Journal: Advanced Science

Article Title: Engineering a 660 nm‐Responsive Optogenetic Inducer of Pyroptosis for Precision Cancer Therapy

doi: 10.1002/advs.76768

Figure Lengend Snippet: Spatiotemporally controlled tumor cell ablation in vitro. (A) Design of adenoviral vector for all‐in‐one delivery of PyroRACS. Expression cassettes for RACS and GSDMD NT were incorporated into a single adenoviral vector. ITR, inverted terminal repeat; pCMV, cytomegalovirus promoter; pCBH, CBH promoter; IRES, internal ribosome entry site. (B) Titer determination of the adenovirus encoding PyroRACS. Data are presented as mean ± SD; unpaired t ‐test, n = 3 independent replicates. PFU, plaque‐forming units. (C,D) Pyroptosis induction in bladder cancer cells. Bladder cancer cells 5637 and BIU87 were transduced with adenovirus at specified multiplicity of infection (MOI). Pyroptotic cells were stained with Annexin V‐FITC/PI 24 h post‐illumination (660 nm, 1 mW/cm 2 , 30 s). Cell death was quantified by flow cytometry. Representative fluorescence images of pyroptotic 5637 and BIU87 cells (C), scale bar: 20 µm. Flow cytometry quantification of pyroptotic cells (D); data are presented as mean ± SD, unpaired t ‐test, n = 4 biological replicates. (E,F) DAMPs release kinetics during pyroptosis. ATP (E) and LDH (F) levels in culture supernatants were quantified at indicated time points post‐illumination, with non‐illuminated cells serving as negative controls. Data are presented as mean ± SD (F); unpaired t ‐test, n = 4 biological replicates. RLU, relative luminescence units; LDH, lactate dehydrogenase. (G,H) Assessment of cytotoxicity under non‐induced conditions. Following adenoviral transduction, 5637 or BIU87 cells were cultured in the dark for 48 h. The levels of ATP (G) and LDH (H) in the culture supernatant were quantified, with non‐transduced cells (UT) as negative controls. Data: mean ± SD, n = 3 (5637) or 4 (BIU87) biological replicates. (I) Spatio‐specific pyroptosis induction. 5637 cells transduced with adenovirus at a MOI of 50. At 48 h post‐transduction, cells were illuminated through a custom‐designed striped photomask to induce localized cell death, followed by SYTOX Green staining 24 h post‐illumination (660 nm, 50 µW/cm 2 , 3 min). The upper panel shows the custom‐designed striped photomask used for patterned illumination. Scale bar: 1 mm; n = 2 independent experiments. (J) Light dose‐dependent pyroptosis induction. 5637 cells transduced with adenovirus at a MOI of 100. At 48 h post‐transduction, cells were illuminated (660 nm, 1 mW/cm 2 ) for the indicated durations. Cell death was quantified by flow cytometry 24 h after illumination. Data: mean ± SD; unpaired t ‐test, n = 3 biological replicates; * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Cytotoxic lactate dehydrogenase (LDH) release dynamics were quantified using a commercial LDH assay kit (MCE, Cat. HY‐K1090) according to the manufacturer's instructions.

Techniques: In Vitro, Plasmid Preparation, Expressing, Transduction, Infection, Staining, Flow Cytometry, Fluorescence, Cell Culture