Radiation-Induced EMT of Adipose-Derived Stem Cells in 3D Organotypic Culture via Notch Signaling Pathway

In our previous study, adipose-derived stem cells (ASCs) cultured in a three-dimensional (3D) organotypic system exhibited mesenchymal-to-epithelial transition (MET) features, including cobblestone morphology and increased expression of E-cadherin and CK18. In this study, we investigated whether ionizing radiation could reverse this phenotype via epithelial-mesenchymal transition (EMT) and examined the involvement of Notch signaling. Mouse ASCs were cultured in Matrigel-based 3D organotypic conditions and exposed to 8 Gy of γ-radiation, and EMT- and Notch-related gene and protein expression were assessed 96 h post-irradiation using ATP viability assays, RT-qPCR, and Western blotting. Exposure to 8 Gy significantly reduced cell viability in 2D ASCs to 49.50 ± 6.50% compared with 61.02 ± 5.77% in 3D organoids (p < 0.0001). Irradiated 3D organoids showed EMT-like changes, including an increase of ~2.5-fold in fibronectin and an increase of ~2.0-fold in Twist1 expression, while epithelial CK18 was modestly elevated. Notch signaling was concurrently activated, with Notch1 and Jagged1 increasing by more than twofold and Fra-1 being significantly upregulated. Pretreatment with 20 μM of the γ-secretase inhibitor (GSI) kept cell viability above 90% and suppressed radiation-induced fibronectin, Twist1, Notch1, and Jagged1 expression. These findings indicate that ionizing radiation promotes EMT in 3D-cultured ASCs and reverses prior epithelialization, with Notch signaling playing a key regulatory role. The 3D ASC organoid model may thus provide a physiologically relevant platform for investigating radiation-induced plasticity and potential antifibrotic interventions.