Methodologies for monitoring alkaliptosis in cancer cells

Alkaliptosis is a pH-dependent form of regulated cell death induced by the small molecule JTC801, characterized by disruption of lysosomal and cytoplasmic pH homeostasis. Unlike classical forms of cell death such as apoptosis, necroptosis, or ferroptosis, alkaliptosis is driven by dysregulation of intracellular pH-regulatory proteins, leading to aberrant alkalinization and metabolic dysfunction. This mechanism is particularly effective against apoptosis-resistant cancers, including pancreatic ductal adenocarcinoma, and represents a promising strategy for overcoming therapeutic resistance. Monitoring alkaliptosis remains challenging, as it is not detected by conventional cell death markers such as caspases or pore-forming proteins. This chapter introduces methodologies for assessing alkaliptosis, including dynamic pH measurement, analysis of alkaliptosis-related protein expression, and evaluation of lysosomal function. Improved detection strategies will advance the understanding of non-canonical cell death pathways and support the development of novel therapeutic interventions targeting alkaliptosis.