High-throughput engineered tumor organoids reveal ROCK signaling as an immunotherapeutic target in triple-negative...

Triple-negative breast cancer (TNBC) remains resistant to immunotherapy because of its profoundly immunosuppressive tumor microenvironment. Here, we establish a droplet-engineered organoid (DEO) platform that preserves endogenous TILs and supports rapid, immunocompetent drug evaluation. Optimizing ROCK pathway modulation reveals that early withdrawal of Y-27632 maintains TIL viability, whereas ROCK activation by pentanoic acid (PA) substantially enhances CD8⁺ T cell infiltration and cytotoxicity within DEOs. In 4T1 tumor-bearing mice, PA monotherapy or alternating PA/Y-27632 treatment significantly reduces tumor volume without detectable systemic toxicity. To validate translational relevance, we applied PA to patient-derived organoids, which exhibited increased T cell activation and abundance, along with a higher proportion of apoptotic cells within the organoid. Integrated transcriptomic and protein analyses reveal that PA induces a cytolytic program coupled to ROCK-dependent effector pathways. Clinical dataset analyses further associate ROCK activation signatures with improved overall survival in basal-like immune-suppressed (BLIS) subtype TNBC. Collectively, our findings highlight that alternating ROCK pathway modulation, in particular ROCK activation, is a promising strategy to convert TNBC into an immune-responsive state.