We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to targetMTAP(a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5 crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5 CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5 CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5 CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5 CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5 CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

The development of resistance to chemotherapy is a major obstacle for lasting effective treatment of cancer. Here, we demonstrate that endogenous mesenchymal stem cells (MSCs) become activated during treatment with platinum analogs and secrete factors that protect tumor cells against a range of chemotherapeutics. Through a metabolomics approach, we identified two distinct platinum-induced polyunsaturated fatty acids (PIFAs), 12-oxo-5,8,10-heptadecatrienoic acid (KHT) and hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)), that in minute quantities induce resistance to a broad spectrum of chemotherapeutic agents. Interestingly, blocking central enzymes involved in the production of these PIFAs (cyclooxygenase-1 and thromboxane synthase) prevents MSC-induced resistance. Our findings show that MSCs are potent mediators of resistance to chemotherapy and reveal targets to enhance chemotherapy efficacy in patients.

Background & AimsCongenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids.MethodsWe collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7.ResultsIn the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids.ConclusionsWe identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.

Quantitative proteomics, lifespan analysis, and biochemical assays were utilized to show that Insulin/IGF-1-mediated longevity in C. elegans is strongly associated with a daf-16 dependent global reduction in protein metabolism.

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SummaryOxidative damage is thought to be a major cause in development of pathologies and aging. However, quantification of oxidative damage is methodologically difficult. Here, we present a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach for accurate, sensitive, and linear in vivo quantification of endogenous oxidative damage in the nematode Caenorhabditis elegans, based on F3-isoprostanes. F3-isoprostanes are prostaglandin-like markers of oxidative damage derived from lipid peroxidation by Reactive Oxygen Species (ROS). Oxidative damage was quantified in whole animals and in multiple cellular compartments, including mitochondria and peroxisomes. Mutants of the mitochondrial electron transport proteins mev-1 and clk-1 showed increased oxidative damage levels. Furthermore, analysis of Superoxide Dismutase (sod) and Catalase (ctl) mutants uncovered that oxidative damage levels cannot be inferred from the phenotype of resistance to pro-oxidants alone and revealed high oxidative damage in a small group of chemosensory neurons. Longitudinal analysis of aging nematodes revealed that oxidative damage increased specifically with postreproductive age. Remarkably, aging of the stress-resistant and long-lived daf-2 insulin/IGF-1 receptor mutant involved distinct daf-16-dependent phases of oxidative damage including a temporal increase at young adulthood. These observations are consistent with a hormetic response to ROS.