Proteomic analysis of bioprinted pancreatic tumor models under different nutrient conditions

Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stromal microenvironment that restricts nutrient availability and contributes to tumor adaptation. Cancer-associated fibroblasts (CAFs), extracellular matrix components, and tumor cells participate in reciprocal interactions that influence the metabolic state of PDAC tumors. Objective This study investigated nutrient-associated proteomic changes in three-dimensional (3D) bioprinted PDAC–CAF tumor constructs and examined alterations in metabolic and cell-junction-related pathways under nutrient-avid and nutrient-deprived conditions. Methods Three-dimensional bioprinted pancreatic tumor constructs composed primarily of PANC1 cells and cancer-associated fibroblasts (CAFs) were cultured under nutrient-avid (N) and nutrient-deprived (N−) conditions. Constructs were monitored at Days 0, 6, and 12, and Day 6 was selected for comparative proteomic analysis as an intermediate stage at which nutrient-associated structural divergence was evident while both tumor and stromal compartments remained detectable. Label-free high-resolution LC–MS/MS proteomic profiling was followed by KEGG pathway enrichment and complementary bioinformatic analyses. Selected junction-associated findings were further examined by quantitative real-time PCR. Results Distinct nutrient-associated proteomic signatures were observed in the bioprinted tumor constructs. Nutrient-avid conditions were associated with enrichment of pathways including pantothenate and CoA biosynthesis, β-alanine metabolism, and arginine and proline metabolism. Nutrient-deprived conditions showed enrichment of pathways related to unsaturated fatty-acid biosynthesis, fatty-acid elongation, riboflavin metabolism, folate-associated metabolism, and terpenoid backbone biosynthesis. Nutrient-associated differences were also observed among proteins assigned to gap-junction, adherens-junction, focal-adhesion, tight-junction, and central-carbon-metabolism pathways. Selected junction-associated proteomic observations were supported at the transcript level by qPCR, although concordance between mRNA and protein abundance varied among targets. Conclusion Nutrient availability was associated with distinct metabolic and junction-related proteomic signatures in 3D bioprinted PDAC–CAF constructs. These findings provide hypothesis-generating evidence of nutrient-dependent molecular remodeling within a multicellular PDAC model. Functional perturbation studies will be required to determine the causal and therapeutic relevance of the candidate proteins and pathways identified here.