Recent evidence appears to indicate that neurons, responsible for our perception of the world around us, are not only electrically excitable, but may have mechanical triggers as well. This is well supported through the growing number of observations of focused ultrasound (FUS) perturbations of the neurons located in our central nervous system (CNS). However, while the CNS is largely responsible for turning electrical signals from the periphery into thoughts and understanding, less is known about the effect of which FUS has upon the peripheral signals themselves: our peripheral nervous system (PNS). Given the non-invasive nature of FUS - were it be discovered to influence neuronal signaling, FUS would become a powerful tool for therapy and medicine, especially in conditions involving pain. Thus, we ponder the question, "How can FUS modulate nerve activity and furthermore, what are the interactions on pain signaling?" In this dissertation, a road-map is described for translating insights acquired through pre-clinical study of ultrasound PNS stimulation to clinical investigation on neuropathic pain modulation in humans. More specifically, methods and tools to study excitation of the sciatic nerve bundle and the dorsal root ganglia (DRG) were built and optimized in rodent models. In turn, these methods and findings enabled investigation into pain signaling and translation to human studies. Finally, FUS was shown to mitigate pain sensations in human patients with neuropathic pain. First, using a newly developed in vivo nerve displacement imaging technique, mechanical deformations of the nerve from FUS stimulation were noninvasively mapped in a two-dimensional plane centered at the sciatic nerve. Nerve displacements were positively correlated with downstream compound muscle activation from FUS sciatic nerve stimulation. Furthermore, by focusing ultrasound waves to the DRGs directly in an ex vivo preparation, additional parameters were identified to modulate spike transmission, effectively regulating high frequency signaling. Next, we investigated the feasibility translating FUS nerve stimulation to clinical studies. We first looked at effects on upstream cortical activity and pain signaling from somatosensory stimuli using high-frequency functional ultrasound (fUS) imaging. FUS was shown to both stimulate somatosensation and suppress pain signaling in the cortex. Secondly, nerve displacement imaging was scaled-up for human investigation, essential for in-procedure localization and stimulation of the targeted nerve bundle. Using a combination of imaging and therapeutic excitation, simultaneous nerve targeting, stimulation, and monitoring was established at pressures required for stimulation. Lastly, clinical feasibility was investigated using previously optimized FUS pulse schemes and scaled-up neuromodulation technologies. Specifically, we applied simultaneous FUS to the median nerve and thermal stimulation to the corresponding dermatome in healthy human subjects. Furthermore, patients with robust and repeatable mechanically-assessed neuropathic pain were similarly stimulated with FUS to assess pain suppression. Based on the findings presented herein, noninvasive FUS peripheral stimulation has the potential for radically shifting the traditional pharmaceutical paradigms in chronic and acute pain treatment by altering signals before being processed in the spinal cord and ultimately the brain. The studies outlined herein serve to elucidate mechanisms of FUS in the PNS, as well as provide the starting foundations for further development of FUS as an effective pain treatment.

