Glutamine is a central nutrient for pancreatic cancer, supporting various metabolic programmes linked to cell growth and survival [1]. Inhibition of the enzyme glutamic-oxaloacetic transaminase 1 (GOT1) has emerged as a promising strategy to disrupt glutamine metabolism and selectively target cancer cells [2]. However, it remains unclear how GOT1 inhibition impacts elements of the tumour microenvironment, and vice versa. Using tumour tissue engineering, we developed a multicellular 3D cancer model to recreate pancreatic cancer ‘on a dish’ and investigate the interplay between glutamine metabolism and the extracellular matrix. Human pancreatic cancer cells, together with cancer-associated fibroblasts and peripheral blood mononuclear cells, were embedded in bioengineered hydrogels and cultured as spheroids. To evaluate metabolism-targeting therapies, multicellular 3D cultures were treated with a GOT1 inhibitor, both alone and in combination with the chemotherapeutic drugs gemcitabine and nab-paclitaxel. Proteomic and metabolomic profiling were conducted using liquid chromatography-mass spectrometry. Cell metabolic activity, proliferation, and mechanical properties were assessed using PrestoBlue, CyQUANT assays, and indentation testing, respectively. Our pancreatic 3D model exhibited an elastic modulus of 15 kPa and upregulated the expression of key matrisome proteins, including collagen I, collagen VI, and fibronectin. Multicellular 3D cultures showed enhanced glutamine metabolism, central carbon metabolism, and oxidative phosphorylation. Cancer cell responses to GOT1 inhibition were influenced by the presence of collagen I and hyaluronic acid in the tumour microenvironment, resulting in a 50% reduction in cell proliferation. In the presence of stromal cells, GOT1 inhibition decreased proliferation by 25% and increased the expression of collagens, laminins, and lysyl oxidases, thereby remodelling the extracellular matrix architecture. Treatment with the GOT1 inhibitor in combination with gemcitabine and nab-paclitaxel reduced cell viability by 80%. Our findings reveal that GOT1 inhibition effectively targets glutamine metabolism and reshapes the extracellular matrix in pancreatic cancer.