Proteomic Characterization of Primary Human Pancreatic Cancer Cell Lines Following Long-Term Exposure to Gemcitabine

Background: Gemcitabine (GEM) remains a cornerstone in the treatment of pancreatic cancer. Upon exposure to GEM, pancreatic cancer cells (PCCs) tend to adapt quickly to outcompete drug-induced cytotoxicity, thereby contributing to treatment failure. Thus, understanding GEM-induced molecular changes in PCCs is important. Methods: Three primary PCC lines (PCC-1, PCC-2, PCC-7) and Mia PaCa-2 cultured for 40 passages (p) in the absence (control) or presence of GEM (GemR) were assessed for phenotypic changes. Proteome profiles for all PCCs at p10, p20, p25, p30, p35, and p40 were obtained using mass spectrometry (MS). Protein expression was determined using immunoblotting. Differentially abundant proteins (DAPs) were evaluated for enrichment of functional and biological attributes and protein–protein interactions. Results: GEM sensitivity and growth were both reduced in GemR versus paired controls for all four PCC lines. MS mapped > 7000 proteins in each PCC line, and the abundance of 70–83% of these was found to be significantly altered when comparing all sample groups. Proteomic changes in GemR versus paired controls differed remarkably among the PCCs and were affected by passaging and treatment duration. DAPs at p40 were mostly related to metabolic pathways, including nucleotide metabolism and diverse cell growth processes. Several closely related DAPs and multiple hub proteins in each PCC line were identified. Conclusions: Overall, this study revealed cell-line-specific, heterogeneous changes in proteome profiles of PCCs following their long-term exposure to GEM, and these were likely affected by treatment duration, dosage, and passaging.