Glioblastoma multiforme (GBM) is the most aggressive type of primary brain tumor. Even after patients undergo maximum and safe surgical resection followed by adjuvant chemotherapy and radiation therapy, residual GBM cells form secondary tumors which lead to poor survival times and prognoses for patients. This tumor recurrence can be attributed to the inherent GBM heterogeneity that makes it difficult to eradicate the therapy-resistant and tumorigenic subpopulation of GBM cells with stem cell-like properties, referred to as glioma stem cells (GSCs). Additionally, the migratory nature of GBM/GSCs enable them to invade into the healthy brain parenchyma beyond the resection cavity to generate new tumors. In an effort to address these challenges of GBM recurrence, this research aimed to develop a biomaterials-based approach to attract, capture, and eradicate GBM cells and GSCs with chemical and physical stimuli. Specifically, it is proposed that after surgical removal of the primary GBM tumor mass, an injectable hydrogel can be dispensed into the resection cavity for crosslinking in situ. A combination of chemical and physical cues can then induce the migration of the residual GBM/GSCs into the injectable hydrogel to localize and concentrate the malignant cells prior to non-invasively abating them. In order to develop this proposed treatment, this dissertation focused on 1) characterizing and optimizing the thiol-Michael addition injectable hydrogel, 2) attracting and entrapping GBM/GSCs into the hydrogel with CXCL12-mediated chemotaxis, and 3) assessing the feasibility of utilizing histotripsy to mechanically and non-invasively ablate cells entrapped in the hydrogel. The results revealed that hydrogel formulations comprising 0.175 M NaHCO3(aq) and 50 wt% water content were the most optimal for physical, chemical, and biological compatibility with the GBM microenvironment on the basis of their swelling characteristics, sufficiently crosslinked polymer networks, degradation rates, viscoelastic properties, and interactions with normal human astrocytes. Loading the hydrogel with 5 µg/mL of CXCL12 was optimal for the slow, sustained release of the chemokine payload. A dual layer hydrogel platform demonstrated in vitro that the resulting chemotactic gradient induced the invasion of GBM cells and GSCs from the extracellular matrix and into the synthetic hydrogel with ameboid migration and myosin IIA activation. This injectable hydrogel also demonstrated direct therapeutic benefits by passively eradicating entrapped GBM cells through matrix diffusion limitations as well as decreasing the GBM malignancy and GSC stemness upon cancer cell-hydrogel interactions. Research findings revealed the hydrogels can be synthesized under clinically relevant conditions mimicking GBM resection in vitro, and hydrogels were distinguishable with ultrasound imaging. Furthermore, the synthetic hydrogel was acoustically active to generate a stable cavitation bubble cloud with histotripsy treatment for ablation of entrapped red blood cells with well-defined, uniform lesion areas. Overall, the results from this research demonstrate this injectable hydrogel is a promising platform to attract and entrap malignant GBM/GSCs for subsequent eradication with chemical and physical stimuli. Further development of this platform, such as by integrating electric cues for electrotaxis-directed cell migration, may help to improve the cancer cell trapping capabilities and thereby mitigate GBM tumor recurrences in patients. / Doctor of Philosophy / Glioblastoma multiforme (GBM) is the deadliest type of primary brain cancer. Upon GBM diagnosis, patients first undergo surgery to remove the tumor from the brain. After waiting several weeks for the wound healing process due to surgery, patients are administered chemotherapy with drugs and radiation therapy to eradicate any remaining GBM cells. Even after undergoing these combinatorial treatments, the cancer returns and leads to median survival times of only 15 months in 90% of patients. Complete GBM eradication is difficult, since the cancer cells can migrate into healthy brain tissue beyond the original tumor site. Additionally, GBM is highly heterogenous and composed of different cell types that can resist chemotherapy and radiation therapy, which lead to secondary tumors and cancer relapse. To address these challenges, this dissertation aimed to develop a polymer-based material (specifically a hydrogel) that can attract, entrap, and localize the GBM cells into the material to subsequently eradicate them with chemical and physical signals. This hydrogel platform would have important clinical implications, as it can potentially be dispensed into the empty cavity after surgical removal of the tumor in the brain. The hydrogel can then be harnessed to attract residual GBM cells for directed migration into the hydrogel to concentrate and localize the cancer cells for their subsequent destruction with a non-invasive technology. In order to develop this proposed treatment, this dissertation investigated the following three aims: 1) to study and optimize the injectable hydrogel for chemical, physical, and biological compatibility with the GBM therapy; 2) to utilize chemical signals to attract and entrap the GBM cells into the hydrogel; and 3) to apply focused ultrasound with high amplitude, short duration negative pressure pulses to mechanically fractionate and destroy the cells entrapped in the hydrogel. The results revealed that the hydrogel comprising 0.175 M NaHCO3(aq) and 50 wt% water content was the most optimal formulation. CXCL12 chemokine proteins loaded into the hydrogel at 5 µg/mL released slowly from the hydrogel to generate a chemical gradient and thereby attract GBM cells to promote their invasion into the hydrogel matrix. The hydrogel was demonstrated to respond well to focused ultrasound treatment, which was capable of mechanically fractionating and destroying red blood cells in the hydrogel uniformly. Overall, the results from this research provide support that this hydrogel platform can attract, entrap, and eradicate GBM cells with chemical and physical stimuli. Hence, further improvement of this platform and implementation of this novel GBM treatment may in the future help minimize GBM cancer relapse in patients who undergo conventional therapies, thereby extending their survival times.

