Abstract A030: Investigating the feasibility of in-vivo histotripsy ablation for osteosarcoma using an orthotopic murine model and a canine model of spontaneous disease
Authors: Hay Alayna N. , Ruger Lauren , Hixson Haleigh , Gannon Jessica , Simon Alex , Sheppard Hannah , Coutermarsh-Ott Sheryl , Davis Elaina , Kierski Katharine , Ciepluch Brittany , Neufeld Nathan , Vlaisavljevich Eli , Tuohy Joanne 
Published: September 2022

Abstract
Osteosarcoma (OS) is the most commonly occurring primary bone tumor in pediatric cancer patients and dogs, with estimated incidences of 3-4 cases per 1 million and 14 cases per 100,000, respectively. The biological similarities shared between human and canine OS and increased OS prevalence in dogs allows the dog to serve as an effective comparative oncology research model. Current standard of care treatment results in a 70% median 5-year survival for humans and a 10-12-month median survival for dogs, and these survival expectations have not improved in 30 years. There is a critical need for novel treatment options to improve the prognosis for OS patients. The objective of this study was to investigate the ablative (murine, canine) and immunological (canine) effects of histotripsy, a novel focused ultrasound tissue ablation technique, for OS. We utilized orthotopic xenograft murine models with induced canine (D17) or human (143B) OS tumors. Ablative outcomes were assessed grossly and microscopically via histopathological evaluation, and treatment effects on limb functionality were evaluated by monitoring lameness, grip capacity, nesting behavior, and body weight for the duration of the study. Fifteen client-owned dogs with suspected OS were enrolled into a clinical trial and underwent histotripsy treatment 24 hours prior to standard of care limb amputation surgery. Ablative outcomes were assessed grossly and microscopically via histopathological evaluation after surgical excision of treated tumors. Immunological outcomes were evaluated by peripheral blood samples collected pretreatment, 24 hours, and 2 weeks post treatment for immune cell phenotyping via flow cytometry to evaluate the systemic immune response. Locally, changes in the tumor immune microenvironment were assessed at both the cellular and molecular levels. Targeted ablation of the tumor tissue was achieved in both the murine models and the canine patients. Ablation was evident by gross and microscopic evaluation where loss of cellular architecture, lytic and coagulative necrosis and delineation of treated and untreated tumor regions were observed. In the mice, decreased limb function due to inflammation after histotripsy ablation resolved within 5-7 days post treatment. The dogs did not exhibit any increase in lameness after histotripsy ablation. The only clinical adverse event experienced by canine patients was mild transient dermal erythema at the treatment site. Our results suggest systemic immunomodulation evident by increases in CD4+ and CD8+ T lymphocyte populations post treatment and changes in monocyte expression of CD62L and CD80. Locally, we observed an upregulation in genes related to chemotaxis, regulation of inflammation, and immunogenic cell death in treated tumor samples compared to untreated samples. In conclusion our preliminary results suggest that histotripsy treatment has both immunotherapeutic and ablative potential and support the development of histotripsy as a tumor ablation technique and immunotherapeutic for OS.
Citation Format: Alayna N. Hay, Lauren Ruger, Haleigh Hixson, Jessica Gannon, Alex Simon, Hannah Sheppard, Sheryl Coutermarsh-Ott, Elaina Davis, Katharine Kierski, Brittany Ciepluch, Nathan Neufeld, Eli Vlaisavljevich, Joanne Tuohy. Investigating the feasibility of in-vivo histotripsy ablation for osteosarcoma using an orthotopic murine model and a canine model of spontaneous disease [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A030.