Abstract B020: Investigating the immunomodulatory effects of combined histotripsy and N-dihydrogalactochitosan: A novel non-invasive therapeutic strategy for osteosarcoma.

Abstract Background: Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, known for its aggressive nature and high potential for metastasis. Despite standard of care, the 5-year survival rate in OS patients remains at 60%-70%, with the primary cause of death being metastatic disease. Histotripsy, an emerging non-invasive, non-thermal, and non-ionizing ablation technique, uses high- intensity focused ultrasound to mechanically destroy tumor tissues. This process can release damage-associated molecular patterns (DAMPs) and tumor antigens, which may stimulate an anti-tumor immune response. N-dihydrogalactochitosan (GC) is a novel immunostimulant previously reported to enhance systemic immune responses when administered intratumorally following local thermal tumor ablation. However, the combination of GC with a non-thermal ablation modality such as histotripsy remains unexplored. This study aimed to investigate the safety and feasibility of combining histotripsy and GC as a novel treatment modality to induce immunomodulation, which is crucial for mitigating metastatic disease. Methods: The syngeneic C3H/HeN mouse model with the DLM8 OS cell line was utilized for this study. Bilateral subcutaneous flank tumors were inoculated into the mice, with one tumor treated with histotripsy and GC, while the contralateral tumor was left untreated. Histotripsy was performed using a 1 MHz histotripsy transducer with 1-2 cycle pulses, a pulse repetition frequency (PRF) of 250 Hz, and 500 pulses per point. GC was administered intratumorally at a dose of 10 mg/mL, determined through a preliminary pilot study to ensure optimal immune response activation without adverse effects. Tumor growth in both treated and contralateral tumors was monitored. At 7-10 days post treatment the local tumor immune response was evaluated through histological analysis for overall immune cell infiltration and flow cytometry for immunophenotyping of T cells, dendritic cells and macrophages. Control groups included bilateral tumor-bearing mice treated with histotripsy only, GC only, or left untreated to evaluate the outcomes of histotripsy + GC treatment. Results: Initial data indicate that the combination of histotripsy and GC led to stabilization of tumor growth in both treated and contralateral tumors within the 7–10- day post-treatment observation period. In contrast, tumors in the control groups exhibited 80%-100% increase in their largest diameter during the same timeframe. We observed increased infiltration of neutrophils, eosinophils, and macrophages in the treated tumors of the combination group compared to the control groups, indicating robust local immune activation. Conclusion: Our preliminary findings suggest that combining histotripsy with GC may offer a therapeutic strategy for OS by stabilizing tumor growth and enhancing immune cell infiltration in the tumor microenvironment. Future directions will include long-term evaluations to assess the sustained efficacy and potential systemic immune responses induced by the combination therapy. Citation Format: Ny T.C. Luong, Elliana R Vickers, Alayna Hay, Sheryl L Coutermarsh-Ott, Samuel S.K. Lam, Wei R Chen, Eli Vlaisavljevich, Joanne Tuohy. Investigating the immunomodulatory effects of combined histotripsy and N-dihydrogalactochitosan: A novel non-invasive therapeutic strategy for osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B020.