Background: Carbohydrate antigen 19-9 (CA19-9) is the most widely used biomarker for pancreatic cancer. Since CA19-9 closely correlates with patient outcome and tumor stage in pancreatic cancer, the deciphering of CA19-9 biosynthesis provides a potential clue for treatment. Methods: Concentration of amino acids was detected by ultrahigh-performance liquid chromatography tandem mass spectrometry. Metabolic flux of glutamine was examined by isotope tracing untargeted metabolomics. Label-free

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... itative N-glycosylation proteomics was used to examine N-glycosylation alterations. Results: Among all amino acids, glutamine was higher in CA19-9-high pancreatic cancers (> 37 U/mL, 66 cases) than in CA19-9-normal clinical specimens (≤ 37 U/mL, 37 cases). The glutamine concentration in clinical specimens was positively correlated with liver metastasis or lymphovascular invasion. Glutamine blockade using diazooxonorleucine suppressed pancreatic cancer growth and intraperitoneal and lymphatic metastasis. Glutamine promotes O-GlcNAcylation, protein glycosylation, and CA19-9 biosynthesis through the hexosamine biosynthetic pathway. UDP-N-acetylglucosamine (UDP-GlcNAc) was an intermediate product between glutamine and CA19-9. Conclusions: Pancreatic cancer cells crave glutamine for CA19-9 biosynthesis. Glutamine blockade may be a potential therapeutic strategy for pancreatic cancer.