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.