbles can induce blood flow shutdown and ischemic necrosis at higher pressures in an approach termed antivascular ultrasound. Combined with conventional therapies of chemotherapy, immunotherapy, and radiation therapy, this approach has demonstrated tumor growth inhibition and profound synergistic antitumor effects. However, the lower cavitation threshold of microbubbles can potentially yield off-target damage that the polydispersity of clinical agent may further exacerbate. Here we investigate the use of a monodisperse nanodroplet formulation for achieving antivascular effects in tumors. We first develop stable low boiling point monodisperse lipid nanodroplets and examine them as an alternative agent to mediate antivascular ultrasound. With synchronous intravital imaging and ultrasound monitoring of focused ultrasound-stimulated nanodroplets in tumor microvasculature, we show that nanodroplets can trigger blood flow shutdown and do so with a sharper pressure threshold and with fewer additional events than conventionally used microbubbles. We further leverage the smaller size and prolonged pharmacokinetic profile of nanodroplets to allow for potential passive accumulation in tumor tissue prior to antivascular ultrasound, which may be a means by which to promote selective tumor targeting. We find that vascular shutdown is accompanied by inertial cavitation and complex-order sub- and ultraharmonic acoustic signatures, presenting an opportunity for effective feedback control of antivascular ultrasound.

Microbubble-enabled focused ultrasound (MB-FUS) has revolutionized nano and molecular drug delivery capabilities. Yet, the absence of longitudinal, systematic, quantitative studies of microbubble shell pharmacokinetics hinders progress within the MB-FUS field. Microbubble radiolabeling challenges contribute to this void. This barrier is overcome by developing a one-pot, purification-free copper chelation protocol able to stably radiolabel diverse porphyrin-lipid-containing Definity® analogues (pDefs) with >95% efficiency while maintaining microbubble physicochemical properties. Five tri-modal (ultrasound-, positron emission tomography (PET)-, and fluorescent-active) [64 Cu]Cu-pDefs are created with varying lipid acyl chain length and charge, representing the most prevalently studied microbubble compositions. In vitro, C16 chain length microbubbles yield 2-3x smaller nanoprogeny than C18 microbubbles post FUS. In vivo, [64 Cu]Cu-pDefs are tracked in healthy and 4T1 tumor-bearing mice ± FUS over 48 h qualitatively through fluorescence imaging (to characterize particle disruption) and quantitatively through PET and γ-counting. These studies reveal the impact of microbubble composition and FUS on microbubble dissolution rates, shell circulation, off-target tissue retention (predominantly the liver and spleen), and FUS enhancement of tumor delivery. These findings yield pharmacokinetic microbubble structure-activity relationships that disrupt conventional knowledge, the implications of which on MB-FUS platform design, safety, and nanomedicine delivery are discussed.

Ultrafast ultrasound imaging enables the visualization of rapidly changing blood flow dynamics in the chambers of the heart. Singular value decomposition (SVD) filters outperform conventional high pass clutter rejection filters for ultrafast blood flow imaging of small and shallow fields of view (e.g., functional imaging of brain activity). However, implementing SVD filters can be challenging in cardiac imaging due to the complex spatially and temporally varying tissue characteristics. To address this challenge, we describe a method that involves excluding the proximal portion of the image (near the chest wall) and divides the reduced field of view into overlapped segments, within which tissue signals are expected to be spatially and temporally coherent. SVD filtering with automatic selection of cut-off singular vector orders to remove tissue and noise signals is implemented for each segment. Auto-thresholding is based on the coherence of spatial singular vectors, delineating tissue, blood, and noise subspaces within a spatial similarity matrix calculated for each segment. Filtered blood flow signals from the segments are reconstructed and then combined and Doppler processing is used to form a set of blood flow images. Preliminary experimental results suggest that the spatially segmented approach improves the separation of the tissue and blood subsets in the spatial similarity matrix so that automatic thresholding is significantly improved, and tissue clutter can then be rejected more effectively in cardiac ultrafast imaging, compared to using the full field of view. In the case studied, spatially segmented SVD improved the rate of correct automatic selection of thresholds from 78% to 98.7% for the investigated cases and improved the post-filter power of blood signals by an average of more than 10 dB during a cardiac cycle.

