Temporally programmed engineered bacteria orchestrate tumor microenvironment remodeling and nitroxidative amplification for enhanced cuproptosis immunotherapy

Cuproptosis-based cancer immunotherapy is severely limited by hypoxia-associated metabolic resistance, immunosuppressive tumor microenvironments, and the lack of tumor-selective delivery of cuproptosis inducers. Herein, we developed a temporally coordinated therapeutic strategy by integrating a tumor-targeting nitric oxide (NO)-producing bacterial platform (ECN-NO) with ultrasound-responsive ES-Cu-loaded microbubbles (ES-Cu MBs) to sequentially remodel the tumor microenvironment and potentiate cuproptosis immunotherapy. ECN-NO selectively colonized tumors and continuously released NO, thereby normalizing tumor vasculature, alleviating hypoxia, and facilitating the intratumoral generation, delivery, and penetration of ES-Cu nanotherapeutics following ultrasound-responsive microbubble destruction (UTMD). The enhanced intratumoral accumulation of ES-Cu amplified cuproptosis-induced oxidative stress, which triggered bacterial lysis to both establish an arginine-dependent nitroxidative amplification cascade and intrinsically limit bacterial persistence, resulting in robust peroxynitrite (ONOO⁻) generation. Consequently, ONOO⁻ disrupted glutamine metabolism, reinforced cuproptosis, and potentiated immunological activation. This temporally coordinated strategy of tumor microenvironment remodeling, cuproptosis amplification, and immune activation elicited potent systemic antitumor immunity and durable immunological memory. Together, these findings establish a self-regulating bacterial–nanomedicine strategy for enhancing cuproptosis-based cancer immunotherapy.