(A) Relative mRNA expression levels of CAF-marker genes in WT and Ras^V12 mouse fibroblasts determined by quantitative RT-PCR and analyzed using 2-way ANOVA (n = 3). (B) EpCAM-positive cancer epithelium was quantitated by FACS of 3D cocultures of human epithelial (CWR22Rv1) cells and WT or Ras^V12 mouse fibroblasts. Statistical analysis was performed using 2-tailed Student’s t test (n = 3). (C) ATP was measured in CWR22Rv1 after incubation with CM human NAF or CAF or mouse WT or Ras^V12 fibroblast or oligomycin in glutamine-free media (n = 3). Statistical analysis was performed using 1-way ANOVA with multiple comparisons. (D) Diagram represents the TCA cycle in CWR22Rv1 cells treated with NAF or CAF CM for 72 hours prior to metabolome analysis (n = 3). (E) Metabolome analysis further indicated differential flux to aspartate, oxidized GSH, and GSH reduced in CWR22Rv1 cells incubated with NAF or CAF CM. (F) Glutamine concentrations (Gln conc.) were measured in the CM from indicated mouse fibroblasts cultured for 72 hours. cond, conditioned. (D–F) Statistical analysis was performed using 2-tailed Student’s t test (n = 3). (G) CWR22Rv1 proliferation was measured by cell counting following incubation with NAF and CAF CM for 72 hours in glutamine-free media. NAF CM was supplemented with 0.4 mM glutamine to mimic the glutamine levels expressed by CAF (see Supplemental Figure 4B). Statistical analysis was performed using 1-way ANOVA with multiple comparisons (n = 3). (H) Proton magnetic resonance spectroscopy data were acquired from orthotopically xenografted mice before and after EIPA administration. Spectra of the unfiltered data are superimposed using simulated echo acquisition (n = 3–4 per group). Glu, gllutamate; ppm, parts per million. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.