Correct measurement of the shell properties of coated microbubbles (MBs) is essential to understanding and optimizing their response to ultrasound (US) exposure parameters in diagnostic and therapeutic ultrasound. MBs are surrounded by blood; however, the influence of the surrounding medium charges on the MB properties is poorly understood. This study aims to measure the medium charge interactions with MB shells by measuring the frequency-dependent attenuation of the same size MBs in mediums of varying charge density. In-house lipid-coated MBs with C3F8 gas core were made and were isolated to a mean size of 2.35um. MBs were diluted to ~8*10^5 MBs/mL in distilled water (DW), and two different concentrations of phosphate-buffered saline solution (PBS-1x and PBS-10x). The frequency-dependent attenuation of the MBs solutions was measured using an aligned pair of PVDF transducers with a center frequency of 10MHz and 100% bandwidth. The MB shell properties were estimated by fitting the linear equation to experiments. Using a pendant drop tensiometer, the surface tension of mm-size drops was measured inside DW, PBS-1x and PBS-10x. The frequency of the peak attenuation changes at different salinity levels was 13, 7.5 and 6.25MHz in DW, PBS-1x and PBS-10x, respectively. The attenuation peak increased by ~140% with increasing ion density. MBs' estimated shell elasticity decreased by 64% between DW and PBS-1x and 36% between PBS-1x and PBS-10x. Reduction in the shell stiffness is in qualitative agreement with the drop surface tension measurements. The shell viscosity was reduced by ~40% between DW and PBS-1x and 42% between PBS-1x and PBS-10x. The reduction in the stiffness and viscosity is possibly due to the formation of a densely charged layer around the shell, further reducing the effective surface tension on the MBs.

Therapeutic focused ultrasound in combination with encapsulated microbubbles is being widely investigated for its ability to elicit bioeffects in the microvasculature, such as transient permeabilization for drug delivery or at higher pressures to achieve 'antivascular' effects. While it is well established that the behaviors of microbubbles are altered when they are situated within sufficiently small vessels, there is a paucity of data examining how the bubble population dynamics and emissions change as a function of channel (vessel) diameter over a size range relevant to therapeutic ultrasound, particularly at pressures relevant to antivascular ultrasound. Here we use acoustic emissions detection and high-speed microscopy (10 kframes/s) to examine the behavior of a polydisperse clinically employed agent (Definity®) in wall-less channels as their diameters are scaled from 1200 to 15 µm. Pressures are varied from 0.1 to 3 MPa using either a 5 ms pulse or a sequence of 0.1 ms pulses spaced at 1 ms, both of which have been previously employed in an in vivo context. With increasing pressure, the 1200 µm channel - on the order of small arteries and veins - exhibited inertial cavitation, 1/2 subharmonics and 3/2 ultraharmonics, consistent with numerous previous reports. The 200 and 100 µm channels - in the size range of larger microvessels less affected by therapeutic focused ultrasound - exhibited a distinctly different behavior, having muted development of 1/2 subharmonics and 3/2 ultraharmonics and reduced persistence. These were associated with radiation forces displacing bubbles to the distal wall and inducing clusters that then rapidly dissipated along with emissions. As the diameter transitioned to 50 and then 15 µm - a size regime that is most relevant to therapeutic focused ultrasound - there was a higher threshold for the onset of inertial cavitation as well as subharmonics and ultraharmonics, which importantly had more complex orders that are not normally reported. Clusters also occurred in these channels (e.g. at 3 MPa, the mean lateral and axial sizes were 23 and 72 µm in the 15 µm channel; 50 and 90 µm in the 50 µm channel), however in this case they occupied the entire lumens and displaced the wall boundaries. Damage to the 15 µm channel was observed for both pulse types, but at a lower pressure for the long pulse. Experiments conducted with a 'nanobubble' (<0.45 µm) subpopulation of Definity followed broadly similar features to 'native' Definity, albeit at a higher pressure threshold for inertial cavitation. These results provide new insights into the behavior of microbubbles in small vessels at higher pressures and have implications for therapeutic focused ultrasound cavitation monitoring and control.

