Boiling Histotripsy with 1.1/3.3 MHz Dual-Frequency Inducing Long-Duration Anti-Tumor Immunity in Mice Breast Cancer.

Triple-negative breast cancer (TNBC) has a poor prognosis and high risk of recurrence and metastasis. Histotripsy is an ultrasound-guided, non-invasive, non-thermal ablation method that mechanically lyses target tissues. A boiling histotripsy (DF-MS BH) technique combining 1.1/3.3 MHz superposition with a multi-stage ultrasound pulse scheme was proposed, aiming to enhance anti-tumor immune response by optimizing pulse dose and number of treatments. A custom-designed 1.1-/3.3-MHz two-element confocal-annular array and multi-stage ultrasound pulses were used for treatment. For 6-mm-diameter tumors, the ablated volume accounted for 60-70 % of the total tumor volume. The anti-tumor immune results showed increasing pulse dose from 85 to 115 elevated DAMPs release and reduced tumor growth rate. After the first high-dose pulse treatment, the infiltration level of CD8 + T cells gradually increased and then decreased on the 7th day, which contributed to a 9-day period of tumor growth arrest. Following the second treatment, the release of danger signals was further augmented. Meanwhile, the high-level infiltration of CD8 + T cells persisted for 35 days, extending the tumor growth arrest period to 25 days and prolonging the median survival period of mice to 50 days. In addition, this treatment strategy effectively reduced the number of lung metastases, confirming its ability to enhance systemic anti-tumor immune response. Furthermore, DF-MS BH ameliorated the immunosuppressive microenvironment of TNBC through first and second enhanced treatments. Ultimately, the combination therapy of DF-MS BH and immune checkpoint inhibitors (ICIs) further suppressed tumor recurrence and metastasis.