Figure 7.

Graphical abstract—timespan of immunogenicity of ferroptotic cancer cell death. After receiving ferroptotic stimuli, cancer cells start to undergo cell death (Phase 0) and release DAMPs (eg, ATP and HMGB1). After 3 hours of ferroptosis induction, cancer cells are in phase I, which is the early ferroptotic cell death stage, where the highest levels of ATP and HMGB1 are reached. At this time point, early ferroptotic cancer cells are engulfed by BMDCs and promote their activation and maturation, as evidenced by analyses of their phenotypic markers (MHCII, CD80, and CD86) and the pro-inflammatory cytokine IL-6. In addition, if early ferroptotic cells in Phase I are injected into mice, they lead to protective immunity in the mouse tumor prophylactic vaccination model if the adaptive immune system is intact. In the late ferroptotic phase II, cancer cells are no longer immunogenic. Because all the supernatants (SN) from the late ferroptotic cancer cells (treated with RSL3 for 24 hours) are removed and no DAMPs (ie, HMGB1, ATP) are present anymore, which are required for their immunogenicity. Notably, although late ferroptotic cells were engulfed by BMDCs, they failed to induce BMDC phenotypic activation and maturation in vitro or to induce an effective antitumor immune response. It is crucial that phase I ferroptotic cell death occurs in the presence of antigen-presenting cells and should preferably proceed in vivo. During that stage (ie, phase I), most of ATP and HMGB1 and possibly other DAMPs are released or reaching the maximal levels, thereby creating the best adjuvanticity effect of early ferroptotic cancer cells. These data show that the stage of cell death is a key aspect of the immunogenicity of ferroptotic cancer cells and demonstrate that ATP and HMGB1 released after ferroptosis induction may act as immunogenic signals. BMDCs, bone-marrow derived dendritic cells; DAMPs, damage-associated molecularpatterns; HMGB1, high-mobility group box 1.