Histotripsy is a non-invasive and non-thermal focused ultrasound therapy that can be used to ablate tissue within the body while overcoming many of the limitations of thermal ablation. Histotripsy utilizes short-duration, high pressure ultrasound pulses to create a cavitation bubble cloud of numerous rapidly expanding and collapsing bubbles, which cause mechanical stress on the targeted region. Histotripsy contains multiple subtypes including intrinsic threshold, shock scattering, and boiling histotripsy, where intrinsic threshold histotripsy utilizes single cycle pulses focused to a single point to create a bubble cloud from the peak negative pressure (p- ≥ 25 MPa for water-based tissues). Nanoparticle-mediated histotripsy (NMH) uses perfluorocarbon-filled nanoparticles to create bubble clouds at lower pressures than that of the intrinsic threshold of histotripsy. Prior studies have shown that nanodroplets (NDs) and nanocone clusters (NCCs) both reduce the cavitation threshold, but further investigation on different parameters to optimize treatments have not fully been studied. Additional research is needed for the characterization of these nanoparticles with different pulsing parameters such as cycle number and frequency in order to better predict and understand the mechanisms underlying NMH. In this thesis, I investigate the ability of new nanodroplets and nanocone clusters to reduce histotripsy cavitation threshold with NMH. I also investigate the effect that multi-cycle pulsing parameters have on NMH and stable bubble formation from acoustic droplet vaporization (ADV) for nancone clusters. The culmination of this thesis will advance our understanding of the behavior of acoustically-active nanoparticles when exposed to varied pulsing schemes and frequencies. This knowledge will allow for the further investigation of more efficient, effective, and safe methods for clinical focused ultrasound therapies. / Master of Science / Histotripsy is a non-invasive and non-thermal focused ultrasound therapy that can be used to destroy targeted tissue within the body. Histotripsy is currently being developed for non-invasive and non-thermal cancerous tissue destruction with the first-in-man trial having been conducted within the last year for the treatment of liver tumors. Histotripsy utilizes high-pressure, short-duration pulses focused to a single region to create a cloud of bubbles that are rapidly expanding and collapsing which causes mechanical damage to the targeted cells. Nanoparticle-mediated histotripsy (NMH) has been developed to utilize nanoparticles to reduce the pressure needed to induce cavitation. Despite many studies and advances in histotripsy, there are many areas within the topic that need additional research to better understand the capabilities of the treatment method. This additional research is crucial in allowing for the development of new nanoparticles, faster treatment times, and new parameters that could allow for more precision near critical structures. In this thesis, I investigate the ability of new nanoparticles to reduce histotripsy cavitation threshold with NMH. I also investigate the effect that multi-cycle pulsing parameters have on NMH and stable bubble formation for nanoparticles. The culmination of this thesis will advance our understanding of the behavior of acoustically-active nanoparticles when exposed to varied pulsing schemes and frequencies. This knowledge will allow for the further investigation of more efficient, effective, and safe methods for clinical focused ultrasound therapies.

Histotripsy is a nonthermal ultrasound therapy used to treat cancer noninvasively by tissue mechanical fractionation with cavitation bubble clouds. Histotripsy is conducted through focused ultrasound transducers, where the piezoceramic (PZT) plate or disc, which emits the ultrasound wave, is the fundamental unit of the transducer. For modular prototype histotripsy designs, these PZTs are housed in a 3D printed focused lens. However, 3D printing transducer components can be time consuming and expensive when scaling up manufacturing, and 3D printing is limited in material selection for transducer applications. This thesis investigates the use of a novel fabrication process for prototype focused ultrasound transducers, injection molding, with an in-house benchtop injection molding machine. Acoustic material properties for investigated injection molded materials, ABS, GPPS, 30% glass filled nylon, nylon 6/6, and nylon 101, are quantified experimentally. Single elements are constructed with injection molded lenses made from ABS, 30% glass-filled nylon, nylon 6/6, and nylon 101 on an in-house benchtop machine. Results show that injection molding is a novel feasible method for applications in focused ultrasound devices and the investigated plastics have favorable properties for developing prototype histotripsy transducers, comparable to 3D printed transducer housings. Future work aims to apply injection molding to various transducer designs and additional materials for focused ultrasound therapy devices. / Master of Science / Histotripsy is a cancer therapy that can noninvasively treat tumors without surgery. This is done through devices called focused ultrasound transducers which emit ultrasound waves to administer treatment to ablate tumors. These transducers are constructed using 3D printing methods, but this can be limiting when scaling up manufacturing or in material selection for transducer applications, therefore additional fabrication methods are needed. This thesis presents injection molding as a novel method for making transducer components with an in-house benchtop injection molding machine. Five plastic materials are investigated to determine ultrasound properties that would identify preferred transducer materials. Single element transducers are made from injection molded materials, tested, and compared with 3D printed single element transducers. Results of this thesis show that injection molding is a feasible manufacturing method capable of producing transducers for histotripsy, and researched materials have favorable properties for this application. In future research, additional injection molded materials should be investigated and multiple transducer designs created for injection molding fabrication. These injection molded transducers can be applied to histotripsy or applied to other focused ultrasound therapies.

