ATP released by ultrasound targeted microbubble cavitation induces vascular inflammation and improves immune checkpoint blockade efficacy.

Extracellular ATP (eATP) is a potent immune stimulant that functions as a damage-associated molecular pattern. The regulation of eATP is primarily mediated by cell surface ecto-nucleotidases (CD39 and CD73) which hydrolyze ATP into adenosine, a potent immune suppressor. CD39 and CD73 are upregulated in most cancers. Therapeutic strategies aimed at increasing ATP release in the tumor microenvironment or inhibiting adenosine activity are active areas of research in immuno-oncology. Ultrasound-Targeted Microbubble Cavitation (UTMC) is an externally applied, spatially targeted approach that has demonstrated synergy with immune checkpoint blockade (ICB) in solid tumors. However, the underlying mechanisms and optimal therapeutic combinations remain under investigation. We hypothesized that modulating purinergic signaling by UTMC could further leverage ICB efficacy. Here, we investigated non-ablative and flow-preserving UTMC to enhance ATP release and induce inflammatory responses in a murine syngeneic colorectal tumor model (MC38) with and without CD39 inhibition. We compared two UTMC pressures (400 and 850 kPa), evaluating their impact on tumor blood flow by contrast perfusion imaging, their ability to release ATP using bioluminescence, their effect on vascular inflammation and cancer cell death through histological analysis, their synergy with aPDL1 to improve ICB efficacy, immune cell infiltration to the tumor, and immune cell drainage to the tumor-draining lymph nodes (TDLN). UTMC at 850 kPa and in CD39 knockout model released higher eATP concentrations, which correlated with increased vascular inflammation, enhanced cancer cell death, and reduced cancer cell proliferation. The combination of aPDL1 with UTMC and CD39 blockade significantly reduced tumor growth. This treatment also increased cytotoxic T cells (CTL), the CTL/Treg ratio, dendritic cells, and M1-prototype tumor-associated macrophages, while reducing M2-prototype macrophages within the tumor. In the TDLNs, the fully combined treatment elevated CTLs, dendritic cells, and M1-prototype macrophages, with a concurrent reduction in M2-prototype macrophages. Our findings support that purinergic signaling can be leveraged in combination with UTMC to improve ICB therapy.