Leveraging Mitochondria‐Endoplasmic Reticulum Functional Interplay With an On‐Demand Nanoparticle to Boost mtDNA‐Based STING Immunotherapy

ABSTRACT Endogenous STING activation by mitochondrial DNA (mtDNA) offers a tumor‐context‐dependent strategy with potentially reduced off‐target toxicity, but is often limited by weak and transient mtDNA‐driven STING signaling that fails to sustain robust STING clustering. Here, we developed an on‐demand nanoparticle system that harnesses mitochondrial–ER functional interplay by concurrently inducing mtDNA release and ER stress, thereby relieving the STIM1‐mediated brake on STING and enabling robust STING–TBK1 assembly and downstream signaling. This strategy markedly increased the phosphorylation levels of STING, TBK1, and IRF3 by 11.50‐, 9.70‐, and 8.95‐fold, respectively, compared with PBS, outperforming the commercial STING agonist MSA‐2 by more than 2‐fold. In addition, the nanoparticles enabled spatially controlled co‐delivery, allowing extracellular release of a PD‐1/PD‐L1 inhibitor and intracellular release of mtDNA‐releasing and ER stress‐inducing agents. Consequently, this on‐demand nanoparticle system potently enhanced both innate and adaptive antitumor immunity in vivo, significantly increasing CD8 + and CD4 + T cell infiltration while reducing Tregs, ultimately suppressing tumor progression, metastasis, and recurrence in mouse models of breast and colon cancer. This strategy advances STING‐based immunotherapy by integrating spatially staged drug release with organelle‐level immune modulation.