Enteroendocrine cell lineages that differentially control feeding and gut motility.

Enteroendocrine cells are specialized sensory cells of the gut-brain axis that are sparsely distributed along the intestinal epithelium. The functions of enteroendocrine cells have classically been inferred by the gut hormones they release. However, individual enteroendocrine cells typically produce multiple, sometimes apparently opposing, gut hormones in combination, and some gut hormones are also produced elsewhere in the body. Here, we developed approaches involving intersectional genetics to enable selective access to enteroendocrine cells in vivo in mice. We targeted FlpO expression to the endogenous Villin1 locus (in Vil1-p2a-FlpO knock-in mice) to restrict reporter expression to intestinal epithelium. Combined use of Cre and Flp alleles effectively targeted major transcriptome-defined enteroendocrine cell lineages that produce serotonin, glucagon-like peptide 1, cholecystokinin, somatostatin, or glucose-dependent insulinotropic polypeptide. Chemogenetic activation of different enteroendocrine cell types variably impacted feeding behavior and gut motility. Defining the physiological roles of different enteroendocrine cell types provides an essential framework for understanding sensory biology of the intestine.

A brainstem map for visceral sensations.

The nervous system uses various coding strategies to process sensory inputs. For example, the olfactory system uses large receptor repertoires and is wired to recognize diverse odours, whereas the visual system provides high acuity of object position, form and movement1-5. Compared to external sensory systems, principles that underlie sensory processing by the interoceptive nervous system remain poorly defined. Here we developed a two-photon calcium imaging preparation to understand internal organ representations in the nucleus of the solitary tract (NTS), a sensory gateway in the brainstem that receives vagal and other inputs from the body. Focusing on gut and upper airway stimuli, we observed that individual NTS neurons are tuned to detect signals from particular organs and are topographically organized on the basis of body position. Moreover, some mechanosensory and chemosensory inputs from the same organ converge centrally. Sensory inputs engage specific NTS domains with defined locations, each containing heterogeneous cell types. Spatial representations of different organs are further sharpened in the NTS beyond what is achieved by vagal axon sorting alone, as blockade of brainstem inhibition broadens neural tuning and disorganizes visceral representations. These findings reveal basic organizational features used by the brain to process interoceptive inputs.

Area Postrema Cell Types that Mediate Nausea-Associated Behaviors.

Nausea, the unpleasant sensation of visceral malaise, remains a mysterious process. The area postrema is implicated in some nausea responses and is anatomically privileged to detect blood-borne signals. To investigate nausea mechanisms, we built an area postrema cell atlas through single-nucleus RNA sequencing, revealing a few neuron types. Using mouse genetic tools for cell-specific manipulation, we discovered excitatory neurons that induce nausea-related behaviors, with one neuron type mediating aversion imposed by multiple poisons. Nausea-associated responses to agonists of identified area postrema receptors were observed and suppressed by targeted cell ablation and/or gene knockout. Anatomical mapping revealed a distributed network of long-range excitatory but not inhibitory projections with subtype-specific patterning. These studies reveal the basic organization of area postrema nausea circuitry and provide a framework toward understanding and therapeutically controlling nausea.

Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer.

Diagnosis of pancreatic ductal adenocarcinoma (PDAC) is associated with a dismal prognosis despite current best therapies; therefore new treatment strategies are urgently required. Numerous studies have suggested that epithelial-to-mesenchymal transition (EMT) contributes to early-stage dissemination of cancer cells and is pivotal for invasion and metastasis of PDAC. EMT is associated with phenotypic conversion of epithelial cells into mesenchymal-like cells in cell culture conditions, although such defined mesenchymal conversion (with spindle-shaped morphology) of epithelial cells in vivo is rare, with quasi-mesenchymal phenotypes occasionally observed in the tumour (partial EMT). Most studies exploring the functional role of EMT in tumours have depended on cell-culture-induced loss-of-function and gain-of-function experiments involving EMT-inducing transcription factors such as Twist, Snail and Zeb1 (refs 2, 3, 7-10). Therefore, the functional contribution of EMT to invasion and metastasis remains unclear, and genetically engineered mouse models to address a causal connection are lacking. Here we functionally probe the role of EMT in PDAC by generating mouse models of PDAC with deletion of Snail or Twist, two key transcription factors responsible for EMT. EMT suppression in the primary tumour does not alter the emergence of invasive PDAC, systemic dissemination or metastasis. Suppression of EMT leads to an increase in cancer cell proliferation with enhanced expression of nucleoside transporters in tumours, contributing to enhanced sensitivity to gemcitabine treatment and increased overall survival of mice. Collectively, our study suggests that Snail- or Twist-induced EMT is not rate-limiting for invasion and metastasis, but highlights the importance of combining EMT inhibition with chemotherapy for the treatment of pancreatic cancer.

Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.

Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1(+) circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1(+) crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1(+) crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1(+) crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1(+) crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.