Mechanistic Insight into Serine Flux Regulation through Nanoscale Organization of Glucose and Serine Transporters by Substrate Probe-Based Direct Stochastic Optical Reconstruction Microscopy Imaging.

Serine serves as a metabolic nexus in tumors, coordinating one-carbon metabolism, nucleotide synthesis, and redox regulation. While serine transporters (SerTs) are known to be dysregulated in cancer, their functional nanoscale organization remains unresolved due to the limitation of resolution imaging and available probes. Here, we developed a substrate-based fluorescent probe (Ser-probe) enabling direct stochastic optical reconstruction microscopy of SerTs, revealing malignancy-associated clustering assembly of SerTs that correlates with transport capacity. Compared to MDA-MB-231 cells, MCF7 cells with higher endogenous serine biosynthetic capacity exhibited more pronounced SerT/glucose transporter (GluT) co-clustering, suggesting that their spatial assemblies closely correlate with serine transport and biosynthetic functions in maintaining serine homeostasis. Their cluster morphology and co-assembly were revealed to depend critically on lipid rafts and glycan cross-linking, identifying the key determinants of spatial distribution to enable mechanistic understanding and potential regulation. Glucose deprivation weakened SerT/GluT clustering and their colocalization, which may be caused by their attenuated functional cooperativity in serine homeostasis maintenance under glucose-dependent suppression of serine synthesis. Pharmacological inhibition of phosphoglycerate dehydrogenase (PHGDH) initially enhanced SerT/GluT aggregation and colocalization, but this effect gradually attenuated as doses increased. The strategic combination of a PHGDH inhibitor with glucose restriction or free sialic acid synergistically disrupted SerT/GluT nanoscale organization, amplifying the anti-tumor efficacy of the PHGDH inhibitor and establishing the metabolic plasticity of transporter assemblies as a targetable vulnerability. This work establishes a fundamental link between transporter spatial assembly and tumor serine metabolic reprogramming, providing a new perspective to better understand SerT dysfunction in tumor metabolic reprogramming, offering novel therapeutic avenues for targeting serine metabolism in cancer.