Converting focused ultrasound-based boiling histotripsy into a systemic cancer vaccine using antigen-capturing microparticles.

Background: Tumors with an immunosuppressive tumor microenvironment (TME) limit effective anti-tumor immunity. Focused ultrasound-based boiling histotripsy (HT) is a noninvasive technique that mechanically ablates tumors while generating acellular lysates with preserved tumor antigens in situ. However, HT alone elicits insufficient anti-tumor immune activation to induce regression of both local and metastatic tumors, particularly in immunoresistant settings. To address this limitation, the objective of this study was to engineer biodegradable polymeric microparticles (MPs) loaded with an agonistic CD40 antibody (CMP) to capture HT-released tumor antigens, enabling sustained uptake and presentation by antigen-presenting cells (APCs) while activating dendritic cells (DCs) to enhance APC priming. Methods: Microparticles were synthesized via a water-in-oil-in-water double emulsion method using PLGA/PCL polymers and an agonistic CD40 antibody. Boiling HT was delivered using the Alpinion HIFU system (VIFU 2000) with a 1.5 MHz transducer, millisecond pulses at 5 Hz, 1% duty cycle, and 600 W power to generate antigen-rich lysates in murine tumors at low (<10%) and high (>50%) ablation. Multiple murine tumor models (melanoma, head and neck, colon) were used to assess local efficacy, immunomodulation, and abscopal responses following HT+CMP therapy. Additionally, in vitro HT lysate adsorbed onto CMPs was administered subcutaneously as a vaccine to promote systemic immunity. Results: Across multiple murine tumor models, local HT+CMP therapy elicited robust local and abscopal immune responses at both low and high ablation volumes. These were associated with increased functional immune cell infiltration and reversal of resistance to immune checkpoint inhibitors (ICIs). Similar effects were observed using in vitro HT-generated lysate adsorbed onto CMPs and administered subcutaneously as a vaccine to promote systemic immunity. Conclusions: By coupling sustained antigen availability with enhanced APC activation, HT-CMP therapy represents a promising strategy to achieve local and abscopal immune responses and overcome ICI resistance, particularly in immunologically "cold" tumors.