Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Surface‐exposed calreticulin (ecto‐CRT) and secreted ATP are crucial damage‐associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)‐based (reactive oxygen species (ROS)‐regulated) pathway for ecto‐CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS‐mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80high, CD83high, CD86high, MHC‐IIhigh) and functional stimulation (NOhigh, IL‐10absent, IL‐1βhigh) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto‐CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK‐orchestrated pathways that require a functional secretory pathway and phosphoinositide 3‐kinase (PI3K)‐mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase‐8 signalling are dispensable for this ecto‐CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto‐CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase‐8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK‐dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS‐mediated ER stress. There is a [Have you seen?][1] (March 2012) associated with this Article. [1]: http://dx.doi.org/10.1038/emboj.2012.2