Organoid-based profiling identifies TGFB1 and PADI2 as key modulators of pancreatic cancer growth

Objective: This study aimed to systematically evaluate the effects of human cytokines on the growth and transcriptional regulation of pancreatic ductal adenocarcinoma (PDAC) cells, using 3D tumor spheroid models to identify key cytokine–gene interactions relevant to tumor progression. Methods: Human PDAC cell lines (PANC-1, CFPAC-1) were cultured in Matrigel-based 3D tumor spheroid systems and treated with a panel of 365 human cytokines. Tumor spheroid number and size were quantified over time. RNA sequencing and quantitative polymerase chain reaction (PCR) were performed to profile transcriptional changes induced by transforming growth factor beta 1 (TGFB1) in 2D versus 3D cultures. Peptidyl arginine deiminase 2 (PADI2) function was validated using short hairpin RNA (shRNA)-mediated knockdown, followed by growth assays and western blot analysis. Results: TGFB1 suppressed pancreatic tumor spheroid formation and growth in 3D culture but showed no effect in 2D monolayer systems. Transcriptomic profiling revealed distinct TGFB1-regulated pathways in 3D spheroids, including phosphoinositide 3-kinase (PI3K)–protein kinase B (AKT), wingless and int-1 (WNT), and amebiasis signaling, while proteoglycan and focal adhesion pathways predominated in 2D. PADI2 expression was selectively upregulated by TGFB1 in 3D spheroids but not in 2D cultures. Functionally, PADI2 knockdown inhibited 3D tumor spheroid growth and potentiated TGFB1-mediated suppression, indicating that PADI2 supports tumor cell survival under TGFB1-induced stress. Conclusions: 3D tumor spheroid models better capture physiologically relevant cytokine responses than conventional 2D systems. TGFB1 acts as a potent inhibitor of pancreatic tumor spheroid growth through model-specific transcriptional reprogramming, while PADI2 serves as a TGFB1-induced survival factor and potential therapeutic target in PDAC.