In addition to protecting the brain from circulating pathogens and neurotoxins, the blood-brain barrier (BBB) limits both the delivery of drugs to the brain and the migration of neurological disease biomarkers from the brain into the blood. Focused-ultrasound blood-brain barrier opening (FUS-BBBO) addresses both of these transport limitations by transiently and noninvasively opening the BBB. Although originally designed as a drug delivery method, FUS-BBBO has also been shown to be an effective neuroimmunotherapy and method of improving liquid biopsy specificity for neurological disease. Prior to the work presented herein, the mechanism of FUS-BBBO neuroimmunotherapy remained poorly characterized and FUS-BBBO liquid biopsy remained poorly optimized. Initially, we present the temporal response of brain macrophages to FUS-BBBO. Due totheir role as the main phagocyte in the brain and the well-documented association between their dysfunction and neurodegenerative disease progression, we hypothesized that FUS-BBBO affects brain macrophage population composition and phenotype. Utilizing temporal single-cell RNA sequencing, we establish that treatment remodels the immune landscape via a number of processes including microglia proliferation, disease-associated microglia population size increase, and central-nervous-system associated macrophage recruitment. To further elucidate the functional role of the brain macrophage response to FUS-BBBO, we find that their depletion is associated with significantly decelerated BBB restoration. Secondly, we compare FUS-BBBO with two other methods of focused ultrasound neuroimmunotherapy, focused ultrasound neuromodulation (FUS-N) and focused ultrasound with microbubbles without BBBO (FUS+MB). FUS-N utilizes FUS parameters that alter neuronal connectivity via a combination of mechanosensitive receptor interactions and transient hypothermia without the injection of microbubbles (MB). FUS+MB is the combination of MB and FUS below the pressure threshold for BBBO (FUS+MB). FUS+MB has been shown to trigger morphological activation of brain macrophages and has proven efficacious as a method of immunotherapy within the peripheral nervous system. Due to the findings of brain macrophage modulation in response to FUS-BBBO, we compare brain macrophage modulation between all three paradigms both in the presence and absence of Alzheimer’s Disease (AD) pathology. We identify FUS-BBBO as the paradigm which maximizes brain macrophage modulation including an increase in the population of neuroprotective, disease-associated microglia and direct correlation between FUS cavitation dose and brain macrophage phagocytosis. Next, we combine spatial and single-cell transcriptomics with immunohistochemical validation to characterize the effect of FUS-BBBO on brain macrophage distribution in both wild-type and Alzheimer’s disease animals. Given their relevance within neurodegeneration and perturbation response, we emphasize the distribution of three brain macrophage populations - disease- and interferon-associated microglia and central-nervous-system-associated macrophages. We find a genotype-specific redistribution of each population, with an overall trend towards increased interaction with the brain-cerebrospinal fluid barrier after FUS-BBBO, an effect that is found to be more pronounced in the presence of disease pathology. Finally, we investigate the role of extracellular vesicles (EVs) in both the mechanism ofFUS-BBBO neuroimmunotherapy and as a method of improving FUS-BBBO liquid biopsy. EVs are lipid vesicles that are responsible for the transport and exchange of diverse cargo between cells and have been reported to modulate the immune system. Isolation of EVs has emerged as a method of improving biomarker detection. Prior to this study, the effect of FUS-BBBO neuroimmunotherapy on EV concentration and content remained unexplored. We investigate the concentration and content of isolated EVs from the serum of mice and Alzheimer’s Disease patients prior to and after treatment with FUS-BBBO. We illustrate a 100% increase in EV concentration one hour after treatment in both mice and patients. Furthermore, we illustrate an increase in murine EV RNA that is associated with the previously reported neuroimmunotherapeutic responses to FUS-BBBO including synaptic remodeling and neurogenesis. Finally, we illustrate an increase in AD biomarker concentration within the patient EVs three days after treatment that is proportional to the volume of blood-brain barrier opening. Overall, we establish that FUS-BBBO drug-free neuroimmunotherapy triggers complex brain macrophage modulation in a manner incomparable by other FUS neuroimmunotherapy paradigms. Furthermore, we illustrate the effect of FUS-BBBO on EV concentration and content in both preclinical and clinical experiments, indicating the role of EVs in FUS-BBBO neuroimmunotherapy and their utility as a method of improving liquid biopsy specificity. The results presented herein support the potential of FUS-BBBO as both a method of neuroimmunotherapy and a method of amplifying liquid biopsy specificity in Alzheimer’s Disease.

