Fig. 2: Spatiotemporal monitoring of caspase-3/-7 activity in 3D spheroid and organoid models. | Cell Death Discovery

Fig. 2: Spatiotemporal monitoring of caspase-3/-7 activity in 3D spheroid and organoid models.

From: Integrated real-time imaging of executioner caspase dynamics, apoptosis-induced proliferation, and immunogenic cell death using a stable fluorescent reporter platform

Fig. 2

A Schematic overview of the experimental workflow for real-time monitoring of caspase activity in 3D culture systems using stable caspase-3/-7 reporter cells derived from the established cell culture model MiaPaCa-2 growing in spheroidal structures, primary patient derived organoid (PDO) and human umbilical vein endothelial cells (HUVECs) growing in spheroids. B, C MiaPaCa-2 caspase reporter cells embedded in Cultrex™, forming grape-like 3D spheroidal structures. B Representative phase-contrast and fluorescence images at 48 h post-treatment with Carfilzomib (50 nM) or DMSO as vehicle control. Scale bar: 50 µm. C Quantification of GFP fluorescence over time, normalized to baseline (t0h), DMSO control, and mCherry signal to account for cell number. Data represent mean ± SD of three independent experiments (n = 3). Statistical significance between treatment groups at each time point was assessed using two-way ANOVA with Šidák’s multiple comparisons test (t96h p < 0.0001). D, E Patient-derived organoid (PDAC-PDO) cultures embedded in Cultrex™ and treated with carfilzomib (50 nM) or DMSO. D Representative images at 48 h showing phase-contrast, mCherry, and GFP channels. Scale bars: 100 µm (panels 1–4); 30 µm (panel 5). E Quantification of GFP fluorescence intensity over 96 h after treatment with carfilzomib or DMSO, with measurements taken every 24 h. Data represent mean +SD (carfilzomib) and -SD (DMSO) of four independent experiments (n = 4). Statistical significance between treatment groups at each time point was assessed using two-way ANOVA with Sidak’s multiple comparisons test (t24h p < 0.05, t48h p < 0.001, t72h p < 0.01, t96h not significant). F, G Primary HUVEC spheroids, generated by the hanging drop method, embedded in hydrogel and treated with carfilzomib (100 nM) or DMSO. F Representative images at 24 h post-treatment. Scale bar: 100 µm. G Quantification of relative GFP fluorescence intensities after 24 h treatment with carfilzomib or DMSO (n = 1). All fluorescence quantifications (C, E, G) were normalized to baseline (t0h), DMSO control, and mCherry signal.

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