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 1×10⁶ 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.