Abstract Abstract: Background: HER2-positive breast cancer accounts for about 20% of all breast cancer cases and is one of the most aggressive subtypes of breast cancer (1,2). These patients are often resistant to current antitumor treatments including immunotherapy. Even with new therapeutic strategies currently available, around 30% of patients relapse, often develop metastatic disease, and the 5-year survival rate for this patient cohort is only about 22%. Histotripsy (HT) is a non-invasive, non-thermal, non-ionizing ultrasound ablation method that utilizes high-pressure, microsecond ultrasound pulses at low-duty cycle to create cavitation at a focal point (3). It has been reported that HT can effectively inhibit the tumor at its target site and also arrest tumor growth at the remote site (3,4). The mechanism of the abscopal effect remains unclear but is thought to be related to HT-triggered systemic anti-tumor immune responses. The release of intact or immunogenic tumor antigens is critical in the induction of systemic and specific anti-tumor immune responses. Methods: HER2-positive murine mammary tumor cells were injected into HER2 transgenic mouse's mammary fat pads. Tumor-bearing mice were treated with histotripsy with acute tumor resection. Treated tumors were respected and separated into cell-free and residual cell pellet fractions using centrifugation. Total protein concentration in both fractions was quantified using BCA protein assays. Ultra-performance liquid chromatography (UPLC) and Western blotting were used to evaluate and characterize HER2 protein. Histotripsy was performed using a custom built 8-element, 1 MHz transducer with ultrasound-guided targeting. The tested treatment dose was defined as the number of pulse repetitions per focal location (ppl). Then, 100 ppl was chosen for this study. Other acoustic parameters such as pulse repetition frequency remained fixed at 100 Hz PRF. Results: Our data showed that the HT cavitation can accurately target and disrupt tumor tissues and release more total proteins into the cell free fraction than untreated tumor by BCA assay. HT treatment significantly increases the level of released HER2 protein as measured by UPLC and Western blot analysis. UPLC analysis further showed that the cell-free fractions exhibited a protein peak with a retention time at around 9.3 minutes. This released antigen appeared identical to recombinant HER2 protein, suggesting that the HER2 released by HT is in line with intact protein. Conclusions: Our findings have demonstrated that HT-treated orthotopic breast tumor could trigger the release of intact tumor antigens, which in some manner can stimulate the host immune system. The release of such intact and concealed tumor antigens can create the basis for HT-induced systemic anti-tumor immune responses and a favorable environment for immunotherapies. Finally, this increase in immune activity potential enhances the host’s ability to prevent tumor recurrence and metastasis. References: 1. Huang L, Liu CC, Shao ZM, Yu KD. Breast cancer: pathogenesis and treatments. Signal Transduct Target Ther. 2025 Feb 19;10(1):49. 2. Khan MM, Yalamarty SSK, Rajmalani BA, Filipczak N, Torchilin VP. Recent strategies to overcome breast cancer resistance. Crit Rev Oncol Hematol. 2024 May;197:104351. 3. Xu Z, Hall TL, Vlaisavljevich E, Lee FT Jr. Histotripsy: the first noninvasive, non-ionizing, non-thermal ablation technique based on ultrasound. Int J Hyperthermia. 2021;38(1):561-575. 4. Pepple AL, Guy JL, McGinnis R, Felsted AE, Song B, Hubbard R, Worlikar T, Garavaglia H, Dib J, Chao H, Boyle N, Olszewski M, Xu Z, Ganguly A, Cho CS. Spatiotemporal local and abscopal cell death and immune responses to histotripsy focused ultrasound tumor ablation. Front Immunol. 2023 Jan 23;14:1012799. Citation Format: S. Wang, S. Tang, R. McGinnis, Z. Cao, Z. Xu. Release of tumor antigens from breast cancer cells by a novel ultrasound ablation method abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-13-09.
Wang et al. (Tue,) studied this question.