Histotripsy-initiated immune response synergizes with chemotherapy in a neuroblastoma murine model

High-risk neuroblastoma (NB) is a pediatric malignancy associated with metastases and an immunosuppressive tumor microenvironment. Standard-of-care treatments like chemotherapy are often ineffective, which motivates the investigation of adjuvant approaches. Histotripsy is a noninvasive focused ultrasound therapy that ablates tissue through the mechanical action of bubble clouds. In addition to disruption of the targeted tumor, non-targeted lesions exhibit growth delay after the histotripsy procedure. The primary hypothesis of this study was histotripsy-induced shifts in the tumor microenvironment will improve the response of metastatic NB to chemotherapy. Female A/J mice flanks were inoculated bilaterally with 1x10 Neuro-2a cells. Histotripsy was applied to one tumor (200-500 mm3), with or without concurrent administration of liposomal doxorubicin (LDOX). The contralateral tumor served as a model of non-targeted distal metastases. Following treatment, tumors were monitored indefinitely for growth, or assessed after 5-7 days with flow cytometry, single-cell RNA sequencing, and immunohistochemistry. Histotripsy alone delayed the growth of treated and contralateral tumors relative to controls (p = 0.01 and p < 0.0001, respectively) and increased CD8 T and CD11b+ cells (p < 0.05 for both comparisons). Further, NB cells in targeted and contralateral tumors exhibited a decrease in Myc expression and cell-cycle activity, and upregulation of interferon and apoptosis pathways. Histotripsy combined with LDOX had the longest delay in tumor growth (p < 0.01) and greatest expression of CD8 and MOMA staining. These findings indicate that histotripsy induces a systemic antitumor immune response that potentiates chemotherapy efficacy in this model of metastatic NB. SignificanceMechanical ablation with histotripsy drives systemic antitumor immunity, reshapes the tumor microenvironment, and enhances chemotherapy efficacy in a syngeneic model of metastatic, high-risk neuroblastoma.