Focused ultrasound (FUS) has become an important non-invasive therapy for solid tumor ablation via thermal effects. The cavitation effect induced by FUS is thereby avoided but applied for lithotripsy, support drug delivery and the induction of blood vessel destruction for cancer therapy. In this study, head and neck cancer (FaDu), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS by using an in vitro FUS system followed by single-dose X-ray radiation therapy (RT) or water bath hyperthermia (HT). Sensitization effects of short FUS shots with cavitation (FUS-Cav) or without cavitation (FUS) to RT or HT (45 °C, 30 min) were evaluated. FUS-Cav significantly increases the sensitivity of cancer cells to RT and HT by reducing long-term clonogenic survival, short-term cell metabolic activity, cell invasion, and induction of sonoporation. Our results demonstrated that short FUS treatment with cavitation has good potential to sensitize cancer cells to RT and HT non-invasively.

Focused ultrasound (FUS) can modulate activity in the central nervous system of animals, however the mechanism of action is not yet fully understood. FUS is a promising technique for clinical use in treating both physiological and psychological pathology of the nervous system. FUS can noninvasively penetrate the skull deep into the brain and modulate brain targets with millimeter-scale resolution. FUS is less invasive than deep brain stimulation (DBS) and can target deeper structures with greater resolution than transcranial magnetic stimulation (TMS). Functional ultrasound imaging (fUSI) is an emerging modality for monitoring stimulus-evoked brain activity. However, the thick skull of large animals poses a significant obstacle for the noninvasive translation of the technique to nonhuman primates and humans. In this dissertation, FUS is performed in mice and nonhuman primates and an fUSI technique is developed for transcranially imaging FUS-evoked responses in both species. The first aim of this dissertation established a procedure for performing high-resolution FUS in mice in vivo. FUS-evoked motor responses were evaluated using four-limb electromyography (EMG). A detailed quantitative analysis of several EMG characteristics demonstrated that observed motor responses exhibited brain target-specific differences. FUS in the brain was also shown to modulate cardiorespiratory activity. However, simulations conceded that intracranial reverberations may activate brain structures outside acoustic foci, suggesting that direct detection of brain activity is preferable to responses like EMG and cardiorespiratory activity. The second aim of this dissertation developed an fUSI system for monitoring FUS-evoked responses in mice in vivo. fUSI was validated using electrical peripheral nerve stimulation to elicit somatosensory-evoked responses, a well-characterized approach in established techniques like functional magnetic resonance imaging (fMRI). fUSI was later integrated into an ultrasoundbased optogenetic stimulation procedure. Lastly, a dual FUS-fUSI transducer system for performing neuromodulation and functional activity monitoring was developed and successfully demonstrated in mice in vivo. The final aim of this dissertation was to adapt the FUS-fUSI procedure developed in mice for use in nonhuman primates. Two approaches were developed and tested in vivo. The first approach employed a low-frequency ultrasound array for both neuromodulation and activity monitoring. The second approach implemented a dual FUS-fUSI transducer system similar to that used in mice. Preliminary evidence indicated that the adapted dual transducer system can successfully perform fully noninvasive neuromodulation and functional activity monitoring transcranially in nonhuman primates in vivo. The findings presented in this dissertation provide a framework for performing fully noninvasive ultrasound-mediated neuromodulation and functional activity monitoring in non human primates and describes a road map for further translating the technique for clinical use in human subjects. A fully noninvasive FUS-fUSI technique can provide an invaluable tool for clinicians to treat diseases of the nervous system not indicated for invasive procedures, opening the door to a wide range of therapeutic applications.

