Generation of pulmonary neuroendocrine cells and SCLC-like tumors from human embryonic stem cells.
ABSTRACT: Cancer models based on cells derived from human embryonic stem cells (hESCs) may reveal why certain constellations of genetic changes drive carcinogenesis in specialized lineages. Here we demonstrate that inhibition of NOTCH signaling induces up to 10% of lung progenitor cells to form pulmonary neuroendocrine cells (PNECs), putative precursors to small cell lung cancers (SCLCs), and we can increase PNECs by reducing levels of retinoblastoma (RB) proteins with inhibitory RNA. Reducing levels of TP53 protein or expressing mutant KRAS or EGFR genes did not induce or expand PNECs, but tumors resembling early-stage SCLC grew in immunodeficient mice after subcutaneous injection of PNEC-containing cultures in which expression of both RB and TP53 was blocked. Single-cell RNA profiles of PNECs are heterogeneous; when RB levels are reduced, the profiles resemble those from early-stage SCLC; and when both RB and TP53 levels are reduced, the transcriptome is enriched with cell cycle-specific RNAs. Our findings suggest that genetic manipulation of hESC-derived pulmonary cells will enable studies of this recalcitrant cancer.
Project description:Mammalian lungs have the ability to recognize external environments by sensing different compounds in inhaled air. Pulmonary neuroendocrine cells (PNECs) are rare, multi-functional epithelial cells currently garnering attention as intrapulmonary sensors; PNECs can detect hypoxic conditions through chemoreception. Because PNEC overactivation has been reported in patients suffering from respiratory diseases - such as asthma, chronic obstructive pulmonary disease, bronchopulmonary dysplasia and other congenital diseases - an improved understanding of the fundamental characteristics of PNECs is becoming crucial in pulmonary biology and pathology. During the past decade, murine genetics and disease models revealed the involvement of PNECs in lung ventilation dynamics, mechanosensing and the type 2 immune responses. Single-cell RNA sequencing further unveiled heterogeneous gene expression profiles in the PNEC population and revealed that a small number of PNECs undergo reprogramming during regeneration. Aberrant large clusters of PNECs have been observed in neuroendocrine tumors, including small-cell lung cancer (SCLC). Modern innovation of imaging analyses has enabled the discovery of dynamic migratory behaviors of PNECs during airway development, perhaps relating to SCLC malignancy. This Review summarizes the findings from research on PNECs, along with novel knowledge about their function. In addition, it thoroughly addresses the relevant questions concerning the molecular pathology of pulmonary diseases and related therapeutic approaches.
Project description:Pulmonary neuroendocrine cells (PNECs) are proposed to be the first specialized cell type to appear in the lung, but their ontogeny remains obscure. Although studies of PNECs have suggested their involvement in a number of lung functions, neither their in vivo significance nor the molecular mechanisms underlying them have been elucidated. Importantly, PNECs have long been speculated to constitute the cells of origin of human small-cell lung cancer (SCLC) and recent mouse models support this hypothesis. However, a genetic system that permits tracing the early events of PNEC transformation has not been available. To address these key issues, we developed a genetic tool in mice by introducing a fusion protein of Cre recombinase and estrogen receptor (CreER) into the calcitonin gene-related peptide (CGRP) locus that encodes a major peptide in PNECs. The CGRP(CreER) mouse line has enabled us to manipulate gene activity in PNECs. Lineage tracing using this tool revealed the plasticity of PNECs. PNECs can be colabeled with alveolar cells during lung development, and following lung injury, PNECs can contribute to Clara cells and ciliated cells. Contrary to the current model, we observed that elimination of PNECs has no apparent consequence on Clara cell recovery. We also created mouse models of SCLC in which CGRP(CreER) was used to ablate multiple tumor suppressors in PNECs that were simultaneously labeled for following their fate. Our findings suggest that SCLC can originate from differentiated PNECs. Together, these studies provide unique insight into PNEC lineage and function and establish the foundation of investigating how PNECs contribute to lung homeostasis, injury/repair, and tumorigenesis.
