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. 2025 Aug 6;11(1):368.
doi: 10.1038/s41420-025-02662-y.

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

Selen Selcen  1 , Lena Wieland  1 , Tiago De Oliveira  1   2 , Michael Ghadimi  1   2   3 , Lena-Christin Conradi  1   2 , Günter Schneider  1   2   3 , Leonie Witte #  1 , Matthias Wirth #  4   5   6   7   8
Affiliations

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

Selen Selcen et al. Cell Death Discov. .

Abstract

Regulated cell death plays a central role in tissue homeostasis, disease progression, and therapeutic responses. However, tools to study these processes with high spatiotemporal resolution in physiologically relevant systems remain limited. Here, we present a fluorescent reporter cell system that enables real-time visualization of caspase-3/-7 activity via a DEVD-based biosensor, alongside a constitutive fluorescent marker for assessing successful transduction and cell presence. We generated stable cell lines expressing this reporter and adapted them to both 2D and 3D culture systems, including organoids. This platform allowed dynamic tracking of apoptotic events and viability loss at single-cell resolution. Using a proliferation dye, we also detected apoptosis-induced proliferation in neighboring cells. Furthermore, the system enabled simultaneous detection of immunogenic cell death via an endpoint measurement of surface calreticulin exposure by flow cytometry, supporting its application in studying immunogenic signaling. By measuring and integrating multiple cell death readouts by live-cell imaging, our system is well-suited for high-content screening and mechanistic dissection of different modes of cell death. When combined with complementary markers of pyroptosis and necroptosis, this platform may also be extended to investigate more complex, integrated forms of cell death.

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Conflict of interest statement

Ethics Statement: The use of patient-derived organoids (PDOs) in this study was approved by the appropriate institutional review board or ethics committee of the University Medical Center Göttingen (11/5/17) and conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all donors prior to tissue collection. All procedures involving human samples were performed in compliance with relevant regulatory guidelines and institutional policies. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Generation and assessment of a ZipGFP-based caspase-3/-7 reporter.
A Live-cell fluorescence imaging of reporter cells under basal and apoptotic conditions. A caspase-3/-7 reporter construct was introduced into human cells via lentiviral transduction to achieve stable expression. mCherry is constitutively expressed as a marker for successful transduction and cell presence, while GFP is assembled following caspase-3/-7-dependent cleavage of the DEVD motif, providing a real-time readout of apoptosis at the single-cell level. B Representative images of MiaPaCa-2 caspase reporter cells treated for 48 h with carfilzomib (50 nM) or DMSO (vehicle control), with or without zVAD-FMK (50 µM), showing phase-contrast, mCherry, and GFP channels. Scale bar: 150 µm. C Quantification of GFP fluorescence intensity over 80 h, with measurements taken every 8 h. Displayed is the relative green fluorescence intensity of MiaPaCa-2 caspase reporter cells treated with carfilzomib (50 nM), carfilzomib (50 nM) + zVAD-FMK (50 µM) or DMSO (vehicle control). 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 (t80h p < 0.0001). D, E End-point apoptosis assessment by immunoblotting for cleaved PARP (n = 4) and cleaved caspase-3 (n = 1) (D) and by flow cytometry using Annexin V/PI staining (E) after 48 h of treatment with carfilzomib or DMSO as vehicle control. The results shown are representative of three independent experiments (n = 3). F Relative GFP fluorescence intensity in caspase-3 deficient MCF-7 reporter cells treated with carfilzomib, under the same imaging conditions as (C). 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 Sidak’s multiple comparisons test (t80h p < 0.0001). G–I Quantification of mCherry fluorescence intensity over 80 h after treatment with carfilzomib or DMSO, with measurements taken every 8 h (G) mCherry intensity at 48 h (H) and corresponding live cell counts (I) obtained using the IncuCyte® AI-based health monitoring module. Data represent mean ± SD of three independent experiments (n = 3). Statistical significance for (H) and (I) was assessed by paired t-test (**p < 0.01, ****p < 0.0001).
Fig. 2
Fig. 2. Spatiotemporal monitoring of caspase-3/-7 activity in 3D spheroid and organoid models.
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.
Fig. 3
Fig. 3. Real-time monitoring of apoptosis-induced proliferation.
A Schematic overview of the triple-reporter strategy: dual caspase-3/-7 reporter combined with a far-red succinimidyl ester (SE) proliferation dye to simultaneously monitor viability, caspase activity, and proliferation dynamics. B Representative images of reporter cells treated with 1.25 µM or 10 µM oxaliplatin and vehicle control, showing mCherry, GFP, and far-red proliferation dye fluorescence. Scale bar: 100 µm. C Time-course of relative GFP fluorescence intensity normalized to t0 and vehicle control, demonstrating a dose-dependent increase in caspase activation across oxaliplatin concentrations (1.25; 2.5; 5; 10 µM). Data represent mean ± SD of three independent experiments (n = 3). D Time-course of relative far-red fluorescence intensity normalized to t0h, illustrating dye dilution as a proxy for cell proliferation after treatment with different oxaliplatin concentrations and vehicle control as described in (C). Data represent mean ± SD of three independent experiments (n = 3). E Correlation between GFP and far-red signals for each treatment condition, calculated across individual fields of view from (C) and (D) using Spearman’s rank correlation (*p < 0.05, ***p < 0.001).
Fig. 4
Fig. 4. The caspase reporter system enables parallel quantification of immunogenic cell death and caspase activation.
A Simultaneous flow cytometric quantification of calreticulin (CALR) surface exposure, alongside caspase-3/-7 activation, allows discrimination between different modes of cell death. B, C Simultaneous staining of surface CALR (using rabbit anti-CALR + anti-rabbit-PE) and cell viability (using Zombie Aqua) after treatment with 1,5 µM carfilzomib (B) or 300 µM oxaliplatin for 6 h (C) shows different levels of ICD (upper panels) and caspase induction (lower panels). Data represent mean ± SD of three independent experiments (n = 3). Statistical significance was assessed by paired two-tailed t-test (*p < 0.05; n.s.: not significant).
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