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Osteosarcoma (OS) is the most common primary bone tumor in dogs and humans and is considered an immunologically “cold” tumor. OS exerts devastating impacts on patients and has not seen significant improvements in survival outcomes in decades, with resistant metastatic disease remaining a main cause of death. Innovative therapies are profoundly needed to improve treatment and outcomes in OS. Canine OS shares striking similarities with human OS but occurs at a greater prevalence, allowing the dog to serve as a valuable comparative oncology research model. Histotripsy, a novel non-thermal focused ultrasound technique, has potential to ablate the primary tumor and activate an immune response to mitigate metastatic disease, thus promising to turn OS into an immunologically “hot” tumor. The objective of this study was to evaluate the immunomodulatory effects of histotripsy after in vitro and in vivo OS ablation. We utilized in vitro cell culture models for mechanistic cell death and immune response evaluation. We delivered histotripsy in vivo to canine OS patients and assessed peripheral immune cells and the tumor microenvironment. Across species, after in vitro exposure of immune cells to histotripsy-ablated OS cells, we observed cellular phenotypic changes indicative of activation and upregulation of genes associated with immune cell chemotaxis, immune and inflammatory responses such as NK cell-mediated cytotoxicity. In vivo, histotripsy ablation led to significantly greater expression of activation markers CD80 and CD62L on circulating monocytes at 1 and 5 days post histotripsy (DPH) respectively. We observed significantly higher production of TGFβ in circulating monocytes at 3 DPH compared to baseline. On an independent patient basis, we observed a greater population of infiltrating CD5+CD4+ and CD5+CD8+ T-lymphocytes within the ablated compared to unablated regions of tumor, and noted intratumoral genetic signatures that indicate immune activation at 24 hours post histotripsy. Collectively our results suggest that histotripsy ablation of OS leads to immune activation.
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