Focused ultrasound in combination with circulating microbubbles is being widely investigated as a means to promote spatially targeted drug delivery. This approach has considerable potential in oncology for a range of therapeutic agents. At sufficiently high pressures, above those typically employed in drug delivery, tumor microvessel damage can be induced to an extent that leads to perfusion shutdown and subsequent ischemic tissue necrosis. This approach is often referred to as antivascular ultrasound (AVUS), which has been shown in preclinical work to be capable of enhancing the effects of radiation therapy, antiangiogenic therapy, chemotherapy, and immunotherapy. At present, the mechanisms of AVUS are not well established. It is important to gain a more detailed understanding of the bubble–microvessel interactions that lead to perfusion shutdown, along with the cavitation signatures associated with these behaviors to enable the rational development of effective cavitation based control methods. This talk will provide a brief overview of AVUS therapy in oncology and highlight recent efforts employing two-photon microscopy, high speed optical imaging, and acoustic emission monitoring to gain insights into bubble behavior within small channels and in vivo microvessels under AVUS exposure conditions.

Background Delivery of viral vectors as gene therapies to treat neurodegenerative diseases has been hampered by the inability to penetrate the blood brain barrier (BBB) and invasive or non-targeted delivery options prone to inducing immune responses. MR guided focused ultrasound (MR-g-FUS) and microbubbles have demonstrated safe, temporary, targeted BBB permeabilization clinically. Methods We developed clinically scalable, microbubble drug conjugates (MDCs) for the viral gene therapy, AAV.SIRT3-myc [adeno-associated virus expressing myc-tagged SIRT3], which has previously been shown to have disease modifying effects in animal models of Parkinson's disease (PD). The lipid shells of the perfluorocarbon gas MDCs were covalently conjugated to antibodies with binding specificity to AAVs. Following systemic (iv) delivery of AAV.SIRT3-myc MDCs, MR-g-FUS was used to deliver SIRT3-myc to brain regions affected in PD. SIRT3-myc expression was determined post mortem, using immunohistochemistry. Results An in vitro, SH-SY5Y cell culture model was used to show that the localized destruction of MDCs using ultrasound exposures within biological safety limits dissociated AAV2-GFP (green fluorescent protein) from the MDCs in the targeted area while maintaining their transduction capacity. In rats, MR-g-FUS resulted in BBB permeabilization in the striatum and substantia nigra (SNc). SIRT3-myc was expressed in the striatum, but not the SNc. Conclusion These studies demonstrate that MDCs combined with MR-g-FUS are an effective method for delivery of viral vector gene therapies, such as AAV.SIRT3, to brain regions affected in PD. This technology may prove useful as a disease-modifying strategy in PD and other neurodegenerative disorders.

Lipid-coated microbubbles (MBs) are used in contrast-enhanced ultrasound (CEUS) imaging and MB enhanced therapeutic ultrasound (US). Understanding the MB behavior and the influence of the surrounding medium on its response to the US is necessary to select the suitable US exposure parameters. The MB lipid coating is often charged, however the influence of the ions in the medium on MB behavior is not fully understood. In this work, the influence of the medium salinity on the pressure-dependent MB behavior is investigated for the first time. MBs of different lipid shell compositions are size isolated to achieve the same size distribution. The MBs linear and pressure-dependent attenuation are measured in deionized water, PBS 1×, PBS 2×, and PBS 10× using a system of aligned PVDF 100% bandwidth transducers with a center frequency of 10 MHz and exposures with peak to peak pressure range of 3–140 kPa. With increasing salinity, the linear resonance frequency decreases up to 50% for conventional lipid shell compositions, and the pressure dependence of the resonance frequency is inhibited. By modifying the shell PEG ratio, the salinity effects can significantly be altered. Moreover, the nonlinear pressure-dependent resonance frequency is restored with applications to increased CEUS.