Histotripsy is a noninvasive mechanical ablation method that uses focused ultrasound to disintegrate target tissues into acellular homogenate through the generation of acoustic cavitation and is currently being developed for numerous clinical applications. Histotripsy uses high-pressure (>10 MPa), short-duration (<15 cycles) pulses to cause the rapid expansion and collapse of nuclei at the focus resulting in large applied stress and strain in the adjacent tissue. At a sufficiently high pressure above the target medium's intrinsic cavitation threshold and an adequate number of applied pulses, cavitation "bubble clouds" create precise lesions with high fidelity to the region of the focus. Despite advances in histotripsy, additional research is still needed to better understand the acoustic cavitation nucleation process and its effects on therapies using focused ultrasound. This understanding is critical to better predict and control pulse dose for more rapid and efficient ablation procedures, to reduce off-target cavitation events for safer focused ultrasound therapies, and to localize ablation for high-precision procedures near critical structures or treatments without active imaging guidance. In this dissertation, I investigate the nucleation and dynamics of ultrasonically generated acoustic cavitation for novel applications of focused ultrasound. My Ph.D. thesis focuses on (1) investigating the effect of histotripsy pulsing parameters on bubble cloud cavitation nucleation, bubble dynamics, and ablation efficiency, (2) investigating the effect of nuclei characteristics on the threshold for cavitation nucleation and resulting bubble dynamics for therapeutic applications, and (3) developing methods alter select characteristics and dynamics of acoustic cavitation by adjusting pulsing parameters to optimize ablation efficiency in conventional and nanoparticle-mediated histotripsy. The culmination of this thesis will advance our understanding of the nucleation and behavior of acoustic cavitation from pulsed focused ultrasound and develop innovative systems to improve the efficacy, efficiency, and safety of clinical focused ultrasound therapies. / Doctor of Philosophy / Histotripsy is a noninvasive focused ultrasound method that precisely destroys target tissues such as tumors through the acoustic generation of cavitation and is currently being developed for numerous clinical applications. Histotripsy uses high-pressure, short-duration pulsed soundwaves to cause the bubbles to rapidly expand and collapse within a precise region called the focus. This rapid cavitation results in large mechanical strain in the targeted tissue. With increasingly higher pressure, numerous bubbles form in the shape of cavitation "bubble clouds" that create lesions, closely matching their shape, in the target tissue after a sufficient number of pulses have been applied. Despite advances in histotripsy, additional research is still needed to better understand the initiation of the acoustic cavitation process in histotripsy and its effects on focused ultrasound therapies. This understanding is critical to better predict and control ablation procedures, improve procedure efficiency, reduce off-target cavitation events for safer focused ultrasound therapies, and further increase ablation precision for procedures near critical structures or treatments without active image guidance. In this dissertation, I investigate the initiation, growth, and collapse of ultrasonically generated acoustic cavitation for novel applications of focused ultrasound. My Ph.D. thesis focuses on (1) investigating the effect of histotripsy pulsing parameters on bubble cloud cavitation initiation, bubble growth and collapse, and treatment efficiency, (2) investigating the effect of particle characteristics on the threshold for cavitation initiation and resulting bubble behavior for therapeutic applications, and (3) adjusting pulsing parameters to optimize ablation efficiency in conventional and particle mediated histotripsy. The culmination of this thesis will advance our understanding of the initiation and behavior of acoustic cavitation from pulsed focused ultrasound and develop innovative systems to improve the efficacy, efficiency, and safety of clinically focused ultrasound therapies.

