Histotripsy therapy elicits distinct anti-tumor immune responses unique from radiotherapy

Radiotherapy (RT) is a non-invasive approach that uses high-dose radiation to damage tumor cell DNA, leading to cell death. Histotripsy (HT) is the first non-invasive, non-thermal therapy that uses ultrasound pulses to destroy tumor tissue. It is unknown how RT and HT may differentially modulate the tumor immune microenvironment to enable tumor control. Therefore, we directly compared the immune responses following RT and HT in a murine model of pancreatic adenocarcinoma. While both RT and HT treatments induced local tumor control, single-cell RNA sequencing of CD45 + tumor-infiltrating lymphocytes revealed that RT and HT induce distinct immune responses. Within the myeloid compartment, only RT promoted a type I IFN response, while both RT and HT induced a TNF response. Within the T cell compartment, RT promoted the proliferation and type I IFN response of CD8 + T cells, while HT promoted expansion of immunosuppressed CD8 + and CD4 + T cells. Combination of RT or HT with anti-PD-L1 therapy enhanced local tumor control and delayed abscopal tumor growth through distinct mechanisms. Addition of anti-PD-L1 during HT treatment enhanced myeloid antigen presentation and TNF response pathway in myeloid, CD4 + , and CD8 + T cells compared to RT + anti-PD-L1. Furthermore, while RT + anti-PD-L1 promoted the expansion of proliferative CD8 + T cells, HT + anti-PD-L1 promoted the expansion of central memory and early effector CD8 + T cells with an enhanced T cell activation signature. Therefore, this study shows that combination of HT with anti-PD-L1 enhances anti-tumor immune mechanisms unique from RT.