Ultrasound-guided spatial delivery based on acoustic bacteria to enhance cancer immunotherapy.

Intratumoral injection is a well-established strategy for local cancer immunotherapy. However, its efficacy is critically limited by the spatially heterogeneous tumor microenvironment (TME), particularly the presence of hypoxic-necrotic regions that induce immunosuppression. The lack of reliable image guidance for precise intratumoral injection site selection remains a major challenge. Here, we developed an ultrasound-guided intratumoral delivery method based on aptamer-modified acoustic bacteria (AAB). AAB were genetically engineered to express gas vesicles (GVs) for enhanced ultrasound contrast and surface-modified with AS1411 aptamer via amide condensation to promote tumor accumulation. Following intravenous administration, AAB preferentially accumulate to hypoxic-necrotic tumor niches, enabling contrast-enhanced ultrasound (CEUS) imaging of these immunosuppressive niches. Guided by AAB-based CEUS imaging, therapeutic bacteria (TB), which were engineered to express a bacteriolytic protein and release CD47 nanobodies, were accurately injected either inside or outside the hypoxic-necrotic regions. Injection outside these regions yielded significantly superior antitumor efficacy. This enhanced therapeutic effect was mediated by preserved availability of functional CD47 targets on viable tumor cells, coupled with robust induction of M1 macrophage polarization and a potent pro-inflammatory immune microenvironment within the tumor. This study provides a practical image-guided strategy to overcome the therapeutic barriers imposed by intratumoral heterogeneity and maximize the efficacy of bacterial cancer immunotherapy.