Abstract A029: Histotripsy induces an anti-tumor immune response with an abscopal effect in a syngeneic neuroblastoma mouse model

Abstract

Background: Despite recent therapeutic advances, patients with high-risk neuroblastoma (NB) remain in need of improved treatment options. Histotripsy is a focused ultrasound therapy for nonthermal-tissue ablation via bubble activation. Recent pre-clinical and clinical reports suggest histotripsy stimulates an anti-tumor immune response in the adult population. We previously demonstrated that histotripsy ablation of NB xenograft tumors enhances tumor necrosis factor-alpha secretion, inducing apoptosis. We hypothesized that histotripsy induces both local and distal abscopal anti-tumor immune response in neuroblastoma, resulting in increased killing of tumor cells. Methods: Myc-expressing neuro-2a cells were subcutaneously and synchronously injected into each flank of immunocompetent A/J mice. Histotripsy was applied with a custom system to approximately 80% of one flank tumor when it reached 200 – 300 mm3. Six days later, single cell RNA sequencing (scRNAseq) libraries were prepared for histotripsy treated tumors, synchronous contralateral tumors, and tumors from untreated control mice. Sequencing files were processed with Cell Ranger and downstream analyses were done with Seurat, followed by Kruskal-Wallis test for cell proportions comparison. Flow cytometry was used to corroborate these findings using anti-CD8 (T cells) and CD11b (macrophages). Results: Tumors from untreated control mice were over two-fold larger than both histotripsy-treated tumors six days after treatment (p=0.03) as well as tumors contralateral to the histotripsy treated tumors (p<0.05), suggesting an abscopal effect. High-quality scRNAseq data were obtained from 3 untreated tumors and 2 each from histotripsy and contralateral tumors. The proportion of cytotoxic CD8+ T cells were significantly higher in histotripsy as well as contralateral tumors compared to untreated controls (p=0.029). Similarly, anti-tumor M1 macrophages had a non-significant trend higher (p=0.27) while pro-tumor M2 macrophages were significantly decreased (p=0.029) in controls compared to treated and contralateral tumors. These findings were validated with flow cytometry of both CD8 and CD11b markers (p<0.05, respectively). NB cells from treated and contralateral tumors had decreased Myc expression (p<0.001) in addition to suppression of cell cycle and growth-related pathways relative to control tumors, including Myc targets, G2M checkpoint, and E2F targets pathways (p<0.001, respectively). Treated and contralateral tumor cells also had activated expression pathways of inflammatory response, regulation of chemokines and cytokines, response to interferon, and antigen processing and presentation when compared to controls (p<0.001, respectively). Conclusions: Our findings show that histotripsy can generate an immune cell activation response in treated and distal tumors in NB models, reducing proliferation of the targeted tumor as well as having an abscopal effect. These findings suggest that histotripsy may sensitize NB tumors to T cell dependent targeted strategies such as PD-1 inhibition. Citation Format: Yuqing Xue, Natalia Antonides-Jensen, Fernando Flores-Guzman, Lydia L. Wu, Jacky Gomez-Villa, Muskan Singh, Kenneth B. Bader, Sonia L. Hernandez, Mark A. Applebaum. Histotripsy induces an anti-tumor immune response with an abscopal effect in a syngeneic neuroblastoma mouse model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2): nr A029.