Fig. 1: Chemotherapy-induced epiCaspase-1 correlates with neutrophil-dominant inflammation and poor clinical outcome.

Gene set enrichment analysis (GSEA) demonstrating the enrichment of NOD-Like Receptor pathway in patients with chemo-resistant bladder cancer, comparing matched pre- and post-chemotherapy cancer tissues (n = 20) (a) and gemcitabine treated T24 (3 biological replicates) (b). c Heatmap of common differentially expressed genes from (a) and (b). Scale bar represents normalized expression. Column represents genes and row represents matched patients pre- and post- treatment/ independent T24 samples. d Representative of three western blot replicates evaluating the caspase-1 pathway components [full length (GSDMD-FL) and cleaved N-terminus gasdermin D (GSDMD-N)] in T24, G69, and 4T1 after gemcitabine chemotherapy treatment at 0.5 μM, 120 μM and 0.1 μM, respectively. e Representative fluorescent microscopy evaluating caspase-1 dimerization using caspase-1-specific bimolecular fluorescence complementation (BiFC, green) reporter, and dsRed2-mito7 (red) in T24. f Representative real-time imaging demonstrating the temporal increase of caspase-1 dimerization in T24 and G69 cancer cells during chemotherapy (1 of 3 biological replicates shown here). g Representative scanning electron microscopy displaying cellular morphology of cancer cells undergoing apoptotic (top panel) and lytic cell death (bottom panel), respectively, in response to gemcitabine chemotherapy. Lactate dehydrogenase (LDH) release assay upon gemcitabine and co-treatment with the caspase-1 inhibitor VRT-043198 (40 μM) in T24 (h), G69 (i), and 4T1 (j) cancer cells (3 biological replicates per model). k qPCR showing relative IL1B mRNA expression post LPS priming (4 biological replicates). l Representative of three western blot replicates of molecular markers characterizing caspase-1 dependent lytic cell death after LPS priming and nigericin treatment. m Methodology for the derivation of the CASCADE signature (Chemotherapy-Activated Signaling reflecting Caspase-1 Associated Death Effect) Kaplan-Meir survival curve of patients with high and low CASCADE expression score in TCGA Bladder Cancer (BLCA) (n = 404) (n) and GSE25066 breast cancer cohort (n = 374) (o). p 10X Xenium spatial profiling in responder (n = 1) and non-responder (n = 1) patient tissues. Images represent full panel (left panel, ROI: yellow box), magnified ROI (middle panel; yellow box in full panel), and single channels (right panels). q UMAP analysis of epithelial cell clusters from patient tissues in (p), showing eleven distinct sub-clusters (left panel) and the relative expression of CASCADE signature in each sub-cluster (right panel). Bubble plot illustrating the relative expression CASCADE signature in each sub-clusters; clusters 4, 5 and 10 are designated as CASCADE^high while clusters 1, 6, 9 are designated as CASCADE^low (bottom). A total of 44,935 cells were analyzed. r Bar plot showing the proportions of CASCADE^high (red) and CASCADE^low (blue) epithelial clusters in responder (n = 1) and non-responder (n = 1) patients. s CIBERSORTx neutrophil signature score of CASCADE^high and CASCADE^low patients in TCGA BLCA (n = 404). Statistics: Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test (h–k), log-rank test (n, o) and two-tailed Wilcoxon rank sum test (s) were performed. Error bars represent standard error of mean (s.e.m.) unless otherwise stated. Source data are provided as a Source Data file.