Adaptive response to serine and glycine deprivation enhances the stemness potential of pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) develops within a nutrient-poor microenvironment, where pancreatic cancer stem cells (PaCSCs) can persist despite limited oxygen and nutrient availability. This resilience partially relies on mitochondrial metabolic flexibility. Serine and glycine are conditionally essential amino acids in PDAC, and many human PDAC models depend on their exogenous supply. However, whether serine/glycine metabolism and the serine synthesis pathway (SSP) contribute to PaCSC maintenance and the consequences of their deprivation for stemness remain unclear. Primary cultures from PDAC patient-derived xenografts (PDXs) were used to model CSC enrichment and adaptation to serine and/or glycine deprivation. SSP gene expression, serine/glycine metabolism, and mitochondrial function were assessed using bioinformatic analyses, RNA and protein measurements, 13 C-glucose tracing, and real-time metabolic assays. Functional consequences were evaluated through in vitro and in vivo assays measuring viability, metabolism, stemness, invasion and tumorigenicity. Bioinformatic analyses showed that expression of the SSP genes PHGDH and PSAT1 was inversely correlated with stemness-related signatures in human PDAC and was reduced in CSC-enriched PDX cultures. Nevertheless, CSC-enriched cultures showed higher SSP pathway activity, inferred from a higher relative contribution of glucose-derived carbon to serine and glycine synthesis and increased resistance to acute serine or glycine deprivation. We next examined how PDAC cells adapt to long-term limited extracellular serine and/or glycine availability. Resistant cultures acquired increased mitochondrial respiratory flexibility, characterized by higher maximal respiration, spare respiratory capacity and resistance to the complex I inhibitor rotenone, but also showed reduced mitochondrial efficiency, as indicated by increased proton leak and lower mitochondrial transmembrane potential. Adaptation to single amino acid deprivation was associated with features consistent with increased fatty acid utilization, whereas resistance to combined serine and glycine deprivation was linked to greater glucose dependence. Functionally, deprivation-resistant cultures showed increased CSC frequency, migration and invasion, and in vivo tumorigenicity, although these effects were less pronounced after combined serine and glycine deprivation. These findings reveal a functional link between serine/glycine availability, mitochondrial plasticity and PaCSC behavior. Adaptation to serine and/or glycine deprivation promotes metabolic rewiring that enhances the stemness potential of PDAC cells, supporting the concept that amino acid metabolic flexibility contributes to PaCSC persistence under nutrient-limited conditions.