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.

Fig 3: Prostaglandin E2 release hinders ICD-induction by gemcitabine.a Western blot analysis of COX-2 and corresponding ELISA of PGE2 from G69 cells treated with gemcitabine in vitro (representative plot shown with two technical replicates of n = 3 independent experiments). b A schematic depicting the proposed interventional strategy that exploits the immunostimulatory/inhibitory DAMP balance to harness immunogenic cell death. c, d Flow cytometry of cell surface CRT and HSP70 of single cells (example gating depicted in Supplementary Fig. 3), as well as western blot of HMGB1 from G69 and Panc02 cells treated with gemcitabine plus celecoxib to achieve iDAMP blockade (representative plot shown with two technical replicates of n = 3 independent experiments). e, f Western blot of COX-2 and ELISA of PGE2 from murine G69 and Panc02 cells to demonstrate the efficacy of celecoxib in abrogating gemcitabine-induced PGE2/ iDAMP release in vitro (representative plot shown with two technical replicates of n = 3 independent experiments). g Temporal changes in tumor volume and h tumor-free survival of mice after challenge (n = 9 per treatment group) resulting from a gold-standard vaccination assay. i Temporal changes in tumor volume and j tumor-free survival of mice after challenge (n = 5 per treatment group) resulting from a gold-standard vaccination assay using the murine Panc02 PDAC model. celex celecoxib, CTx chemotherapy, gem gemcitabine. Statistics: two-tailed, unpaired T test (a; p = 0.0107); two-tailed, one-way ANOVA-Tukey’s multiple comparisons test (c; p = 0.002 [top] and p = 0.008 [bottom]), (d; p = 0.0011 [top] and p = 0.0007 [bottom]), (e; **p = 0.0076 and **p = 0.0028), (f; p = 0.0033), (g; **p = 0.015 and ***p = 0.0003), and (i; *p < 0.0486); Kaplan–Meier survival analysis using Mantel–Cox test (h; p = 0.0001) and (j; p = 0.004); and where appropriate, data are presented as mean values ±SEM.

Fig 4: The release of hallmark DAMPs from gemcitabine-induced dying cancer cells.Representative peptide chromatogram illustrating differential enrichment of a CRT, b HSP70, c HSP90aa1, and d PDIA3 on the cell surface, as well as e ANXA and f HMGB1 released into the cultured media by cancer cells (murine G69 bladder cancer cells) treated with gemcitabine (n = 2 independent mass spectrometry proteomic profiling experiments). ANXA1 annexin A1, CRT calreticulin, gemCTx gemcitabine chemotherapy-treated, HMGB1 high-mobility group protein box 1, HSP heat-shock protein, PDIA3 protein disulfide isomerase A3. Extracted chromatograms for a representative peptide of each protein was plotted using the Skyline software; precursor (blue color), precursor [M + 1] (purple color), precursor [M + 2] (red color) represent the three isotopic peaks for each indicated peptide.

Fig 5: USP18 loss-induced atypical ISG PLK2 regulates GSDME cleavage dependent pyroptosis.a Western blot of WT, PLK2-/-, USP18-/-, and USP18-/-PLK2-/- THP-1 cells treated with or without IFNa (1000 U/ml) for 48 h with indicated antibodies. b Percentage of live cells in (a) (n = 3 independent samples each). p-value was determined by one-way ANOVA test. c Analysis of ICD markers (LDH release and ecto-CRT) in (b) (n = 3 independent samples each). p-value was determined by one-way ANOVA test. d Western blot of PLK2-/- THP-1 cells infected with Mock or PLK2 after two days of blasticidin selection using the indicated antibodies. e; Percentage of live cells in (d) (n = 3 independent samples each). p-value was determined by two-tailed Student t-test. f Analysis of ICD markers (LDH release and ecto-CRT) and IL-1ß secretions in (d) (n = 3 independent samples each). p-value was determined by two-tailed Student t-test. g; THP-1 and Kasumi-1 cells were transduced with MIP, MIP-PLK2 WT, or MIP-PLK2 enzyme activity mutants. Two days after puromycin selection, cell lysates were analyzed by western blot with indicated antibodies. h Ectopic PLK2 expression induces cell death in several AML cell lines independent of its kinase activity. THP-1, Kasumi-1, and MV4-11 cells were infected with MIP, MIP-PLK2 WT, or MIP-PLK2 enzyme activity mutants. Two days after puromycin selection, live cells were analyzed by FACS (n = 3 independent samples each). p-value was determined by one-way ANOVA test. i THP-1 or OCI-AML3 cells were treated with DMSO or TC-S7005 (final 10 µM) for 48 h, then cell lysates were analyzed. j THP-1 or OCI-AML3 cells were treated with DMSO or TC-S7005 for indicated times, then PI (%) and Annexin V+ (%) were analyzed by FACS (n = 3 independent samples each). p-value was determined by one-way ANOVA test. k Two different PDX AML cells were cultured with different dose of TC-S7005, and analyzed cell growth (n = 3 independent samples each conditions). All data represent mean ± s.d, except where indicated. n.s. not statistically significant. p-value was determined by one-way ANOVA test. Source data are provided as a Source Data file.