Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions.DOI: http://dx.doi.org/10.7554/eLife.10250.001

Reactive stromal cells are an integral part of tumor microenvironment (TME) and interact with cancer cells to regulate their growth. Although targeting stromal cells could be a viable therapy to regulate the communication between TME and cancer cells, identification of stromal targets that make cancer cells vulnerable has remained challenging and elusive. Here, we identify a previously unrecognized mechanism whereby metabolism of reactive stromal cells is reprogrammed through an upregulated glutamine anabolic pathway. This dysfunctional stromal metabolism confers atypical metabolic flexibility and adaptive mechanisms in stromal cells, allowing them to harness carbon and nitrogen from noncanonical sources to synthesize glutamine in nutrient-deprived conditions existing in TME. Using an orthotopic mouse model for ovarian carcinoma, we find that co-targeting glutamine synthetase in stroma and glutaminase in cancer cells reduces tumor weight, nodules, and metastasis. We present a synthetic lethal approach to target tumor stroma and cancer cells simultaneously for desirable therapeutic outcomes.

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low‐invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high‐invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high‐invasive OVCA cells by blocking glutamine's entry into the TCA cycle, along with targeting low‐invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs.

To study the effect of dietary N level on urea kinetics and recycling, four Holstein heifers (267 +/- 3.6 kg) were used in a Youden square design. Isocaloric diets with a N content of 1.44, 1.89, 2.50, 2.97, and 3.40% were fed at approximately 1.8 times maintenance intake. Increasing the N content of the diet increased urinary N excretion (P < 0.001) and N balance (P < 0.01), but did not affect the fecal N excretion (P = 0.21). Increasing the level of dietary N, increased urea production (P < 0.001) and excretion (P < 0.001), but no effect (P = 0.24) could be detected in the amount of N recycled to the gut. Urea recycled with the saliva, however, increased (P < 0.001) both in absolute and relative terms, with increasing dietary N. No difference could be detected on the amount of recycled N that was used for anabolism or returned to the ornithine cycle, but less (P = 0.001) N originating from urea was excreted in feces as dietary N increased. Ruminal ammonia concentration increased (P < 0.001) with increasing N intake, but total tract neutral detergent fiber digestibility was depressed only on the lowest N intake diet. No difference (P = 0.30) was detected in ruminal microbial yield among diets, but more (P < 0.003) N was derived from blood urea at low N intakes, and the efficiency of use of the recycled N decreased (P < 0.001) with increasing levels of dietary N. Adaptive changes to low-N diets were a decrease (P < 0.003) in the renal clearance of urea and an increase (P < 0.001) in the gastrointestinal clearance of urea. Urea transporters were present in the rumen wall of the heifers and differentially expressed depending on dietary N content, but their role in the transfer of urea into the rumen remains uncertain. Different mechanisms of N salvage and recycling were involved when animals were fed low-N diets that ensured a supply of endogenous N to the gastrointestinal tract and, due to the reduced contribution of dietary N, an increased efficiency of the N recycled was observed.

Nitric Oxide (NO) plays a critical role in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (Asl) deficiency exhibits a distinct phenotype manifest by multi-organ dysfunction and NO deficiency. Loss of Asl leads to reduced NO synthesis due to decreased endogenous arginine synthesis as well as reduced utilization of extracellular arginine for NO production in both humans and mice. Hence, ASL as seen in other species through evolution has a structural function in addition to its catalytic activity. Importantly, therapy with nitrite rescued the tissue autonomous NO deficiency in hypomorphic Asl mice, while a NOS independent NO donor restored NO-dependent vascular reactivity in subjects with ASL deficiency. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as treatment of NO-related diseases.