Extended Data Fig. 7: OAT maintains in vivo tumor polyamine pools supporting PDA growth.
From: Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer
a, Generation of mouse orthotopic PDA transplants lacking Odc1 or Oat. iKras clonal cell lines with CRISPR/Cas9 knockout of Odc1 (clones #3 and #10) or Oat (clones #10 and #11) using single guide (sg) RNAs each, or sgControl (sgCtrl) were injected (5 x 105 cells) into the pancreas of non-Cre-expressing iKras mice. All mice were treated with Dox (2g l−1 drinking water) and monitored for tumor growth by ultrasound over 3 weeks, prior to subjecting them to a 3-hour infusion with 15N-(amine)Gln. b, Levels of ODC1 and OAT proteins in iKras cells from a. c,d, Relative abundance of 15N-labeled (c) or total (d) ornithine and putrescine in iKras cells from a that were fed for 24 h 15N-(amine)Gln or 15N4-Arg. n = 4 biological replicates. e, Proliferation of iKras cells described in a. n = 8 biological replicates. f–h, Volumes (ultrasound) of orthotopic iKras tumors with control (Ctrl), Odc1 or Oat knockout from a, injected into non-tumor-bearing mice (f), with representative ultrasound images (g), and weights (h), 3 weeks post-injection of iKras cells. n = 9 (sgCtrl); n = 10 (sgOdc1 #10; sgOat #10); n = 6 (sgOdc1 #3; sgOat #11). T, Tumor; S, Spleen. i, 15N enrichment in plasma glutamine of tumor-bearing mice in f–h, that were infused with 15N-(amine)Gln over 3 h, 3 weeks post-tumor cell injection (related to Fig. 2g). n = 4 mice per group. j, Relative abundance of indicated metabolites in orthotopic tumors derived from iKras cells with knockout for Odc1, Oat, or control, described in a. n = 6 mice per group. k, Relative abundance of putrescine and spermidine in human orthotopic PDA tumors derived from AsPC-1 cells with ARG2 or Scramble (Scr) knockdown, that were injected (105 cells) into the pancreas of Rag1−/− mice and grown for 6 weeks. n = 7 mice per group. l, Relative abundance of the indicated metabolites in mouse orthotopic transplant tumors derived from KPC cells (see Methods) expressing or lacking Arg2 (Arg2+/+ or Arg2−/−, respectively) that were injected (2.5 x 105 cells) into the pancreas of mice of the same strain and grown for 2 weeks. n = 6 mice per group. In j–l, Box plots represent medians ± 10-90 percentile and whiskers span minimum and maximum values. m, Levels of OAT and ODC1 proteins in iKras cells with control (sgCtrl) or Odc1 or Oat knockout (sgOdc1 or sgOat) that are overexpressing GFP control or the respective gene cDNA (Odc1 or Oat). n,o, Relative abundance of 15N-labeled (n) or total (o) ornithine and putrescine levels in iKras cells described in m, that were fed 15N-(amine)Gln for 24 h. n = 4 biological replicates. p, Proliferation of iKras cells described in m. n = 8 biological replicates. q, Volumes of syngeneic orthotopic tumor transplants derived from iKras cells described in m that were grown and quantified by ultrasound, 2 and 3 weeks post-cell injection (related to Fig. 2h). n = 8 mice (sgCtrl + GFP); n = 7 (sgCtrl + Oat); n = 9 (sgOat #10 + GFP; sgOat #10 + Oat). r, Metabolite abundance in TIF or plasma of mice related to Fig. 2i. n = 8 except sgCtrl + Oat (7). In i,j, m–r, sgOdc1 clone #10 and sgOat clone #10 from a–h were used. Data represent the mean ± s.d. (c,d,f,h,i,n,o,q,r) or mean ± s.e.m. (e,p). p-values were obtained by one-way (c,d,h,j,n,o) or two-way (e,f,i,p,q,r) ANOVA followed by Tukey test, or by unpaired two-tailed t-test (k,l). In e, n–p, statistical significance is for each condition vs. control knockout “sgCtrl” (e) or each condition vs. “sgCtrl + GFP” (n–p). In b,m, Arrowhead indicates non-specific band detected by ODC1 antibody (ab97395) and β-Actin was used as a loading control. Data are representative of two (b–d, m–o) or three (e,p) independent experiments.