Histotripsy is an emerging non-invasive, non-thermal, image-guided cancer ablation modality that has recently been approved for its first clinical trial in the United States (NCT04573881). Histotripsy utilizes focused ultrasound to generate acoustic cavitation within a tumor to mechanically fractionate targeted tissues. While pre-clinical work has demonstrated the feasibility of applying histotripsy to solid tumors including primary liver and renal tumors, there is still a need to investigate the potential of histotripsy to treat additional malignancies. In investigating the potential for treating other malignancies there are two avenues that need to be considered: 1) the feasibility for treating tissues with more complex stromal structures and 2) the ability of histotripsy to modulate the tumor microenvironment. To determine the safety and feasibility of additional applications of histotripsy, we conducted dose studies ex vivo on human tumors and human liver to establish dosimetry metrics for applying histotripsy to more fibrotic tumors such as cholangiocarcinoma while sparing nearby critical structures, such as bile ducts and blood vessels. Learning the safety dose-margins from the excised tissues, we performed an in vivo study using mice bearing patient-derived xenograft cholangiocarcinoma tumors. With this model, we were able to demonstrate our ability ablate the stiff cholangiocarcinoma tumors without causing any debilitating off- target damage. To gain a more robust understanding of the effects of histotripsy ablation on potentially difficult to treat tumors, we developed a porcine xenograft tumor model and utilized veterinary cancer patients. These studies have helped established protocols for utilizing histotripsy with ultrasound guidance to treat tumors that are more difficult to treat and can withstand mechanical ablation, including pancreatic adenocarcinoma, osteosarcomas, and soft tissue sarcomas. Pigs share many similarities with human anatomy and physiology, making them an ideal model organism for testing new medical devices and regimes for treating new targets. Using pigs, we were able to establish a procedure to utilize histotripsy to target the pancreas in vivo without causing any lasting or major side effects, such as off-target damage or pancreatitis. One limitation to the porcine model and veterinary patients, is the limitation of gaining rapid insight into the immunological effects of histotripsy. Established cancer mouse models offer the opportunity to rapidly test many organisms with an intact immune system. We used these mice to study pancreatic adenocarcinoma to determine the immune response after histotripsy ablation. For these tumors the general response was an increase in immune cell infiltration post-treatment and a shift in the tumor microenvironment to a more anti-tumor environment. The results of this dissertation provide insight into establishing protocols for treating new types of tumors with histotripsy and immunological effects that lay groundwork for improving future co-therapeutic treatment planning. Future work will aim to translate histotripsy into clinical applications and determining co-therapies that can help control metastasis. / Doctor of Philosophy / Histotripsy is a new medical therapy that can remove tumors without the need for surgery, with the first clinical trial in the United States starting this year, 2021. This therapy uses focused ultrasound waves to generate powerful microscopic bubbles that can rapidly destroy targeted tissues with a high-degree of precision. Early studies on histotripsy have demonstrated the ability of histotripsy to ablate tumors of the liver and kidneys. In order to be able to fully use this therapy on more difficult to target and treat cancers more studies are needed. Given that histotripsy uses physical forces to destroy targets, stronger, more fibrotic tumors and cancers that have begun to spread throughout the body will be more difficult to treat will need more than simple tumor removal to better treat these patients. Therefore, when investigating new cancer applications of histotripsy, it is important to consider the physical features of the tumors as well as the ability of histotripsy to initiate an immune response against the cancer. To determine the safety and feasibility of additional applications of histotripsy, we conducted dose studies on excised human tumors and human liver to see what doses of histotripsy are required to ablate stronger tumors, such as bile duct tumors. Learning the potential safety margins of doses from the excised tissues, we conducted a study using a mouse model to grow stiff, human tumors. With this model, we were able to show that it is possible to ablate the stiffer tumors without causing any major off-target damage. While it is useful to prove in excised tissues and mice that we can treat certain tumors, there is an additional need to study the therapy in a model that is more similar in size and anatomy to humans. Therefore, to gain a better understanding of the effects of histotripsy on potentially difficult to target and ablate tumors, we developed a novel porcine tumor model that can support the growth of human tumors and utilized veterinary cancer patients. These studies have helped established protocols for utilizing histotripsy to treat difficult to physically ablate tumors and difficult to ultrasound target tumors, including pancreatic and bone cancers. Established cancer mouse models offer the opportunity to rapidly test many organisms with an intact immune system. We used these mice to study pancreatic cancer to determine the immune response after histotripsy ablation. For this tumor type, while there were slight differences, the general response was an increase in immune cell infiltration of the tumors post-treatment and a shift to a stronger immune response against the tumor. The results of this dissertation provide insight into establishing protocols for treating new types of tumors with histotripsy and immune effects that lay groundwork for improving future co-therapeutic planning. Future work will aim to translate histotripsy into clinical applications and determining co-therapies that can help control body-wide disease.