Tendon injuries are prevalent, debilitating and difficult to treat. Common interventions such as anti-inflammatory medication, growth factor injections and surgery are associated with short-term efficacy and long rehabilitation periods. Tendons possess an incomplete healing response which is reparative (scar-mediated) rather than regenerative, resulting in a 'healed' tissue that is mechanically inferior to the native tendon. While it is widely accepted that mechanical-loading based treatments offer long-term symptomatic resolution and improved functionality, the exact mechanisms of action of such mechanotransduction-based healing cascades remain unclear. Nevertheless, there is significant motivation for the development of non-invasive and efficient rehabilitative treatments that mechanically stimulate the injured tendons to achieve functional healing responses. Focused Ultrasound (FUS) methods are an attractive treatment option as they are non-invasive, utilize higher intensities for shorter durations and are targeted to a very specific treatment volume, hence inducing significant bio-effects in the tissue without affecting surrounding structures. Herein, we present a body of work that includes the development of FUS pulsing to precisely target murine Achilles tendons and emphasize distinct bioeffects (thermal-dominant and mechanical-dominant). We investigated the feasibility of applying FUS pulsing to murine Achilles tendons ex vivo and in vivo and demonstrated that FUS can be safely applied without any deleterious effects in the tendons and surrounding tissues. The animals showed no symptoms of distress after multi-session treatments. Overall, results suggest that tendon material properties are not adversely altered by FUS pulsing. Histological analyses showed mild matrix disorganization, suggesting the need for slight modifications in the ultrasound pulsing parameters and treatment durations. When applied to injured tendons, mechanical dominant schemes seemed to drive larger improvements in material properties compared to thermal-dominant pulsing, confirming our original hypothesis that mechanical stimulation may play a bigger role in tendon healing compared to purely thermal-dominant stimulation. Additionally, feasibility of histotripsy ablation in murine Achilles tendons was successfully investigated ex vivo and in vivo and experimentation to further optimize these methods are ongoing. Such (non-thermal) ablative paradigms will be extremely useful when conservative treatment options are unavailable and debridement of scar tissue is warranted to interrupt the degenerative process and stimulate healing. Finally, a pilot investigation into FUS-induced strains was performed to guide our parameter selection process and deliver controlled strains to achieve healing responses (similar to current clinical rehabilitation protocols). We were able confirm that strains between 1% and 6% (or higher) can be induced by manipulating ultrasound treatment parameters. Overall, or results reiterate the potential of FUS in eliciting the desired bioeffects and thus achieve healing in tendons and provide a snapshot of the expected effects of using such pulsing methods to treat tendon injuries. / Doctor of Philosophy / Tendons are tissues that connect muscles to bones, and are unfortunately prone to injuries. Such injuries are prevalent and difficult to treat. Effective treatment options remain limited, as common methods such as surgery, anti-inflammatory medications and corticosteroid injections do not provide long-term relief. One of the few treatments that has been proven to provide symptomatic relief and improved the functionality of chronically (over a long period of time) injured tendons is physical therapy. However, researchers are still investigating the reasons for this successful healing response. Some limitations of physical therapy are long rehabilitation and recovery periods, and the need for patient compliance (i.e., performing painful exercises while already being under significant pain). In this research, we explore the use of a non-invasive modality known as ultrasound to treat tendon injuries. Ultrasound is commonly thought of as a diagnostic tool, i.e., to detect injuries in musculoskeletal medicine. It, however, is also an attractive therapeutic (treatment) modality, as sound waves can be concentrated in the required area of interest which results in different types of effects in the chosen tissue, such as heating. A huge advantage is that ultrasound is non-invasive, painless, and safe, as the energy is only applied to the chosen volume of interest and surrounding structures are unaffected. To examine the utility of therapeutic ultrasound in treating tendon injuries, we used a mouse model that has been previously used in our lab, and designed different types of ultrasound treatments that elicit two main types of effects in the tissue, namely, thermal, or heating effects and mechanical, or physical therapy-like effects. Prior to applying these treatments, we