Long-term confinement triggers RIPK3-independent necroptosis in amoeboid cancer cells to shape tumor evolution

The mesenchymal-amoeboid transition (MAT) enhances cancer cell migration and therapy resistance during dissemination through mechanically confined spaces. Using an in vitro MAT induction system combining physical confinement with a non-adhesive environment to mimic interstitial migration, we introduce "long-term MAT" (L-MAT) to describe cells sustaining MAT under prolonged pressure and show that L-MAT triggers receptor-interacting protein kinase 3 (RIPK3)-independent necroptosis. Mechanistically, the PIEZO1-STAT1 axis upregulates mixed lineage kinase domain-like pseudokinase (MLKL), whose membrane accumulation drives necroptosis in cells otherwise resistant to death-receptor signaling. Before membrane rupture, L-MAT cells show progressive migration restriction; MLKL-knockout cells bypass this checkpoint and persist as surviving clones. Spatial transcriptomics of patient tumors shows MLKL expression correlates with PIEZO1 activation and immunogenicity in less aggressive regions, while advanced malignant and metastatic clones show reduced MLKL. These findings establish MLKL as a conditional tumor checkpoint and identify the PIEZO1-STAT1-MLKL axis as a predictive biomarker and therapeutic target.