Fig. 2: Ferroptotic cells are not immunogenic regardless of the stage of cell death.

a Induction of GPX4 knockdown by doxycycline administration (1 µg/ml) in iGPX4KD MCA205 cell line measured by western blotting. One of two independent experiments is presented. b Scheme of ferroptosis induction in iGPX4KD cells. c Analysis of lipid ROS, cytosolic ROS accumulation in the cells, levels of calreticulin on the surface of dying, non-permeabilized cells, and ATP, HMGB1, LDH, IFN-β, TNFα, and CXCL release from cells during ferroptosis. Three stages of ferroptosis can be distinguished: initial ferroptosis where cells experience accumulation of lipid ROS; intermediate ferroptosis involving partial permeabilization and release of ATP and exposure of calreticulin, and terminal ferroptosis with complete permeabilization and release of LDH, HMGB1, and cytokines. Data obtained from n = 3 independent samples are presented as mean. Microscopy pictures represent one of n = 2 independent live cells imaging experiments. d The analysis of calreticulin exposure during ferroptosis. Data generated from n = 3 biologically independent samples are presented as floating bars with bounds as the range and center as median of the relative MFI prior to membrane permeabilization; histograms represent the shift of calreticulin fluorescence in non-permeabilized cells. One-way ANOVA with Dunnett’s post-hoc test in comparison to the ‘0 h’ sample. e The % of live cells exposing calreticulin during the process of ferroptotic cell death. Data generated from n = 3 biologically independent samples are presented as floating bars with bounds as the range and center as median of CRT⁺ cells. One-way ANOVA with Dunnett’s post-hoc test in comparison to the ‘0 h’ sample. f Establishing the point of no return for ferroptosis in iGPX4KD cells. Re-adding ferroptosis inhibitor Fer1 2 h or later after cell death induction does not rescue cells from dying. Data presented as mean ± SEM from n = 3 independent experiments. g Prophylactic vaccination model using iGPX4KD cells at the initial and terminal stage of cell death. Mitoxantrone-treated (1 μM, 24 h) iGPX4KD cells undergoing immunogenic apoptosis served as a positive control. Kaplan–Meier curves represent the effectiveness of dying iGPX4KD cells in preventing tumor growth on the challenge site. The experiment was performed n = 2 and analyzed by Kaplan–Meier simple survival analysis. h The tumor growth on the challenge site in prophylactic vaccination model. Only data presented as mean ± SEM from animals that developed the tumor is shown.