Ultrasound-targeted microbubble cavitation enhances anti–PD-L1 therapy in TNBC via eNOS-mediated reoxygenation

Hypoxia critically restricts the effectiveness of immunotherapy in triple-negative breast cancer (TNBC). Comprehensive bioinformatics analyses demonstrated that highly hypoxic TNBC tumors exhibited elevated T cell exhaustion, increased immune checkpoint molecule expression, and diminished responsiveness to immune checkpoint blockade (ICB). Consequently, strategies aimed at alleviating tumor hypoxia may effectively augment ICB therapy. Although ultrasound-targeted microbubble cavitation (UTMC) has been shown to reduce tumor hypoxia, the precise molecular mechanisms remain unclear. Here, we provided evidence that UTMC activated endothelial nitric oxide synthase (eNOS) through G protein-coupled signaling, resembling pathways induced by fluid shear stress. UTMC-induced eNOS activation was largely Ca²⁺-dependent and resulted in increased nitric oxide production. Enhanced nitric oxide generation was associated with improved tumor perfusion and reduced hypoxia. Combining UTMC with anti-PD-L1 therapy markedly improved the tumor immune microenvironment, characterized by increased CD8+ T cell infiltration, reduced T cell exhaustion, diminished regulatory T cell infiltration, increased macrophage polarization from an M2 to M1 phenotype, and elevated production of pro-inflammatory cytokines. Collectively, our findings identified UTMC as a promising adjunctive therapeutic approach to mitigate hypoxia and enhance the efficacy of anti-PD-L1 immunotherapy in TNBC. These results support further translational evaluation of UTMC-based combination strategies in hypoxic TNBC.