measured how much heating is produced in mouse Achilles tendons via these treatments, to establish safety. Once we identified safe thermal and mechanical treatment sets, we treated mouse Achilles tendons ex vivo, i.e., after euthanasia. We tested the mechanical properties of the treated tendons and determined that treatments do not alter the mechanical properties of tendons, which is encouraging, given that we do not want treatments to interfere with the properties of native tendons. We also examined the influence of treatments on structure of Achilles tendons after treatments and deducted that the structure was not damaged due to treatments. We followed up these studies with treatments conducted in live mice, which received four treatment sessions in one week. These studies were conducted to further determine the safety and tolerance to these procedures and also examine the healing effects of treatments in injured Achilles tendons. Results suggest that focused ultrasound treatments are safe and tolerable to mice and seem to elicit improvements in tendon properties. In other studies, we also examined a different ultrasound method named histotripsy, as a non-invasive alternative to dry needling (which is another methodology used to treat tendon injuries) and scar debridement (removal of scar tissue to stimulate a new healing response). This research establishes that therapeutic ultrasound is a novel, non-invasive alternative with good potential to treat tendon injuries. Future studies will investigate the effects of ultrasound treatments over longer durations and also aim to clarify the exact type and magnitude of physical therapy-like forces that are produced by ultrasound treatments. This understanding will enhance our treatment design process to be able to mimic clinical treatments that are known to be effective.

Focused ultrasound (FUS) mediated neuromodulation has become a trending topic due to its promising attributes that enable precise and transcranial neuromodulation. Despite multiple reports of FUS effects on neurons, nervous systems, and the human brain, the mechanisms underlying such excitation or inhibition remain controversial. In our previous study, we showed that 2.1 MHz FUS induced membrane depolarizations on single crayfish motor axons in the presence of voltage-gated channel blockers, which led to a nanopore hypothesis: FUS triggered lipid molecule reconfiguration and form ion-permeable nanopores on the axonal membrane. Based on this hypothesis, stretching of the axonal membrane due to swelling in low osmolarity should increase the probability of nanopore formation under FUS. As predicted, exposure to 75% hypotonic saline induced significant increases in amplitude and frequency of occurrence of those FUS-induced depolarizations (FUSD) while the onset latency of the FUSD showed a significant decrease. Those results support the hypothesis that FUSD can be modulated by mechanically altering membrane properties. Since FUS inevitably perturbs cell membranes, we examined the role of mechanosensitive K2P channels at the crayfish opener neuromuscular junction. At ultrasound intensity lower than those used to evoke FUSD, FUS consistently induced membrane hyperpolarization (FUSH) in motor axons but not muscle fibers, which may lack K2P. Since K2P channels are also thermosensitive, we varied the temperature from 12 to 32 °C. However, there was no significant correlation between FUSH amplitudes and temperature. Furthermore, FUSH was not inhibited by the K2P channel blockers, although the presence of the channels was confirmed by K2P blockers which increased input resistance and depolarized axonal resting membrane potential. Thus, it is unlikely that K2P channels underlie FUSH. We also studied the impact of FUS on propagating action potentials (APs) in the crayfish motor axons. APs recorded during FUS took off from a hyperpolarized membrane potential and exhibited larger amplitudes and shorter duration. Three hypotheses were examined and eliminated. The US modulated AP shape changes cannot be due to: (1) alterations in microelectrode characteristics, (2) the increase in the fraction of sodium channels in the closed and not-inactivated state due to the hyperpolarization and (3) US activation of K2P channels which in turn altered AP shapes. One potential mechanism that requires further investigation is that FUS may accelerate the activation of sodium channel opening. Other factors that may indirectly modulate AP shapes are discussed. In summary, results presented in this thesis suggest that FUS-mediated membrane responses in a single cell could vary depending on the FUS intensity and the type of ion channel a given cell expresses. Furthermore, ultrasound not only evokes changes membrane potential but also modulates action potentials. Collectively, these results represent significant contribution to the understanding of mechanisms underlying ultrasound neuromodulation at the cellular level.

No description available.

No description available.