Histotripsy is a non-thermal, non-invasive, focused ultrasound ablation method that uses acoustic cavitation to mechanically break down tissues [1-8]. Histotripsy is heavily dependent on the mechanical properties of the tissue, allowing it to mechanically ablate tissues of lower mechanical stiffness while preserving the stiffer critical structures [15]. However, the mechanical properties of clinically relevant abdominal tissues and critical structures have not yet been adequately quantified under uniform testing parameters. Previous studies have tested and modeled the tissue selectivity of histotripsy, but these studies have been limited by the lack of mechanical property data available for these tissue types. In addition, there remains a need for additional experimental studies directly comparing the differential treatment doses required to induce histotripsy tissue damage in intra-abdominal tissue types. This thesis investigates the mechanical properties of intra-abdominal tissues under uniaxial tension, the effect of histotripsy treatment dose on intra-abdominal soft tissues and critical structures, and the potential of inducing damage to critical structures along the acoustic path pre-focal to the targeted histotripsy treatment. Results show that there are significant differences between the parenchymal tissues (liver, kidney) and the critical structure (stomach, gallbladder, small intestine, ducts, and vessels) elastic modulus, yield stress, yield strain, post-yield strain, energy to yield, and maximum stress and strain at yield. In general, histology analysis from the histotripsy experiments showed that there was an increase in tissue damage with increasing histotripsy pulses/point for all tissues. Critical structures with higher mechanical strength were more resistant to ablation compared to tissues with lower mechanical strength. Pre-focal studies showed damage to gallbladder and small intestine only in cases in which pre-focal cavitation was observed, while no damage occurred in skin and stomach for any samples treated at varying distances from the bubble cloud. Overall, this work improves our understanding of tissue selectivity of histotripsy and provides mechanical properties measurements for clinically relevant tissues that can be used to improve predictive models of tissue-selective histotripsy treatments. This work can be used in the planning of histotripsy treatments to establish proper margins of safety for treating intra-abdominal tumors. / Master of Science / Histotripsy is a non-invasive cancer treatment that mechanically breaks down tissues by rapidly forming and bursting bubbles within the tumor [1-8]. Histotripsy is heavily dependent on the mechanical properties of the tissue, allowing it to destroy weaker tissues while preserving the stiffer tissues in the surrounding area [15]. The mechanical properties of clinically relevant intra-abdominal tissues have not been quantified under uniform testing parameters. Previous studies have tested and modeled the tissue selectivity of histotripsy, but these studies have been limited by the mechanical property data available. This thesis investigates the mechanical properties of intra-abdominal tissues under tension, the effect of histotripsy treatment dose on intra-abdominal tissue damage, and the damage to critical structures from histotripsy treatment at varying distances from the tissue. Results show that there are significant differences between the liver and kidney mechanical stiffness and strength compared to the other tissues. In general, histology analysis showed that there is an increase in tissue damage with increasing histotripsy dose. Tissues with higher mechanical strength were more resistant to damage at lower doses compared to tissues with lower mechanical strength. Histotripsy damage to critical structures that are along the beam path, set distances in front of the focal point of the cavitation bubble cloud was studied. This study showed damage to gallbladder and small intestine only in cases in which pre-focal cavitation, cavitation bubbles that are not within the focal point of the cloud but are in contact with the tissue, was observed, while no damage occurred in skin and stomach for any samples treated at varying distances from the bubble cloud. Overall, this work improves our understanding of tissue selectivity of histotripsy and provides mechanical properties for clinically relevant tissues that can be used to improve predictive models of tissue-selective histotripsy treatments. This work can be used in the planning of histotripsy treatments to establish proper margins of safety for treating intra-abdominal tumors.

