Systemorientierte Einzelzell-RNA-Sequenzierungsanalyse von Zellzustandsübergängen und Chemoresistenz in 3D-pankreatischem Tumorgewebe

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, largely due to its intrinsic heterogeneity and pronounced resistance to chemotherapy. To dissect the cellular mechanisms underlying these adaptive responses, this study employed single-cell RNA sequencing (scRNA-seq) of 3D PDAC cultures exposed to gemcitabine (GEM), transforming growth factor β1 (TGFβ1), or their combination. Using computational analyses encompassing clustering, differential gene expression, and pseudotime trajectory reconstruction, we mapped the transcriptional transitions driving therapy resistance and phenotypic plasticity. Static clustering revealed distinct subpopulations defined by proliferative and quiescent transcriptional programs. GEM-treated cells predominantly localized along a proliferative G₂/M-associated branch characterized by the upregulation of cell-cycle regulators such as CDK1, AURKA, and CENPF, suggesting that chemoresistance arises from pre-existing cycling states rather than de novo resistant lineages. Chronic TGFβ1 exposure induced a separate epithelial–mesenchymal transition (EMT)-like trajectory enriched for extracellular matrix remodeling genes (FN1, COL5A1, THBS1) and metabolic reprogramming signatures, indicating a shift toward invasive and stress-tolerant phenotypes. Importantly, pseudotime inference revealed that the combination of GEM and TGFβ1 produced hybrid EMT–proliferative states, co-expressing mitotic and mesenchymal markers (CDK1, FN1, S100A2, THBS1). These transitional phenotypes maintained replicative capacity while acquiring migratory and survival advantages, reflecting the dynamic nature of PDAC plasticity. Gene set enrichment analyses confirmed coordinated activation of pathways involved in cell-cycle progression, TGFβ signaling, and hypoxia-related metabolism across treatment conditions. Together, these results demonstrate that resistance in PDAC is a trajectory-dependent and temporally regulated process, emerging through the integration of proliferative and EMT programs rather than fixed genetic alterations. By reconstructing these transcriptional trajectories at single-cell resolution, this work provides mechanistic insight into how TGFβ1 signaling and GEM exposure cooperate to promote phenotypic diversification, chemotolerance, and invasive potential in pancreatic cancer.