BACKGROUND/OBJECTIVES: Pancreatic adenocarcinoma (PDAC) remains one of the most lethal cancers, with limited advancements in treatment efficacy due to high rates of chemoresistance. Circulating tumor cells (CTCs) derived from liquid biopsies offer a non-invasive approach to monitoring tumor evolution and identifying molecular mechanisms of resistance. This study aims to longitudinally collect, culture, and characterize CTCs from PDAC patients to elucidate resistance mechanisms and tumor-specific gene expression profiles. METHODS: Blood samples from 10 PDAC patients were collected across different treatment stages, yielding 16 CTC cultures. Differential gene expression, pathway dysregulation, and protein-protein interaction studies were utilized, highlighting patient-specific and disease progression-associated changes. Longitudinal comparisons within five patients provided further insights into dynamic molecular changes associated with therapeutic resistance. RESULTS: CTC cultures exhibited the activation of key pathways implicated in PDAC progression and resistance, including TNFα/NF-kB, hedgehog signaling, and the epithelial-to-mesenchymal transition. Longitudinal samples revealed dynamic changes in signaling pathways, highlighting upregulated mechanisms of chemoresistance, including PI3K/Akt/mTOR and TGF-β pathways. Additionally, protein-protein interaction analysis emphasized the role of the immune system in PDAC progression and therapy response. Patient-specific gene expression patterns therefore suggest potential applications for precision medicine. CONCLUSIONS: This proof-of-concept study demonstrates the feasibility of longitudinally capturing and analyzing CTCs from PDAC patients. The findings provide critical insights into molecular drivers of chemoresistance and highlight the potential of CTC profiling to inform personalized therapeutic strategies. Future large-scale studies are warranted to validate these findings and further explore CTC-based approaches in PDAC management.