RHOT1/2-driven mitochondrial membrane vesicles confer tumor-homing selectivity and enable in situ cancer vaccination.

The development of in situ tumor vaccines has been limited by the lack of delivery systems with precise tumor selectivity. Here, we show that mitochondria from cancer-associated fibroblasts (CAFs) exhibit an efficient tumor-homing property. We find that enrichment of the RHOT1/2 complex on the mitochondria mediates selective uptake by cancer cells through ITSN1-dependent clathrin endocytosis. Using this insight, we engineer RHOT1/2-enriched mitochondrial outer membranes into nanoscale vesicles (RMNPs) that preserve this intrinsic targeting capacity. In multiple mouse cancer models, RMNPs loaded with an immune-stimulating agent (TLR7/8 agonist) and gold nanoparticles enable photothermal therapy, which releases tumor antigens and activates antigen-presenting cells. This generates strong CD8⁺ T cell responses and long-lasting protection against tumor rechallenge. These findings reveal a fundamental organelle-based mechanism of selective intercellular targeting and establish a mechanism-driven platform with translational potential for cancer immunotherapy, highlighting how natural cellular and molecular biology can be harnessed for precision medicine.