Histotripsy Dose Impacts Tumor Cellular Damage and Treatment Outcomes in a Preclinical Model of Hepatocellular Carcinoma

Clinically, histotripsy utilizes a fixed, non-optimized dose (defined by the number of histotripsy pulses) to non-invasively ablate liver tumors. This study investigates the impact of histotripsy doses on tumor cellular damage, immune response, and treatment outcomes. Single orthotopic rat HCC liver tumors were established in immunocompetent rats by injecting McA-RH7777 cells into the liver and were classified as intermediate-stage or late-stage. Tumors were treated with doses 20, 50, 100 and 200 pulses per location (ppl) at 20 Hz, 50 Hz, 100 Hz and 200 Hz pulse repetition frequency (PRF) respectively using 1–2 cycle length histotripsy pulses delivered via a 1 MHz custom histotripsy transducer with an estimated peak negative pressure > 30 MPa. Cellular damage and immune response were evaluated in late-stage tumors. Local tumor control, metastasis inhibition, and survival were evaluated and compared across tumor stages. Abscopal response was evaluated by generating multicentric tumors in separate liver lobes but treating only one tumor. As the dose increased from 20 to 200 ppl, histologic cellular disruption increased from sparse-treatment to overtreatment. Dose 100 ppl (intermediate dose resulting in complete treatment) had the greatest infiltration of CD3 + T-cells and NK-cells, highest TUNEL+ area, lowest Ki67 index, and the highest percentage of animals with complete regression in late-stage tumors. Overtreatment dose (200 ppl) did not have the best treatment outcomes for any tumor stage. Dose 100 ppl also demonstrated improved abscopal response in distant untreated tumor. Histotripsy dose impacts histological tumor damage, immune infiltration, local tumor progression, metastasis inhibition, abscopal response, and survival. An intermediate dose had the best outcomes, but overtreatment resulted in worse outcomes, highlighting the need for histotripsy dose optimization.