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.