Fig. 2: Hallmark DAMP release is insufficient to induce immunogenic cell death.

a, b Flow cytometry analysis and validation of DAMPs (i.e., CRT and HSP70) on the cell surface of human T24 and murine G69 bladder cancer cells treated with gemcitabine in vitro (representative plot shown with two technical replicates of n = 3 independent experiments; example gating depicted in Supplementary Fig. 3). c, d Western blot of HMGB1, an extracellular DAMP, released into cultured media that were collected from gemcitabine-treated human T24 cells and murine G69 cells, respectively. e A schematic depiction of the in vivo vaccination assay treatment schedule for assaying immunogenic cell death. f Tumor volume and g corresponding tumor-free survival Kaplan–Meier plot (n = 3 mice per vaccination group; tumor volume calculation as indicated in Methods section) resulting from mice vaccinated with dying G69 cells via various chemotherapy and subsequently challenged with live G69 murine bladder cancer cells. cis cisplatin, gem gemcitabine, mitox mitoxantrone. Statistics: two-tailed, unpaired T test (a; p = 0.0013 [top] and p = 0.0006 [bottom]) and (b; p = 0.0044 [top] and p = 0.0123 [bottom]); two-tailed, two-way ANOVA-Tukey’s multiple comparisons test (f; p < 0.0240); Kaplan–Meier survival analysis using Mantel–Cox test (g; p = 0.0123); and where appropriate, data are presented as mean values ±SEM.