Abstract 133: From waste to nutrient: ammonia's recycling by fibroblasts to sustain collagen production

Abstract Collagen and other extracellular matrix (ECM) proteins are highly abundant in tumors, including pancreatic and breast cancers, and are involved in tumor progression and therapy resistance. Given its amino acid composition, collagen production imposes unique biosynthetic demands on cancer-associated fibroblasts (CAFs), particularly a high need for proline. Understanding how CAFs meet the metabolic requirements for collagen synthesis could provide opportunities to modulate ECM production in cancer. CAFs synthesize proline from glutamine; however, glutamine and other nutrients are often depleted in poorly perfused tumors. How CAFs maintain collagen synthesis under such nutrient-limiting conditions remains unclear, and addressing this question may reveal unique metabolic vulnerabilities in cancer. Previous work from our laboratory showed that CAFs recycle extracellular lactate as an alternative carbon source to sustain non-essential amino acid (NEAA) biosynthesis for collagen production. However, NEAA synthesis also requires nitrogen, and how CAFs fulfill the nitrogen demands for NEAA synthesis under nutrient limitations remains poorly understood. To address this gap, this study aims to identify the nitrogen source used by CAFs for collagen production. To define the nitrogen source for collagen synthesis, we performed metabolic tracing of potential nitrogen donors using fibroblasts stimulated to deposit ECM in the presence of 15N-labeled nutrients. Stable isotope tracing with GC-MS was used to quantify 15N-labeled hydroxyproline in the decellularized and hydrolyzed ECM. We further used CRISPR/Cas9 to delete rate-limiting enzymes to elucidate the pathways involved. Our findings indicate that ammonia, a waste product of cellular metabolism, is recycled for NEAA biosynthesis. Ammonia’s contribution to collagen was dependent on the concentrations of glutamine and ammonia, and could rise to up to 50% of nitrogen in ECM hydroxyproline. All tested fibroblast cell lines exhibited similar ammonia-derived nitrogen labeling. Through genetic and chemical inhibition, we identified glutamate dehydrogenase (GDH) as a key enzyme in ammonia recycling via reductive amination. In conclusion, ammonia, a waste product of cancer metabolism, can be recycled by CAFs to sustain collagen production. This discovery highlights the metabolic plasticity of fibroblasts to maintain ECM synthesis under nutrient-limited conditions and offers new avenues for targeting adaptive pathways to reduce CAF-mediated pro-tumoral effects. Citation Format: Julien Guillard, Shan Cao, Marta Storl-Desmond, Brandon Faubert, Simon Schwörer. From waste to nutrient: ammonia's recycling by fibroblasts to sustain collagen production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 133.