Project description:Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non-small cell tumors and secondary transitions from non-small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of "small cell-ness" based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT-signaling in the context of mutual bi-allelic RB1 and TP53 lesions. Additionally, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH-ASCL1-RB-p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon-based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well.
Project description:Expansion of pulmonary neuroendocrine cells (PNECs) is a pathological feature of many human lung diseases. Human PNECs are inherently difficult to study due to their rarity (<1% of total lung cells) and a lack of established protocols for their isolation. We used induced pluripotent stem cells (iPSCs) to generate induced PNECs (iPNECs), which express core PNEC markers, including ROBO receptors, and secrete major neuropeptides, recapitulating known functions of primary PNECs. Furthermore, we demonstrate that differentiation efficiency is increased in the presence of an air-liquid interface and inhibition of Notch signaling. Single-cell RNA sequencing (scRNA-seq) revealed a PNEC-associated gene expression profile that is concordant between iPNECs and human fetal PNECs. In addition, pseudotime analysis of scRNA-seq results suggests a basal cell origin of human iPNECs. In conclusion, our model has the potential to provide an unlimited source of human iPNECs to explore PNEC pathophysiology associated with several lung diseases.
Project description:Pulmonary neuroendocrine cells (PNECs) are the only innervated airway epithelial cells. To what extent neural innervation regulates PNEC secretion and function is unknown. Here, we discover that neurotrophin 4 (NT4) plays an essential role in mucus overproduction after early life allergen exposure by orchestrating PNEC innervation and secretion of GABA. We found that PNECs were the only cellular source of GABA in airways. In addition, PNECs expressed NT4 as a target-derived mechanism underlying PNEC innervation during development. Early life allergen exposure elevated the level of NT4 and caused PNEC hyperinnervation and nodose neuron hyperactivity. Associated with aberrant PNEC innervation, the authors discovered that GABA hypersecretion was required for the induction of mucin Muc5ac expression. In contrast, NT4-/- mice were protected from allergen-induced mucus overproduction and changes along the nerve-PNEC axis without any defects in inflammation. Last, GABA installation restored mucus overproduction in NT4-/- mice after early life allergen exposure. Together, our findings provide the first evidence for NT4-dependent neural regulation of PNEC secretion of GABA in a neonatal disease model. Targeting the nerve-PNEC axis may be a valid treatment strategy for mucus overproduction in airway diseases, such as childhood asthma.-Barrios, J., Patel, K. R., Aven, L., Achey, R., Minns, M. S., Lee, Y., Trinkaus-Randall, V. E., Ai, X. Early life allergen-induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion.
Project description:BACKGROUND:Histologically, SCLC are classified as pure (P-SCLC) and combined subtypes (C-SCLC). Currently, few studies compare the clinicopathological characteristics and explore the treatment strategies applied to them. METHODS:Between July 2005 and April 2016, the clinical records of 297 postoperative patients with pathologically confirmed SCLC were retrospectively analyzed. Kaplan-Meier method and Cox regression model were separately used for stratified univariate and multivariate survival analysis. RESULTS:A total of 46 cases (15.5%) of C-SCLCs and 251 cases (85.5%) of pure SCLCs (P-SCLCs) were included in this study. The average age of C-SCLCs was a little higher than that of P-SCLCs (59.65?±?8.72 vs. 56.56?±?10.12; P = 0.053). More patients had a history of smoking in C-SCLC (78.3% vs. 63.3%; P = 0.074). The five-year overall survival (OS) rate for P-SCLCs and C-SCLCs was 65.1% and 56.7%, respectively (P = 0.683). For P-SCLC, stage and an intervention of prophylactic cranial irradiation (PCI) were independent factors that affected OS. In C-SCLCs cases, performing sublobectomy was an independent risk factor for poor prognosis. CONCLUSIONS:We identified no significant difference in clinical characteristics and outcome between C-SCLCs and P-SCLCs. However, the factors affecting the prognosis of the two subtypes were slightly inconsistent. For C-SCLCs, the extent of resection had a greater impact on survival, and lobectomy combined with systemic lymph node dissection should therefore be performed as extensively as possible. In addition, PCI was beneficial in improving the SCLC OS rate. KEY POINTS:This study demonstrated the prognosis of C-SCLCs did not significantly differ from that of P-SCLCs, but was more susceptible to the extent of resection. Patients with C-SCLC who underwent limited resection had a significantly increased risk of shorter OS. This study highlighted the importance of performing lobectomy for resectable C-SCLC patients. This study also proved the benefit of PCI in improving the OS rate for both P-SCLC and C-SCLC patients.
