Abstract 1700: Immune effects of high intensity focused ultrasound ablation with checkpoint inhibitors in murine neuroblastoma
Authors: Tydings Caitlin , Eranki Avinash , Sharma Karun V. , Kim AeRang , Srinivasan Priya , Lazarski Christopher , Rossi Christopher 
Published: July 2021

Abstract
Background: Pediatric solid tumors have not shared in the success of immunotherapy, possibly due to mechanisms impeding an effective immune response. High intensity focused ultrasound (HIFU) ablation uses ultrasound energy to non-invasively and precisely destroy targeted tumor tissue and can cause local inflammatory responses. Our group previously showed a significant survival benefit with increased antitumor cytokine and cellular response treating refractory, murine neuroblastoma (N2a) with HIFU boiling histotripsy (BH) and αCTLA-4 + αPD-L1. We sought to reproduce these bioeffects using HIFU ablation with αCTLA-4 + αPD-L1.
Methods: N2a tumors were established in the hindlegs of A/J mice and when tumors reached longest diameter of 12-15mm, mice were treated with 1) HIFU ablation alone (n =36), 2) HIFU ablation with αCTLA-4 + αPD-L1 (n = 35) or 3) no treatment (n = 14). Mice were treated with HIFU ablation (1.5MHz transducer, 35W acoustic power, 3 foci x 5 sec) under ultrasound imaging guidance. αCTLA-4 + αPD-L1 were injected on days 1, 4 and 7 after HIFU ablation. Survival endpoints were euthanasia due to poor clinical status or tumor size &gt;20mm. ELISA (serum), and flow cytometry (tumors, draining and contralateral lymph nodes (DLN , CLN), and spleen) were performed at 24, 48 and 72 hour time points after HIFU ablation alone or with αCTLA-4 + αPD-L1.
Results: HIFU ablation alone or with αCTLA-4 + αPD-L1 did not provide a significant survival benefit. Following HIFU ablation alone, there was significant downregulation of CD8α+CD11c+ cells in tumor and DLN but no other statistically significant changes in immune cell markers compared to control. After HIFU ablation and αCTLA-4 + αPD-L1, CD8α+Ki67+ increased in tumor and DLN, but higher expression of CD3+CD8+ was not observed. CD4+FoxP3- cells remained unchanged but CD4+FoxP3+ population increased in the tumor, DLN, CLN and spleen. Interestingly, PD-1 expression was upregulated on CD3+ and CD8α+ cells in tumor, DLN, CLN and spleen. IFN-Γ, CXCL-10 and TNF-α were unchanged after HIFU ablation alone but increased following the combination therapy. IL-10 and VEGF-A were not affected by either treatment and KC/GRO decreased steadily after HIFU ablation with αCTLA-4 + αPD-L1.
Conclusion: Unlike BH with immunotherapy, HIFU ablation with immunotherapy did not improve survival in this N2a model. HIFU ablation and αCTLA-4 + αPD-L1 did not produce a profound immune response and favored a regulatory T cell response with protumor activity. This is in stark contrast to the immune response seen with BH and immunotherapy despite the same tumor model and experimental set up. It will be important to understand the role of PD-1 and other protumoral factors, to investigate the effect of other HIFU parameters on systemic immune antitumor response and to evaluate this approach in other murine cell line models for broader clinical applicability of this combination therapy.
Citation Format: Caitlin Tydings, Avinash Eranki, Karun V. Sharma, AeRang Kim, Priya Srinivasan, Christopher Lazarski, Christopher Rossi. Immune effects of high intensity focused ultrasound ablation with checkpoint inhibitors in murine neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1700.