Pancreatic cancer is a significant cause of cancerrelated deaths in the United States with an abysmal 5-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, non-invasive, non-thermal 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 non-thermal 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 hours post-treatment 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.

Histotripsy is a novel, non-thermal, image guided ablation modality that can rapidly kill cells in a targeted region with millimeter precision. Previous work established that there is an immune response to histotripsy ablation, however a mechanism behind this response has not been established. For the current study, we utilized an in vitro model, Pan02 (pancreatic adenocarcinoma), transfected with a known antigen (hemaglutanin/HA), and verified findings in vivo. Cells were ablated at therapeutic and sub-therapeutic doses to determine the release of damage associated molecular pattern (DAMP). For both treatments, gel electrophoresis confirmed the release of damaged DNA, and western blots verified the release of intact HMGB1 and HA proteins. To determine the efficacy of the released DNA to stimulate an immune response, we exposed naïve macrophages to isolated DNA from the treatment supernatant and showed activation by 24 hours. Additionally, to show the potential for a systemic immune response, complete supernatant was given to naïve dendritic cells, that were in turn co-cultured with naïve T cells. CD4+ and CD8+ T cell activation was confirmed with IL-2 and INFg ELISAs as well as flow cytometry. To verify these findings, mice with Pan02 tumors were treated, and demonstrated similar DAMP and antigen release. Further, changes in immune cell populations within the tumor were consistent, with increased macrophages and dendritic cells 24 hours and 7 days and in T cell populations at 7 and 14 days after treatment. These results show the role of specific DAMPs and immune cell populations in responding to histotripsy ablation. Overall, these results indicate a robust immunomodulatory response to histotripsy tumor ablation.

While surgery, chemotherapy, and radiotherapy are available as treatments for osteosarcoma, there is only a 60% 5 year overall survival rate. To address this need, novel therapies are being developed and investigated. Histotripsy is a non-invasive, non-thermal focused-ultrasound ablation therapy that uses acoustic cavitation to mechanically lyse targeted cells. Recent evidence suggests that it is immunomodulatory, and can shift the tumor microenvironment to being more pro-inflammatory. However, given histotripsy’s mechanical mechanism, it is not established to be able to treat stronger tissues such as osteosarcomas at a dose that will allow for the preservation of immune stimulating molecules. In this study, mice with subcutaneous osteosarcoma DLM8 tumors were treated with histotripsy and acute changes to the local tumor immune response were analyzed 72 hours later. Using partial ablation to avoid off-target damage, a 40% average reduction in tumor size was achieved with targeted tissues being fully ablated. Through rtPCR, the levels of various cytokines associated with wound healing were significantly increased in the treated tumors following histotripsy treatment, with the greatest fold change observed in the concentrations for IL-6 and IL-13 over a given period of time. An increase in fold change for CD274, also known as PD-L1, was also observed, implicating the potential to pair histotripsy with checkpoint inhibitors for osteosarcoma therapy. The overall results of the study suggest that histotripsy is able to shift the tumor microenvironment following partial-ablation treatment. Thus, upon activation of the immune system, wound and tissue repair mechanisms are subsequently activated and responses are regulated.