Fig 1: HER2‐targeted NIR‐pyroptosis induces antitumor effect and immunologic memory. A) Validation of HER2‐ICG by SDS‐PAGE (left: Colloidal Blue staining, right: fluorescence). Diluted HER2 mAb was used as a control. B) The in vitro cytotoxicity of HER2‐ICG on OE19 cells with a serial dose of NIR light irradiation using propidium iodide (PI) staining. C) Representative phase‐contrast cell images of OE19 cells treated with NIR‐pyroptosis. Arrows mark the cells showing pyroptotic morphology. D) Western blot analysis of GSDMD cleavage of OE19 cells with different treatments. GSDMD‐F, full‐length GSDMD; GSDMD‐N, N‐terminal cleavage product of GSDMD. E) Schematic design of in vivo efficacy for HER2‐targeted NIR‐pyroptosis in a CT26.WT‐hHER2 Balb/c mouse model, and the schematic design of the CT26.WT‐hHER2 tumor rechallenge model. Laser3: 1 W cm−2, 180 s; Laser4: 1 W cm−2, 300 s. F) Tumor progression in the CT26.WT‐hHER2 mouse model treated with PBS (n=7), PD‐1 (n=7), NIR‐pyroptosis (n=8) or combination (n=7), respectively, and monitored by tumor volume measurement. G) Individual CT26.WT‐ hHER2 tumor growth curves. H) Mouse body weight in the CT26.WT‐hHER2 model. I) Tumor progression in cured mice rechallenged with CT26.WT‐hHER2 tumor and monitored by tumor volume measurement. J) Individual CT26.WT‐hHER2 tumor growth curves. K) Mouse body weight in the rechallenge model. L) IFN‐γ secretion from splenocytes co‐cultured with CT26.WT‐hHER2 cells, as detected by ELISA. In L), data are presented as mean ± SD, and differences between each group are determined by One‐way ANOVA. In F) and I), data are presented as mean ± SD. The differences between each group are determined by Two‐way ANOVA.
Fig 2: Pyroptotic effects of ICAM1‐ICG under NIR light irradiation on ATC cells. A) Schematic illustration of an ICAM1‐ICG (Left). Chemical structures of ADC linkers and warheads used in ICAM1‐ICG (Right). B) Validation of ICAM1‐ICG by SDS‐PAGE (left: Colloidal Blue staining, right: fluorescence). Diluted ICAM1 mAb was used as a control. C)The in vitro cytotoxicity of ICAM1‐ICG on ATC cells with a serial dose of NIR light irradiation, using propidium iodide (PI) staining. D) Representative phase‐contrast cell images of ATC cells treated with NIR‐pyroptosis. Arrows mark cells that show pyroptotic morphology. E) Representative TEM images of 8505C cells treated with NIR‐pyroptosis (Scale bars, 2 µm). F) Representative fluorescence images of intracellular ROS detection using DCFH‐DA staining (Scale bars, 50 µm). G) Heatmap of top 5000 genes with significant expression in 8505C cells treated with ICAM1‐ICG, NIR‐pyroptosis, Laser, or PBS, respectively, followed by RNA‐seq analysis (n = 3). H) GSEA of the pyroptosis signaling pathway, comparing the NIR‐pyroptosis and PBS groups. I) Western blot analysis of GSDMD cleavage in 8505C cells with different treatments (Up). NAC was used to eliminate intracellular ROS. Quantitative analysis of GSDMD‐N relative expression (Down) (n = 3, mean ± SEM). J) Representative phase‐contrast cell images of 8505C cells treated with solvent‐based ICG under NIR light. K) Western blot analysis of GSDMD cleavage in 8505C cells treated with solvent‐based ICG under NIR light. L) Flow cytometry analysis of NIR‐pyroptosis cytotoxicity after adding NAC. In C) and I), data are presented as mean ± SEM, and differences between each group are determined by One‐way ANOVA.
Fig 3: Recombinant GSDMD cleavage by cathepsin S. A) Confocal immunofluorescence images of lysosomes and ICAM1‐ICG after NIR‐pyroptosis (Scale bars, 10 µm). B) Confocal immunofluorescence images of LAMP1 and Gal‐3 after NIR‐pyroptosis (Scale bars, 10 µm). C) In vitro cleavage of GSDMD by recombinant cathepsin. D) Inhibitory effect of cathepsin S‐specific inhibitor on cathepsin S mediated cleavage of GSDMD. E) Western blot analysis of GSDMD cleavage mediated by cathepsin S. F) HiS‐SIM immunofluorescence images of GSDMD and cathepsin S in 8505C‐GSDMD‐EGFP cells after NIR‐pyroptosis (Scale bars, 5 µm). G) PI staining analysis after co‐expression of cathepsin S and GSDMD in 293T cells. H) In vitro cleavage of IL‐18 and IL‐1β by recombinant cathepsin S.
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