TSPAN4 Regulates the Effects of Radiation-Induced Pyroptosis in Non-Small Cell Lung Cancer

Abstract Migrasome, a recently discovered cellular component in migrating cells, are gradually gaining recognition for their role in tumor initiation and development. TSPAN4, identified as a marker protein for migrasome, is deemed essential for their formation. In this study, we present compelling evidence that knockout of TSPAN4 significantly inhibits the migration and proliferative capabilities of non-small cell lung cancer (NSCLC) A549 cells. TSPAN4 depletion enhances the radiation sensitivity of A549 cells. Intriguingly, our findings demonstrate that effective inhibition of TSPAN4 in combination with radiation induces pyroptosis in A549 cells, as evidenced by cell-specific manifestations observed through microscopy (including cell swelling and the formation of large bubbles from the plasma membrane), increased lactate dehydrogenase (LDH) release, and a higher proportion of Annexin V/PI double-positive cells. Importantly, these effects are associated with caspase 3/GSDME cleavage. Furthermore, we observed the involvement of the ROS pathway in these processes. Our findings unveil, for the first time, previously unacknowledged connections between TSPAN4, radiotherapy (RT), and pyroptosis, thereby substantiating the regulatory role of TSPAN4 in radiation-induced pyroptosis in NSCLC through the ROS-caspase 3-GSDME signaling axis.