Pancreatic cancer is a significant cause of cancer-related deaths in the United States with an abysmal five-year overall survival rate that is under 9%. Reasons for this mortality include the lack of late-stage treatment options and the immunosuppressive tumor microenvironment. Histotripsy is an ultrasound-guided, noninvasive, nonthermal tumor ablation therapy that mechanically lyses targeted cells. To study the effects of histotripsy on pancreatic cancer, we utilized an in vitro model of pancreatic adenocarcinoma and compared the release of potential antigens following histotripsy treatment to other ablation modalities. Histotripsy was found to release immune-stimulating molecules at magnitudes similar to other nonthermal ablation modalities and superior to thermal ablation modalities, which corresponded to increased innate immune system activation in vivo. In subsequent in vivo studies, murine Pan02 tumors were grown in mice and treated with histotripsy. Flow cytometry and rtPCR were used to determine changes in the tumor microenvironment over time compared to untreated animals. In mice with pancreatic tumors, we observed significantly increased tumor-progression-free and general survival, with increased activation of the innate immune system 24 h posttreatment and decreased tumor-associated immune cell populations within 14 days of treatment. This study demonstrates the feasibility of using histotripsy for pancreatic cancer ablation and provides mechanistic insight into the initial innate immune system activation following treatment. Further work is needed to establish the mechanisms behind the immunomodulation of the tumor microenvironment and immune effects.

Cancer is the second leading cause of death worldwide despite major advancements in diagnosis and therapy over the past century. One of the most debilitating aspects of cancer is the burden brought on by metastatic disease. Therefore, an ideal treatment protocol would address not only debulking larger primary tumors but also circulating tumor cells and distant metastases. To address this need, the use of immune modulating therapies has become a pillar in the oncology armamentarium. A therapeutic option that has recently emerged is the use of focal ablation therapies that can destroy a tumor through various physical or mechanical mechanisms and release a cellular lysate with the potential to stimulate an immune response. Histotripsy is a non-invasive, non-ionizing, non-thermal, ultrasound guided ablation technology that has shown promise over the past decade as a debulking therapy. As histotripsy therapies have developed, the full picture of the accompanying immune response has revealed a wide range of immunogenic mechanisms that include DAMP and anti-tumor mediator release, changes in local cellular immune populations, development of a systemic immune response, and therapeutic synergism with the inclusion of checkpoint inhibitor therapies. These studies also suggest that there is an immune effect from histotripsy therapies across multiple murine tumor types that may be reproducible. Overall, the effects of histotripsy on tumors show a positive effect on immunomodulation.

Histotripsy is a noninvasive, nonionizing, and nonthermal focused ultrasound ablation method that is currently being developed for the treatment of liver cancer. Promisingly, histotripsy has been shown for ablating primary [hepatocellular carcinoma (HCC)] and metastatic [colorectal liver metastasis (CLM)] liver tumors in preclinical and early clinical studies. The feasibility of treating cholangiocarcinoma (CC), a less common primary liver tumor that arises from the bile ducts, has not been explored previously. Given that prior work has established that histotripsy susceptibility is based on tissue mechanical properties, there is a need to explore histotripsy as a treatment for CC due to its dense fibrotic stromal components. In this work, we first investigated the feasibility of histotripsy for ablating CC tumors in vivo in a patient-derived xenograft mouse model. The results showed that histotripsy could generate CC tumor ablation using a 1-MHz small animal histotripsy system with treatment doses of 250, 500, and 1000 pulses/point. The second set of experiments compared the histotripsy doses required to ablate CC tumors to HCC and CLM tumors ex vivo. For this, human tumor samples were harvested after surgery and treated ex vivo with a 700-kHz clinical histotripsy transducer. Results demonstrated that significantly higher treatment doses were required to ablate CC and CLM tumors compared to HCC, with the highest treatment dose required for CC tumors. Overall, the results of this study suggest that histotripsy has the potential to be used for the ablation of CC tumors while also highlighting the need for tumor-specific treatment strategies.

New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely noninvasive, nonthermal, and nonionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant preclinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients.

Pericytes are critical for microvascular stability and maintenance, among other important physiological functions, yet their involvement in vessel formation processes remains poorly understood. To gain insight into pericyte behaviors during vascular remodeling, we developed two complementary tissue explant models utilizing ‘double reporter’ animals with fluorescently-labeled pericytes and endothelial cells (via Ng2:DsRed and Flk-1:eGFP genes, respectively). Time-lapse confocal imaging of active vessel remodeling within adult connective tissues and embryonic skin revealed a subset of pericytes detaching and migrating away from the vessel wall. Vessel-associated pericytes displayed rapid filopodial sampling near sprouting endothelial cells that emerged from parent vessels to form nascent branches. Pericytes near angiogenic sprouts were also more migratory, initiating persistent and directional movement along newly forming vessels. Pericyte cell divisions coincided more frequently with elongating endothelial sprouts, rather than sprout initiation sites, an observation confirmed with in vivo data from the developing mouse brain. Taken together, these data suggest that (i) pericyte detachment from the vessel wall may represent an important physiological process to enhance endothelial cell plasticity during vascular remodeling, and (ii) pericyte migration and proliferation are highly synchronized with endothelial cell behaviors during the coordinated expansion of a vascular network.

Pancreatic cancer is a malignant disease associated with poor survival and nearly 80% present with unresectable tumors. Treatments such as chemotherapy and radiation therapy have shown overall improved survival benefits, albeit limited. Histotripsy is a noninvasive, non-ionizing, and non-thermal focused ultrasound ablation modality that has shown efficacy in treating hepatic tumors and other malignancies. In this novel study, we investigate histotripsy for noninvasive pancreas ablation in a pig model. In two studies, histotripsy was applied to the healthy pancreas in 11 pigs using a custom 32-element, 500 kHz histotripsy transducer attached to a clinical histotripsy system, with treatments guided by real-time ultrasound imaging. A pilot study was conducted in 3 fasted pigs with histotripsy applied at a pulse repetition frequency (PRF) of 500 Hz. Results showed no pancreas visualization on coaxial ultrasound imaging due to overlying intestinal gas, resulting in off-target injury and no pancreas damage. To minimize gas, a second group of pigs (n = 8) were fed a custard diet containing simethicone and bisacodyl. Pigs were euthanized immediately (n = 4) or survived for 1 week (n = 4) post-treatment. Damage to the pancreas and surrounding tissue was characterized using gross morphology, histological analysis, and CT imaging. Results showed histotripsy bubble clouds were generated inside pancreases that were visually maintained on coaxial ultrasound (n = 4), with 2 pigs exhibiting off-target damage. For chronic animals, results showed the treatments were well-tolerated with no complication signs or changes in blood markers. This study provides initial evidence suggesting histotripsy's potential for noninvasive pancreas ablation and warrants further evaluation in more comprehensive studies.