Investigating the immunomodulatory effects of histotripsy ablation for osteosarcoma

Abstract

Abstract Osteosarcoma (OS) is the most common primary bone cancer in humans and dogs. Canine OS shares many biological similarities with human OS and is a valuable comparative oncology research model. In both species, metastasis remains a major hurdle against improved survival, thus warranting a novel therapeutic approach such as histotripsy, a non-thermal, non-invasive, focused ultrasound ablation modality. Histotripsy can mechanically disintegrate gross tumor and potentially induces an anti-tumor immune response to inhibit metastatic development. The objectiveof this in vitro study was to investigate the immunomodulatory effects of histotripsy ablation of canine OS. We hypothesizedthat histotripsy ablation of OS promotes a pro-inflammatory immune signature in primary monocytes and macrophages. We isolated CD14+ primary monocytes using positive selection immunomagnetic cell separation from peripheral blood mononuclear cells of healthy dogs, and differentiated primary macrophages by culturing primary monocytes with canine GM-CSF for 72 hours. We co-cultured primary monocytes and macrophages with histotripsy-ablated canine D17 OS cells for 24 hours, then characterized their cell surface markers using flow cytometry and performed multiplex supernatant cytokine analysis. We observed upregulation of CD80 and CD62L on primary monocytes and a downregulation of CD206 on macrophages exposed to histotripsy-ablated D17 cells compared to controls, suggesting immune activation. We observed decreased levels of the pro-tumor cytokine, TGF-β, from primary monocytes exposed to histotripsy-ablated D17s compared to controls. Our results indicate that histotripsy ablation of OS can induce a pro-inflammatory immune signature. This work is supported by grants from the American Kennel Club Canine Health Foundation, the Focused Ultrasound Foundation.