Histotripsy is a non-thermal, non-invasive focused ultrasound therapy using controlled acoustic cavitation to mechanically disintegrate tissue into an acellular homogenate. Histotripsy applies microsecond-length, high pressure (> 10 MPa) pulses to initiate the rapid expansion and collapse of nuclei in a millimeter-scale focal region, applying large stresses and strains to targeted tissues. The cavitation "bubble cloud" generated during histotripsy treatment can be visualized in real time on ultrasound imaging, assisting with treatment guidance and monitoring. Past studies have demonstrated histotripsy's potential for a variety of applications, but histotripsy has not yet been investigated for superficial musculoskeletal tumor ablation. Additionally, preliminary investigations using histotripsy to ablate brain tumors are underway, but require advanced histotripsy devices capable of overcoming attenuation of the therapeutic ultrasound signal by the skull and rely on MRI for real-time guidance. As a result, open questions remain regarding ultrasound-guided histotripsy for brain tumors. Early evidence also suggests that histotripsy ablation may induce immunogenic changes in the tumor microenvironment. Continued research is needed to explain and corroborate these findings under conditions more immunologically representative of human cancers, such as in large animal models with spontaneous tumors. This dissertation investigates the safety and feasibility of using ultrasound-guided histotripsy to ablate superficial soft tissue sarcomas (STS), osteosarcomas (OS), and brain tumors and considers the immunological impacts of histotripsy treatment for STS and OS. The research described herein (1) investigates the ability of histotripsy to treat superficial STS tumors in companion animals with spontaneous tumors, (2) investigates the feasibility of treating bone tumors with histotripsy through a series of ex vivo and in vivo studies, and (3) applies histotripsy for the minimally invasive treatment of superficial brain tumors. The completion of this dissertation will provide significant insight into the ability of ultrasound-guided histotripsy to treat novel tumor types (i.e., STS, OS, and brain tumors) and the potential role of histotripsy in veterinary medicine. Future work will build upon the studies detailed in this dissertation to optimize ultrasound-guided histotripsy for the treatment of complete STS, OS, and brain tumors in veterinary and human patients. / Doctor of Philosophy / Histotripsy is a non-invasive focused ultrasound therapy that mechanically breaks down targeted tissues through acoustic cavitation. Histotripsy is currently being developed for a number of clinical applications, including tumor ablation, but its potential for treating many cancer types remains unknown. Histotripsy uses very short, high pressure ultrasound pulses to initiate the nucleation of bubbles in the target region. These bubbles then expand and rapidly collapse to impart large stresses and strains on surrounding tissues, leaving behind only acellular debris. The cavitation "bubble cloud" generated during histotripsy treatment can be visualized on ultrasound imaging, offering real-time treatment guidance and monitoring. Histotripsy has not yet been investigated for superficial musculoskeletal tumor ablation, and preliminary studies using histotripsy to ablate brain tumors are underway, but require advanced histotripsy devices still under development. As a result, open questions remain regarding histotripsy ablation as a treatment for musculoskeletal and brain tumors. Additionally, early evidence suggests that histotripsy ablation may be able to stimulate an immune response, treating not only the targeted tumor but also multifocal or metastatic disease. Continued research is needed to explain and corroborate these findings under conditions more similar to human cancers, such as in large animal models with naturally-occurring tumors. This dissertation investigates the safety and feasibility of using ultrasound-guided histotripsy to ablate superficial soft tissue sarcomas (STS), osteosarcomas (OS), and brain tumors and considers the immunological impacts of histotripsy treatment for STS and OS. This research (1) investigates the ability of histotripsy to treat superficial STS tumors in companion animals with spontaneous tumors, (2) investigates the feasibility of treating bone tumors with histotripsy through a series of ex vivo and in vivo studies, and (3) applies histotripsy for the minimally invasive treatment of superficial brain tumors. The completion of this dissertation will provide significant insight into the ability of ultrasound-guided histotripsy to treat novel tumor types (i.e., STS, OS, and brain tumors) and the potential role of histotripsy in veterinary medicine. Future work will build upon the studies detailed in this dissertation to optimize ultrasound-guided histotripsy for the treatment of complete STS, OS, and brain tumors in veterinary and human patients.