Figure 1.

Glutamine deprivation induces EMT and promotes aggressive behaviors in PDAC cells. (A) Quantification of amino acids in orthotopic KPC tumors relative to normal murine pancreatic tissue. NEAA, nonessential amino acids; EAA, essential amino acids. Data are presented as box and whiskers plots. Vertical lines extend to the minimum and maximum values. (B) Immunohistochemical staining using antibodies specific to ASNS and SESN2 in normal murine pancreas, KPC orthotopic pancreatic tumor, and human PDAC specimen. Scale bar, 50 µm. (C) GSEA plots derived from RNA-seq data of glutamine-deprived versus control (glutamine-replete) KPC or AsPC-1 cells. NES, normalized enrichment score; FDR, false discovery rate. (D) Relative mRNA expression of the indicated genes measured by qPCR in KPC or AsPC-1 in glutamine-replete or glutamine-free medium for 24 h. Results are expressed relative to control cells in glutamine-replete conditions (+Q). Data are expressed as mean ± SEM; n = 5 (KPC) and n = 4 (AsPC-1) independent experiments. (E and F) Migration (E) and invasion (F) capacity of the indicated PDAC cells cultured in glutamine-replete medium (High Q, 4 mM) or glutamine starvation conditions (Low Q, 0 or 0.2 mM). Quantification is shown relative to the High Q condition and expressed as mean ± SEM; n = 3–5 independent experiments, each performed in triplicate. Representative images of migrating/invading cells stained with crystal violet are shown. Statistical significance was determined using unpaired two-tailed Student’s t test. The quantification of amino acids in A was generated from one experiment with n = 6 mice per cohort. The GSEA plots in C were generated from a single RNA-seq experiment that included n = 5 replicates per condition. Results are representative of two independent experiments in B and a pool of three to five independent experiments in D, E, and F. *, P < 0.05; **, P < 0.01; ***, P < 0.001.