Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify nongenetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.

30Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly 31 desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving 32 PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism 33 of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for 34 clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find 35 that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. 36Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino 37 acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity 38 that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for 39 glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open 40 chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and 41 mitigates tumor growth. These findings identify non-genetic adaptations to nutrient deprivation in PDA 42 and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients. 43 44 3 stroma restrict nutrient access, remains unknown. 53Because nutrient availability largely dictates metabolic behavior 10 , the relevance of studying 54 tumor metabolism within the native environment has been recently underscored [11][12][13][14][15] . Although culture 55 media composition can be modulated to mimic circulating metabolite levels 16, 17 , this modulation may 56 not accurately reflect the tumor metabolic microenvironment. In particular, levels of glucose and 57 glutamine, two of the most abundant and tumor-preferred nutrients in the circulation, can be limiting in 58 PDA tumors, compared to benign adjacent tissue 7 , and significantly lower within tumor cores, compared 59 to the periphery 18, 19 . Paradoxically, both glucose and glutamine are routinely supplemented in culture 60 media at levels that are significantly higher than the circulation: 11mM glucose and 2mM glutamine in 61 RPMI compared to ~ 5.5mM glucose and 0.6mM glutamine in serum 20 . Although prior studies focused 62 on depriving tumor cells of either glucose or glutamine alone 9, 21-24 , how PDA cells survive and 63 proliferate under limiting levels of both major nutrients is not well understood. 64To investigate this, we selected for clonal PDA cells that survive and adapt to limiting levels of 65 both glucose and glutamine. We find that the adapted clones have enhanced proliferation in vitro and 66 tumor-forming capacity in vivo. They also share common signaling, transcriptional and metabolic 67 alterations that are acquired upon adaptation. These include a post-translational role for mTORC1 68 signaling in the stabilization of glutamine synthetase (GS), and the use of amino acids for the synthesis...

Background/AimsStudies of human cadaveric pancreas specimens indicate that pancreas inflammation plays an important role in type 1 diabetes pathogenesis. Due to the inaccessibility of pancreas in living patients, imaging technology to visualize pancreas inflammation is much in need. In this study, we investigated the feasibility of utilizing ultrasound imaging to assess pancreas inflammation longitudinally in living rats during the progression leading to type 1 diabetes onset.MethodsThe virus-inducible BBDR type 1 diabetes rat model was used to systematically investigate pancreas changes that occur prior to and during development of autoimmunity. The nearly 100% diabetes incidence upon virus induction and the highly consistent time course of this rat model make longitudinal imaging examination possible. A combination of histology, immunoblotting, flow cytometry, and ultrasound imaging technology was used to identify stage-specific pancreas changes.ResultsOur histology data indicated that exocrine pancreas tissue of the diabetes-induced rats underwent dramatic changes, including blood vessel dilation and increased CD8+ cell infiltration, at a very early stage of disease initiation. Ultrasound imaging data revealed significant acute and persistent pancreas inflammation in the diabetes-induced rats. The pancreas micro-vasculature was significantly dilated one day after diabetes induction, and large blood vessel (superior mesenteric artery in this study) dilation and inflammation occurred several days later, but still prior to any observable autoimmune cell infiltration of the pancreatic islets.ConclusionsOur data demonstrate that ultrasound imaging technology can detect pancreas inflammation in living rats during the development of type 1 diabetes. Due to ultrasound’s established use as a non-invasive diagnostic tool, it may prove useful in a clinical setting for type 1 diabetes risk prediction prior to autoimmunity and to assess the effectiveness of potential therapeutics.

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