Histotripsy is a non-invasive, non-thermal, and non-ionizing therapy that utilizes converging high-pressure ultrasound waves at a focal point to produce cavitation and induce mechanical tissue destruction. Currently, rapid prototyped histotripsy transducers consist of multiple elements and are made using 3D printing methods. Multi-element transducers introduce size constraints and 3D printing has limitations in material choice, cost, and time for larger scale manufacturing. This thesis investigates the development of rapid prototyped single element histotripsy transducers and the use of injection molding for transducer fabrication, utilizing an in-house metal CNC mill for mold manufacturing and a desktop injection molding machine. Nylon 101 and 30% glass-filled nylon were chosen as the plastics to inject as these were found to have the most similar acoustic properties to WaterShed, an ABS-like plastic currently used. Six single-element transducers were constructed with a 2 MHz curved Pz26 piezoceramic disc: two with SLA 3D printed housing, two with SLS 3D printed housing, and two with injection molded housing. Electrical impedance, beam dimensions, focal pressure output, and cavitation were characterized for each element. The results show that rapid prototyped single element transducers can generate enough pressure to perform histotripsy. This marks the development of the first rapid prototyped single element histotripsy transducer and further confirms that injection molding can produce transducers comparable, if not identical or potentially superior, to 3D printed counterparts. Future work aims to further characterize these transducers, explore more material options, and apply injection molding to various transducer designs while optimizing both CNC and injection molding parameters. / Master of Science / Histotripsy is a form of cancer therapy that can non-invasively treat tumors using focused ultrasound waves. Focused ultrasound transducers are used to achieve this and are currently prototyped using 3D printing. However, these methods are limiting in material options and upscale manufacturing. Many of these devices currently used tend to be larger in size, comparable to the size of a mixing bowl, which limits its applications. This thesis investigates the development of single element histotripsy transducers and the use of injection molding for transducer fabrication, using an in-house metal CNC mill for mold manufacturing and desktop injection molding machine. Nylon 101 and 30% glass-filled nylon were chosen as the plastics to inject due to their ideal acoustic properties. Six single-element transducers were constructed: two with SLA 3D printing, two with SLS 3D printing, and two with injection molding. All transducers were tested and compared against each other. The results show that 3D printed single element transducers can perform histotripsy and that injection molding can produce comparable results. Future work should continue to test and characterize these transducers, explore more material options for injection molding, apply injection molding to other transducer designs, and optimize CNC and injection molding parameters.

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.

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.

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.

The heterogeneity of pancreatic cancer renders many available general therapies ineffective holding the five-year survival rate close to 10% for decades. Surgical resection eligibility, resistance to chemotherapy and limited efficacy of immunotherapy emphasize the dire need for diverse and innovative treatments to combat this challenging disease. This study evaluates co-therapy strategies that combine non-thermal, minimally invasive ablation technology and targeted drug delivery to enhance treatment efficacy. Our research begins by uncovering the multifaceted potential of Irreversible Electroporation (IRE), a cutting-edge non-thermal tumor ablation technique. This study demonstrates IRE-mediated ability to trigger programmed necrotic cell death, induce cell cycle arrest, and modulate immune cell populations within the tumor microenvironment. This transformation from a pro-tumor state to a proinflammatory milieu, enriched with cytotoxic T lymphocytes and neutrophils. IRE-induced proinflammation in the tumor site renders immunologically "cold" tumor into immunologically "hot" tumor and holds significant promise of improving treatment efficacy. Notably, IRE-treated mice exhibited an extended period of progression-free survival, implying clinical potential. The transient nature of these effects suggests potential mechanisms of tumor recurrence highlighting the need for further studies to maximize the efficacy of IRE. Our mechanistic studies evaluated the IFN-STAT1-PD-L1 feedback loop as a possible reason for pancreatic tumor recurrence. Our data also suggest a stronger IFN-PD-L1 feedback loop compared