There has been growing interest in nanobubbles (NBs) for vascular and extravascular ultrasound contrast imaging and therapeutic applications. Studies to date have generally utilized low frequencies (<12 MHz), high concentrations (>109 mL-1), and uncalibrated B-mode or contrast-mode on commercial systems without reporting investigations on NB signatures upon which the imaging protocols should be based. We recently demonstrated that low concentrations (106 mL-1) of porphyrin-lipid-encapsulated NBs scatter nonlinearly at low (2.5, 8 MHz) and high (12.5, 25, 30 MHz) frequencies in a pressure threshold-dependent manner that is advantageous for amplitude modulation (AM) imaging. Here, we implement pressure-calibrated AM at high frequency on a commercial preclinical array system to enhance sensitivity to nonlinear scattering of three phospholipid-based NB formulations. With this approach, improvements in contrast to tissue ratio relative to B-mode between 12.4 and 22.8 dB are demonstrated in a tissue-mimicking phantom, and between 6.7 and 14.8 dB in vivo.

Abstract Checkpoint inhibitor (CI) therapies are playing an increasingly prominent role in the treatment of cancer but are only effective and durable in subsets of patients. This has prompted the investigation of combining multiple CIs or their use in conjunction with conventional therapies such as chemotherapy to achieve complementary effects. A significant consideration with combinatorial approaches is the associated increase in toxicity. This provides a compelling motivation to couple CI therapy with locally applied physical methods such as radiotherapy or therapeutic ultrasound. Therapeutic ultrasound is undergoing rapid development for oncological applications and can elicit therapeutically relevant effects through ablation or impacting vascular function. It is also known to evoke immune responses, though their use in combination with immunotherapy remains to be established. One therapeutic ultrasound approach is to acoustically stimulate systemically injected “microbubbles” to undergo violent oscillations in targeted tumor regions to shut down the vasculature. We have previously shown that this approach can potentiate the antitumor effects of a range of chemotherapeutic agents. In the present study, we investigate if this form of “antivascular” ultrasound can enhance the efficacy of anti-antagonistic PD-1 (aPD-1) therapy. To test this paradigm, we used a mouse colorectal cancer line (CT26.wt), which was initiated subcutaneously in the right hind limb of 8- to 12-week-old female Balb/c mice. Mice were split into four groups: MBs (control), aPD-1 (drug group), US (ultrasound plus microbubbles group) and US + aPD-1 (combo group). Experiments were initiated on mice when tumors were in the range of 50-100 mm3. The immunotherapy drug used in this study is an anti-mouse PD-1 (clone: RMP1-4, Bioxcell). The drug aPD-1 was administered intraperitoneally at a dosage of 200 μg to respective groups prior to treatment and subsequently administered every 3 days for a total of 5 doses. Longitudinal studies were done where tumor growth was monitored every 3 days until animals reached endpoint (tumor size>1000 mm3). Acute experiments were done, which included flow cytometry and ELISPOT to assess how T-cell populations/activity changed with each respective treatment. Longitudinal experiments (n=5-6) showed that US + aPD-1 treatment significantly inhibited tumor growth relative to MB-only, US-only group and aPD-1 group at Day 6 (p<0.0001, p<0.01, p<0.01, respectively) and at Day 9 (p<0.0001, p<0.01, p<0.05, respectively). This inhibition of tumor growth with combinatorial treatment translated to longer survival times compared to MBs (p<0.01), US (p<0.01) and aPD-1 (p<0.005). Flow cytometry (n=5-7) and ELISPOT (n=6) data did not clearly show a T cell-dependent mechanism for the inhibition of tumor growth. In conclusion, these results demonstrate the ability of “antivascular” US therapy to enhance CI therapy, while the specific mechanisms of enhancement remain to be elucidated. Citation Format: Sharshi Bulner, Aaron Prodeus, Jean Gariepy, Kullervo Hynynen, David E. Goertz. Enhancing checkpoint inhibitor therapy with ultrasound stimulated microbubbles [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A20.