Fig 2: Tryptophan-derived one-carbon units are incorporated into nucleotides in pancreatic tumors in vivo and can substitute for serine(A) Schematic diagram outlining in vivo13C11-tryptophan tracing.(B) KPC cell line 2 (see Figures 1E and S1A) from C57Bl6/J Pdx1-Cre;KrasG12D/+;Trp53R172H/+ mice were engineered to stably express IDO1 (KPC-IDO1) or empty vector control (KPC-EV). Cells were injected into the flanks of C57Bl6/J mice. Once tumors had formed, the mice were given a single intraperitoneal injection of 800 µL 120 mM 13C11 tryptophan and harvested after 3 h. The tumor tissue was excised and analyzed by LC-MS (EV n = 7, IDO1 n = 7, p values calculated with unpaired 2-sided t test, bars are SDs). y axis = 13C labeled fraction for the stated isotopologues.(C) KPC-IDO1 cells were grown in culture medium with or without serine, with or without epacadostat (1 µM). The cell number was counted every 24 h for 5 days; n = 3 biological replicates, bars are SDs.(D) KPC-IDO1 cells were grown in culture medium with or without serine, with or without epacadostat (1 µM) for 24 h, and intracellular metabolites were analyzed by LC-MS; n = 3 biological replicates, bars are SDs.(E and F) KPC-IDO1 cells were injected as subcutaneous allografts into BALB/c nude mice and received vehicle or epacadostat (50 mg/kg twice per day) as oral gavage. Mice either received a control diet containing serine and glycine (E) or a matched diet lacking serine and glycine (F). Bars are SDs. p values calculated using unpaired 2-sided t test.

Fig 3: Knocking out Sox2 triggers TRC exit from dormancy. (A) Cell cycles of Sox2-knocked out (KO) cells were analyzed by flow cytometry after seeding in 90-Pa fibrin gels for 5 days. (B) Cells were cultured in 90-Pa fibrin gels for 5 days and total proteins were extracted for analyses of PCNA or CD133 expression by western blotting. Left: representative blotting images; right: quantitative data (mean ± SEM; n = 3 independent experiments). (C) Both control cells and Sox2 knockout cells were treated with 0.1% DMSO, Tazarotene (100 µM), or all-trans retinoic acid (ATRA) (100 µM) for 3 days in 90-Pa fibrin gels. Similarly, cells were cultured in 900 Pa fibrin gels for 2 days and then treated with IFN-? (100 ng/ml) for 24 h. Colony sizes were measured on day 5 (mean ± SEM; n = 3 independent experiments). (D) Cell apoptotic ratio was measured by flow cytometry ( mean ± SEM; n = 3 independent experiments). (E) Representative westernblotting images of IDO1 expression in control B16 cells or Sox2-knocked out cells, either treated with IFN-? or with PBS. Quantification results are shown in Figure S4C.*P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: IDO1 is also regulated by attachment-independent growth via JAK/STAT signaling(A) Schematic diagram of the kynurenine pathway.(B–D) Protein expression was analyzed after 24 h of (B) culture under either normoxic (20% O2) or hypoxic (1% O2) conditions, (C) treatment with rotenone (1 µM) or vehicle-only control, or (D) culture in media containing either glucose (Glc) (10 mM) or galactose (Gal) (10 mM).(E and F) Cell lines were cultured in 2D or 3D conditions for 24 h and cell lysates analyzed for protein expression, and (F) band intensity of IDO1 relative to actin (loading control) was quantified with a Li-Cor infrared scanner (data from n = 4 independent experiments, p value calculated with paired 2-sided t test, bars are SDs).(G) CFPAC-1 cells were cultured in 2D or 3D (attachment-independent) conditions for 24 h and treated with epacadostat (1 µM) or vehicle-only control for 16 h before media (extracellular) kynurenine was analyzed by LC-MS (n = 3 biological replicates, bars are SDs).(H) CFPAC-1 and HPAF-II cells were cultured in either 2D or 3D conditions for 24 h and then treated for 16 h with JAKi or vehicle-only control (veh.) and/or human IFN? (1 ng/mL). Cells were then lysed and protein expression analyzed.

Fig 5: Tryptophan-derived one-carbon units are incorporated into serine and purine nucleotides in pancreatic cancer cells(A and B) CFPAC-1 cells were grown with or without human IFN? (1 ng/mL) or with IFN? and IDO1 inhibitor epacadostat for 24 h (A), or in 2D versus 3D conditions for 24 h (B), in the presence of 13C11-tryptophan. Intracellular metabolites were analyzed by LC-MS and corrected for natural background 13C abundance. y axis = 13C labeled fraction for the stated isotopologues. Labeled isotopologues are defined as mass + n (m+n), where n = number of 13C incorporated. The potential positions of tryptophan-derived carbons are labeled in red in the structural diagrams.See Figure S3C for explanation of labeling in acetyl-CoA. n = 3 biological replicates, error bars are SDs.