Project description:Pulmonary neuroendocrine cells (PNECs) are sensory cells within the lung airway epithelia. Here, we provide a detailed protocol for generating induced PNECs (iPNECs) from human induced pluripotent stem cells (iPSCs). The cellular and molecular profile of iPNECs resembles primary human PNECs. Primary human PNECs are exceedingly rare, comprising only 1% of the adult lung. Therefore, a self-renewing source of patient-specific iPNECs facilitates the creation of reproducible human cellular models to study lung diseases characterized by PNEC dysfunction. For complete details on the use and execution of this protocol, please refer to Hor et al. (2020).
Project description:Expansion of pulmonary neuroendocrine cells (PNECs) is a pathological feature of many human lung diseases. Human PNECs are inherently difficult to study due to their rarity (<1% of total lung cells) and a lack of established protocols for their isolation. We used induced pluripotent stem cells (iPSCs) to generate induced PNECs (iPNECs) which express core PNEC markers, genes therby recapitulating known functions of primary PNECs. Single-cell RNA sequencing (scRNA-seq) of these iPNEC revealed a PNEC-associated gene expression profile that is concordant between iPNECs and human fetal PNECs. In conclusion, our model has the potential to provide an unlimited source of human iPNECs to explore PNEC pathophysiology associated with several lung diseases. Overall design: Examination of 2 iPSC replicates for their differentiation to iPNEC.
Project description:Pulmonary neuroendocrine cells (PNECs) are rare airway epithelial cells whose function is poorly understood. Here we show that Ascl1-mutant mice that have no PNECs exhibit severely blunted mucosal type 2 response in models of allergic asthma. PNECs reside in close proximity to group 2 innate lymphoid cells (ILC2s) near airway branch points. PNECs act through calcitonin gene-related peptide (CGRP) to stimulate ILC2s and elicit downstream immune responses. In addition, PNECs act through the neurotransmitter ?-aminobutyric acid (GABA) to induce goblet cell hyperplasia. The instillation of a mixture of CGRP and GABA in Ascl1-mutant airways restores both immune and goblet cell responses. In accordance, lungs from human asthmatics show increased PNECs. These findings demonstrate that the PNEC-ILC2 neuroimmunological modules function at airway branch points to amplify allergic asthma responses.
Project description:The lung is constantly exposed to environmental atmospheric cues. How it senses and responds to these cues is poorly defined. Here, we show that Roundabout receptor (Robo) genes are expressed in pulmonary neuroendocrine cells (PNECs), a rare, innervated epithelial population. Robo inactivation in mouse lung results in an inability of PNECs to cluster into sensory organoids and triggers increased neuropeptide production upon exposure to air. Excess neuropeptides lead to an increase in immune infiltrates, which in turn remodel the matrix and irreversibly simplify the alveoli. We demonstrate in vivo that PNECs act as precise airway sensors that elicit immune responses via neuropeptides. These findings suggest that the PNEC and neuropeptide abnormalities documented in a wide array of pulmonary diseases may profoundly affect symptoms and progression.