One-way ANOVA; b, *=0.0134; c, *=0.0129. we uncover a previously undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel source decreases the tumours dependence on glucose and serum-derived nutrients, which are limited in the pancreatic tumour microenvironment4,11. Moreover, we demonstrate that alanine secretion by PSCs is dependent on PSC autophagy, a process that is stimulated by cancer cells. Thus, our results demonstrate a novel metabolic conversation between PSCs and cancer cells, in which TGFB2 PSC-derived alanine acts as an alternative carbon source. This finding highlights a previously unappreciated metabolic network within pancreatic tumours in which diverse fuel sources are used to promote growth in an austere tumour microenvironment. We previously exhibited that metabolism is usually rewired in pancreatic cancer cells to facilitate biosynthesis and maintain redox balance in the Gilteritinib hemifumarate nutrient-poor conditions of a pancreatic tumour2,14,15. While extracellular protein can provide nutrients to the starved cancer cells11,13, we hypothesized that this stroma may provide additional avenues of metabolic support for the tumour. Pancreatic stellate cells (PSCs) are a predominant cell type in the pancreatic tumour stroma and are important mediators of the desmoplastic response. Their abundance suggests that they may contribute to the metabolism of cancer cells. To test this idea, we assessed changes in the oxygen consumption rate (OCR) and extracellular media acidification rate (ECAR), steps of mitochondrial activity and glycolysis, respectively, in PDAC cells treated with conditioned medium from a well characterized human PSC (hPSC) line16 (Fig. 1a, b and Extended Data Fig. 1aCe). PDAC glycolysis showed minimal changes when cells were treated with PSC-conditioned medium, as measured by ECAR (Extended Data Fig. 1d, e). By contrast, we observed a consistent increase of 20C40% in the basal OCR after treatment with hPSC medium (Fig. 1a, b and Extended Data Fig. 1aCc), a feature Gilteritinib hemifumarate that was impartial of serum during the conditioning process (Extended Data Fig. 1f, g) and reproducible with multiple primary specimens (Fig. 1b and Extended Data Fig. 1h, i). Notably, this metabolic phenotype was specific to pancreatic cancer cells; non-transformed pancreatic ductal epithelial cells did not exhibit increased OCR in response to PSC medium (Extended Data Fig. 1j). Open in a separate window Physique 1 Pancreatic stellate cells secrete metabolites that fuel pancreatic cancer metabolisma, Conditioned medium (CM) from hPSCs increases PDAC OCR (green line), as compared to cells treated with PDAC CM (red line) or control (DMEM with 10% serum, black line). A representative trace showing change in OCR during a mitochondrial stress test. Error bars depict s.d. of 6 impartial wells from a representative tracing from 6 impartial experiments (depicted in b). b, Per cent change in basal OCR for 8988T cells treated with conditioned medium from different cell lines relative to 8988T cells treated with standard culture medium. Error bars depict s.e.m. of pooled impartial experiments (= 3 for primary hPSC #1, #2, primary mPSC; =4 for hPSC#2, IMR90 and MiaPaCa2; = 6 for 8988T, hPSC#1). c, OCR activity of PSC-conditioned medium is retained after heating at 100 C for 15 min. Error bars, s.e.m. of impartial experiments (=4). d, Metabolites that were significantly elevated in PSC-conditioned medium, decreased in double-conditioned medium (PSC-conditioned medium added to 8988T cells and then collected), and elevated intracellularly in PDAC cells treated with PSC-conditioned medium. Error bars, s.d. (=3). e, A mixture of NEAAs (1 mM alanine, aspartate, asparagine, glycine, glutamate, proline and serine) or alanine alone increases PDAC OCR. Data are normalized to cells treated with standard culture medium. Error bars, s.e.m. of impartial experiments (=4). f, The concentration of Gilteritinib hemifumarate alanine was measured in conditioned medium samples using liquid chromatography with tandem mass spectrometry (LCCMS/MS). Error bars, s.d. (=3). Significance decided with one-way.