to mammary, osteosarcoma and glioblastoma tumors rendering pancreatic cancer immunologically "cold". This study also investigates the use of histotripsy (a non-thermal, noninvasive, nonionizing ultrasound-guided ablation modality) to treat pancreatic cancer utilizing a novel immunocompromised swine model. We successfully generated human orthotopic pancreatic tumors in the immune deficient pigs, which allowed for consequent investigation of clinical challenges presented by histotripsy. While rigorous clinical studies are indispensable for validation, the promise of histotripsy offers new hope for patients. In parallel, we used our immunocompromised swine model of orthotopic pancreatic cancer to investigate the SonoTran® system, which employs ultrasound-activated oscillating particles to enhance drug delivery within hard-to-reach tumors. Our study demonstrates that SonoTran® significantly enhances the intratumoral penetrance of therapeutic agents, including commonly used chemotherapy drugs like paclitaxel and gemcitabine. Additionally, SonoTran® improved delivery of the anti-epidermal growth factor (EGFR) monoclonal antibody, cetuximab- which is frequently used in cancer immunotherapy. Together, our findings address challenges in the delivery of a range of therapeutics while simultaneously exposing challenges like off-target damage. In conclusion, this study presents a multifaceted approach to confront the complex characteristics of pancreatic cancer. Given the variations in patient response and the complexity of the disease, it is clear that a singular solution is unlikely. Our research, which combines IRE, histotripsy, and SonoTran®, to interrogate a promising array of tools to tackle different challenges to provide tailored treatments. In the ever-evolving landscape of pancreatic cancer therapy, this research opens new avenues to investigate deeper into molecular mechanisms, co-therapy treatment options, future preclinical and clinical studies which eventually encourage the potential for improved patient outcomes. / Doctor of Philosophy / Pancreatic cancer is a formidable disease, known for its late-stage diagnosis and limited treatment options with a poor 5-year survival rate of ~10%. However, a promising frontier in the battle against this lethal disease has emerged through combining mechanical, cell based and immunotherapies to attack the cancer from multiple angles at once. In my PhD research, I explored novel approaches to transform the landscape of pancreatic cancer treatment. We began by investigating Irreversible Electroporation (IRE), a non-thermal method to ablate tumors. Beyond its known function of reducing tumor size, IRE initiated programmed necrotic cell death, halted tumor cell division, and triggered changes in the immune landscape within the tumor. In response to IRE treatment, the immune environment shifted from pro-tumor to proinflammatory state, showing potential for clinical use. Mice treated with IRE experienced extended cancer progression-free survival temporarily, followed by eventual relapse. During relapse, we found that immune cells reverted back to their original, pre- IRE treated state. This observation logically implies combining IRE and immune checkpoint inhibitors aimed towards maintaining the IRE-altered immunological environment. Next, we developed and used novel pig models that closely resemble human pancreatic cancer patients to test histotripsy, a first phase toward making histotripsy as a non-invasive treatment approach for pancreatic cancer. Use of orthotopic tumor in a large animal model and clinical device allowed us to expose some challenges of ultrasound guidance of histotripsy. Notably, the treatment results in partial ablation and a reduction in stroma materials, which play a role in the tumor's resistance to commonly used treatments. While rigorous clinical studies are needed for validation, this approach offers hope in the quest for innovative pancreatic cancer treatment. Another promising approach we investigated involves SonoTran® particles, ultrasound-activated oscillating particles that can increase drug absorption in a targeted fashion. Our study demonstrated increased concentrations of commonly used therapeutic agents within tumors through SonoTran®-facilitated delivery, providing an effective means to overcome drug delivery issues within pancreatic tumors. There is no one size fits all treatment to address the complexity of pancreatic cancer. The future of treatment lies in the integration of IRE, histotripsy and SonoTran® into clinical practice. In summary, this PhD research identified promising novel technologies and combinations of treatments for pancreatic cancer, reaffirming the importance of exploring innovative solutions to combat pancreatic cancer. The dynamic nature of the pancreatic tumor microenvironment underscores the importance of further research to extend the positive impacts of these treatments and improve tumor debulking.